Engineering bispecific for targeted delivery of cytotoxin- loaded nanoparticles to tumour cells

by Karin Taylor

Bachelor of Science (Genetics) Bachelor of Science Honours (Genetics) Graduate Certificate in Business

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 The Australian Institute for Bioengineering and Nanotechnology (AIBN) Abstract

First-line treatments, such as surgical removal of tumours, are necessary but highly invasive and can only be of therapeutic benefit if the cancer has not yet spread to other organs. and radiotherapy can help to slow the spread of cancer, but the systemic exposure leads to cumulative and cytotoxic effects, which leave the patient immune-compromised and susceptible to organ failure. This highlights the need to develop targeted therapies capable of delivering such drugs directly to the cancer cells, to overcome drug resistance and limit the cytotoxic effects associated with chemotherapeutics.

Monoclonal antibodies (mAbs) provide a means to target conjugated drugs or radio-labels while also having therapeutic benefits in their own right. Cancer cells are often characterised by the overexpression of particular cell surface biomarkers, and these biomarkers make ideal targets for delivery of drugs via specific mAbs. The epidermal growth factor receptor (EGFR) is a validated cell surface that has been extensively evaluated in the literature. EGFR is associated with a number of different including breast and colon, and anti-EGFR mAbs are approved for therapeutic use (e.g. and ). Drug-conjugated anti-EGFR mAbs are also under pre- clinical and clinical evaluation. However significant challenges remain, as some cancers are refractive to mAb therapy due to pre-existing and acquired resistance to a given treatment, both mAb and drug related. The drug dosage carried by conjugated antibodies is limited to the number of molecules that can be attached to the without compromising the antibody’s binding affinity.

Another novel approach lies in the development of nanoparticles capable of carrying a high therapeutic payload, including cytotoxins, DNA, siRNA or other therapeutic agents. Inclusion of a targeting system on such drug delivery vehicles provides a means to deliver concentrated doses directly to sites of interest while simultaneously protecting the payload from the environment and vice versa. The EnGeneIC drug delivery vehicle (EDVTMnanocell) is a novel 400 nm anucleate bacterial nanoparticle, which can be loaded with high concentrations of various drugs and utilises bispecific antibodies (BsAbs) as a targeting moiety. These BsAbs bind both the EDVTMnanocell and the cancer cell biomarker. Initial clinical testing of the BsAb-EDVTMnanocell system has been shown to exhibit promising outcomes in vitro and in initial clinical trials. In preliminary studies, i EDVTMnanocells were targeted to EGFR-expressing cells utilising a BsAb produced by linking two separate mAbs via A/G. However, problems were identified relating to product uniformity, with a propensity for forming cross-linked aggregates impacting the ability to produce these BsAb preparations economically.

In this project we have created engineered BsAbs to facilitate the targeting of EDVTMnanocells directly to a cancer cell surface antigen, EGFR, allowing improved product quality and consistency. We have engineered a number of BsAb formats for production in the CHO-S expression system, and investigated their ability to bind to the EDVTMnanocell and subsequently facilitate targeting. We have related changes in BsAb structure to final BsAb product yield, stability and ability to bind recombinant and native targets in vitro and in vivo following purification by chromatography techniques. While all formats were capable of EDVTMnanocell targeting, differences in product yield and stability were observed.

Development of processes to produce high levels of BsAbs have proved to be more complicated than standard production techniques required for mAb development owing to the lower expression levels of BsAbs and their inherent downstream instability. Slight modifications of the BsAb amino acid sequence resulted in two similar constructs showing a 6-fold difference in product yield. However, the same changes in sequence resulted in improved downstream and long-term stability of the product, strengthening the need to develop optimal systems for BsAb production. Surface Plasmon Resonance analyses showed that these engineered BsAbs retain high binding affinities for EGFR comparable to the parent mAb. Furthermore, BsAbs are able to bind EGFR-overexpressing MDA-MB-468 breast cancer cells both independently and while bound to EDVTMnanocells. Selection of an engineered BsAb that has shown optimal value in the ability to consistently produce a uniform, predictable amount of BsAb has enabled further in vivo product characterisation. Tumour regression data produced with our research partners EnGeneIC has shown that in Balb/C athymic nude mouse xenograft models, doxorubicin-loaded EDVTMnanocells with bound BsAbs have a positive effect on tumour regression. The optimised format selected by EnGeneIC has now enabled the commencement of scale-up BsAb production for use in a Phase IIa clinical trial.

The modular design of the BsAb optimised during this work allows the substitution of the EGFR targeting domain for other specificities. As such, this design is now being ii investigated by other members of our laboratory for the targeting of EDVTMnanocells to other cancer cell biomarkers such as mesothelin and c-kit. This allows the EDVTMnanocell to be tailored to particular cancer types, leading to a personalised medicine approach for the patient.

iii 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, 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 research higher degree 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.

iv Publications during candidature

Peer-reviewed journal papers

K. Taylor, C. B. Howard, M. L. Jones, I. Sedliarou, H. Brahmbhatt, J. A. MacDiarmid ,T. P. Munro and S. M. Mahler. Nanocell targeting using engineered bispecific antibodies. mAbs 2015; 7:53-65. DOI: 10.4161/19420862.2014.985952. 2013 Impact Factor: 4.726

Oral conference presentations

K. Taylor, C. B. Howard, M. L. Jones, I. Sedliarou, S. M. Mahler and T. P. Munro. Bispecific Antibody Production: Addressing problems with stability. Annual Bio Processing Network (BPN) Conference, Gold Coast, Australia. October 2013.

K. Taylor, C. B. Howard, M. L. Jones, I. Sedliarou, S. M. Mahler and T. P. Munro. Next generation bispecific antibody design and the influence thereof on product yield and stability. The 23rd European Society for Animal Cell Technology (ESACT), Lille, France. June 2013

Poster conference presentations

K. Taylor, C. B. Howard, M. L. Jones, I. Sedliarou, S. M. Mahler and T. P. Munro. Engineering bispecific antibodies and the implications of design on expression levels, stability and binding of targets. Poster presentation delivered at the 9th Annual Essential Protein Engineering Summit (PEGS), Boston, MA, USA. May 2013.

K. Taylor, C. B. Howard, M. L. Jones, S. M. Mahler and T. P. Munro. Development and characterisation of bispecific antibodies. Poster presentation delivered at the IMB Student Symposium, Brisbane, Australia. November 2012.

K. Taylor, C. B. Howard, M. L. Jones, S. M. Mahler and T. P. Munro. Development and characterisation of bispecific antibodies. Poster presentation delivered at the International Conference on BioNano Innovation (ICBNI), Brisbane, Australia. July 2012.

v Publications included in this thesis

Peer-reviewed journal papers

K. Taylor, C. B. Howard, M. L. Jones, I. Sedliarou, H. Brahmbhatt, J. A. MacDiarmid ,T. P. Munro and S. M. Mahler. Nanocell targeting using engineered bispecific antibodies. mAbs 2015; 7:53-65. DOI: 10.4161/19420862.2014.985952. 2013 Impact Factor: 4.726 • Incorporated as part of Chapters 2, 3, 4 and 5 Contributor Statement of contribution K. Taylor (Candidate) Designed experiments (80%), wrote and edited the paper (80%) and completed physical experimental work (85%). C.. B. Howard Advised on experimental design (20%) and helped with physical experimental work (10%). M. L. Jones Designed experiments (10%), edited the paper (10%), advised on experimental design (25%). I. Sedliarou Advised on (EDV related) experimental design (5%), completed physical (mouse xenograft ) experimental work (5%). H.. Brahmbhatt Commercial partner – final approval for experiments (50%) J. A. MacDiarmid Commercial partner – final approval for experiments (50%) T. P. Munro Designed experiments (5%), advised on experimental design (25%) S. M. Mahler Designed experiments (5%), edited the paper (10%), advised on experimental design (25%).

vi Contributions by others to the thesis

The nanoparticles used in this project are the proprietary property of EnGeneIC Pty Ltd. As such all EnGeneIC delivery vehicle (EDV)TMnanocells were produced at the company’s facilities in Sydney. BsAb binding to EDVs and in vitro experiments were completed jointly by myself and Ilya Sedliarou at EnGeneIC when possible. If not Ilya Sedliarou would prepare slides and sample aliquots and courier products to Brisbane for further analysis by myself. Mouse xenograft models were completed and results analysed by experienced staff based at EnGeneIC after collaborative planning with regards to which sample populations and controls needed to be included in the in vivo experiments. Non-routine work such as dynamic light scattering and mass spectrometry were outsourced due to timing constraints and facilities being unavailable to us at the time. Martina Jones and Stephen Mahler provided substantial amounts of input and advice regarding the editing of the journal article to make it suitable for publication in mAbs.

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

None

vii Acknowledgements

On final completion of this PhD thesis I would firstly like to thank all members of the National Biologics Facility (NBF) and the Gray group at the Australian Institute for Bioengineering and Nanotechnology (AIBN) that have helped train and facilitate me to complete my project in a timely manner.

I would like to thank the members of the Mahler group for their advice during the course of this project relating to all aspects of the experience. In particular, Dr Chris Howard for always being ready and available to help with experiments in the lab; particularly production based work and maintaining experiments when I was away. My past and present advisors, Dr Trent Munro, Dr Martina Jones and Associate Professor Stephen Mahler for their patience and guidance with the planning and “fine-tuning” of my project.

Thanks are also due to our collaborators, EnGeneIC Ltd, without whom there would not have been a project. I give thanks that they provided me with access to their facilities when I needed to characterise my antibodies. Dr Ilya Sedliarou is owed particular thanks for performing repeat experiments for me at EnGeneIC and for conscientiously providing me with many prepared slides for confocal microscope analyses.

The Australian National Fabrication Facility, Queensland node (ANFF-Q), is also owed thanks for providing access to their confocal microscope to enable me to complete many hours of confocal microscopy. I would like to say a very big thank you to Dr Elena Taran who trained me to use the confocal microscope and was always able to find a moment to help and advise us on any experimental ideas and outcomes we might have had.

Furthermore I’d like to thank all funding bodies that have contributed to this project. Firstly the Australian Government for providing support by way of an ARC Linkage Grant and my personal scholarship an Australian Postgraduate Award. I also thank the AIBN for providing a top-up scholarship throughout my candidature. Lastly I’d also like to acknowledge the bursary provided to me by the European Society for Animal Cell Technology (ESACT) conference organising committee, which assisted me in travelling to and presenting at the conference.

viii Keywords bispecific antibody, nanoparticle, aggregation, mammalian expression, CHO-S, epidermal growth factor receptor, confocal microscopy, active targeting

Australian and New Zealand Standard Research Classification (ANZSRC)

100403, Medical Molecular Engineering of Nucleic Acids and , 70%

060199, Biochemistry and Cell Biology not elsewhere classified, 30%

Fields of Research (FoR) Classification

1004, Medical Biotechnology, 70% 0601, Biochemistry and Cell Biology, 30%

ix Table of contents

Abstract ...... i Declaration by author ...... iv Publications during candidature ...... v Publications included in this thesis...... vi Contributions by others to the thesis ...... vii Statement of parts of the thesis submitted to qualify for the award of another degree ...... vii Acknowledgements ...... viii Keywords ...... ix Australian and New Zealand Standard Research Classification (ANZSRC) ...... ix Fields of Research (FoR) Classification ...... ix Table of contents...... x List of figures ...... xiii List of tables ...... xv List of abbreviations ...... xv i

1. Introduction ...... 1

1.1. Cancer therapeutics ...... 1 1.2. Cell surface markers ...... 3 1.2.1. Epidermal Growth Factor Receptor...... 6 1.3. Monoclonal antibodies ...... 8 1.3.1. Historical perspective ...... 8 1.3.2. mAb types and structure ...... 9 1.3.3. Biopharmaceutical product market...... 11 1.4. Bispecific antibodies ...... 14 1.4.1. Historical perspective ...... 14 1.4.2. Modes of Action ...... 16 1.4.3. Current development ...... 18 1.4.4. Future prospects and hurdles to development ...... 30 1.5. Nanoparticles ...... 32 1.5.1. Passive and active targeting ...... 32 1.5.2. Types of nanoparticles ...... 36 1.5.3. The EnGeneIC delivery vehicle...... 37 1.5.4. BsAbs as facilitators of therapeutic NP delivery ...... 40 1.6. Conclusion and research aims...... 43 1.6.1. The future of nanoparticles ...... 43 1.6.2. Research aims and strategy ...... 44

x 2. Materials and methods ...... 46

2.1. EnGeneIC delivery vehicle (EDVTMnanocell) production ...... 46 2.2. Routine molecular biology techniques ...... 46 2.2.1. Vector design ...... 46 2.2.2. Bacterial transformation ...... 46 2.2.3. Colony PCR ...... 47 2.2.4. E. coli culture for maintenance of DNA stocks ...... 47 2.2.5. DNA Sequencing set up ...... 48 2.3. Sequence design ...... 49 2.3.1. EGFR-ecd ...... 49 2.3.2. EGFR-targeting and negative-control scFv ...... 49 2.3.3. EDV-targeting scFv ...... 49 2.3.4. BsAb sequences ...... 51 2.3.5. Commercial mAbs – positive and negative controls ...... 57 2.4. Mammalian cell culture ...... 58 2.4.1. Cell resuscitation and passaging ...... 58 2.4.2. Transient cell culture media and techniques ...... 58 2.5. Qualitative western blot for BsAb expression analysis ...... 59 2.6. Affinity chromatography ...... 59 2.6.1. IMAC and TFF ...... 59 2.6.2. ...... 60 2.7. Protein quality ...... 60 2.7.1. Quantification ...... 60 2.7.2. PAGE gels ...... 61 2.7.3. Size exclusion chromatography (SEC) HPLC ...... 61 2.8. Stability monitoring ...... 62 2.8.1. Storage buffer formulation ...... 62 2.8.2. Dialysis into formulation buffers ...... 62 2.8.3. DLS performed at CSL ...... 63 2.8.4. Analysis of tandem scFv by differential scanning calorimetry ...... 63 2.9. Functional binding ...... 64 2.9.1. Surface plasmon resonance (SPR)...... 64 2.9.2. Biolayer interferometry kinetic characterisation of LPS binding ...... 64 2.9.3. Flow cytometry against MDA-MB-468 breast cancer cells ...... 64 2.9.4. BsAb- EDVTMnanocell preparation ...... 65 2.9.5. BsAb- EDVTMnanocells aggregation analysis using fluorescence microscopy ...... 65 2.9.6. Co-localisation of BsAbs alongside EGFR-ecd and EDVTMnanocells ...... 66 2.9.7. Flow cytometry of BsAb-EDVTMnanocells in vitro binding to MDA-MB-468 cells...... 66 2.9.8. Confocal microscopy of in vitro binding of BsAb-EDVTMnanocells to MDA-MB-468 cells ...... 67 2.9.9. Effect of ABX-EGF tandem scFv-EDVTMnanocell on tumour regression in vivo ...... 67

xi 3. Bispecific antibody design, production and function ...... 69

3.1. Chapter summary ...... 69 3.2. Introduction ...... 69 3.3. Results ...... 73 3.3.1. Production, yield and stability ...... 73 3.3.2. Functional binding ...... 88 3.4. Discussion and Conclusion ...... 94

4. Targeting EnGeneIC drug delivery vehicles (EDVTMnanocells) ...... 97

4.1. Chapter summary ...... 97 4.2. Introduction ...... 97 4.3. Results ...... 101 4.3.1. Characterisation of stable BsAbs binding to EDVTMnanocells ...... 101 4.3.2. In vitro binding of BsAb-EDVTMnanocells to MDA-MB-468 breast cancer cells ...... 101 4.3.3. EDVTMnanocell-mediated tumour regression in a mouse xenograft model ...... 105 4.4. Discussion and Conclusion ...... 107

5. Concluding remarks and future work ...... 110

6. List of References ...... 113

7. Appendices ...... 136

7.1. mAbs paper published online December 2014 ...... 136

xii List of figures

Figure 1.1: Early discoveries enabling current therapeutic mAb and development. Figure 1.2: Types of monoclonal antibodies. Figure 1.3: Basic or IgG structure. Figure 1.4: BsAb production by chemical conjugation or somatic hybridisation. Figure 1.5: Hybridoma produced antibody combinations. Figure 1.6: Historical BsAb development timeline. Figure 1.7: Applications of Bispecific Antibodies (BsAbs). Figure 1.8: BsAb formats. Figure 1.9: Antibody structural engineering for modified pharmacological effects. Figure 1.10: Crossmab development through Fab domain exchange and KIH technology.  Figure 1.11: TandAb BsAb formation. Figure 1.12: Format variations of ADAPTIR multi-specifics. Figure 1.13: Nanoparticle drug delivery through passive and active targeting. Figure 1.14: Nanoparticle and targeting ligand properties affecting biodistribution and function. Figure 1.15: Loading and functionalisation of EDVTMnanocells.

Figure 2.1: Recombinant Epidermal Growth Factor Receptor (EGFR) sequences. Figure 2.2: Variable chain sequences for BsAb design. Figure 2.3: Bispecific antibody sequence information. Figure 2.4: Diagram representing sequence orders for the design of KIH BsAb constructs.

Figure 3.1: Gel electrophoresis images of the development of the Fc-1H10 sequence. Figure 3.2: Qualitative western blot of BsAb transient expression in CHO-S over time. Figure 3.3: SDS-PAGE gels of purified bispecific antibodies under reducing and non- reducing conditions. Figure 3.4: Size exclusion chromatography results of purified BsAb products stored in PBS buffer. Figure 3.5: Tandem scFv stability data. Figure 3.6: Size exclusion chromatography comparing Vectibix-Fc-1H10 (NBF 715) stability. xiii Figure 3.7: Concentration monitoring of Fc-containing BsAbs in different formulations. Figure 3.8: SDS-PAGE gels of Fc-containing BsAb after 3 months storage at either 4°C or -20°C in different formulation buffers. Figure 3.9: SDS-PAGE gels of purified stability engineered BsAbs under reducing and non-reducing conditions. Figure 3.10: SEC HPLC chromatograms corresponding to Table 3.3. Figure 3.11: Dynamic Light Scattering (DLS) of disulphide engineered BsAbs. Figure 3.12: ABX-EGF (Panitumumab or Vectibix) and 1H10 representative binding curves. Figure 3.13: In vitro binding of BsAbs and IgG1 mAbs at 1µg/ml to EGFR overexpressing MDA-MB-468 Breast cancer cells. Figure 3.14: Co-localisation imaging by confocal microscopy of recombinant EGFR-His (DL650) and EDVTMnanocells (AF488) with or without specific BsAb.

Figure 4.1: Diagrammatic representation of a BsAb created through Protein A/G linkage. Figure 4.2: BsAb format binding to EnGeneICTMnanocells. Figure 4.3: Analysis of BsAb-EDVTMnanocell aggregation post-conjugation. Figure 4.4: In vitro binding of BsAb-EDVTMnanocells to EGFR overexpressing MDA-MB- 468 breast cancer cells. Figure 4.5: Confocal in vitro imaging of stable BsAb-EDVTMnanocells binding to the surface of MDA-MB-468 breast cancer cells. Figure 4.6: Mouse MDA-MB-468 xenograft results following EDVTMnanocell (EDV) treatment.

xiv List of tables

Table 1.1: Therapeutic cell surface targets Table 1.2: Antigen properties for consideration in targeting moiety affinity design. Table 1.3: mAbs targeting EGFR approved and in clinical development. Table 1.4: BsAbs undergoing clinical trials. Table 1.5: Companies developing symmetric, bivalent BsAbs. Table 1.6: Summary of monovalent, BsAbs in development.

Table 2.1: BsAb format and sequence design parameters. Table 2.2: Splicing overlap extension. Table 2.3: Vessels for cell culture. Table 2.4: BsAb vector and protein data.

Table 3.1: Product yield. Table 3.2: Formulation study to evaluate the stability of Fc-containing BsAb Vectibix-Fc- 1H10 at different storage temperatures over a 3 month period. Table 3.3: SEC HPLC evaluation of BsAb stability under varying conditions. Table 3.4: Surface plasmon resonance binding affinities for recombinant EGFR-mFc collected from a Biacore T-200. Table 3.5: Average EDV nanosight counts post BsAb binding.

Table 4.1: Experimental sample breakdown.

xv List of abbreviations

ABX-EGF Vectibix or Panitumumab monoclonal antibody ADC Antibody-drug conjugate AML Acute myelogenous leukemia ANG Angiopoietin BLA Biological licence application BsAb Bispecific antibody BiTE Bispecific T-cell engager CD Cluster of differentiation CDR Complementarity-determining region CEA Carcinoembryonic antigen CHO Chinese hamster ovary CRC Colorectal cancer DART Dual-affinity re-targeting Db Diabody DPBS Dulbecco’s phosphate buffer saline Dox Doxorubicin DPCs Dynamic polyconjugates ECD Extracellular domain EDV EnGeneIC delivery vehicle or EDVTMnanocell EpCAM Epithelial cell adhesion molecule FDA US Food and drug administration Gp Glycoprotein HER Human epidermal growth factor receptor HGF Hepatocyte growth factor HPLC High performance liquid chromatography IGF Insulin-like growth factor IL Interleukin IND Investigational new drug ka Association constant KD Binding affinity kd Dissociation constant KIH Knob-in-hole LRR Leucine rich repeat xvi mAb Monoclonal antibody MOA Mode of action mPEG methoxy PEGylated ng Nanogram nM Nanomolar NME New molecular entity NSCLC Non-small cell lung carcinoma mg Milligram ml Millilitre PSMA Prostate specific membrane antigen RA Rheumatoid arthritis RANKL Receptor activator of nuclear factor kappa-B ligand RTK Receptor tyrosine kinase scDb Single chain diabody scFv Single-chain variable fragment SEC Size exclusion chromatography SLE Systemic lupus erythematosus SMIP Small modular immunopharmaceutical taFv Tandem variable fragment TNF Tumour necrosis factor µg Microgram µl Microlitre VEGF Vascular endothelial growth factor VH Variable heavy VL or VK Variable light or variable kappa VLR Variable lymphocyte receptor

xvii 1. Introduction

1.1. Cancer therapeutics

Current cancer therapies have shown variable potency levels due to their lack of tumour- specificity. Early, and some current methods in treating cancer have been said to be reminiscent of military tactics – finding and destroying the target entity and its immediate environment with subsequent off-target injury or destruction (bystander effect) being an unfortunate but unavoidable consequence. Surgical procedures sometimes even involved near-complete removal of entire organs to ensure removal of a cancer.1 Even in today’s high-tech society chemotherapeutic drug administration leads to the destruction of rapidly dividing cells principally though interfering with DNA synthesis or replication, and so, not only cancer cells but some normal cells are affected, causing side-effects such as hair-loss and nausea. Furthermore, cancer treatments such as surgical removal of tumours are highly invasive and have limited therapeutic benefit if the cancer has spread to other organs.2, 3

Although the common belief is that prevention is better than cure,4 the nature of and expanse of cancers that exist is as yet not fully understood. As such, early detection has been identified as a major contributing factor in a clinician’s ability to manage and prevent detrimental disease onset in a patient. In aid of early detection numerous tools for improved diagnostics have been developed over the years such as various imaging technologies (MRI and CT scans), endoscopic techniques, and in the case of non- skin cancer - optical coherence tomography.5-7 However barring a complete understanding of the biological scope and manifestation of all cancers (Cancer Genome Atlas), the full potential of diagnostic tools has not been fully realised and probably won’t be for some time.

With confirmed disease diagnosis there is the obvious necessity to take action in the eradication of the cancerous manifestation. The first avenue for preventing the spread of a cancer tends to be surgery, in which case a benign tumour is excised from the surrounding tissue with minimal damage to the local environment. However rates of long-term patient survival following surgery, whether due to surgical complications or unpredicted spread of the disease, have not been overwhelmingly successful.8 If surgery isn’t expected to be the optimal treatment option it may be combined with radiotherapy or chemotherapy.

1 Depending on the circumstances the latter options may also be used as stand-alone alternative treatments.

Radio- and have been used with varying levels of success. Radiotherapy involves the use of x-rays, gamma rays or other charged particles that are able to disrupt the DNA sequence relating to the cancer and so shrink the tumour. A patient can either be subjected to radiation by a machine called a linear accelerator delivering an external beam, or through systemic means whereby radioactive isotopes travel through the blood stream to attack cancer cells. Treatment of this nature also affects normal cell populations therefore clinicians need to plan a course of treatment carefully to maintain a patient’s overall well-being. Further to the original mentality that treating cancer equated to military strategies – one of the original drugs used as a chemotherapeutic agent was mustard gas.9 The reason for this was that physicians realised that the gas resulted in patients having very low counts following gas exposure. They therefore inferred that if cancer patients were treated with the same it should result in a decrease in the rapidly increasing cancerous cells.

Today chemotherapy covers a wide range of anti-neoplastic and cytotoxic drug treatment regimens, including antimetabolites, alkylating agents, anthracyclines, DNA methyltransferase inhibitors, platinum compounds and spindle poisons to name a few categories.9, 10 However, as these treatments aren’t selective towards particular tumour cells themselves, they are associated with many side-effects that can be more detrimental to the patient’s health.3 Furthermore, patients can also develop resistance to particular drug treatments rendering the drugs completely therapeutically ineffective. The ability of cancer cells to evade the immune system and acquire resistance to current treatments strengthens the need to develop targeted therapies capable of overcoming drug resistance and eliminating the emergence of multiple drug resistant tumour cells, associated with chemotherapeutics.1

2 1.2. Cell surface markers Breast, lung and prostate cancer are the most common cancers diagnosed in both developing and developed countries and account for the majority of cancer-related deaths. These prevalent cancer indications are followed by colorectal, stomach, liver, lung and cervical cancers.2 An obvious progression from the traditional global, “whole-person” cell treatment by radiotherapy and chemotherapeutic agents is to provide a means to specifically target a therapeutic agent directly to a cell or tumour of interest.3 By analysing markers specifically associated with or overexpressed on tumour cells compared with healthy cell populations, a tumour cell 'signature' can be defined, and these markers can become targets for therapeutics, while sparing the surrounding healthy cells from the cytotoxic effects of the treatment. Targeted therapeutics can therefore be used to specifically treat pancreatic, breast, prostate, colorectal, or any other cancer with a therapeutically beneficial identified cell surface marker.

Many cell surface markers are currently under investigation as therapeutic targets, not only in cancer but also in inflammatory diseases and other indications.11-14 Amongst these are common validated targets such as the epidermal growth factor receptor (EGFR), CD20 and Human epidermal growth factor 2 and 3 (Her2 and Her3).15-18 The table below provides a list of some of the common targets currently under investigation (Table 1.1).

Table 1.1: Therapeutic cell surface targets Surface marker Cancer type Targeting treatment example Neovascular endothelial cells (Tumour Various peptides carrying therapeutic Aminopeptidase 19, 20 angiogenesis) payloads. Folate receptor Various cancers including ovarian cancer Folate conjugates21-23 Various monoclonal antibodies (mAbs) Various cancers including breast and colorectal EGFR such as Panitumumab and bispecific cancer 24, 25 affibodies Various mAbs such as , Her 2 Various cancers including ovarian and breast cancer 26-29 ankyrin repeat proteins and Fynomabs CD19, 20, 22, 25, Various mAbs including Blood cancers such as Leukemia and lymphomas 12, 14 and 33 (CD20) and other engineered formats Carcinoembryonic Carcinoembryonic antigen. Colorectal, small-cell 30 Bispecific antibodies antigen (CEA) lung, ovarian cancer and adenocarcinomas CD133 Head and neck squamous cell carcinomas Various mAbs and fusion proteins31

A number of factors need to be considered when deciding on a disease-specific marker as a target for therapeutics. These factors include whether the target antigen is a secreted or cell membrane antigen, and whether it has a high surface density limited to a cancer cell, for example or whether it is also present at high levels on healthy cell populations. If the latter is true then the therapeutic benefit of the target most likely won’t be an improvement on current systemic drug administration regimens. There also needs to be a low 3 heterogeneity in antigen expression amongst cell populations of a certain type – otherwise the treatment will fail due to natural resistance at the target epitope. The target moiety should not be one that is naturally down-regulated or released from the cell surface; this would result in a decreased efficacy as the therapeutic molecules would simply be cleared from circulation.

The researcher needs to determine whether or not the marker is internalised and whether either situation poses a problem to the proposed therapeutic system. As an example, if therapeutic benefit is achieved by the agent being delivered to the cell surface to block a particular site or to recruit immune cells then it may not be necessary for the marker to be internalised. If however the therapeutic mode of action (MOA) involves cessation of internal cellular pathways following uptake or internalisation of the therapeutic molecule, as is the case with siRNA delivery, then ligand internalisation is a major contributing factor to the system’s success.

The majority of cancers consist of solid tumours; making factors like penetration capabilities and diffusion into cells through tumour vasculature important during the product design phase.32, 33 Binding affinities are another important factor for consideration. If for example a receptor has a very high affinity for its natural ligand, then any therapies that target this receptor would have to out-compete and therefore have a stronger binding affinity for the receptor. However, if a therapeutic agent’s binding affinity is too strong then the products will only bind to the outermost regions of the tumour environment and not be able to penetrate deep enough into the tumour – limiting the reach of the treatment.34 Table 1.2 adapted from Tabrizi, 2012, summarises some of the most important considerations to be taken into account with regards to target selection for optimal affinity requirements. Understanding these characteristics would enable developers to critically predict the required balance between product binding affinity and dosage requirements to optimise therapeutic benefit without overspending on the cost of development and production.35

4 Table 1.2: Antigen properties for consideration in targeting moiety affinity design. Adapted from excerpts published in Tabrizi, 2012; Springer.35 Antigen type Properties Explanation In serum and comparison between diseased and normal individuals. Is the antigen Antigen levels readily available and produced in the body at high or low amounts? Low concentration antigen may require high affinity binders. If a secreted target is cleared from a patient’s system rapidly, a targeting agent may be cleared from the system within a short time-frame as well depending on Antigen serum antigen/therapeutic complex size – providing little if any therapeutic benefit. A clearance product could however be designed to bind a product such as PEG, BSA or Secreted or natural immune system components which would increase product retention times. soluble Affinity for natural ligands – targeting therapeutic would need a higher binding Antigen affinity for the receptor than the natural ligand (antigen) to compete and provide a receptor(s) benefit. Receptor density/cell – low concentration antigens may benefit from a therapeutic with a very high binding affinity. Serum antigen Do other targeting moieties or antibodies exist naturally that could compete with binding proteins the developed therapeutic? Comparisons of available products on the market and in clinical trial with regards Other products to affinity and biodistribution can inform developers as to what improvements are in development required to perform competitively in the biopharmaceutical market. Location and cell density – Does the blood brain barrier, a solid tumour environment or other area which is difficult to reach need to be penetrated? A solid tumour environment may benefit from lower binding affinities to ensure that the therapeutic can disengage and travel deeper into the tumour micro-environment. Expression The surface antigen density will affect the avidity of the binding interaction and the pattern possibility of receptor cross-linking could have a positive or negative effect on the proposed therapeutic outcome. Avoid or limit product interaction with healthy tissues. If a target is highly prevalent in normal tissues as well as diseased targets, adverse off-target effects may occur. If the receptor is internalised it may impact antibody clearance rates and ability to Cell Receptor provide a therapeutic benefit if the product is then degraded inside the cell. Unless membrane mediated internalisation would provide a benefit a product may need to be designed that antigens internalisation would limit or retard the occurrence of internalisation events. Shed Could lead to high clearance rates or unavailability of the receptor for a therapeutic extracellular to provide a benefit at the cell surface – therapeutic may need to target a structural domain or component of the membrane protein, rather than external ligand-binding sites. receptors Competition between natural ligands for receptor would require that the Ligand affinity therapeutic have a higher affinity. Comparisons of products on the market and in clinical trial with regards to affinity Other products and biodistribution can inform developers as to what improvements they would in development have to include in order to perform well in the biopharmaceutical market.

5 1.2.1. Epidermal Growth Factor Receptor Levels of EGFR expression have been associated with poor prognosis of disease progression in cancer patients. Mutations in EGFR, overexpression thereof and autocrine stimulation can lead to EGFR activation and altered activity of various intracellular compounds, which can lead to cancer. Overexpression of EGFR has been associated with tumours in colorectal, gastric, breast, glioblastoma and lung cancers to name a few.16, 36-38 The widespread occurrence of EGFR-related tumours has made it one of the most commonly utilised validated cell surface markers in the development of targeting agents.

EGFR is part of the family of receptor tyrosine kinases (RTKs) which when overexpressed confers a cancerous phenotype to cells, providing longevity to solid tumours.36, 39 There are four receptors in this family, EGFR, ErbB2, ErbB3 and ErbB4. EGFR (also known as HER1 or ErbB1) is normally activated by up to seven known ligands including EGF, TGFα, amphiregulin and heparin binding EGF-like growth factor (HB-EGF). When the ligands bind to EGFR, receptor-mediated dimerisation occurs and activates related intracellular pathways.40 A review by Burgess and colleagues noted that EGFR cell surface domains bind targets with variable affinities. EGF is bound by 2 – 5% of receptors at binding affinities (KD) corresponding to lower than 0.1 nM while the majority of receptors (92 – 95%) bind the ligand at a higher 6 – 12 nM. This variation could possibly be explained by the presence of different conformational changes in cell surface receptors.37

ErbB2 (Her2/neu) does not have any ligands that could induce its dimerisation but it has been suggested by Burgess that the monomer exists in a constantly active state, behaving as a heterodimerisation motif for other EGF receptors that are already bound to their respective ligands.37, 41 Overexpression of ErbB2 dimers is associated with many aggressive tumours, particularly in breast cancer. The ErbB2 receptor has a permanently mutated ligand binding site resulting in the receptor being in a constant state where it is able to either form homo- or heterodimers with nearby EGF receptors. Research has not been able to show that this permanently “active” state is the reason why the receptor causes cancer.37 ErbB3 (Her3) and ErbB4 (Her4) also undergo ligand binding to induce dimerisation to proteins called neuregulins (NRGs) . Ligand binding then allows these receptors to either form homodimers or interact with available ErbB2 receptors to form heterodimers.42

6 Targeting EGFR for therapeutic purposes has been achieved through the use of tyrosine kinase inhibitors as well as monoclonal antibodies (mAbs) while many other potential treatments are also in the clinical pipeline.15, 16, 38, 43-54 Several inhibitors directed against intracellular EGFR tyrosine kinase activity have been developed as therapeutic agents.52 Tyrosine kinase inhibitors (TKIs) are small molecule drugs, and are therefore capable of penetration into solid tumours and are more cost effective to produce. Small molecules can however be cleared from a patients’ body through the renal system rather rapidly. The most well-known therapeutic agents are Gefitinib (Iressa) and Erlotinib (Tarceva).36, 53, 54 mAbs are another therapeutic treatment option which has provided benefits in not only treating EGFR-related cancers but many other carcinomas and disease indications as well.

7 1.3. Monoclonal antibodies

1.3.1. Historical perspective The development of antibodies as therapeutic agents began in the late 1800s with serum therapy, and has evolved through the development of technologies such as hybridoma formation, PCR, restriction enzyme cloning, site-directed mutagenesis, recombinant protein production, and transgenic mice (Figure 1.1).55

Figure 1.1: Early discoveries enabling current therapeutic mAb and fusion protein development. Adapted from illustration in chapter 1 of Therapeutic mAbs: from bench to clinic, ed. Zhiqiang An. Copyright, Wiley.55

8 Serum therapy was the first treatment relating to the use of antibodies as a therapeutic in indications such as diphtheria and tetanus. The therapeutic basis of these treatments was the presence of antibodies against disease-target antigens in the serum of immunised non-human sources such as mice, rabbits and horses. Low product purity of serum treatments caused adverse side-effects in patients which led to various techniques being developed to standardise quality and remove impurities. The subsequent discovery of antibiotics and chemotherapeutics as successful alternative treatments with less associated side-effects led to the decline of serum therapy as a therapeutic option. The development of hybridoma technology allowed standardised production of defined mAbs in cell culture, after mouse immunisation, and led to the first therapeutic mAb (Muromonab or Orthoclone).56, 57

1.3.2. mAb types and structure The most-common drawback in the use of murine mAbs (derived from mouse hybridomas) as a human therapeutic is in the widespread occurrence of the HAMA (Human anti-mouse antibody) response whereby the patient’s body produces antibodies to attack the murine components of the mAb (principally the Fc-domain).58 Recombinant antibody technology has endowed researchers with the ability to move away from using pure murine antibodies, and to introduce increasingly greater human-derived sequences to minimise immunogenicity (Figure 1.2).

58 Figure 1.2: Types of mAbs. As illustrated in Brekke and Sandlie. Copyright Nature Reviews Drug Discovery

9 The basic structure of IgG (mAb) molecules remains constant whether they are murine, chimeric, humanised or human. An IgG structure consists of both constant and variable heavy and light chain domains. The heavy chain consists of one variable domain (VH) connected to the first constant heavy domain (CH1). A hinge domain connects CH1 to the Fc domain which consists of CH2 (responsible for complement activation and FcRn binding) and CH3 (facilitates IgG dimerisation). The light chain has two domains, namely variable light, Lambda or Kappa (VL or VΚ respectively) and constant light – in combination with VH and CH1 a Fab molecule is formed. The association of a VH and VL chain constitutes the formation of an scFv fragment which contains a mAb’s complementarity-determining regions (CDRs). Three CDRs, responsible for the molecules ability to specifically bind targets, are located within the VH and VL (VK) domains (Figure 1.3).59

A. B.

Figure 1.3: Basic mAb or IgG structure. Adapted from illustrations as published in Elgert, 1996; Copyright John Wiley & Sons, Inc. .59 A. The structure of an IgG molecule and B. the sequence of heavy and light chains including the location of the two sets of three CDRs.

Chimeric antibody production through cloning murine variable domains onto human antibody chains was an initial improvement as are humanised antibodies developed by grafting murine CDRs into human mAbs.60, 61 Phage, ribosome and yeast display technologies allowed the in vitro development of fully human mAbs from cloned repertoires of human naive variable region genes.62-66 However, these mAbs tend to have lower binding affinities due to the lack of in vivo affinity maturation, and therefore need to undergo in vitro affinity maturation to be more effective as therapeutics. Development of

10 transgenic humanised mice provided in vivo means of isolating fully human mAbs that already have high affinities for their targets.62, 67

1.3.3. Biopharmaceutical product market The protein therapeutics (biologics) market encompasses a large variety of products including coagulation factors, growth hormones, human insulin and mAbs – to name but a few. Monoclonal antibodies are currently estimated to be the fastest growing component of the protein therapeutic market owing to their superior ability to target disease, and their diversity – both as a product in their own right and in the number of diseases that mAbs can target.58, 64, 68-71 Global sales revenue of mAb-based products (inclusive of Fc-fusions, antibody fragments, antibody-drug conjugates and single mAbs) was estimated to be around $75 billion in 2013 – accounting for 50% of total biopharmaceutical product sales.72

The first mAb approved for clinical use was Orthoclone (OKT3 – prevention of transplant rejection) in 1986 and since then a vast number of mAb-based products have entered clinical evaluation and been subsequently approved.14, 69, 72, 74, 75 As summarised in Ecker, fifty-eight mAb-based products have been approved since 1986, eleven of which were approved and subsequently withdrawn from the market. All of the currently marketed full- length mAb products have been expressed using mammalian cell culture techniques.72 The bi-annual reports published by Reichert provides extensive summaries of “antibodies to watch” with regards to being close to market release, regulatory review stage and late phase 3 antibody-products that are likely to progress to market approval. Also provided in each article is a link to a table describing a number of mAb-based products approved since 1986 (http://www.tandf.co.uk/journals/pdf/announcements/kmab-antibodies.pdf).75

Monoclonal antibody-based treatments have been developed for both cancer and non- cancer related indications encompassing asthma, anthrax, breast cancer and lymphoma, to name a few. A number of EGFR-targeting mAbs have been approved for clinical use and are currently in development.75-79 The three most notable anti-EGFR mAbs include two approved mAbs (Cetuximab and Panitumumab) and a third () currently under priority review at the FDA (Table 1.3).53, 54, 75, 80 These three mAbs are distinguished according to species, isotype and various biological characteristics including binding affinity and IC50.

11 Table 1.3: mAbs targeting EGFR approved and in clinical development.75, 76, 81 Monoclonal antibody ID Species Isotype Clinical indication Clinical phase CRC, NSCLC, head Cetuximab (Erbitux) Chimeric IgG1 Approved, 2009 and neck Panitumumab (Vectibix) Human IgG2 CRC Approved, 2006 3, under review by Necitumumab Human IgG1 NSCLC FDA (Theracim, 1 and 2, approved Humanised IgG1 Head and Neck Theraloc) in India and China (HuMax- 1, 2 and 3; Human IgG1 Head and Neck EGFR) development halted

Panitumumab was the first fully human mAb derived from transgenic Xenomouse technology approved for clinical use.24, 67, 82, 83 It is an IgG2 mAb which distinguishes it from the other IgG1 formats and therefore has a low affinity for Fc-receptors. Although this can be viewed as a limitation in that an immune response cannot be mounted at the target antigen, an immune response is not always required and can be a hindrance given some MOAs. Utilising an antibody isotype which naturally has a low affinity for Fc-receptors circumvents the need to engineer a mAb with reduced affinity which could risk product instability.84 Panitumumab has a higher binding affinity (increased on-rate and slower off- rate) for EGFR than both Cetuximab and Necitumumab; nevertheless it has been suggested that the latter two mAbs retain a greater functional “anti-cancer” effect on EGFR.80

As an extension of mAbs being used as therapeutic products in their own right, it is also possible to enhance their therapeutic capabilities by re-engineering the antibody or attaching various moieties to its structural framework. Attaching peptides, radiolabels, drugs or any number of other particles to the mAb, combines the ability of the mAb to impose disease regression and also the therapeutic action of the attached ligand.85-88 However the development of antibody-drug conjugates (ADCs) is hampered by factors including conjugation chemistry, attachment method, choice of linker, location on the mAb and the number of drug particles per mAb; all affecting potential therapeutic benefit and product stability.89 ADC development is a rapidly growing field with two ADCs currently approved for clinical use, namely Adcetris () and Kadycla (ado- trastuzumab emtansine); in addition a radioimmunotherapeutic conjugate – Zevalin (), and many other new molecular entities based on mAbs are entering the clinical pipeline.89-97 Mylotarg (), an ADC, was

12 originally approved for clinical use in AML patients but was subsequently withdrawn in 2010 following new safety concerns raised by the FDA.92

Monoclonal antibody-based technology has become well established as a therapeutic alternative to standard treatment regimens, and the production has become routine and reproducible. Recombinant DNA technologies provide developers with the opportunity to endow their product with higher, very specific affinity, potency and variable effector functions as applicable to the proposed purpose.84 Most mAbs have a longer serum half- life than standard drug-based treatments thereby improving bioavailability. However mAb production is renowned for being time-consuming and very expensive and the follow-on costs to patients are also high. The larger size of mAbs also means that they have a decreased ability to penetrate tissues and the BBB.68-70, 72, 98-102 In addition to these factors, some patients may not receive a benefit from a selected mAb-product due to natural or acquired resistance at a selected marker.77, 79, 90, 103 The development of next- generation biological therapeutics such as bispecific antibodies (BsAbs) provides a means to improve on some of these deficiencies and further enhance the positive attributes associated with mAbs.29, 30, 104-106

13

1.4. Bispecific antibodies BsAbs are a next-generation protein product with applications spanning both diagnostic and therapeutic fields. The most important factor classifying a protein as a BsAb lies in its ability to bind more than one target for which it has previously engineered specificities, unlike traditional mAbs which are limited to monovalent target binding.

1.4.1. Historical perspective The development of processes relating to hybridomas, improved cell culture and recombinant protein engineering technologies has been a major driving force in the presently expanding field of BsAb engineering.107-109 Although BsAbs have only recently started gaining popularity amongst big pharmaceutical companies the theory and concept behind their development has been around for more than fifty years.110, 111 Original efforts in BsAb production were plagued with problems relating to aggregation, low yield, inactivity and heterogeneity in BsAb preparations. Academic interest surrounding the original concepts in producing BsAbs were initially realised by Nisonoff and Rivers around 1961. Their BsAbs were produced through oxidative recombination of pepsin digested and reduced rabbit antibody preparations.110 However, problems such as chemical protein denaturation and high reagent wastage associated with BsAb formation through chemical means were major barriers to production. In the early eighties, Milstein and Cuello reported a means to produce BsAbs that did not require chemical methods but would negatively impact final product binding affinity and cause protein denaturation (Figure 1.4).

Milstein and Cuello’s process involved the fusion of two myeloma cell lines which enabled expression of hybrid molecules due to the mixed availability of different VH and VL chains from both parental cell lines. They noted that out of ten possible antibody combinations that could be produced, only one was the desired BsAb (Figure 1.5), but they found that only a total of four out of ten were actually produced in their study. Their theoretical BsAb yield was expected to be 17% - 50% of total IgG produced.112, 113 During purification they found that the optimal chromatographic trace showed 3 peaks where the two flanking peaks were of parental mAbs and the middle peak of BsAbs, however this central peak would be a combination of inactive and active BsAbs (Figure 1.5). F(ab’γ)2 bispecific heterodimers were produced using a thioether-linkage and evaluated for their performance by Glennie et al.114 Purified product was heterogenous but 50 – 70% was found to be the bispecific heterodimers; although they reported relatively good bispecific product yields this was hampered by less efficient levels of retrieving starting parental Fab’γ fragments. 14 The benefit in the system is the lack of a murine Fc-domain to illicit a HAMA (human anti- mouse antibody) immune response in patients.

Figure 1.4: BsAb production by chemical conjugation or somatic hybridisation. Adapted from illustration published 107, 115 in Bispecific antibodies, ed. Kontermann; Copyright, Springer

Figure 1.5: Hybridoma produced antibody combinations. Theoretical antibody recombinations that can be produced from hybridomas assuming random association of heavy and light chains as predicted by Suresh, Cuello and Milstein; Copyright, Academic Press, Inc.112, 113

15 Early efforts in BsAb clinical development also had a number of problems including patients exhibiting immune responses due to adverse reactions to Fc-components and HAMA being produced due to the presence of murine components in the constructs.107, 116 The relatively recent advent of being able to produce fully human mAbs such as Panitumumab (Xenomouse technology)67 and recombinant methodologies such as the removal of Fc-domain function84, 87 have been successful endeavors in the future of BsAb development (Figure 1.6).

Figure 1.6: Historical BsAb development timeline. Diagram illustrates the path towards the development of next- generation BsAbs for clinical development adapted from an illustration in Bispecific antibodies, ed. Kontermann; Copyright, Springer.107

1.4.2. Modes of Action By binding two targets, patients who are resistant to drugs that bind one extracellular target can be provided with a therapeutically significant outcome following the independent binding of the second BsAb arm to a separate target, circumventing the need for multiple doses of different drugs and thereby lowering costs (Figure 1.7.A).117, 118 High affinity binding of one extracellular target can promote high avidity binding of the second target due to proximity on the cancer cell surface – another advantage of BsAbs, but this modality is yet to achieve significant success.32, 119 BsAbs can also be used as dual inhibitors of soluble targets to suppress various pathways, as is the case in RG7221 (Ang- 2 x VEGF), ABT-122 (TNFα x IL-17) and 120 (IL-4 x IL-13).121, 122

The most common use of BsAbs to date has been in redirecting an immune response to a target antigen (Figure 1.7.B). BsAbs can bind an extracellular target on T-cells (T-cell 16 engagers), to activate and direct them to a tumour cell where cytolysis can occur, for example, DART, BiTE and FynomAb technology.29, 118, 123-127 Low drug potency of chemotherapeutics associated with the lack of drug localisation at cancer targets can also be improved by BsAbs binding directly to a drug128 (Figure 1.7.C) or drug loaded particle (Figure 1.7.D) while the second arm binds a disease target.103, 118, 129

Figure 1.7: Applications of BsAbs. Therapeutic modalities include: (A) crosslinking separate antigens on the cell surface; (B) T cell engagement by cross-linking CD3 on cytotoxic T cells to tumour cells; (C) targeting drugs or radiolabels to cell surface; (D) targeted delivery of drug-laden nanoparticles to tumour cells. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC.

17 1.4.3. Current development Three methods are generally used in BsAb production, namely hybridoma technology, chemical conjugation and the third option which can create an inordinate amount of construct variation – recombinant protein engineering. Expression of recombinant BsAb formats has been completed using both bacterial and mammalian expression systems. Purification requirements can become excessive in situations where heterogenous mixtures of monospecific, multi-specific, active and inactive formats are produced – driving up the time and cost factors associated with BsAb production.

Each method has its associated disadvantages. Hybridomas can potentially produce ten different variants,107, 112, 113, 130 chemical conjugation can cause protein denaturation,110, 111, 131 and both methods have potentially low BsAb product yields. Recombinant antibody engineering may require excessive sequence fine-tuning at times – a particular designed format might not express well in one system (glycosylation requirements limit ability to produce in bacterial systems), or an scFv being in a particular orientation may incur instability to the format and need either stability engineering or to be moved to a different position in the construct sequence.87, 115, 125, 132-143

Many different BsAbs have been studied thus far but for long only one had been approved for clinical use, namely .144-146 The BsAb is produced from a rat/mouse hybridoma and is characterised as a Triomab due to it having binding affinity for CD3 on T cells and the EpCAM antigen in ovarian cancer while still maintaining Fc-mediated activity through recruitment of and NK cells. Recently a second BsAb (; trade name Blincyto), a T-cell engaging tandem scFv, was also approved for clinical use in the treatment of acute lymphoblastic leukemia.75 Although there are only two clinically approved BsAbs, many are currently undergoing clinical evaluation (Table 1.4) and the recent approval of Blinatumomab is a promising occurrence in the clinical progression of next-generation biological therapeutics.147

A multitude of BsAb formats have been developed over the last two decades and been extensively reviewed (Figure 1.8).104-106, 115, 118, 125, 133, 135, 139, 140, 148-156 Format design is of particular interest in how it behaves in a particular application. As an example, using an IgG-like BsAb might not be preferable if the molecule’s large size would negatively impact tumour penetration whereas a small diabody or tandem scFv format in the same system would improve on this in a solid tumour environment.157 18 147 Table 1.4: BsAbs undergoing clinical trials. Information sourced online and from PEGS Europe conference (2014) as well as Weidle et. al. BsAb ID Company Molecule type Targets Clinical phase Indications Blinatumomab (Blincyto) Micromet/Amgen BiTE CD19 x CD3 Approved Dec 2014 Leukemia BAY-2010112 Bayer/Micromet/Amgen BiTE PSMA x CD3 Phase I Prostate cancer (AMG-110) Micromet/Amgen BiTE EpCAM x CD3 Phase I Oncology MEDI-565 (MT-111) Micromet/MedImmune/Amgen BiTE CEA x CD3 Phase I Gastrointestinal MGD006 Macrogenics/Servier Humanised DART CD3 x CD123 Phase I AML AFM13 Affimed Bispecific TandAb CD30 x CD16A Phase II Hodgkin lymphoma AFM11 Affimed Bispecific TandAb CD3 x CD19 Phase I Hematological disorders IMCgp100 Immunocore Fusion protein CD3 x gp100 Phase I/II Melanoma MP0250 Molecular Partners DARPin VEGF x HGF Phase I Oncology MM-141 Merrimack scFv-IgG fusion protein IGF-1R x HER3 Phase I Solid tumours MM-111 Merrimack scFv-albumin fusion protein HER2 x HER3 Phase II Various cancers Ozoralizumab (ATN-103) Ablynx/Eddingpharm Trivalent Nanobody TNF x serum albumin Phase II RA 19 ALX-0061 Ablynx/Abbvie Bispecific Nanobody IL-6R x serum albumin Phase IIa RA/SLE ALX-0141 Ablynx Trivalent Nanobody RANKL x serum albumin Phase I Bone-loss disorders ALX-0761 Ablynx/MerckSerono Trivalent Nanobody IL-17A x IL-17F x serum albumin Phase I RA RG7221 Roche CrossmAb VEGF-A x ANG-2 Phase II Colorectal cancer RG6013 Roche IgG-like BsAb Factor IX/X* Phase II Hemophilia COVA322 Covagen/Johnson & Johnson Bispecific Fynomab TNF x IL-17A Phase I/IIa Psoriasis ABT-122 Abbvie DVD-Ig TNF x IL-17A Phase II RA ABT-981 Abbott DVD-Ig IL-1α x IL-1β Phase IIa Osteoarthritis SAR156597 Sanofi DVD-Ig IL-4 x IL-13 Phase I/II Pulmonary fibrosis MEHD7945A/RG7597 Genentech/Roche Two-in-one IgG EGFR x HER3 Phase II Oncology Acronyms: BiTE, Bispecific T-cell engager; BLA, Biological license application; CD, Cluster of differentiation; PSMA, Prostate specific membrane antigen; EpCAM, Epithelial cell adhesion molecule; CEA, carcinoembryonic antigen; gp, Glycoprotein; VEGF, Vascular endothelial growth factor; HGF, Hepatocyte growth factor; IGF, Insulin-like growth factor; HER, Human epidermal growth factor receptor; TNF, Tumour necrosis factor; RA, Rheumatoid arthritis; IL, Interleukin; SLE, Systemic lupus erythematosus; RANKL, Receptor activator of nuclear factor kappa-B ligand; ANG, Angiopoietin; AML, Acute myelogenous leukemia; DART, Dual-affinity re-targeting. *Mimics coagulation factor VIII. Figure 1.8: BsAb formats. A summarising image of some of the many BsAb formats that have been developed as described by Kontermann; Copyright, Taylor and Francis.118 20 Recently BsAbs have been described as grouped in three different categories (PEGS Europe conference, 2014): 1. Symmetric, bivalent 2. Asymmetric, monovalent 3. Fragments

Symmetric, bivalent A large number of companies are investigating BsAb formats of this type (Table 1.5).104 The most well-known formats are F-mab2, DVD-Ig, IgG-like and scFv-IgG fusion BsAbs. F- star’s BsAb technology is an extension of their monovalent Fcab product which is produced by engineering the CH3 domain of an Fc to be able to bind a specific target antigen.158 Normal mAbs can then have their Fc-domains replaced by a particular Fcab and in so doing the mAb acquires affinity for a secondary target. F-star’s BsAb is still in the pre-clinical stages but they have initiated clinical trials for the monovalent Fcabs. Questions still remain as to how engineering the CH3-domain could affect Fc effector functions and also how or if binding affinity is affected by the presence of a second targeting moiety in the constructs.

Table 1.5: Companies developing symmetric, bivalent BsAbs. Information from PEGS Europe conference, 2014. BsAb format Companies References Two-in-one antibody Genentech 159, 160 Dual-targeting-IgGs GSK/Domantis 161 F-mab2 F-star 158 DVD-Ig Abbvie 162, 163 IgG-like BsAb Eli Lilly 164, 165 scFv-IgG fusions Biogen Idec, Numab, Roche, Zymogenetics/BMS 51, 136, 166-169 Fynomabs Covagen 29, 127 Zybodies Zyngenia 170, 171 Fibronectin-antibody fusions MIT (D. Wittrup) 30

IgG-like and scFv-IgG fusion BsAbs can mediate complement activation and bind other Fc cell-surface receptors, however they can be engineered to limit, in some cases, undesirable functions of the Fc region.84 For example if a particular therapeutic or diagnostic application would be negatively impacted by a mounted immune response which causes toxicity in the patient due to using a functional Fc-linker, a better option is to engineer the domain to be inactive. However in other cases such as when a BsAb is used to deliver drug payloads to targets it may be beneficial to maintain the ability of the Fc to

21 recruit immune system components to provide an added “attack” in the diseased region. Removing Fc-function requires very stringent engineering however and careful decisions need to be made on which functions to maintain or discard (Figure 1.9).104, 119

Figure 1.9: Antibody structural engineering for modified pharmacological effects. Adapted from illustration by Beck; Copyright, Nature Reviews Immunology.119

Important questions that need to be considered during Fc-engineering include the following: which Fc-domains are beneficial in the system – complement activation, protein A binding for purification, dimerisation motifs, half-life extension? Will modification of a sequence result in product aggregation or instability?87, 119, 172-176 The Fc interacts with various cell surface markers which increase the construct’s retention time. One of the markers it interacts with is FcRn which regulates IgG homeostasis; it is a protective carrier of IgG. Selecting the correct IgG isotype is also important because IgG3 doesn’t bind FcRn efficiently and so has a shorter serum half-life, and IgG2 and IgG4 are naturally non- activating.141, 156 To improve BsAb activity in vivo Chames and Baty32 stated that affinity for some Fc-receptors has to be modified: • increase binding to activating receptors such as FcγRI, FcγRIIa and FcγRIIIa • decrease binding to FcγRIIb inhibitory receptors

22 NK cells express FcγRIIIa receptor which is important for tumour cell lysis, whereas the FcγRIIa receptor is beneficial for target cell apoptosis through recruitment of neutrophils, monocytes, macrophages and dendritic cells. and dendritic cell activity can activate cytotoxic T cells through cross-priming when disease-related antigens are presented on the class 1 MHC complex.177 These cells also express inhibitory FcγRIIb however, which possesses high homology with the beneficial receptor FcγRIIa. A review by Presta provides a significant amount of information regarding Fc engineering.156 Binding to FcRn increases half-life at pH 6 in the endosome but not at pH 7.4 in the blood. Antibodies that are not bound to FcRn are catabolised in the endosome. To improve FcRn binding the Fc should only be engineered for enhanced binding at pH 6.0, not 7.4 otherwise there will only be a decreased half-life. Not all published mutations to enhance FcRn activity have the same effect on different mAb constructs. As examples, an anti- hepatitis B mAb had enhanced effector function but an anti-TNFα mAb with the same mutation didn’t show any increase in FcRn activity.178, 179 All of these factors further complicate the process of enhanced Fc-engineering.

IgG fusion proteins also require substantial amounts of stability engineering at times to prevent aggregation.180 Variable chain stability can be enhanced by incorporating cysteine residues for stabilisation at Kabat position 44 of the heavy chain and position 100 of the variable light chain for intramolecular stabilisation.141, 181 Schanzer states that 141 making (G4S)4-6 linker repeats stabilises scFvs. Expression levels reported by Schanzer show that having the scFv linked to the N-terminal region of the IgG results in 4-5 times lower expression levels in HEK cells whereas incorporating disulfide stabilisation into the constructs results in 1.5-1.9 times lower expression levels. There appear to be quite a few trade-offs when it comes to adding the disulfide stabilisation; Schanzer showed that when the scFv isn’t disulfide stabilised it has a higher biological benefit. In addition to this a number of other scFv stability engineering options have been described.136 Sometimes the inclusion of disulphide bridges can increase product yield while still preventing aggregation.182

Another important factor to consider relates to the optimal linker type and length connecting additional scFvs or other binding proteins to an IgG’s Fc domain. Shorter linkers can make a construct more rigid and could potentially interfere with stability and binding function, depending on the included amino acid sequences. It was however 23 reported that varying linker lengths between 15 and 30 residues (3 – 6 G4S repeats) did not affect BsAb expression levels. However, they found that longer linker lengths reduced the tendency of these molecules to aggregate.141 ADCC activity varies amongst different scFv-IgG fusion proteins relating to where the additional targeting moiety is attached to the primary structure.182

DVD-Ig or Dual Variable Domain-Ig technology involves fusion of additional variable domains to the N-terminal end of an antibody.119, 162, 163 The bispecificity of this format is expressed on a single polypeptide, thereby avoiding the problem of other design formats that may undergo hetero- or homodimer miss-pairing of different heavy and light chain components. BsAb development involves the utilisation of two different mAbs and connecting the variable domains. This creates a single molecule for expression which improves single product homogeneity, yield, efficient purification and scalability. However it is noted that the linker design between the two variable domain targeting moieties can significantly influence the ability of a designed construct to bind its target antigen.162 A notable benefit of this BsAb format is that it is very stable and so does not aggregate.

Asymmetric, monovalent One of the most common problems associated with asymmetric bispecifics is that co- expression of four chains (2 Heavy and 2 light) within a single cell can result in the formation of ten potential products.112, 113 Numerous methods have been employed to address this problem. Firstly there is the use of a common light chain to increase theoretical proportions of final product; however this process still produces a heterogenous mixture which makes purification more time consuming.

Another option is through forced heterodimerisation by means of Fc-engineering. Knob-in- hole (KIH) mutations, developed by Paul Carter’s group at Genentech, can be inserted into the two CH3 domains of the individual mAb monomers to be dimerised. 63, 150, 154, 155, 183 The design concept is that an amino acid residue on one chain is substituted for an amino acid with a larger side chain while a second CH3 interface has a hole inserted by substituting large amino acid residues with ones that possess small side chains. A common light chain and different heavy chains were included to cater for the possibility of heavy chain/light chain miss-pairing. KIH technology has been used to produce a number of different formats including Crossmab (Figure 1.10) – involving forced heterodimerisation of heavy and light chains; orthogonal Fab interfaces and truncated monovalent heavy 24 chains, namely MetMabs.150, 165, 184 Crossmab production can result in a heterogenous mixture of products including light chain dimers, knob-knob and hole-hole variants, however stable cell lines can secrete up to 90% of the BsAb product in the supernatant. Final purification involves a four step process to remove all contaminants.

Figure 1.10: Crossmab development through Fab domain exchange and KIH technology. Adapted from illustration in Schaefer et al.150

Moore et al mention a number of different mutation sets that they used to produce knob-in- hole variants.138 According to them their best mutation set (Knob - Y349T:T394F and Hole - S364H:F405A) produces more heterodimers compared with the Atwell et al paper (89% versus 85% heterodimers).63, 138 Moore et al predicted, but didn’t test, that having the variable fragments on the C-terminal end would promote increased heterodimer formation. They also included a mutation of G236R:L328R that prevents binding of the Fc to FcγR’s and the complement. The linkers they used to connect the variable fragments to CH3 included hinge components as well as variable lengths of G4S repeats.

25 Their results suggested that having the C-terminal variable fragments attached to CH3 doesn’t prevent the required heterodimerisation from occurring. They also observed larger aggregates but attributed them to the biophysical properties of the C-terminal scFv because they did not observe the large aggregates when they used other variable domains at this position. The linker used to link the variable fragments to CH3 was:

• For connecting the anti-CD16 fragments: SSD KTH TSP PSP (G4S)3 GGGG • For connecting the anti-CD3 fragments: SSD KTH TSP PSP SG Large aggregates were visible for the CD3 version but not the CD16 version with the longer linker – implying that linker length influences BsAb product stability but this is also dependent on the nature of the linked variable fragments. Another set of mutations used in BsAb design by Merchant et al 154 included the same KIH set as in Atwell et al 63 (Knob - T366W and Hole - T366’S:L368’A:Y407’V) as well as disulfide engineering to stabilise heterodimers using a S354C:Y349’C mutation.137

Electrostatic steering heterodimerisation also produces 89% heterodimers.135 Amgen produced heterodimeric Fc domains by incorporating mutations in the Fc that promote electrostatic interactions.185 This summary on the symposium refers to W. Yan as being the speaker on this topic. Performing a K392’D:K409’D mutation on one CH3 domain and a D356’K:D399’K mutation on the second CH3 arm results in forced heterodimerisation and is therefore useful in bispecific production. Some homodimers do form but at very low levels. This format can otherwise be known as charge pairs. Jin and Zhu 137 mention that by combining the KIH technology and electrostatic steering effects135 a higher degree of heterodimers could be produced during expression. However, multiple mutations could affect immunogenicity.

In vitro assembly can also be used to resolve light chain/heavy chain miss-pairing. In this instance “half-antibodies” are expressed and purified separately and reassembled under redox conditions to produce the final BsAb.152, 186 Overall the method is very efficient in BsAb production and homodimer contaminants only equate to about 5% of total produced product. An efficient purification system is essential to streamline the BsAb production process. Catumaxomab production is completed using Rat/mouse quadroma cell lines and the heavy/light chain pairing happens on a preferential intra-species basis which limits the amount of mispairing during production. Affinity chromatography can be used to purify BsAbs based on specific motifs present in the preferred format present in the supernatant.

26 Alternative fragment BsAb formats Out of a total of 23 BsAbs currently undergoing clinical trials, 15 are fragment-based formats (PEGS, Europe conference, 2014). Tissue penetration is important in therapeutic molecules, especially in solid tumours.157 Molecular size affects penetration; therefore having the Fc might make the molecule too big to penetrate tissues efficiently.132 Small products do tend to have shorter serum half-lives however but this can easily be mitigated by binding to human serum albumin (HSA) or PEG.187, 188 PEGylation can lead to decreased immunogenicity but also partial inactivation and decreased affinity of the antibody. HSA interacts with FcRn and does not decrease binding affinity. Muller et al showed that HSA’s presence increases serum half life/circulation time.189

Variable lengths of G4S linkers are utilised in many different BsAb platforms. Shorter lengths promote a certain amount of rigidity and are therefore ideal in preventing certain components of a construct from interacting. Longer linker lengths at 12-15 nucleotides are more flexible and are therefore useful when you’re trying to build a scFv where you want the VH and VL chains to associate with one another and form a functional scFv. If longer linkers were used to join two separate scFvs the risk would be that mismatched folding would occur resulting in inactive scFvs being formed in the BsAb construct.187

Symmetric bivalent fragment constructs include scFv-Fc fusions such as those produced by Emergent Biosolutions (Trubion), the ADAPTIR platform (previously known as the Scorpion/SMIP platform) and also Affimed’s Tandem Diabodies (TandAbs). TandAbs are homodimeric tetravalent molecules that lack an Fc but still maintain a longer, 12 – 24 hour serum half-life.190 They are designed as two complementary monomeric polypeptide chains that can dimerise without covalent linkages. Self-association of monomers is prevented by limiting the linker length connecting variable chain components on the single polypeptide (Figure 1.11). Co-expression of the two polypeptides enables variable domains to pair up through intermolecular pairing to produce functional, tetravalent TandAb BsAbs.

Emergent Biosolutions’ ADAPTIRTM platform comprises a monospecific 105 kDa molecule (formerly known as a SMIP) with a structure similar to a traditional mAb (150 kDa) but lacking a CH1 domain.191, 192 An extension of the monospecific ADAPTIRTM platform provides a means to produce a BsAb193 (160 kDa) by adding additional binding moieties (scFv, extracellular domains or ligands) to the C-terminal domain (formerly known as 27 SCORPION) (Figure 1.12). Whereas the main mode of action under investigation for TandAbs is to redirect an immune response, ADAPTIR multi-specifics are used to either crosslink two cell surface targets or bind and neutralise two soluble target antigens. Linkers connecting N- and C-terminal ADAPTIR binding domains to the Fc are not substantially described 191, 194 however they are said to change binding moiety spatial orientation compared with mAbs (60-80 A° versus 95-140 A° respectively) which endows the molecule with modified signal responses according to the Emergent Biosolutions website (http://www.emergentbiosolutions.com/node/76).

 190 Figure 1.11: TandAb BsAb formation. Adapted from illustration in McAleese and Esser.

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Figure 1.12: Format variations of ADAPTIR multi-specifics. Adapted from illustration on http://www.emergentbiosolutions.com/node/76

Numerous monovalent bispecific scaffolds are also under development some of which are summarised in Table 1.6. Nanobodies or single-domain antibodies (sdAbs) are derived from variable domains (VHH) of camelid heavy chain antibodies (HcAb) which naturally lack 195, 196 light chains. These VHH are generally 11-12 kDa in size and would therefore have a low serum half-life when administered in isolation. As such one option is to produce then as tandem sdAbs that bind HSA for increased serum half-life in addition to the therapeutic target. Although nanobodies lack Fc-mediated effector functions, not all therapeutic applications benefit from the domain’s presence.84, 104 It has been shown that tandem sdAbs are highly stable and are produced at good yields.197 Furthermore four nanobodies have entered clinical trials so far (Table 1.4; Ablynx).

Table 1.6: Summary of monovalent, bispecific scaffolds in development. BsAb platform Company Reference Nanobodies (camelid) Ablynx 196, 198 scFv-albumin fusions Merrimack 139, 189, 199 scFv fusions Macrogenics, Micromet/Amgen 123, 142, 200, 201 Adnectins Adnexus/BMS 202 Affibodies Affibody 25, 203 Molecular Partners 204-206 Pieris 207

The most commonly utilised monovalent formats are scFv fusions or tandem scFvs. Two well characterized formats of this type are the Bispecific T-cell Engager (BiTETM; Micromet/Amgen) and Dual Affinity Retargeting (DART) molecules (Macrogenics). 126, 199- 201, 208-210 Typically tandem scFv-based formats are recombinant products that consist of two scFvs connected by flexible peptide linkers such as Glycine-Serine repeat motifs.147 DARTs consist of two separate chains, designed to include proprietary disulphide

29 stabilisation linkages, which are co-transfected in either mammalian or bacterial cells. Blinatumomab is an example of a BiTE which has gone through clinical trials and for which a BLA has subsequently been filed (Table 1.4).124, 211

BiTEs are highly potent with an EC50 in the picomolar range but DARTs have been described as being even more potent.200 The enhanced stability of the DART molecule decreases product aggregation levels and consequently improves cell killing due to an increased amount of time being spent in contact with target cells. Nonetheless, numerous BiTEs and one DART is currently in clinical trials and many more DARTs in the preclinical pipeline (http://www.macrogenics.com/products.html) (Table 1.4). The small size of these BsAbs may increase solid tumour penetration levels but their size also means they have a very short in vivo half-life and, in the case of Blinatumomab, have to be administered by continuous infusion over a 28 day period.212

There are also other non-antibody protein scaffolds under development that are able to bind targets. These include DARPins (Designed ankyrin repeat proteins), lipocalins, fynomers and affibodies, to name a few.213 As an example, DARPin technology utilises a synthetic scaffold of 13-17 kDa without any cysteines derived from ubiquitous receptor proteins. Repeat sequences consist of a β-turn and two flanking α-helices arranged so that a binding surface is created that provides access to randomisable loops that can be modified to provide novel target binding surfaces. The size of the DARPin can easily be modified by varying the number of repeat motifs present to enable binding of different targets. DARPins have the potential of being very effective therapeutic products. They have high thermodynamic stability, are soluble up to 100 mg/ml and exhibit high thermodynamic stability and binding affinity.27, 204-206, 214, 215 Boder and Jiang noted that the smaller a designed scaffold is the lower its immunogenicity is expected to be. It is therefore likely that DARPins may not induce significant immune responses in patients but the impact of the synthetic sequences is yet to be ascertained.216 One DARPin is currently in clinical trials and others are also currently in the preclinical pipeline.

1.4.4. Future prospects and hurdles to development Two of the biggest problems that need to be addressed in BsAb development are product stability and the means to upscale manufacturing without significant cost outlays. Furthermore, when designing a BsAb many factors need to be considered to select the optimal characteristics for a proposed therapeutic system. A large bulky format will limit a 30 product’s ability to penetrate deep into the tumour vasculature, and if administered in isolation a very small product such as a DARPin, nanobody or tandem scFv will be cleared from the patient’s system very quickly. In the latter mentioned situation the BsAbs would either have to be modified to have their half-lives extended by binding a larger partner molecule (HSA, PEG, nanoparticles or other drugs) or the benefit of repeat dosages needs to outweigh the high associated costs and time spent administering the drugs.

Careful consideration needs to be given to how multiple binding domains will affect each other in a single BsAb construct. Different targeting pairs, including the order of the moieties, can influence each other on many different levels including stability, binding affinity and mode of action – either as an enhancement or more commonly a decrease in BsAb design fecundity. The combination of binding moieties included in a system is also important when considering targeting two cell surface receptors on a single cell surface – if an incorrect pair is chosen the BsAb might over-stimulate a pathway that is meant to be suppressed. Notable prospects in the use of BsAbs as therapeutics are not limited to them being used as singular therapeutic agents. Besides being used as complementary therapies to chemotherapeutic treatments or in redirecting an immune response, they can also be used as facilitators of drug delivery by binding specific drugs or even drug-laden nanoparticles.

31

1.5. Nanoparticles Nanoparticles (NPs) are substantially larger than other next generation therapeutics with diameters ranging from 1 – 1000 nm with the majority being smaller than 400 nm. The applications of these novel technologies have the potential to provide benefit across a number of fields relating to molecular imaging, diagnostics and targeted gene and drug delivery-based therapeutics.217, 218 A major benefit of NPs over the use of standard chemotherapeutic treatment regimens lies in the ability of NPs to be loaded with high drug doses thereby limiting systemic drug exposure and protecting the patient from associated toxicity.219, 220 Furthermore, NP payloads are not limited to standard drugs and the therapeutic catalog can be expanded to include DNA, siRNA and other peptides. The ability to carry such payloads not only provides a means to combat resistance to various standard treatments (siRNA knock-out of chemotherapeutic resistance traits) but simultaneously protects the small molecules from the environment which would otherwise result in their degradation and inability to provide a therapeutic benefit.103, 221 To understand the main mechanism by which NPs reach a target or tumour cell some knowledge of the physiological characteristics of the tumour microenvironment is required.222, 223

1.5.1. Passive and active targeting The major factors relevant to NP targeting in the human tumour environment were extensively described by Bertrand et al., 2014.224 Improved understanding relating to tumour biology, including leaky vasculature and the enhanced permeability and retention effect (EPR effect), has accelerated the development of NPs for targeted delivery in both passive and active formats.220 As tumours grow their eventual size becomes restricted due to insufficient availability of oxygen. As a result, the tumour cells secrete growth factors which promote the formation of additional vascular structures. These structures are however “leaky” and capillary sizes range from 200 to 2000 nm, enabling oxygenated blood to easily extravasate into the tumour microenvironment; described as enhanced permeability. Enhanced retention is then promoted due to the absence of a functional lymphatic drainage system to drain interstitial fluid from the tissues to the lymphatic system. The process allows molecules smaller than 4 nm to escape but retains anything larger; a process which allows NPs, whether passively or actively targeted, to enter and remain in the tumour microenvironment (Figure 1.13).223-225

32 Figure 1.13: NP drug delivery through passive and active targeting. Adapted from illustration published in Peer, 2007; Copyright, Nature Nanotechnology.226 Passive targeting (A) takes place by utilising the EPR effect associated with leaky vasculature in the tumour microenvironment whereas active targeting involves the same mechanism but provides an improvement due to specific cell-targeting and the potential for enhanced cellular uptake of payload-carrying NPs. Active targeting as illustrated in the inset can result in the NPs i) releasing the payload close to the target cell, ii) binding to the cell-surface and undergoing a slow, controlled payload release without particle internalisation, or iii) particle internalisation and subsequent endosomal degradation and payload release.

Non-targeted (passive) NPs can be rapidly cleared from circulation by opsonisation where NPs are engulfed by macrophages or accumulation in the liver or spleen. However various means have been developed which circumvent this phenomenon such as coating passive NPs with polyethylene glycol (PEG). PEG-layers circumvent NP clearance by preventing protein absorption to NP surfaces (a trigger for opsonisation).225 An obvious factor affecting NP accumulation in a tumour environment is particle size. The optimal particle size is expected to be between 10 nm and 500 nm to avoid both renal clearance and phagocytic internalisation respectively.227 Smaller particles are able to better penetrate the tumour, particularly solid tumours with low permeability, but are then also more prone to being recycled back into the vascular system.

33 NP charge, whether positive or negative affects the efficacy of the particles in vivo. It is expected that charges ranging from -10 mV to +10 mV can aid in avoiding phagocytic internalisation and prevent NP-aggregation.227 Negative particles exhibit varied effects in tumour treatment but positive charged particles are thought to preferentially interact with tumour blood vessels which subsequently limits their ability to penetrate deeper into a tumour or back into circulation. A positively charged particle also associates with the extracellular matrix which further limits particle diffusion. Coating particles with substances such as salt and heparin can mask charges and improve therapeutic efficacy. Lastly, particle shape also impacts levels of tumour accumulation in that elongated products exhibit higher tumour accumulation levels.

The EPR effect is well described in animal models but has not been evaluated as extensively in humans due mainly to the difference in analytical methods (tissue extraction in animals). Available data suggests that the EPR effect may vary between patients and different cancer types exhibit variable levels of the EPR effect. Therefore relying on this mechanism as the primary means of NP delivery will likely not always present in a therapeutic benefit. Without a means to direct payload carrying NPs to specific targets agents such as siRNA and protein-based treatments, which cannot overcome the tumour environment and cross cell membranes, are unlikely to provide a therapeutic benefit.223, 224

The incorporation of targeting moieties into the NP structure, generally referred to as functionalisation, provides an advantage by further minimising off-target effects exuded by the NP-payloads (Figure 1.13).226 Providing NPs with a targeting function does not however overcome the problems associated with NP opsonisation and subsequent clearance although actively targeted NPs may improve the likelihood of a particle and its payload being internalised at a cell surface receptor. Selecting an optimal means to provide NPs with targeting functions, namely ligand type and conjugation methods, plays a major role in the efficacy of the overall proposed actively targeted NP system.224 NPs can be functionalised using a variety of different targeting ligands including antibodies, sugars and many other small molecules. The surface architecture of NPs influences the number of targeting moieties that can be bound on the NP surface. A resulting high surface density of targeting moieties incurs multivalency to the NP and thereby enhances the potential of the particles to exhibit high avidity binding at targets.224

34 An important consideration in active targeting is the strength of the bond between the NP and the targeting moiety. The method by which targeting moieties are bound to NP surfaces plays an important role in the production process.224, 228 Various chemical conjugation methodologies have been employed, however the chemistry involved can sometimes be time-consuming, expensive and inconsistent which is a barrier to up-scaled production of targeted-NPs for clinical evaluation. Furthermore, the timing of conjugation needs to be considered. If it is likely that solvents used in NP-loading may render the targeting moiety non-functional or degraded then conjugation needs to happen after NPs have been pre-loaded with the drugs of choice and purified. Another important consideration, particularly with NP systems where non-covalent ligand attachment is utilised, is whether or not the ligand will change the NP’s immunogenic profile. As described in Bertrand et al. the physiochemical properties of both the NP and the targeting ligand influence the actively-targeted NPs functionality and ability to be internalised at target cell surfaces (Figure 1.14).224

Figure 1.14: NP and targeting ligand properties affecting biodistribution and function. Adapted from illustration published in Bertrand et al.224

The types of targeting ligands (various proteins, peptides, nucleic acids, small molecules and antibody-based fragments) used to actively target NPs have been extensively reviewed; herein we will focus on antibody-based products and their targets.223-225 The first

35 antibody (mAb)-targeted NPs were described in the early 1980s.229, 230 While being a very effective means to give a NP targeting capabilities, the large size of whole mAbs (150 kDa) means that they can significantly alter the size and hydrodynamic radius of a NP, altering the NPs ability to effectively penetrate tissues. Conjugating mAbs to NP surfaces can also be problematic as the mAbs are sensitive to various chemicals and may become denatured during the process. The presence of Fc-components attracts immune cells which can subsequently result in NP clearance before they are able to reach their targets. The action of the Fc-domain can be subdued by total exclusion of the Fc by only using Fab or scFv domains as targeting moieties. Utilising scFvs (25 kDa) provides an additional advantage in that their association with NPs doesn’t increase the hydrodynamic radius as much as whole mAbs.49, 223

1.5.2. Types of nanoparticles Developing the optimal NP is influenced by a number of factors including the primary material composition of the NP, size and biodegradability.231 They have to be able to withstand the surrounding biological environment to control payload release while still being degradable at target locations, be taken up preferentially in areas where a biological benefit is required, possess good pharmacokinetic profiles, be non-immunogenic themselves and not aggregate.226 It may be difficult or impossible to develop NPs that have all of the optimal characteristics required in therapeutic applications. Various published reviews provide a comprehensive list of NPs that have undergone clinical evaluation.218, 226, 227 The two most commonly utilised classes of NPs in clinical development are liposomes and polymeric carriers.218, 223, 224, 226, 227, 231-233

Liposomal nanoparticles Liposomes are formed through association of lipid molecules to form enclosed bilayers and can carry both hydrophilic and hydrophobic compounds.22, 223, 227, 234 An attractive feature of liposomes is their relative ease of production under non-toxic conditions and low immunogenicity in patients. However they tend to be unstable and easily cleared from the bloodstream while also lacking the ability to enable controlled release of payloads. Functionalising liposome surfaces by inclusion of targeting moieties or PEGylation (as in PEGylated Doxil/Caelyx, the first approved liposome-drug for cancer) improves product stability.218, 223, 225, 235 Three non-PEGylated liposomes (DaunoXome, Myocet and Onco TCS) have been approved for cancer treatment while a fourth, Depocyt, is undergoing clinical trials (Phase I-III) against leukemia and glioblastoma. An additional 3 PEGylated 36 liposomes (Thermodox, SPI-77 and NL CPT) are progressing through clinical evaluation to join the approved Doxil/Caelyx liposome formulation as potential cancer treatments.218 Actively targeted liposomal particles are also under development; eight lipid-based particles have been functionalised to target cell surface receptors not limited to cancer- related indications and are currently in various clinical phases of development.219, 224

Polymeric nanoparticles Polymeric NPs are produced by association of many different types of monomers, branched or un-branched. Some polymeric forms are even capable of being responsive to environmental stimuli where a drug-payload can be released when a temperature, pH or other change happens in the particle’s immediate environment.218, 227, 231, 236 It can be difficult to efficiently load drugs into a polymeric NP’s core (albeit more efficient than liposomes), and polymers that self-associate can become too large for therapeutic applications.227 A definite advantage of polymeric NPs over liposomal formats is their ability to undergo controlled drug-release, improved drug-loading capabilities and superior stability.219 Although no polymeric NPs have been approved for clinical use yet, seven passive and three actively targeted polymer-NPs are currently undergoing clinical trials for various cancer indications.218, 225 Even though there are advantages in developing polymeric NPs instead of liposomal formats, there are many more lipid-based NPs in the clinical pipeline. However this is not necessarily due to liposomal NPs possessing superior properties but rather due to the fact that liposomes have been under investigation for a much longer period of time.220, 229, 230 Other NP formats such as quantum dots, gold nanocarriers and carbon nanotubes are also under investigation but have not progressed through to the clinical trial stage yet.225, 226, 237

1.5.3. The EnGeneIC delivery vehicle The EnGeneIC delivery vehicle, otherwise known as the EDVTMnanocell, constitutes another class of NP-based drug delivery system. These particles are bacterially-derived, anucleate, 400nm particles devoid of the genes required for cell division, and can be loaded with very high doses of cytotoxic drugs and siRNA.103, 129, 238, 239 Although many may consider the EDV’s large 400 nm size to be a potential hindrance to therapy, this may not be the case. EDV size is thought to limit the BsAb-EDV system’s ability to penetrate normal tissues (such as skin and gastrointestinal cells) which only have fenestrations ranging between 50 and 100 nm compared with tumours (10 – 1000 nm).239 This attribute

37 would provide an advantage over other smaller NPs which are able to more readily penetrate normal tissues and illicit various side effects in patients.

EDV production and loading EnGeneIC’s bacterial strain of choice for EDVTMnanocell production is Gram- Salmonella typhimurium which has undergone genetic manipulation to produce minCDE- chromosomal deletion mutants. The purification protocol includes various crossflow steps, a salt treatment and subsequent filtration and, ensures that the end product retains its characteristic LPS surface, is sterile and more than 90% pure. Loading nanocells depends on the drug concentration of the incubating solution, time, and occurs down a concentration gradient. Nanocells can be loaded easily with high concentrations of a variety of drugs (Doxorubicin (Dox), Cisplatin (Cis), Paclitaxel (Pac), Vinblastine (Vin), etc) having different charge, hydrophobicity and solubility despite the presence of a bilayer membrane. Drug efflux was shown to be notably absent over a 72 hour period when the EDVs were incubated in buffered saline gelatin.129 To load EDVs with siRNA, plasmids encoding the sequences for siRNAs are transformed into the S. typhimurium strain prior to nanocell purification. Each nanocell was shown to be preloaded with up to 50 copies of plasmids and 14 000 copies of siRNA.103, 239

EDV targeting The EDVs are actively targeted to cancer cell surface targets through the use of BsAbs (designed to bind the nanocell’s natural surface lipopolysaccharide (LPS) and EGFR on cancer cells simultaneously) (Figure 1.15).129, 238, 240, 241 EnGeneIC’s original BsAb format was produced by mixing equimolar amounts of two full mAbs (anti-LPS and anti-EGFR) with a protein A/G molecule. To conjugate the BsAb to the EDV all that is required is a simple mixing step of both the purified BsAbs and EDVs followed by a brief incubation period shaking at room temperature and subsequent spin/wash cycles in phosphate buffered saline (PBS). Thus, using a BsAb to target NPs requires no complicated chemical conjugation steps, allowing for efficient and scalable production without compromising either the NP, drug or antibody integrity. Furthermore, the NPs are loaded with drugs or other therapeutic compounds prior to them being functionalised with the desired targeting moiety. This method is beneficial in ensuring that the BsAb to be used for targeting is not negatively impacted by the harsh conditions associated with drug loading and nanocell production.129

38 239 Figure 1.15: Loading and functionalisation of EDVTMnanocells. As illustrated in MacDiarmid and Brahmbhatt.

In vitro and in vivo results The use of a protein A/G was described as the reason why complement-mediated toxicity through interactions with the Fc were not observed in the original system since the Fc is blocked for complement activation by protein A/G.129, 239 Active targeting of nanocells not only facilitates targeted drug delivery but provides a means for these NPs to be taken up into the cell (endocytosis) and degraded intracellularly for optimal payload release. Although the mechanism (e.g. macropinocytosis) by which the NPs enter the cell is not yet fully understood, EnGeneIC have demonstrated that nanocells targeted using their Protein A/G BsAb format do enter the cell and provide a therapeutic benefit. EnGeneIC’s initial data showed that single cells could carry as many as 40 – 50 nanocells enclosed in 5 – 6 vacuoles. In comparison, non-targeted EDVs do not enter the cell which suggests a definite link to the active targeting being a requirement for particle internalisation. Over time internalised targeted EDV membrane integrity is lost and the payload released, as shown in MacDiarmid et. al.129, 241

In vivo mouse xenograft models and studies of canine lymphomas have shown significant amounts of tumour regression even though drug and antibody concentrations were low. 39 EnGeneIC found that administering 150 µg of free Dox in mouse xenograft models does not illicit the same high tumour growth inhibition as treating with BsAb-EDVTMnanocells loaded with a mere 0.08 µg Dox. Similarly, treatments with Pac required 8000-fold lower amounts compared with standard systemic drug administration. In addition to this lower drug concentration, they also noted that there appeared to be a distinct absence of some of the toxic effects normally associated with chemotherapeutic drug treatments. Data has shown that in comparison to the clinically approved passive liposome, Doxil, nanocells targeting EGFR and loaded with Dox required 100-fold lower doses to achieve similar tumour regression in xenograft models.129, 238

It is also possible to load EDVs with siRNA or DNA as a therapeutic means to knock out genes responsible for drug resistance in tumour cells which subsequently increases the therapeutic benefit of treating with chemotherapeutic agents which would otherwise have been ineffective.103, 221, 239 The importance of this attribute lies in that siRNA is normally poorly membrane permeable, easily degradable by serum nucleases, illicit off-target effects and intracellular delivery is a requirement for therapeutic efficacy. The nanocell provides a platform whereby siRNA can be used as a viable therapeutic option by overcoming all of these potential drawbacks.103, 239 Sequential treatments using firstly siRNA-loaded targeted nanocells followed by a drug a tumour was known to be resistant to, showed evidence of the reversal of the resistance phenotype.

Furthermore, a study on trophoblast overgrowth associated with various gynaecological disorders, including ectopic pregnancies, confirmed the ability of EGFR-targeting BsAb- EDVs to effectively treat trophoblast tissue with Dox. The actively targeted NP-system was much more effective than both a non-specific and non-targeted EDV format.241 Initial biodistribution studies showed a definite advantage in actively targeting the EDVs to cancer cells in that a much higher drug doses was localised at the site of interest. Although the results also indicated that EDVs were present in the lungs, spleen and liver, these were rapidly cleared and were only detected at very low levels after 24 hours.

1.5.4. BsAbs as facilitators of therapeutic NP delivery The EDV is unique amongst other targeted NPs currently in clinical trials by its use of BsAb for targeting rather than chemically conjugated moieties. EnGeneIC’s original BsAb format was quite large, and consisted of two mAbs (150kDa each) plus the additional protein A/G molecule used to connect the individual mAbs. The method of BsAb 40 production could even result in products of up to six mAbs. Although their original work did not note any problems with penetration associated with the increased hydrodynamic radius of the nanocell, it is logical to expect that in a clinical setting where the EPR effect isn’t as well understood the larger product size may be a hindrance in therapeutic efficacy. Thus the development of a smaller NME to functionalise the nanocell surface could prove to be a much more potent endeavour.103, 129, 239, 240

Schneider et al. described a means to utilise BsAbs to deliver siRNA to tumour cells. They found that although the siRNA was effectively delivered and internalised, the siRNA was unable to escape the endosome to provide a therapeutic benefit. To overcome this barrier to therapy they formulated the siRNA into polyconjugate (Dynamic polyconjugates or DPCs) or liposomal NPs targeted to cells with BsAbs.242, 243 They found that using the BsAbs as facilitators to deliver siRNA carrying DPCs of a endosomolytic nature to target cells, resulted in a highly specific and effective means to incur mRNA knockdown in targeted cells. However the process involved conjugating digoxigenin (BsAb scFv target on NP) to siRNA which is then formulated into the NP or polymer surface, in addition to the inclusion of the BsAbs that have to undergo proteolytic cleavage; all of which makes the production process less efficient. Next they evaluated the use of PEGylated lipid-based NPs. Here the PEG-surfaces were functionalised with a target for the C-terminal end of the BsAbs which also made the functionalisation process less efficient. They also noted that a decrease in mRNA knockdown was evident possibly due to the presence of PEGylated surfaces. Comparatively the means by which the EDVTMnanocell is functionalised is far simpler and as siRNA can be housed inside the nanocell system it is protected further from possible environmental degradation.103, 128, 242, 243

Another actively targeted format, which utilises diabodies as the targeting moieties for polymerised lipid NPs (PLNs), required that cysteines be added to the Db. This enables the Db to be conjugated to the PLN using maleimide chemistry.244 A BsAb targeting methoxy PEGylated (mPEG) liposomal NPs and EGFR on cancer cells was produced by Kao et al.228 Although this BsAb binds the NP non-covalently by the same means as in the EDVTMnanocell, the use of liposomal NPs is in this instance the drawback in comparison with the nanocell system. As previously mentioned, EDVTMnanocells can be loaded with higher drug concentrations and exert a greater therapeutic benefit than other liposomal NPs (e.g. Doxil). However the system has many attractive features similar to that of EDVTMnanocells. A lack of chemical conjugation steps circumvents the risk of degradation 41 of drugs, NPs and targeting moieties. The modular nature of the BsAbs enables a means to effectively substitute one disease-targeting moiety for another and because the BsAbs can only attach in a specific orientation, the NP surface can be relatively homogenous given a uniform surface antigen distribution.103, 129, 228, 238, 239, 241, 245 Another major difference however is that the format described by Kao et al. is applicable across a wide variety of NPs as long as they possess mPEG on their surfaces, whereas the BsAb in the EDVTMnanocell system is designed specifically for the LPS on its surface.129, 228, 240

42 1.6. Conclusion and research aims

1.6.1. The future of nanoparticles Factors important in NP design and development include characterisation of the degree of accumulation in tumour environments, drug loading capabilities, controlled versus rapid payload release (is the drug housed internally or on the NP surface) and immunogenic profiles. Couvreur noted that developing products that mimic natural products and are able to escape the endosome once inside a cell could be an ideal means to enable effective NP delivery to target surfaces.220 Given that the EDVTMnanocell is based on a naturally occurring entity and has been shown to efficiently release payloads intracellularly following endosomal escape, nanocells may well be a highly promising technological advance amongst developed NP systems.103, 129, 240, 241, 245

NP development for clinical use is still in its infancy and a lot of work remains to be done to fully evaluate all aspects of NPs and how their physical characteristics (charge, surface material, ligand density, shape etc.) affect biological and therapeutic outcomes in patients. The benefit of active targeting also needs to be fully elucidated given that it is believed that the main method by which both passively and actively targeted NPs reach tumour cells is via the EPR effect. Nonetheless, the inclusion of targeting moieties in NP-delivery systems seems to enhance particle uptake and internal release of drug payloads, which is of particular benefit in the case of siRNA delivery where external degradation would have precluded any potential therapeutic benefit. However, the degree to which particle uptake and controlled payload release varies between different types of NPs still needs to be determined. The most important benefit of actively targeted NPs however is in that they provide a means to avoid non-specific activity of drug payloads on normal tissues; thereby increasing the therapeutic benefit at a targeted site where treatment is actually required.218, 219, 223, 224, 227, 228, 231, 238

The wide variety of targeting moieties available to functionalise NP surfaces endows different products with various physiological characteristics. Moreover the means by which these targeting moieties are conjugated to the NP surface is of significant consequence. The use of chemical treatments to conjugate ligands to NP surfaces can be costly, inconsistent and time-consuming; thus making the use of alternative options such as engineered BsAbs specific for a disease cell surface target and the NP itself very appealing. BsAbs provide a means to simply and efficiently “label” NPs with targeting 43 moieties in a homogenous orientation on its surface to present to target antigens, thereby completely avoiding the drawbacks of chemical techniques.

1.6.2. Research aims and strategy The BsAb format utilised in the original EDVTMnanocell system was produced by conjugation of two mAbs (anti-EGFR and anti-LPS) to Protein A/G. Protein A/G has six binding sites for mAbs, so can therefore bind multiple specificities.129 Although this design showed proof of principle in the use of BsAbs to direct EDVTMnanocells to their targets, manufacture of this design is cumbersome and inconsistent. This is because there is no control of how many of each mAb binds the Protein A/G molecule, resulting in a heterogeneous product. A more homogeneous and scalable approach to producing BsAbs is to produce them recombinantly. Different recombinant formats need to be evaluated to determine how the design influences production and final product quality.129, 240

The aim of this project is to develop novel, modular, BsAb formats that can be used in conjunction with the novel drug delivery vehicle technology developed by EnGeneIC Ltd; the EDVTMnanocells, to target specific cancer cell surface markers. To achieve this aim a number of objectives needed to be achieved, namely:

1. Design a number of novel BsAb formats, with specificites towards a tumour cell surface marker (EGFR) and a surface antigen on the EDVTMnanocells (LPS). 2. Construct and express the BsAb formats, and evaluate for product yield, stability and in vitro binding affinities. 3. Perform bioassays on BsAbs to evaluate their suitability in targeting cancer cells when bound to EDVTMnanocells.

By engineering modular BsAb formats we can develop a technology that will utilise mAbs and scFvs that are either novel or have already passed clinical trials to be inserted into the BsAb-EDVTMnanocell system with ease for simple target redirection, enhancing the potency of the targeting moieties via the linked cytotoxic payload.225, 246 The research described herein will inform researchers across multiple fields as to a means to efficiently evaluate and optimise targeting moieties of an actively targeted drug delivery system. This research improves on EnGeneIc's original protein A/G BsAb format by using recombinant technologies to produce a more scalable and cGMP compliant targeting moiety, which avoids the use of chemical conjugation and multistep NP functionalisation. By 44 incorporating these new BsAb designs into the EDVTMnanocell, EnGeneIC have developed an active NP delivery system ahead of its counterparts.

45 2. Materials and methods

2.1. EnGeneIC delivery vehicle (EDVTMnanocell) production Protocols outlining the production process involved in EDVTMnanocells (EDVs) manufacturing are described in the supplementary material of MacDiarmid et al. (2007).129 Initially a number of different EDV-producing bacterial strains were created and analysed to select the optimal version.129 Finally a Salmonella typhimurium minCDE¯ strain was selected for EDV production.103, 129 Currently the production of EDVs has been scaled up to be completed in a fermenter using conditions similar to that previously described in MacDiarmid et al. (2007).129 EDV purification is completed using a number of cross-flow filters instead of the centrifugation steps previously described to ensure product homogeneity and the complete removal of contaminating matter such as parental bacterial cells, endotoxin and other cellular debris.129

The production process currently takes 3 days to complete and further technical details relating to the method are subject to confidentiality and cannot be revealed by EnGeneIC Pty Ltd (personal communication I. Sedliarou).

2.2. Routine molecular biology techniques

2.2.1. Vector design Vectors containing BsAb sequences were developed by either GeneArt (Life Tachnologies) or staff at the National Biologics Facility (NBF) based at the Australian Institute for Bioengineering and Nanotechnology (AIBN). For the purpose of transient mammalian expression BsAbs were cloned into pcDNA 3.1+ (Invitrogen).

2.2.2. Bacterial transformation Restriction enzyme (New England Biolabs or Roche Life Science) digested vectors and fragments of interest were cleaned up (Qiagen QiaExII gel extraction kit) and subsequently underwent ligation (Roche Rapid DNA Ligation kit) to produce new plasmids containing recombinant protein DNA sequences for eventual mammalian expression. For bacterial transformation to take place ligation mix was added to either TOP10 or α-select gold efficiency (Bioline) competent cells that were thawed on ice. The mixture was left to incubate on ice for 15 minutes then underwent a heat shock treatment in a 42 °C waterbath for 30 seconds and placed back on ice for another 2 – 5 minutes. Sterile LB was 46 subsequently added to the mix and the samples placed at 37 °C, shaking at 220 rpm for 1 hour after which the sample was spread out on agar plates containing either LB-Ampicillin or LB-Kanamycin depending on the required selective pressure for correct clone growth to take place. Plates were incubated at 37 °C overnight.

2.2.3. Colony PCR To screen whether or not clones from bacterial transformation carried the fragment of interest colony PCRs were routinely performed. A mastermix was set up for the PCR that contained 0.5 µM (final concentration) of each primer (T7-Forward and BGH-Rev), sterile water and Promega 2× mastermix, from which 10µl was aliquoted out per colony to be screened. Selected colonies were picked using a sterile, short, 10 µl pipette tip and dipped in the PCR mix containing tube. The tip was then transferred to an appropriately labelled sterile 1.5 ml eppendorf tube and stored until a positive clone is selected to be cultured for DNA stock preparation.

The PCR tubes were placed in the thermocycler and a pre-programmed colony PCR program loaded with the following conditions:

94 °C 10 min 25 cycles of: • 94 °C 1 min • 50 °C 30 sec • 72 °C 2 min 72 °C 10 min 15 °C hold

On completion of the run 1.5 µl of loading dye was added to each reaction and 5 µl of the samples run on a 1% agarose gel for 30 – 40 minutes. Positive clones were identified according to the expected insert size.

2.2.4. E. coli culture for maintenance of DNA stocks For the purpose of maintaining DNA stocks prepared either as a miniprep for molecular biology processes (Qiagen QiaQuick Miniprep kit) or a maxiprep for transfection (Invitrogen HiPure Filter Maxiprep kit), E. coli cultures were grown using LB broth at either 5 ml or 400 ml volumes respectively. LB broth was prepared by adding 25 g/L of premixed 47 LB powder to RO water after which the media was autoclaved at 121 °C for sterilisation purposes. Selective pressures were applied to ensure growth of the correct clones by the addition of either 100 µg/ml Ampicillin or 30 µg/ml Kanamycin, to the sterile LB broth. A single colony or starter culture prepared from a single colony was then added to the prepared growth media for minipreps or maxipreps, respectively. Cultures were incubated at 37 °C for 8 – 18 hours depending on the application, and 220 rpm.

On completion of incubation period cultures were spun at 5 200 g at room temperature for 20 minutes. The supernatant was then discarded and miniprep and maxipreps completed using manufacturers’ recommended reagents and conditions. DNA concentrations were determined using a nanodrop spectrophotometer and stored at -20 °C.

2.2.5. DNA Sequencing set up Sequencing reactions were set up following plasmid DNA extraction from E. coli culture using a Qiagen QiaQuick Miniprep kit. DNA concentrations were determined using a nanodrop. Each reaction contained a single primer and therefore 2 reactions were set up per plasmid sample as follows:

200 – 250 ng plasmid DNA 10 pmol Primer (Forward or Reverse) Sterile water to make up a 12 µl total volume

Prepared samples were submitted to the Australian Genome Research Facility (AGRF), Brisbane node, for sequencing on AB3730xl Sequencers following completion of a BigDye Terminator (BDT) v3.1 (Life Technologies) labelling reaction and subsequent sample cleanup using CleanSeq beads. Traces received from the AGRF were analysed for the presence of the DNA sequence of interest and absence of any mutations such as deletions, frame-shifts and point mutations. Positive confirmation of the plasmid quality led to maxipreps being completed to prepare high quality amounts of DNA template for the purpose of sterile transient transfection.

48 2.3. Sequence design

2.3.1. EGFR-ecd The DNA sequence of the extracellular domain (amino acid residues 25-645) of wildtype human EGFR (Swiss-Prot P00533) was synthesised by Geneart, incorporating a mammalian leader sequence from IgK and a C-terminal His tag (Figure 2.1.a), with codon optimisation for expression in CHO cells. Further constructs consisting of amino acids residues 25-525 followed by either human or mouse Fc tags were created (Figure 2.1.b-c), similar to that described by Adams, 2009.48

2.3.2. EGFR-targeting and negative-control scFv The sequence information for the heavy and light chain variable regions for the commercial anti-EGFR mAb panitumumab (Vectibix) was obtained from US Patent 6 235 883. The sequence for an unrelated control antibody (anti-RSV) palivizumab (Synagis) was obtained from the RCSB Protein Data Bank file, 2HWZ (Figure 2.2).

2.3.3. EDV-targeting scFv The heavy and light chain variable region sequences of the anti-LPS mAb (Figure 2.2) were determined by isolating and sequencing cDNA isolated from the hybridoma 1H10, using established protocols.247

49

Figure 2.1: Recombinant EGFR sequences. The a) initial EGFR sequence included a C-Terminal His-tag and 116 additional amino acids (green) not included in the b) human-Fc and c) mouse-Fc tagged recombinant EGFR sequences.

50 Figure 2.2: Variable chain sequences for BsAb design. The a) anti-EGFR (panitumumab) sequences were reverse engineered from the US patent while the b) negative control, anti-RSV (palivizumab), variable fragment details were sourced from a Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank File. The final variable sequences for c) the anti-LPS, EDVTMnanocell targeting, scFv designs were determined by sequencing cDNA from a 1H10 hybridoma.

2.3.4. BsAb sequences The DNA templates for all BsAb formats except the original IgG-like format were synthesised by Geneart with codon optimisation for CHO expression. Construct designs are shown in Figure 2.3, showing the location of the immunoglobulin leader sequence, glycine-serine linkers, Fc domains, stabilisation linkers and engineered cysteine residues.

51 The tandem scFv included 6xHis and c-myc tags for purification and detection, whilst the other constructs utilised the Fc domain for purification and detection.

Tandem scFv

The DNA template for BsAb Vectibix-G4S-1H10 was synthesised by GeneArt (Figure 2.3.a) and was subsequently cloned into a mammalian expression vector (pcDNA 3.1+) to produce a tandem scFv format. This BsAb was designed to include a 5 amino acid glycine- serine linker (G4S) between the Vectibix- and 1H10-scFv; the shorter linker reduces flexibility and therefore prevents miss-folding from occurring between different scFv components (Table 2.1). The sequence was flanked by a N-terminal His- and C-terminal myc-tag to aide in detection and downstream purification; creating a 55kDa construct.

A second tandem scFv was designed to be used as a negative control that would be unable to bind EGFR receptors. Here we used the variable chains of a commercial anti- RSV mAb, namely palivizumab (Synagis). Any binding to EGFR from this construct would suggest non-specific binding by the BsAb format or EDVTMnanocells depending on the context of the experiment.

52 Table 2.1: BsAb format and sequence design parameters. (A) Tandem scFv, (B) Fc-containing and (C) Knob-in-Hole (KIH) BsAbs. (A) The tandem scFv, incorporated a G4S linker as well as N-terminal His and C-terminal myc tags. Homodimeric Fc-containing variants (B) incorporated an IgG1-Fc linker; variants of (B) included either G4S or hinge-like (Longlink) linkers to connect αEDV scFv’s to the CH3 domain. Variants also included or excluded engineered cysteine residues in scFv’s for enhanced stability. KIH constructs (C) are heterodimers with αEDV heavy and light variable regions on separate chains. Format design included a hinge-like (Longlink) linker connecting CH3 to αEDV variable fragments and again either included or excluded engineered cysteine residues in scFv’s. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. Additional CH3-αEDV Linker BsAb ID Sequence information disulphide Structural Illustration G4S Longlink bridges

A Tandem scFv His-αEGFR-G4S-αEDV-myc NA NA

Fc-containing αEGFR-G4S-Fc-G4S-αEDV Fc-containing (Longlink) αEGFR-G4S-Fc-Longlink-αEDV Fc-containing (Cys) αEGFR(cys)-G4S-Fc-G4S-αEDV(cys)

53 B Fc-containing (Cys αEGFR(cys)-G4S-Fc-Longlink-αEDV(cys) Longlink)

αEGFR(cys)-G4S-Fc(Knob)-Longlink-VHαEDV(cys) KIH 1 (Cys) + αEGFR(cys)-G4S-Fc(Hole)-Longlink-VLαEDV(cys) C αEGFR-G4S-Fc(Knob)-Longlink-VHαEDV + KIH 2 αEGFR-G4S-Fc(Hole)-Longlink-VLαEDV Figure 2.3: BsAb sequence information. a) Engineered sequence sent to GeneArt to develop Vectibix-G4S-1H10 tandem scFv BsAb. b) Fc-containing Vectibix-Fc-1H10 sequence developed by splicing overlap extension (SOE) PCR and restriction enzyme digestion. c) Sequence designs sent to GeneArt to develop Knob-in-Hole formats for co- transfection. Both a) b) and c) show the locations of restriction enzyme sites used during cloning and to produce a modular structure which would enable scFvs to be exchanged for different ones easily when the need arises to target different markers.

54 Fc-containing BsAb through Splicing Overlap Extension (SOE) PCR The second BsAb (Fc-containing) format was designed to incorporate a longer linker between the scFvs (Figure 2.3.b) and give it the ability to function as a quadromer by binding two of both the EGFR (cancer cell) and LPS (EDV) antigens. We used an IgG1 Fc- domain to link and separate the two scFvs spatially thereby developing the Vectibix-Fc- 1H10 homodimeric Fc-containing BsAb (Table 2.1), similar to the scFv-Fc-scFv BsAb dimer described in Jendreyko et al, 2003 among others.118, 151, 169, 193 Fc-domains (Hinge- CH2-CH3) were amplified from in-house materials. To create the Fc-containing BsAb construct we firstly produced a plasmid that contained the sequence for the Fc-domain and 1H10-scFv. The only scFv that needs to be interchangeable is the cancer cell-specific scFv; therefore no restriction enzyme sites were included between the Fc and 1H10-scFv because the anti-LPS component is considered to be a permanent component of all the construct designs for this project. The Fc sequence and 1H10 sequences were PCR amplified from their original vectors, using primers which incorporated complementary overhang sequences to allow subsequent SOE PCR (Table 2.2). After purifying the individual bands a number of splicing overlap extension (SOE) PCRs were performed to produce the required Fc-1H10 sequence.248 The cycling conditions were:

98 °C 30 sec 10 cycles of: • 98 °C 10 sec • 50 °C 30 sec • 72 °C 1 min 20 cycles of: • 98 °C 10 sec • 68 °C 30 sec • 72 °C 1 min 72 °C 10 min 15 °C hold

55 Table 2.2: Splicing overlap extension. Primer sets as used in the development of the homodimeric Fc-containing BsAb format from IgG1 Fc and Panitumumab (Vectibix) tandem scFv sequence components.

Primer name Primer sequence (5’ – 3’) Tm (°C) Description

TTA ATT GAG CTC ATA Fc individual PCR primer including a 5’ Fc_Fc-link_ TAT TCC GGA GGA GGC 73 overhang with Sac1 and BspE1 RE sites and a Forward GGA TCA ACC CAC ACC TGC CCT sequence for a SG4S linker. CCA GCT GCA CCT CTG Fc individual PCR antisense primer sequence Fc_longoverlap_ ATC CAC CAC CTC CTG 76 for 3’ end of Fc region and overhang for SOE Reverse ACT TGC CAG GAG ACA GG PCR to connect to 1H10-scFv. No RE sites. 1H10-scFv individual PCR antisense primer 1H10_Fc-link_ sequence for 3’ end of 1H10 region and GGC GCC TCT AGA TCA 66 Reverse GAT GAT TTC CAG GCG overhang to include a stop codon followed by a Xba1 RE site.

CCT GTC TCC TGG CAA To create a long overlap between the 3’ end of 1H10_longoverlap_ GTC AGG AGG TGG TGG 76 the Fc and 5’ end of the 1H10-scFv. No RE Forward ATC AGA GGT GCA GCT GG sites. Vectibix (Panitumumab) scFv individual PCR Vect_Fc-link_ AAT AAT GAG CTC CAG 63 primer including a 5’ overhang with a Sac1 RE Forward CTC CAG CTG CAG G site. Vectibix (Panitumumab) scFv individual PCR Vect_Fc-link_ antisense primer sequence for 3’ end of GGC CCG TCC GGA CTT 67 Reverse GAT TTC CAC CTT GG Vectibix region and overhang to include a BspE1 RE site.

To complete this part of the experiment I then transformed the Fc-1H10 sequence into a mammalian expression vector following restriction enzyme (RE) digestion and performed a colony PCR of the resulting colony. Next the Vectibix (Panitumumab)-scFv component was PCR amplified from the tandem scFv construct using primers: Vect_Fc-link_Forward and Vect_Fc-link_Reverse (Table 2.2); and transformed into the new Fc-1H10 plasmid to produce Vectibix-Fc-1H10. No additional tags were added onto the construct as the presence of the Fc-domain would enable identification and purification through the Fc- domain’s ability to bind protein A.

Knob-in-hole (KIH) BsAbs The third designed format was developed to limit the number of EDVs that one BsAb can bind by only having a single 1H10-scFv present on the construct (Figure 2.4). Heterodimeric BsAb formats were based on the common Knob-in-hole constructs and partly on the TandAb BsAb designed by Affimed where variable linker lengths are used to

56 determine chain association for functional BsAb formation (Table 2.1).138, 140, 155 Knob-in- hole (KIH) engineering of the CH3 sequence facilitates heterodimeric association as described in Atwell et al. 1997.63 A “knob” was produced in one heavy chain by amino acid substitution for a longer side chain (T366’W) whereas a “hole” was produced in the second heavy chain by amino acid substitutions for shorter side chains (T366’S:L368’A:Y407’V); sequence positions as indicated by Kabat et al. 1991.63 As in Vectibix-Fc-1H10 (Fc- containing); here the Fc constitutes the linker region that effectively separates the two scFvs – two DNA templates were required, one containing the 1H10 Vh region and “knob” mutation and one containing the 1H10 Vk region and “hole” mutation (Figure 2.3.c). To ensure that the VH- and VL-chains associate with one another to form a functional 1H10- scFv, linker length between the Fc CH3 domain and the 1H10-scFv component was designed to include only the longer linker as described below (Figure 2.4).

Figure 2.4: Diagram representing sequence orders for the design of BsAb construct 3. a) Sequence of construct “light” chain. b) Sequence of construct “heavy” chain.

Stability engineered formats Cysteine stabilisation substitutions were made at Kabat position VH44 and VL100 of the 141, 181 scFvs. Fc-containing variants included either a short (G4S) linker or a longer linker (SSDKTHTSPPSPGGGGSGGGGSGGGGSGGGG) as described in Moore et al. 2011; connecting the CH3 domain to the 1H10-scFv (Table 2.1).138

2.3.5. Commercial mAbs – positive and negative controls Whole mAbs for panitumumab (Vectibix) and palivizumab (Synagis) were created by reformatting the BsAb sequences using a previously described method.249

57 2.4. Mammalian cell culture

2.4.1. Cell resuscitation and passaging Aseptic techniques were maintained in all cell culture related experiments and processes completed in Class II biosafety cabinets that were cleaned using 80% (V/V) ethanol and UV sterilised at the end of each day. Media prepared in the cabinets were heated to 37 °C in a water bath located external to the cabinet but still within an area dedicated to large- scale quality protein production.

Chinese Hamster Ovary cells,250 in this instance the suspension adapted CHO-S (Invitrogen), were stored in cryovials located in liquid nitrogen. Resuscitation involved transporting selected vials on dry ice and thawing the cells at 37 °C after which 1 ml of pre- warmed media (CD-CHO + 8 mM Glutamax) was added directly to the cells. The cell suspension was transferred to a 125 ml shaker flask to make up a total volume of 20 – 30 ml (Table 2.3) so that cells equated to a viable density of around 0.2 × 106 cells/ml in the prepared media. Flasks were maintained for 2 - 3 days shaking in a humidified incubator at 37 °C with 5 % carbon dioxide and 220 rpm. For passaging cells were routinely seeded at 0.2 – 0.4 × 106 cells/ml in 20 – 30 ml media, and densities were maintained to not exceed 3.4 × 106 cells/ml. CHO-S cell counts were performed using a Cedex HiRes (Roche) which also provided values such as cell viability, size and aggregation rate.

Table 2.3: Vessels for cell culture. Maximum cell number Growth type Vessel Supplier or culture volume 6 8 x 10 cells, 2 25 cm T-flask 4-8 ml media 6 20 x 10 cells, 2 Corning Inc (Corning, Adherent 75 cm T-flask 10-40 ml media NY, USA). 6 40 x 10 cells, 30- 2 150 cm T-flask 60 ml media 8-40 ml 125 ml shake flask Corning Inc. 40-100 ml 250 ml shake flask Suspension 100-180 ml 500 ml shake flask 180-300 ml 1 L shake flask 0.3-1 L 3 L shake flask

2.4.2. Transient cell culture media and techniques Transient expression of all BsAbs was performed using PEI-mediated transfection of suspension adapted CHO cells using CD-CHO (Gibco, Life Technologies) chemically defined media supplemented with 8 mM Glutamax and 0.4% anti-clumping agent (ACA), as previously described (Table 2.3).251 Knob-in-hole chains were co-transfected at a 1:1 58 DNA ratio (1H10-Vh:1H10-Vk). Culture supernatants were harvested 7-10 days post- transfection by centrifugation, then filtered and stored frozen at -80 °C until purification could be completed.

2.5. Qualitative western blot for BsAb expression analysis 300ml transient transfections were completed for all BsAb constructs, and cell pellet and supernatant samples taken at 2, 5, 7 and 9 days post-transfection. Transfections were all harvested at Day 9 and supernatants stored at -80 °C until purification could take place. Pellet samples were collected by spinning down 1ml of each sample at 9 000 g, removing supernatant, and washing 3 times with sterile PBS and storing at -20 °C. The removed supernatant was collected to ascertain whether or not BsAb had been secreted from the cells at the various time points. Pellet samples were resuspended in 50µl Dulbecco’s PBS. For western sample preparation 10µl of supernatant and pellet samples were added to 4 µl 4× LDS Sample Buffer (Invitrogen) and 2 µl 10× Reducing Agent (Invitrogen). Supernatant samples were incubated at 95 °C for 5 minutes whereas pellet samples were incubated for 15 minutes before loading onto a Novex 4-12 % Bis-Tris PAGE gel and run for 35 minutes at 200 V in 1× MES buffer (Invitrogen).

Separated proteins were transferred to PVDF membrane using a BioRad Trans-Blot Turbo Transfer System and PVDF Transfer packs, with a 7min mixed molecular weight transfer protocol, as per the manufacturer guidelines. Following transfer, the membrane was rinsed with PBS-T and blocked in PBS-T + 2 % milk, then secondary antibody added for 30 minutes. For the tandem scFv BsAb, a mouse anti-6xHis-HRP conjugated (BD ’ Biosciences) secondary antibody was used, and an anti-human IgG (Fab) 2-HRP (Sigma- Aldrich) was used for the Fc-containing and Knob-in-Hole variants. Both secondaries were at 1mg/ml and used at a 1:10 000 dilution in PBS-T. Membranes were then washed 3 times in PBS-T at 5 minute intervals while shaking and 5mL ImmunStar WesternC solution added to each membrane for imaging using a BioRad ChemiDoc MP.

2.6. Affinity chromatography

2.6.1. IMAC and TFF His-tagged constructs (tandem-scFv and EGFRecd-His) were initially purified by IMAC chromatography. Mammalian cell culture media contains chelating agents that interfere

59 with this mode of purification therefore it was first buffer exchanged using tangential flow filtration (TFF).

Optimised protocol for His-tagged tandem scFv purification Optimisation of the tandem-scFv purification protocol led to the use of two sequential IMAC chromatography steps. First step using a HisTrap Excel (GE Healthcare) column which tolerates nickel chelating agents present in mammalian cell culture media, followed by HisTrap FF (GE Healthcare) which further removes impurities. Manufacturer- recommended buffers were used for equilibration and loading – no Imidazole used for equilibration and a 20 mM Imidazole wash with the HisTrap column, 20 mM Imidazole for equilibration of HisTrap FF and 500mM Imidazole for all elution steps. The eluted product was buffer exchanged into PBS using a HiPrep desalting column after each IMAC step. Tandem scFv BsAb monomer was isolated using a GE gel filtration column (HiPrep 26/60 Sephacryl S-200 HR) and the monomer subsequently used for kinetic experiments and long-term stability studies. Products were stored at 4 °C unless specified otherwise.

2.6.2. Protein A For constructs containing an Fc domain, purification was performed in one step using a MabSelect SuRE Protein A HP column (GE Healthcare). Buffers used include 1× PBS for loading and 0.1 M Glycine, pH 3.0 for elution. Following purification the BsAbs were desalted into Dulbecco’s PBS buffer for storage using the HiPrep desalting column. Products were stored at 4 °C unless specified otherwise.

2.7. Protein quality Post-purification quality control was routinely completed. All samples were analysed by nanodrop, PAGE gels and SEC HPLC.

2.7.1. Quantification A Nanodrop 1000 (ThermoFisher) was used to determine protein concentrations post- purification by incorporating extinction coefficients (Table 2.4) and using 1 x Dulbecco’s PBS as a blank sample unless the protein was eluted into a different buffer.

60 Table 2.4: BsAb vector and protein data. The NBF designation of each vector ID refers to the reference in the National Biologics Facility (NBF) plasmid database. Insert Insert AA Co- Molecular pcDNA 3.1 size (bp) length Extinction Theoretical BsAb ID transfection Weight vector ID (incl. (without coef. pI Vector ID (kDa) Leader) leader) Tandem NBF 713 - 1593 509 54.39 1.63 5.91 scFv Fc- NBF 715 - 2179 723 77.96 1.54 6.31 containing NBF732 NBF733 1922 615 65.89 1.34 6.38 KIH 1 (Cys) NBF733 NBF732 1940 621 66.85 1.68 6.39 NBF734 NBF735 1940 621 66.81 1.69 6.53 KIH 2 NBF735 NBF734 1922 615 65.75 1.33 6.38 Fc- containing NBF736 - 2246 723 78.04 1.53 6.20 (Cys) Fc- containing NBF737 - 2321 748 80.04 1.49 6.24 (Cys Longlink) Fc- containing NBF738 - 2321 748 79.95 1.49 6.34 (Longlink) Non- specific NBF748 - 1595 509 54.49 1.80 6.63 tandem scFv - pcDNA 3.1 - 80 - - - -

2.7.2. PAGE gels Pre-cast protein gels were used for all routine PAGE gels to determine sample purity and protein size. The gels used were NuPAGE Novex Bis-Tris 4-12 % gels (Invitrogen) with either 10 or 15 wells run in 1 x MES buffer (Invitrogen) alongside a SeeBlue2 pre-stained protein size marker. Samples were prepared according to manufacturers recommendations with the addition of 4 x LDS sample buffer, 10 x reducing agent and Milli-Q water and heated for 10 minutes at 70 °C. The gel runs were completed at 200 V and 400 mA for 45 – 50 minutes before being removed, washed in Milli-Q water and stained using SimplyBlue SafeStain (Invitrogen) for final imaging.

2.7.3. Size exclusion chromatography (SEC) HPLC A mobile phase of 0.1 M sodium phosphate buffer, 0.2 M sodium chloride, pH 6.8 was prepared and filtered using a 0.22 µm filter. A gel filtration standard was included on each run of the HPLC to calculate molecular weights by their retention times. Samples were not

61 concentrated prior to performing SEC so as to avoid the potential of concentration based aggregation that might occur. The guard column and SEC-HPLC column were connected to the HPLC system, flushed and equilibrated in mobile phase. 100 µl sample injections were used in all cases with a flow rate of 0.8 ml/min. Columns were stored in 20 % ethanol pre- and post- run completion.

2.8. Stability monitoring

2.8.1. Storage buffer formulation Buffers composed of a variety of different components have been used in designing optimal formulation buffers. The incorporation of excipients into formulation buffers allows the added benefit of being able to optimise the composition even further by tailoring the buffer to exactly suit the inherent structure of a single antibody format.

In the present study ten different buffer systems were investigated to determine the optimal means of stabilising an engineered Fc-containing BsAb through multiple freeze/thaw cycles. Purified Fc-containing BsAb, Vectibix-Fc-1H10 desalted into Dulbecco’s PBS was separated into aliquots of which some was left in PBS and others dialysed into either a Tris- or Histidine-based buffer. Various excipients were added to aliquots of the soluble BsAb (See results Table 3.2) and each one split in half and stored at either 4 °C or -20 °C. The concentrations of the Fc-containing BsAb in the different formulation buffers were measured at least once weekly over a 3 month period at both 4 °C and -20 °C storage temperatures. Regular SEC HPLC runs were also performed to monitor product stability as were SDS-PAGE gels.

2.8.2. Dialysis into formulation buffers The dialysis was completed in one of 2 stock solutions initially prepared as 5 x stock solutions:

50 mM Tris-HCl + 150 mM NaCl pH 8.0 50 mM Histidine pH 7.4 + 150 mM NaCl

In preparation for dialysis, bottles of Milli-Q water was autoclaved as were the stock solutions, beakers and stirrers to ensure that all components were sterile. Buffers and water were stored at 4 °C. For dialysis 3 ml dialysis cassettes (Pierce/Thermo Scientific 62 Slide-A-Lyzer) were immersed in 1 x buffer solutions and 3 ml Fc-containing BsAb injected into the cassette’s entry-port. A float was placed over the cassette and the beaker covered with foil and transferred to magnetic stirring blocks located at 4 °C. Samples were left stirring for 2 hours before the first buffer change was done followed by another buffer change after 16 hours. A final buffer change was then done 7 hours later and left to stir overnight for 17 hours at which point each 3 ml sample was removed from the dialysis cassette using a syringe and needle and aliquoted out appropriately into eppendorf tubes to which excipients could then be added.

Excipient stock solutions: 2 M L-Arginine 1 M Sucrose 1:100 of Polysorbate-80 (Tween-80)

Sample buffers were prepared as in Table 3.2 and in each case a blank buffer sample was also prepared. Each sample was halved and 1 tube stored at 4 °C and the other at -20 °C.

2.8.3. DLS performed at CSL Analysis was completed by CSL, Melbourne, Australia. Molar mass and hydrodynamic radius (Rh) was determined using SEC-MALS. A Wyatt WTC-030-N5 4.6 column was used with buffer composed of 100 mM phosphate/200 mM Sodium chloride, pH 6.8. The column was equilibrated in the buffer and flow rate set to 0.2 ml/min with 100 µl sample injections. A DAWN Heleos MALS detector was normalised using BSA and used in series with an Agilent 1200 series UV diode array detector and an Optilab T-rEx RI detector. Wyatt’s Astra VI software was used to calculate the weight-averaged molar mass and hydrodynamic radius.

2.8.4. Analysis of tandem scFv by differential scanning calorimetry The tandem scFv BsAb was concentrated to 1mg/ml in PBS using membrane based centrifugal concentrators with nominal molecular weight cutoff (NMWCO) of 10Kda (Millipore). 500 µl of 1 mg/ml tandem scFv was vacuum degassed and loaded into the sample chamber of the VP differential scanning calorimeter (VP-DSC, Microcal). The same volume of PBS was loaded into the reference chamber. The sample and reference chambers were scanned once from 25 °C to 90 °C for 1 h to determine the melting profile

63 of the tandem scFv. The data was analysed using Origin 7.0 software and graphed as time (min) (x axis) versus heat capacity (Cp) (y axis).

2.9. Functional binding

2.9.1. Surface plasmon resonance (SPR) To evaluate binding affinities of the EGFR-targeting Vectibix-scFv’s on all constructs, kinetic data was collected from High performance (Multi-cycle) Kinetic Assays (HPKA) performed on a Biacore T-200. A CM5 chip was coated with an anti-mouse IgG antibody on flow cells 1 and 2 as described in the Mouse Antibody Capture Kit guidelines (GE Healthcare). Recombinant EGFR-mFc was captured on flow cell 2 at 10 µg/ml for 8-15 seconds and variable concentrations of BsAb (1, 3, 10, 30 and 60 nM) flowed over both flow cells 1 and 2. Recommended flow rates (30 µL/min) and regeneration conditions (10 mM Glycine pH 1.7; 10 µL/min for 180 sec) were used. The dissociation phase was 1800 seconds. Reference subtractions of flow cells 2-1 were incorporated in the analysis as were blank and buffer only sample runs. Kinetic analyses were performed using a 1:1 fit of binding curves using BiaEvaluation software (GE Healthcare).

2.9.2. Biolayer interferometry kinetic characterisation of LPS binding The binding of mAbs and BsAbs to LPS molecules was tested utilising Biolayer Interferometry and the ForteBio Octet module. Aminopropylsilane (APS) biosensors (ForteBio) were briefly hydrated in PBS and coated with 1mg/ml LPS (Salmonella enterica, serotype typhimurium - Sigma L7261) diluted in PBS. A concentration range of the 1H10- mAb (anti-LPS) or tandem-scFv BsAb was set up using 2 fold dilutions starting at 40 ug/ml and subsequently tested for binding to immobilised LPS. PBS was added to LPS coated sensors as a reference.

2.9.3. Flow cytometry against MDA-MB-468 breast cancer cells All BsAb and mAb samples had been stored at 4 °C prior to commencement of the experiment. For the indirect flow cytometry (FACS) protocol cell viability was determined to be greater than 90% for each experiment completed. All components were added to a cell suspension of washed, 2 × 105 MDA-MB-468 cells in 100 μl of 10 % FBS/DPBS and kept at 4 °C to prevent receptor-mediated endocytosis from occurring. No sodium azide was added to the cell suspensions. Primary BsAb and mAb stock dilutions to 10 μg/ml were made in ice cold 5 % BSA + Dulbecco's PBS buffer as were secondary antibody dilutions. Final 64 BsAb and mAb concentrations were at 1 μg/ml in the final cell suspension volume. After adding primary antibodies to the cells, samples were incubated for 30 minutes at 4 °C in the dark. Cells were washed 3 times by cycles of centrifugation at 400 g for 5 minutes, supernatant removal and cell resuspension in cold PBS until the final wash step at which point appropriate concentrations of secondary antibody was added to the cells resuspended in cold 5 % BSA + Dulbecco's PBS buffer. Samples were again incubated for 30 minutes at 4 °C in the dark. FITC-conjugated anti-myc (AbD Serotec; Bio-Rad) was used as the secondary for detecting binding shifts when the Vectibix tandem scFv and nonspecific tandem scFv were incubated with MDA-MB-468 breast cancer cells; whereas APC- conjugated F(ab)’2 fragment goat anti-human IgG (Jackson Immunoresearch) was used for detection of all mAb, Fc-containing and Knob-in-hole constructs. Three final wash cycles were completed following incubation and the cells resuspended in cold, 5 % BSA + Dulbecco's PBS buffer. Cells were stored in the dark, on ice and fluorescence shifts subsequently analysed on the Accuri C6 flow cytometer. Cells were gated using forward and side scatter, and the cell sample incubated with secondary antibody only was used to determine the cut-off fluorescence for non-specific binding. Events were limited to 100 μl or 100 000 events.

2.9.4. BsAb- EDVTMnanocell preparation 1 x 1012 Non-targeted EDVTMnanocells were incubated with 1 mg of Invitrogen’s NH2- reactive AlexaFluor488 (EnGeneIC internal protocol) and excess dye was removed by 4 spin wash cycles in sterile PBS. The fluorescently labeled EDVTMnanocells were counted using a NanoSight at EnGeneIC Pty Ltd and found to be 5 × 1010 EDVs/ml. Doses were prepared as 2.5 × 1010 EDV (AF488) particles in 0.5 ml, which were then incubated with 6 µg of various BsAb constructs at room temperature for 30 min while shaking at 300 rpm. Samples then underwent 3 PBS spin wash cycles at 9 000 g for 8 min each to remove any excess BsAb. BsAb-EDVTMnanocells were resuspended to concentrations as required for specific experiments.

2.9.5. BsAb- EDVTMnanocells aggregation analysis using fluorescence microscopy Microscope slides were cleaned and labelled appropriately after which 5 – 10 µl of BsAb- EDVTMnanocells were placed on the slides and sealed with clean cover slips. The AlexaFluor 488 (AF488) was used as an indication of EDVTMnanocell aggregation. Visualisation was completed on an Olympus IX81 confocal microscope.

65 2.9.6. Co-localisation of BsAbs alongside EGFR-ecd and EDVTMnanocells AF488-labelled, non-targeted and targeted EDVTMnanocells were prepared as previously mentioned and observations made regarding clumping of the BsAb-EDVTMnanocells during each resuspension. BsAb-EDVTMnanocells were resuspended to an approximate concentration of 4 × 1010 EDVs/ml in 500 µl. Counts and aggregation analysis were again performed using established techniques on the nanosight. EGFR-His (1 mg) labelling was completed using a DyLight650 amine-reactive labelling kit (Thermo Scientific) as per the manufacturer's instructions after having been concentrated to 1.6 mg/ml using a 10 kDa spin column.

To set up samples for co-localisation imaging, 2 µg EGFR-His (DyLight650) was added to 5 × 109 particles of each of the conjugated BsAb-EDVTMnanocells. The resulting suspension was then incubated at RT for 30 min on a shaker at 300 rpm to allow sufficient time for recombinant EGFR binding to occur. Samples underwent 3 spin wash cycles as before to remove excess unbound labelled EGFR-His and checked for evidence of clumping resulting from BsAb-nanocell interaction with recombinant EGFR. EGFR-BsAb- nanocell suspensions were resuspended to a concentration of 0.4 × 1010 EDVs/ml. For analysis on an Olympus IX81 confocal microscope using Xcellence RT software, slides were prepared by pipetting 10 µl sample onto a clean slide which is then spread out by addition of a cover slip.

2.9.7. Flow cytometry of BsAb-EDVTMnanocells in vitro binding to MDA- MB-468 cells EnGeneIC staff maintained MDA-MB-468 cells as part of their routine internal processes in their laboratories. 1 × 105 MDA-MB-468 cells grown on coverslips in 400 µl RPMI + 5% FBS + 1% PC-SM were treated with 1 × 109 pre-targeted and AF488-labeled EDVTMnanocells and returned to 37 °C for 3 h. This was repeated for the non-targeted EDVTMnanocell and each of the five BsAb-EDVTMnanocells – non-specific and ABX-EGF tandem-scFv, KIH 1 (Cys), Fc-containing (Cys) and Fc-containing (CysLonglink); with only the cells only control remaining untreated. On completion of the 3 h incubation period, coverslips were washed 3 times with sterile DPBS and cells scraped for flow analyses on a Beckman FC500 flow cytometer. Flow rate was set to high and cells were again gated using forward and side scatter. The “cells only” sample was used to determine the cut-off fluorescence for non-specific binding. Events were detected over a 250 sec period. Final analyses were completed using CXP and VenturiOne software. 66 2.9.8. Confocal microscopy of in vitro binding of BsAb-EDVTMnanocells to MDA- MB-468 cells Everything except for confocal microscopy, which was performed using ANFF-Q facilities, was completed at EnGeneIC’s premises. MDA-MB-468 cells were incubated on coverslips in the presence or absence of targeted [non-specific and ABX-EGF tandem scFv, KIH 1 (Cys), Fc-containing (Cys) and Fc-containing (CysLonglink)] and non-targeted EDVTMnanocells – 7 slides total. EDVTMnanocells (labeled with AF488) were added to each of 6 samples at a ratio of 10 000 EDVs per cell and the seventh was left as a “cells only” control. Plates were returned to 37 °C for 3 hours which was followed by 3 washes in sterile DPBS.

Fixative volume of 500 µl, or enough to cover each coverslip, of 4% PFA was added to each of the 7 samples and left for 10 minutes for cells to become fixed. This was again followed by 3 DPBS washes. After the final wash 500 µl DPBS was added to each well to cover the coverslips completely. An anti-EGFR mAb, 528 mAb, was labeled with AF647 to be used as a cell membrane stain; 4 µg of which was added directly to each sample. Wells were mixed gently and left for 10 min to allow cells to stain sufficiently. All coverslips were again washed 3 times with DPBS followed by a final wash in Milli-Q water.

Coverslips were removed from the water and placed on tissue paper cell-side-up to allow complete drying to occur, at which point they were mounted on clean slides using Fluka Eukitt (Sigma-Aldrich) quick-hardening mounting medium. Dry slides were stored at 4 °C until confocal microscopy could be completed. Microscopy was completed on a LSM Zeiss 710 confocal using a plan-apochromat 63x/1.40 Oil DIC M27 objective and ZEN 2008 software for image formatting.

2.9.9. Effect of ABX-EGF tandem scFv-EDVTMnanocell on tumour regression in vivo The experiment was performed in compliance with the NHMRC Australian guidelines for the care and use of laboratory animals and with the approval of the EnGeneIC Animal Ethics Committee. Five to six week old female Balb/C athymic nude mice were obtained from the Laboratory Animal Services at the University of Adelaide, South Australia. The animals were housed at the EnGeneIC Animal Facility under specific pathogen free conditions.

67 MDA-MB-468 cells were obtained from ATCC and grown in RPMI with 10% fetal calf serum and penicillin/streptomycin. Each mouse was injected subcutaneously on the left flank with 1 x 107 cells in 100 µl of media together with 100 µl of growth factor reduced matrigel (BD Biosciences). Tumour volume was determined by measuring length (l) and width (w) and calculating volume (V = l x w2 x 0.5). Once the tumours reached 80 – 100 mm3 the mice were randomized into treatment groups which included 7 mice per group. The treatment groups were as follows:

• Group 1 – Saline • Group 2 – Tandem scFv EDV (ABX-EGF or Panitumumab tandem scFv BsAb targeted EDVTMnanocell not loaded with Doxorubicin) TM • Group 3 – EDV Dox (Non-targeted EDV nanocell loaded with Doxorubicin) Protein A/G TM • Group 4 – EDV Dox (Protein A/G linked BsAb targeted EDV nanocell loaded with Doxorubicin)103, 129 Tandem scFv • Group 5 – EDV Dox (ABX-EGF or Panitumumab tandem scFv BsAb targeted EDVTMnanocell loaded with Doxorubicin)

The amount of Doxorubicin was measured by HPLC for all loaded EDVTMnanocells and determined to be 1 ± 0.1 µg per 1 × 109 EDVTMnanocells (one iv dose); method completed as previously described.103, 129 All treatments were injected intravenously via the tail vein and administered three times a week for three weeks. Tumours were measured using a caliper three times a week. The mice were weighed twice a week for three weeks.

68 3. Bispecific antibody design, production and function

3.1. Chapter summary The design of BsAbs impacts expression levels in CHO cells, product stability and antigen binding. The utility of our engineered BsAbs was to target a novel nanocell (EnGeneIC Delivery Vehicle, EDV or EDVTMnanocell) to the EGFR. EDVTMnanocells are coated with LPS, and BsAb designs incorporated the binding site of an anti-LPS antibody (1H10) with the anti-EGFR commercial antibody panitumumab (ABX-EGF or Vectibix). We engineered various BsAb formats including monovalent and bivalent binding arms and linking scFv fragments via either glycine-serine (G4S) or Fc-linkers. Aggregation of BsAbs was a common occurrence with more complex designs. A tandem G4S-linked scFv format was the most stable, but had lower expression yields than complete mAbs. The Fc-containing BsAbs showed increased expression levels compared to a tandem scFv, but a greater propensity for aggregation. Engineering additional disulphide bridges into the scFvs of the Fc-containing BsAbs decreased aggregation, but also expression levels. Various buffer formulations were evaluated with regards to their ability to maintain BsAb stability during short and long term storage at both 4 °C and -20 °C with multiple freeze/thaw cycles. Results suggested that the addition of arginine to PBS was the simplest means to stabilise the tandem scFv at -20 °C however the formulation merely slowed the aggregation rate in the case of the Fc-containing BsAb format. Binding analyses utilising ELISA, surface plasmon resonance, flow cytometry and fluorescence microscopy for antigen co- localisation, showed that binding to LPS and to either soluble recombinant EGFR or MDA- MB-468 cells expressing EGFR, was conserved for all construct designs.

3.2. Introduction Therapeutic mAbs approved for clinical use such as Avastin (anti-VEGF), Humira (anti- TNF α), Herceptin (anti-Her2), Erbitux and Vectibix (anti-EGFR) make up a significant portion of the global pharmaceutical market.246, 252 Of the total mAb sales in 2010, half are attributed to cancer related therapies.17 In 2012 half of the antibody based oncology products under clinical evaluation were complete IgG mAbs. The remaining candidates included drug conjugated or radio-labeled mAbs, protein- and glyco-engineered mAbs, fragment or domain antibodies.195, 253, 254 Also under evaluation are BsAbs12, which are subject to increasing interest in recent years owing to their ability to target multiple antigens.118, 124, 188, 255

69 BsAbs are able to crosslink antigenic determinants and so have value beyond that of single antigen specific mAbs (Figure 1.7).32, 117, 118, 125, 246, 256 An emerging application of BsAbs is active targeting of drug-loaded NPs (NP) to tumour sites by cross-linking the NP to tumour cells, leading to endocytosis, fusion with lysosomes and drug release intracellularly.103, 118, 129, 246 However, the most prominent therapeutic utility for BsAbs in cancer therapeutics is the cross-linking of cell surface antigens or receptors, so that immune cells can be tethered to cancer cells through a BsAb.118, 123-126

Catumaxomab (Removab) was approved for therapeutic use in Europe in 2009 and remains the only approved therapeutic BsAb, although the number of BsAb formats entering clinical trials is increasing steadily.12, 256 Catumaxomab is a T-cell engager, with specificity for both CD3 on cytotoxic T cells and the EpCAM antigen on ovarian cancer cells, for the treatment of malignant ascites.144-146 However BsAbs are not natural immunoglobulins, and unlike the well-understood cellular processes of natural immunoglobulin expression, assembly in the endoplasmic reticulum and secretion in B cells,101, 257 the stability and expression levels of BsAbs are less understood and influenced by design.

Recombinant BsAbs are constructed by combining binding modules such as scFvs or single domain antibodies (sdAbs) – such as shark and camelid antibodies;115, 196, 205, 258 124, into a single polypeptide utilising linkers such as the flexible Glycine-Serine (G4S) motif 126, 188 or Fc (Hinge-CH2-CH3 domains) to tether the binding modules. On their own, scFv components are unstable but by including a linker between them, and if required additional cysteines, stability can be improved.199 Design of recombinant BsAbs is only limited by imagination, and a variety of novel designs have been reported (Figure 1.8).32, 118 Formats used thus far range from non-IgG like varieties containing chemical or protein linkers or various Fc-motifs, through to recombinant Fc-containing, Dock-and-lock and knob-in-hole (KIH) BsAbs.115 Some of the more well-known formats include Triomabs, Dual Affinity Retargeting (DART) molecules, Bispecific T-cell Engagers (BiTEs), and the F-star mAb2 TM BsAbs.124, 143, 144, 158, 200

Product design may endow the BsAb with specific properties, such as enhanced serum half-life through PEG- or BSA-conjugation, complement activation capability through interaction with the Fc domain and increased tissue penetration.156, 193 Single domain antibodies (sdAbs) can bind targets without the need to be connected to heavy and light 70 chains; for example shark and camelid nanobodies. sdAbs are capable of binding targets (extra- and intracellular) with affinities comparable to full mAbs and their small size allows penetration into sites that cannot be reached by standard mAbs. They have high refolding capabilities, are very stable and product yield ranges from 0.5 to 5 mg/L depending on the bispecific design specifications and the type of domain used; but functional BsAbs are not always produced.195

Chemical conjugation can be used to produce BsAbs, however antibody inactivation, low associated yield and product heterogeneity has limited the progress of these formats to the clinic – yields of 10 to 40% dimeric BsAb from hetero-bifunctional reagents and 65 to 75% from homo-bifunctional reagents. In comparison the use of Thiomab technology to produce bis-Fabs, using a bis-maleimide cross-linker, is highly reproducible with high conjugation rates and homogeneity however the biological effect is sometimes altered relative to the parental mAb.131 Bispecific diabody production relies on heterodimeric association of two chains; however as expression is achieved within a single cell inactive homodimers are also produced.187, 199 Incorporating cysteines for stability in the Fv fragments has been shown to improve the proportion of functional heterodimers produced during expression.259 Nonetheless unnatural recombinant BsAb formats remain harder to express in suitable homogenous quantities and as a result a lot of work remains to be done to determine what the most beneficial BsAbs are for potential clinical and diagnostic use.

In this study we designed several BsAbs based on three basic format designs for the purpose of delivering the EnGeneIC delivery vehicle (EDVTMnanocell) to the EGFR and to replace the currently used protein A/G linked mAbs.15, 16, 18 The EDVTMnanocell (a 400 nm particle of bacterial origin capable of carrying a drug payload) was originally targeted using a BsAb moiety produced by incubating two different mAbs with protein A/G (Pierce, USA).129 Although functional BsAbs capable of delivering the EDVTMnanocell were prepared, it was noted that multimer formation and the inability to control the ratio of mAbs bound to the protein A/G (which has 6 potential Fc-binding sites) rendered the targeting entity unsuitable for commercial manufacture and downstream clinical trials.129, 231 Recombinant BsAbs offer an advantage of producing one antibody with dual target binding capability.

The EDVTMnanocell is coated with LPS129, and thus BsAb designs incorporated scFvs derived from an anti-LPS antibody (1H10) with the anti-EGFR commercial antibody 71 panitumumab (Vectibix or ABX-EGF).24, 67, 83 ABX-EGF was developed via transgenic humanised mouse technology by Abgenix Inc.24, 67, 83, and is approved for clinical use in the treatment of colorectal cancer. ABX-EGF has picomolar affinity for EGFR and its properties, including affinity, specificity and immunogenicity; have been extensively evaluated and is an ideal model antibody to incorporate into the design and testing of bispecific formats.129 Three BsAb formats were evaluated for EDVTMnanocell targeting; a G4S-linked tandem scFv format, homodimeric Fc-containing BsAbs and Knob-in-Hole (KIH) engineered formats to promote forced heterodimerisation of the EDV-targeting variable fragments. Additionally, modifications of the latter two formats were investigated, incorporating additional disulfide bridges in the scFv domains and longer linkers between the Fc-CH3 and anti-LPS scFv interface (Table 2.1). We have evaluated these different formats with regard to expression yields, stability post-purification, their ability to bind respective targets and their effect on EDVTMnanocell clumping. The purpose of which is to determine which is the optimal BsAb for use in the proposed delivery system of drug- loaded EDVTMnanocells to target cancer cells.

72 3.3. Results

3.3.1. Production, yield and stability All format sequences except for the Fc-containing BsAb were produced by GeneArt. The Fc-domain and 1H10-scFv sequences were PCR amplified from their original vectors and subsequently purified (Figure 3.1.b) The Fc-containing BsAb was produced through SOE PCR techniques as previously described. The largest band in Figure 3.1.c (~1500 bp) is of the successfully produced Fc-1H10 sequence, the remaining bands of ~800 bp in size are the original DNA templates added into the PCR mix. Colony PCR confirmed the presence of the required sequence (Figure 3.1.d). The gel image shows the presence of two bands following colony PCR, one which is the positive band (~2500 bp) and the other a result of non-specific binding of the primer. The positive result was also confirmed by sequencing the insert at the Australian Genome Research Facility (AGRF).

Figure 3.1: Gel electrophoresis images of the development of the Fc-1H10 sequence. a) Hyperladder size standard run using 5 ul per well. b) Gel following PCR to produce the Fc-sequence using primers: Fc_Fc-link_Forward and Fc_longoverlap_Reverse; and producing 1H10 scFv from the tandem scFv using primers: 1H10_longoverlap_Forward and 1H10_Fc-link_Reverse. c) SOE PCR results to create Fc-1H10 product. d) Gel showing a positive colony PCR containing the Fc-1H10 insert (Table 2.2).

Soluble proteins such as mAbs and BsAbs are notoriously unstable in solution which represents one of the major barriers in therapeutic product development. Herein BsAbs were produced in CHO-S transient cell culture and purified from culture supernatant by either IMAC or Protein A affinity chromatography techniques.

73 BsAb yield and expression We observed that BsAb yields are much lower when compared with standard mAbs (Table 3.1).Tandem scFv BsAb expression routinely yielded 4 - 5 mg/L transient cell culture supernatant, whereas Fc-containing and Knob-in-Hole formats lacking scFv disulphide engineering yielded 9 - 14 mg/L. Engineering disulphide bridges into Fc-containing and Knob-in-Hole variant scFvs dramatically decreased BsAb yield 6 - 9 fold compared to that of non-stabilised formats.141

Table 3.1: Product yield. A comparison of all 7 BsAbs developed in the present study relating to the total amount in milligrams retrieved following purification of either IMAC purification using HisTrap Excel and HisTrap FF for tandem- scFv purification and MabSelect Sure Protein A for all constructs with an Fc followed by desalting into Dulbecco’s PBS for storage at 4 °C. The results show that all bispecific formats tested express at a much lower level than the whole mAb. Additionally, the tandem scFv produces at a much lower level than the Fc-containing format and non-stabilised KIH format and that when disulphide engineering is included there is a significant decrease in overall product yield. *Yield expressed as mg BsAb produced from a theoretical 1 L transient culture volume. Actual culture volume was 300 ml. BsAb ID Yield* (mg/L) IgG1 mAb 52.6 Tandem scFv 4.23 Fc-containing 11.93 Fc-containing (Longlink) 9.57 Fc-containing (Cys) 1.88 Fc-containing (Cys Longlink) 1.09 KIH 1 (Cys) 1.65 KIH 2 14.07

BsAb Secretion Although the secretory pathway for native mAb expression is well documented 101, 257 engineering antibodies through manipulating or deleting the Fc region will inevitably result in altered BsAb flux through the secretory pathway compared to native mAbs, and perhaps to retention in the endoplasmic reticulum.242, 260 To investigate whether the various formats were being efficiently secreted or retained within the cell, culture supernatant and cell lysate samples were taken over a 9 day period post-transient transfection and analysed by a qualitative Western blot (Figure 3.2). Cell numbers weren’t standardised in this experiment therefore each successive time point is of increasing cell numbers; however each similar time point is comparable between formats because cell number and viability were similar on day of sampling for each transient transfection (results not shown). An increase of secreted BsAb was observed over time and there did not appear to be any significant retention of BsAb in the cell pellet during the same period; confirming that each 74 BsAb is being secreted and retention is an unlikely explanation for the decreased production yield recorded in some samples. It may however be due to individual transfection conditions requiring optimisation.

Figure 3.2: Qualitative western blot of BsAb transient expression in CHO-S over time. (A) Secreted BsAb and (B) CHO cell lysate samples were taken at 2, 5, 7 and 9 days post-transfection. BsAbs were expressed in 300ml cultures. Typically, trend shows increase in secreted BsAb (A) and a decrease in intracellular BsAb (B) with increasing cell numbers over time. (1) tandem scFv, (2) Fc-containing, (3) Fc-containing (Longlink), (4) Fc-containing (Cys), (5) Fc- containing (CysLonglink), (6) KIH 1 (Cys) and (7) KIH 2. Smearing in (B) is an indication of higher cell numbers with each subsequent time point.

75 Tandem scFv and Fc-containing BsAb stability Reduced SDS-PAGE analyses for purified tandem scFv and Fc-containing BsAb samples showed the expected monomeric sizes: 55 kDa for tandem scFv and around 80 kDa for Fc-containing BsAbs. Non-reduced PAGE gels showed the tandem scFv maintained its monomeric size of 55 kDa, whilst the Fc-containing BsAbs formed an expected dimer (Figure 3.3). The Fc-containing format forms a dimer via the hinge region of the Fc- domain. A benefit of dimerisation is the increased avidity associated with multiple binding sites being present for the same target.

Figure 3.3: SDS-PAGE gels of purified BsAbs under reducing and non-reducing conditions. Size standard is Seeblue plus 2 in MES running buffer. a) PAGE gel of purified tandem scFv BsAb (Vectibix-G4S-1H10) showing that the format exists as a monomer of 55 kDa under both reducing and non-reducing conditions. b) PAGE gel of purified Fc- containing (Vectibix-Fc-1H10) showing the presence of the monomeric form of the BsAb at ~78 kDa under reducing conditions, and a multimer of ~160 kDa and greater under non-reducing conditions. It is also likely that Vectibix-Fc-1H10 (NBF715) is undergoing Asparagine N-linked glycosylation which also accounts for the increased molecular weight.

Further analysis of the purified product sizes were performed by size exclusion chromatography (SEC) which confirmed the monomeric confirmation of the tandem scFv and the presence of Fc-containing BsAb multimers. However, after storing the Fc- containing BsAb in PBS for a period of time it became apparent that aggregation occurs in solution to form large multimer aggregates at 4 °C and it is unstable at -20 °C (Figure 3.4). A potential drawback associated with multimer formation is that the EDVTMnanocells

76 subsequently aggregate through multiple interactions of the 1H10-scFv and therefore become non-viable as therapeutic agents.

Figure 3.4: Size exclusion chromatography results of purified BsAb products stored in PBS buffer. a) SEC of Vectibix-G4S-1H10 showing that more than 90% of the product exists as the 55 kDa monomer of the BsAb. b) SEC of Vectibix-Fc-1H10 stored at -20 °C in PBS performed within one week of purification showing the presence of ~160 kDa BsAb dimers as the major product but also ~700 kDa products indicating the possibility of aggregation. c) SEC of Vectibix-Fc-1H10 stored at 4 °C in PBS performed approximately 1 month post-purification indicating that the major product present is a ~700 kDa octamer (tetramer of dimers) and that although the dimer still exists, it is a minor component of the BsAb solution.

At 4 °C the BsAb concentration stays constant over time when stored in PBS however at - 20 °C the BsAb appears to reduce in concentration without any visible signs of aggregation to account for the loss of soluble product. Possible causes for the observed product loss could be that the anti-LPS portion of the BsAb might be binding to the plastic tubes or degradation resulting from pH shifts relating to the sample being stored in PBS,261 but this is yet to be confirmed. The instability prompted the need to perform a formulation study (Table 3.2) to determine whether an alternative buffer would improve stability and decrease aggregation levels during storage.262 Various buffer formulations were tested to improve the storage stability of the BsAbs. Homodimeric Fc-containing BsAb formats, without additional stability engineering, formed large aggregates post-purification when stored at 4 °C, and exhibited similar product loss as the tandem scFv when stored in DPBS at -20 °C, possibly explained by pH changes in the buffer.261 From initial buffer formulation evaluations for the Fc-containing BsAb, we determined that the most effective method to stabilise and maintain the BsAb concentration levels was to add 100 mM L- Arginine (Buffer 10) directly to the sample in PBS after purification (Table 3.2).

77

Formulations tested thus far have not shown significantly improved stability of Fc- containing and KIH variants, and there is evidence of concentration-dependent aggregation above 0.1 mg/ml (results not shown). Size exclusion chromatography (SEC) HPLC analyses over a 1 month period indicated that tandem scFv stability, when stored at a concentration of 0.12 mg/ml, is affected by buffer formulation. Product storage in a formulation of PBS/trehalose resulted in slightly increased aggregation levels when stored at -20 °C compared with 4 °C. However, stability was maintained at both 4 °C and -20 °C in a HEPES/Trehalose buffer formulation (Figure 3.5.A). Dynamic light scattering (DLS) data confirmed the tandem scFv to be a monomer of 55 kDa in size, with a hydrodynamic radius of 2.5 nm (results not shown). Thermostability determined by differential scanning calorimetry (DSC) indicated that the tandem scFv has a melting temperature (Tm) of 57 °C (Figure 3.5.B).

Figure 3.5: Tandem scFv stability data. A) Tandem scFv samples were stored in various buffers at different temperatures to evaluate stability during storage over a 3 month period. The representative trace is indicative of stable product stored in Hepes/trehalose buffer for 3 months. B) Thermostability of tandem scFv BsAb in PBS was determined by differential scanning calorimetry and indicates unfolding and subsequent aggregation of the tandem scFv BsAb at 57 °C. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC.

78 Table 3.2: Formulation study to evaluate the stability of Fc-containing BsAb Vectibix-Fc-1H10 at different storage temperatures over a 3 month period. Aliquot volumes were halved and stored at either 4 °C or -20 °C over a 3 month period during which time various observations were made regarding buffer ability to promote product stability.

Buffer ID Buffer Composition Results

Control samples: When stored at -20 °C no visible aggregation occurs but the BsAb “disappears” from solution evidenced by loss of A280 (possible degradation). PBS only 1 x Dulbecco’s PBS Storing the Fc-containing BsAb at 4 °C prevents apparent degradation from occurring but there is evidence of aggregation present following HPLC and PAGE gel analysis.

BsAb concentration level maintained over the first 1.5 months at which point it 50 mM Tris-HCl+150 mM NaCl 1 started decreasing at both 4 and -20 °C. Aggregation also visible on gel at both pH 8.0 + 100 mM Arginine temperatures.

Could not get accurate A280 readings at -20 °C. The concentrations appeared to 50 mM Tris-HCl+150 mM NaCl increase according to the Nanodrop values - attributed to polysorbate interference. 2 pH 8.0 + 0.25 mg/ml Storage at 4 °C showed maintenance of concentration levels but aggregation also Polysorbate 80 evident in gel image.

Storage at 4 °C showed maintenance of concentration levels but aggregation 50 mM Tris-HCl+150 mM NaCl 3 evident in gel image. BsAb concentration level maintained over the first 1.5 months pH 8.0 at which point it started tapering off at -20 °C.

50 mM Histidine + 150 mM Concentration level maintained at both 4 and -20 °C. Aggregation visible on gel at 4 NaCl pH 7.4 + 100 mM Arginine both temperatures.

Could not get accurate readings at -20 °C. The concentrations appeared to 50 mM Histidine + 150 mM increase according to the nanodrop values - attributed to polysorbate interference. 5 NaCl pH 7.4 + 0.25 mg/ml Storage at 4 °C showed maintenance of concentration levels but aggregation also Polysorbate 80 evident in gel image.

50 mM Histidine + 150 mM Concentration level maintained at both 4 and -20 °C. Aggregation visible on gel at NaCl pH 7.4 + 0.25 mg/ml 6 both temperatures. However the use of polysorbate in the buffer was deemed to be Polysorbate 80 + 100 mM unsatisfactory for use with the EnGeneIC delivery vehicle. Arginine

Storage at 4 °C showed maintenance of concentration levels but aggregation 50 mM Histidine + 150 mM 7 evident in gel image. BsAb concentration level maintained over the first 1.5 months NaCl pH 7.4 at which point it started decreasing at -20 °C.

Aggregation present but appears to be less than for other buffers when compared 8 PBS + 100 mM Sucrose on gel image. Storage at 4 °C showed maintenance of concentration levels but apparent degradation occurred soon after excipient addition and storage at -20 °C.

PBS + 100 mM L-Arginine Concentration level maintained at both 4 and -20 °C. Aggregation visible on gel at 9 (pH10.65) both temperatures.

79 Buffers composed of a variety of different components have been used in designing optimal formulation buffers. The incorporation of excipients into formulation buffers allows the added benefit of being able to optimise the composition even further by tailoring the buffer to exactly suit the inherent structure of a single antibody format. In the present study ten different buffer systems were investigated to determine the optimal means of stabilising an engineered Fc-containing BsAb. A number of the buffers enabled longer storage times while maintaining the structural integrity and binding affinities of the BsAb irrespective of the occurrence of multiple freeze/thaw events (results not shown). Addition of 100 mM L-Arginine appeared to be the simplest means of preventing the loss of BsAb when stored at -20 °C. Although adding 100 mM L-Arginine to PBS stabilises the BsAb at - 20 °C, it does not prevent the observed aggregation; it merely slowed the aggregation rate (Figure 3.6).263

Figure 3.6: Size exclusion chromatography comparing Vectibix-Fc-1H10 (NBF 715) stability. a) NBF 715 stored at 4 °C in PBS only for 1.5 months. b) NBF 715 stored at 4 °C in PBS + 100 mM L-Arginine for 1.5 months. c) NBF 715 stored at 4 °C in PBS + 100 mM L-Arginine for 3 months. d) NBF 715 stored at -20 °C in PBS only for 1.5 months. e) NBF 715 stored at -20 °C in PBS + 100 mM L-Arginine for 1.5 months. f) NBF 715 stored at -20 °C in PBS + 100 mM L- Arginine for 3 months. 80 The concentrations of Fc-containing BsAbs in the different formulation buffers (Table 3.2) were measured at least once weekly over a 3 month period at both 4 °C and -20 °C storage temperatures (Figure 3.7). From these results we determined that the easiest method to stabilise the BsAb concentration levels was to add 100 mM L-Arginine (Buffer 9) directly to the sample in PBS after purification.

Figure 3.7: Concentration monitoring of Fc-containing BsAbs in different formulations. Graph shows effects of storage temperature on BsAb stability with respect to aggregation; BsAb samples stored at either 4 °C or -20 °C in different formulation buffers.

SDS-PAGE analysis was performed on samples stored in formulation buffers at 4 °C and - 20 °C at 1 and 3 months post-dialysis and excipient addition. The gel image below gives an indication of apparent concentration levels of Fc-containing BsAb when stored for 3 months in the different formulation buffers listed in Table 3.2 at either 4 °C or -20 °C. Some aggregation appears to be present in all samples however the apparent degradation which occurred to BsAb stored in PBS at -20 °C (Figure 3.8: Gel a and b; lane 3) does not seem to have occurred for any of the formulation samples except when sucrose was added to the PBS as an excipient (Buffer 8; -20 °C). Interestingly however, the larger aggregation

81 products (>200 kDa) are visible in all lanes except the samples stored in PBS only and PBS + 100mM Sucrose (Buffer 8) at -20 °C which were unstable. As aggregation wasn’t eliminated through formulation optimisation a number of stability engineering options for the Fc-containing and KIH BsAb variants were investigated (Table 2.1).63, 138, 141, 155

Figure 3.8: SDS-PAGE gels of Fc-containing BsAb after 3 months storage at either 4 °C or -20 °C in different formulation buffers. Lane number indications exclude the lane containing the size marker. Numbers 1 through 9 on the image indicate the buffer identification number from Table 3.2. Lanes i are of purified Fc-containing BsAb stored in an eppendorf tube in PBS at 4 °C; Lanes ii is of purified BsAb stored in a glass HPLC vial in PBS at 4 °C; Lanes iii is of purified BsAb stored in an eppendorf tube in PBS at -20 °C - in both gel images a) and b). Lane 1 is again of purified Fc- containing BsAb stored in an eppendorf tube in PBS at 4 °C as a control. a) Reduced SDS-PAGE gel of samples stored at 4 °C with regular freeze/thaw cycles over a 3 month period and b) non-reduced SDS-PAGE of the same. c) Reduced SDS-PAGE gel of Fc-containing BsAb formulation samples stored at -20 °C with regular freeze/thaw cycles over a 3 month period; and d) non-reduced SDS-PAGE of the same.

BsAb stability engineering A number of stability engineering strategies including modification of the Fc-containing format and inclusion of Knob-in-hole (KIH) BsAb variants were investigated (Table 2.1).63, 138, 141, 155 Reduced gels of the Fc-containing BsAb, KIH BsAb and variants thereof, showed the expected monomeric sizes (KIH BsAbs at 70 kDa) (Figure 3.9) but there was size variation on non-reduced gels. Non-disulphide stabilised Fc-containing and KIH BsAbs are larger than their stabilised counterparts (possibly a result of larger associated hydrodynamic radii of the unfolded proteins) suggesting a link between disulphide

82 engineering and folding of the molecule. KIH 1 (Cys) appeared as a 140 kDa band whereas the non-disulphide engineered format showed a band at around 180 kDa. Similarly for the homodimeric Fc-containing formats with the non-cysteine stabilised variants having an apparent molecular weight 40kDa larger than expected (Figure 3.9).

Figure 3.9: SDS-PAGE gels of purified stability engineered BsAbs under reducing and non-reducing conditions. Size standard is Seeblue plus 2 in MES running buffer. Lane number indications exclude the lane containing the size marker. Reduced gel a) shows BsAb monomers as expected in lanes 1 – Fc-containing, 2 – KIH 1 (Cys) and 3 – KIH 2; and non-reduced gel b) shows the presence of the Fc-containing multimer in lane 1, and variations in dimer size between the KIH 1 (Cys), lane 2, and KIH 2, lane 3, which were expected to be the same size. PAGE gel c) shows monomer size variations as expected of Fc-containing, Fc-containing (Cys), Fc-containing (Cys Longlink) and Fc-containing (Longlink) in lanes 1 – 4 respectively. Non-reduced gel d) shows a similar variation between multimer sizes where disulphide engineered variants Fc-containing (Cys), lane 2, and Fc-containing (Cys Longlink), lane 3 are smaller in size than their non-stabilised counterparts Fc-containing, lane 1, and Fc-containing (Longlink), lane 4, respectively.

83

Figure 3.10 and Table 3.3 show a summarising comparison of all BsAbs and conditions analysed through SEC HPLC with regards to stability of the tandem scFv versus all Fc- containing and KIH formats. The tandem scFv remained as a stable monomer when stored in Dulbecco’s PBS (DPBS) at 4 °C for up to 12 weeks (Table 3.3.A) and as such a variant on the basic format was not created; however storage at -20 °C for as little as one week resulted in degradation and loss of the BsAb (Table 3.3.B). Homodimeric Fc-containing BsAb formats lacking stability engineering formed large aggregates post-purification which persisted for the 12 week analysis period when stored at 4 °C (Table 3.3.E), and exhibited similar product loss as the tandem scFv when stored in DPBS at -20 °C (Table 3.3.F). Adding L-Arginine to the tandem scFv (Table 3.3.C – D) and Fc-containing (Table 3.3.G – J) BsAb solution prevented BsAb loss when stored at -20 °C; and decreased the aggregation rate of the latter format at 4 °C over time.181, 263, 264

Increasing the linker length 138 connecting the CH3 domain of the Fc to the anti-LPS, EDVTM-targeting scFv did not improve stability when stored at 4 °C in DPBS for up to 3 months unless combined with the inclusion of additional disulphide bridges in the scFv domains (Table 3.3.K and N).141, 181 BsAbs that lack the longer linker at CH3 but contain disulphide bridges engineered into the scFvs remain stable (Table 3.3.L – M) indicating that in this case the longer linker does not influence overall product stability. The same was observed for the KIH formats with disulphide bridge scFv stability engineering, and without. Preliminary data suggests a concentration-based link to the observed aggregation for KIH 1 (Cys) (Table 3.3.P) when concentrated from 0.02 mg/ml to 0.2 mg/ml. In comparison, the same concentration increase for the Fc-containing (Cys) BsAb format resulted in a slight aggregation increase (Table 3.3.M). Dilution post-purification does not improve aggregation levels (data not shown). Although the variants showed evidence of improved stability, observations were made at low concentrations of 0.02 mg/ml at which level protein loss is possible due to adsorption and therefore not an adequate means of storage.

SEC-MALS analysis was used to determine the true molar mass of the Fc-containing (Cys), Fc-containing (Cys Longlink), KIH 1 (Cys), in addition to the previously mentioned tandem scFv BsAb formats. These BsAb formats were analysed at CSL, Melbourne, and were selected due to their superior stability in comparison to the non-stability engineered formats, Fc-containing, Fc-containing (Longlink) and KIH 2. Due to the low protein concentrations of the 3 disulphide stability engineered BsAbs, the hydrodynamic radii (Rh) 84 of these sample could not be accurately determined. Dynamic light scattering (DLS) results indicated that “Peak 1” of all 3 disulphide engineered BsAbs were monodispersed (of a single sized product) of the expected dimeric products: Fc-containing (Cys), 159 kDa, Fc-containing (CysLonglink), 170 kDa and KIH 1 (Cys), 135 kDa (Figure 3.11).

Table 3.3: SEC HPLC evaluation of BsAb stability under varying conditions. Original tandem scFv (A – D) and Fc- containing (E – J) BsAb formats were stored at either 4 °C or -20 °C in 1x Dulbecco’s PBS or with added 100 mM L- Arginine as an excipient. SEC graphs provided a means to compare percentage of stable BsAb versus aggregate in solution at various time points. The formulation study (Table 3.2) did not provide a solution for the aggregation of the Fc- containing format which led to the design and evaluation of stability engineered formats stored at 4 °C in Dulbecco’s PBS over time (K – Q). Aggregate:BsAb ratio remained the same at 0.2 mg/ml for Fc-containing format. L – M) Samples at 0.02 mg/ml were transfected in 300 ml volumes whereas 0.2 mg/ml batch was expressed in a 1 L total volume. O – P) All batches transfected in 300 ml volumes; 0.2 mg/ml sample was concentrated from an aliquot of the 0.02 mg/ml batch. BsAb Storage conditions Stable Aggregate Time Temperature Concentration BsAb ID Buffer (%) (weeks) (°C) (mg/ml) (%) A 1-12 4 0.50 >95 <5 PBS B 1-12 -20 0.10 unstable Unstable Tandem scFv C PBS + 100 mM L- 6 4 0.50 >95 <5 D Arginine 12 -20 0.50 >95 <5 E 1-12 4 0.60 <10 >90 PBS F 1-12 -20 0.10 unstable Unstable G 6 4 0.60 ~60 ~40 Fc-containing H PBS + 100 mM L- 12 4 0.60 <20 >80 I Arginine 6 -20 0.60 ~60 ~40 J 12 -20 0.60 ~40 ~60 Fc-containing K PBS 1-12 4 0.20 <10 >90 (Longlink) L Fc-containing 1-12 4 0.02 >80 <20 PBS M (Cys) 1 4 0.20 ~60 ~40 Fc-containing N PBS 1-12 4 0.02 >80 <20 (Cys Longlink) O 1-12 4 0.02 >85 <15 KIH 1 (Cys) PBS P 1 4 0.20 ~65 ~35 Q KIH 2 PBS 1-12 4 0.20 <10 >90

85 Figure 3.10: SEC HPLC chromatograms corresponding to Table 3.3. Tandem scFv BsAb stored in PBS 1) directy after and 2) 3 months post-purification, all at 4 °C. 3) Storage in PBS at -20 C. 4) PBS + 100 mM L-Arginine as an excipient for storage at -20 °C. Fc-containing BsAb at 5) -20 °C in PBS and at 6) 4 °C in PBS after only 1 week of storage. PBS + 100 mM L-Arginine for storing the Fc-containing BsAb at 7) 4 °C over 6 weeks and 8) 3 months. PBS + 100 mM L-Arginine at -20 °C at 9) 6 weeks and 10) 3 months. Fc-containing (Longlink) stored at 4 °C in PBS for 11) 1 week or 12) 3 months post-purification. Fc-containing (Cys Longlink) format stored at 4 °C in PBS 86 for 13) 1 week and up to 14) 3 months. Fc-containing (Cys) at 4 °C in PBS at 15) 1 week and 16) 3 months storage. KIH 1 (Cys) at 17) 1 week and 18) 3 months storage and KIH 2 for 19) 1 week and 20) 3 months post-purification under the same conditions. Concentrating a KIH 1 (Cys) sample from 0.02 mg/ml to 0.2 mg/ml in PBS and stored at 21) 4 °C. Fc-containing (Cys) BsAb at a higher product yield at 22) 0.2 mg/ml as opposed to 15) 0.02 mg/ml. Figure 3.11: Dynamic Light Scattering (DLS) of disulphide engineered BsAbs. a) Fc-containing (Cys) DLS trace showing a polydispersed peak 3 and monodispersed peak 1 and 2 where peak 1 is of the expected dimer, concentration was 20 µg/ml. b) Fc-containing (Cys Longlink) sample concentration was 70 µg/ml and all 3 peaks were monodispersed with peak 1 the expected dimer. c) KIH 1 (Cys) concentration was 20 µg/ml and both peaks monodispersed where peak 1 is of the expected dimer. 87 3.3.2. Functional binding Binding to recombinant protein targets Binding activity has been maintained for both targets on all engineered BsAb formats (Figure 3.12. and Table 3.4). Surface plasmon resonance (SPR) analysis of recombinant

EGFR binding using Biacore T-200 showed that BsAb association constants (ka) were 5 to 10-fold lower than that of the published Vectibix-mAb (using purified native EGFR).46, 265

Dissociation rate constants (kd) of the Vectibix-scFvs for the tandem scFv, Fc-containing and Fc-containing (Longlink) BsAbs have values similar to the published full mAb whereas all remaining EGFR-targeting formats have off-rates (kd) up to 10-fold slower. A non- specific tandem scFv BsAb showed no specific binding to EGFR. Reformatting the

Vectibix-scFv into an IgG1 mAb showed similar ka, kd and KD values as those reported for the published Vectibix IgG2 mAb. Binding affinities (KD) of the EGFR-targeting BsAbs were all in the nanomolar (nM) range with slight differences relating to different constructs.

Figure 3.12: ABX-EGF (Panitumumab or Vectibix) and 1H10 representative binding curves. SPR sensorgrams illustrative of A) ABX-EGF tandem scFv (1 nM, 3 nM, 10 nM, 30 nM and 100 nM BsAb) and B) ABX-EGF-IgG1 mAb (1 nM, 3 nM, 10 nM and 30 nM BsAb) binding to immobilised recombinant EGFR. C) Biolayer interferometry kinetic curve of ABX-EGF tandem scFv the 1H10-mAb and ABX-EGF IgG1 mAb at 100 nM binding to captured LPS. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC.

88 Table 3.4: Surface plasmon resonance binding affinities for recombinant EGFR-mFc collected from a Biacore T-

200. Surface Plasmon Resonance (Biacore) analysis of BsAb binding to EGFR – ka (association rate constant), kd

(dissociation rate constant), and KD (binding affinity). High performance (Multi-cycle) Kinetic Assay (HPKA) data is shown for all BsAb constructs as well as for a reassembled, complete IgG1 Vectibix-mAb. Standard error values 6 -5 corresponding to binding values of recombinant EGFR is shown as ± x × 10 for ka and ± y × 10 for kd. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. Binding to recombinant EGFR Antibody ID ka (1/Ms) (×106) (±SE) kd (1/s) (×10-5) (±SE) KD (nM) Vectibix IgG2 mAb (published) 1.97 11.3 0.05 Vectibix IgG1 mAb 2.18 ± 0.0025 15.5 ± 0.047 0.07 Non-specific Tandem scFv 0.0034 ± 0.0018 7830 ± 140 22820 Tandem scFv 0.26 ± 0.0004 13.6 ± 0.021 0.52 Fc-containing 0.36 ± 0.0023 9.42 ± 0.028 0.26 Fc-containing (Longlink) 0.19 ± 0.0005 21.6 ± 0.084 1.13 Fc-containing (Cys) 0.48 ± 0.0002 4.89 ± 0.019 0.10 Fc-containing (Cys Longlink) 0.70 ± 0.0004 2.47 ± 0.025 0.04

KIH 1 (Cys) 0.82 ± 0.0005 2.67 ± 0.026 0.03 KIH 2 0.50 ± 0.0021 6.75 ± 0.021 0.13

Although unconfirmed it is possible that the weaker affinity of the Fc-containing (Longlink) BsAb could be related to how the protein is folded. Possibly, the longer linker at the CH3- 1H10 interface provides higher flexibility in this domain, resulting in some interference or miss-folding generated from the anti-LPS scFv entering the Panitumumab-scFv’s region. The Fc-containing (Cys Longlink) BsAb does not show the same weakened affinity; in fact it is one of the best at 0.03 nM, almost 100 times stronger than Fc-containing (Longlink); possibly explained by the presence of the engineered scFv disulphide bridges which ensure that the anti-LPS scFv components associate more closely. A similar observation is reflected in the KIH formats’ KD values albeit a lower difference of approximately 10 times; this can be attributed to the lack of both the anti-LPS VH and VΚ chains being present on each CH3 domain therefore limiting the reach and ability of the anti-LPS components to interfere with the opposite end of the BsAb molecule.

Binding of all BsAbs to LPS was firstly confirmed by ELISA (results not shown). As LPS is naturally adhesive and binds strongly to surfaces (e.g. glass, plastic), performing binding studies by SPR was not compatible with the microfluidics system of the Biacore. The kinetics of 1H10-scFv component of the BsAbs binding to LPS were therefore determined by the Octet system (ForteBio), which circumvents the problems of a fluidics based system. Binding data was obtained for the original, hybridoma-derived 1H10-mAb [KD = 89 0.2 nM, ka = 8.20E+05 (±1.25E+04), kd = 1.36E-04 (±1.72E-05)] and 1H10-scFv of the tandem scFv format [KD = 10.0 nM, ka = 2.20E+04 (±1.76E+03), kd = 5.64E-04 (±2.39E-

05)] (Figure 3.12. C). The 60-fold difference in KD between the two formats is indicative of differences in avidity.

Binding to native EGFR Flow cytometry analyses showed that all EGFR-targeting antibody formats (mAb and BsAb) were able to bind to EGFR-overexpressing MDA-MB-468 cells, whilst non-specific formats showed no binding (Figure 3.13). A decreased shift for the tandem scFv BsAb compared to the Fc-linked formats can be attributed to the higher avidity of the EGFR- bivalent, Fc-linked BsAbs. The lower fluorescence intensity of the tandem scFv could also be due to the different secondary antibody used for detection.

Co-localisation of recombinant EGFR and EDVTMnanocells It was of interest to investigate the ability of BsAbs to crosslink the two antigens LPS and EGFR. A co-localisation experiment, using fluorescently labelled recombinant-EGFR (DL650) and BsAb-targeted EDVTMnanocells (AF488) provided visual confirmation that BsAbs could simultaneously bind their two targets (Figure 3.14). Results show that non- targeted and non-specifically targeted EDVTMnanocells do not co-localise in the presence of recombinant EGFR (green fluorescence only). We included all seven of the BsAb formats, and observed their effect on nanocell clumping both visually and as particle counts on a nanosight (Table 3.5). The EGFR-targeting tandem scFv, KIH 1 (Cys), Fc- containing (Cys) and Fc-containing (Cys Longlink) BsAbs caused less EDVTMnanocell aggregation illustrated by a uniform distribution of co-localised EGFR and targeted EDVTMnanocells, whereas nanocells targeted with KIH 2, Fc-containing and Fc-containing (Longlink) BsAbs produced large, aggregated clumps of co-localised recombinant EGFR and EDVTMnanocells. This illustrates how different recombinant BsAbs affect the EDVTMnanocells during and post targeting, and the final product stability. These findings highlight how BsAb design and properties in solution may influence the performance of the BsAb-EDVTMnanocell formulated product.

90 Figure 3.13: In vitro binding of BsAbs and IgG1 mAbs at 1µg/ml to EGFR overexpressing MDA-MB-468 Breast cancer cells. All specific BsAbs include Vectibix-scFvs (anti-EGFR). Non-specific formats include Synagis-scFvs (anti-RSV). Mean fluorescence intensities correlate well with the observed Biacore binding affinities. A) A non-specific IgG1 mAb (blue) did not result in a binding shift occurring however incubation with a Vectibix-IgG1 mAb (red) resulted in a positive binding shift as expected. B) No binding shift was evident in the presence of a non-specific tandem scFv BsAb (blue) but a shift was present when using the Vectibix tandem scFv format (red). C) All Fc-containing BsAbs resulted in a positive binding shift compared with cells only samples. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. 91 Table 3.5: Average EDV nanosight counts post BsAb binding. EDV’s were prepared and aliquoted into tubes to ensure that roughly equal amounts were present in solution for each sample to be analysed. Once binding of BsAbs to EDVs had been achieved and sample preparations washed; duplicate measurements were made on a nanosight to determine the number of targeted or non-targeted EDVs present. Data showed that stable* BsAbs (2-6) bound to EDVs resulted in the number of EDVs/ml remaining constant when compared with non-targeted EDVs (1) that had undergone the same process of incubation and washing. Binding unstable* BsAbs to EDVs (7-9) resulted in an apparent decrease of EDVs/ml attributable to the presence of BsAb-EDV clumps. *Stable or unstable states were determined previously through SEC HPLC. 10 BsAb-EDV ID Average count (x10 EDVs/ml) 1 Non-targeted-EDV 4.62 2 Non-specific targeted-EDV 5.65 3 Tandem scFv-EDV 4.58 4 KIH 1 (Cys)-EDV 4.76 5 Fc-containing (Cys)-EDV 4.55 6 Fc-containing (CysLonglink)-EDV 4.58 7 Fc-containing-EDV 0.87 8 Fc-containing (Longlink)-EDV 1.42 9 KIH 2-EDV 1.01

92 93

Figure 3.14: Co-localisation imaging by confocal microscopy of recombinant EGFR-His (DL650) and EDVTMnanocells (AF488) with or without specific BsAb. Recombinant EGFR was co-incubated with EnGeneIC Delivery Vehicles (EDVTMnanocells or EDVs) pre-targeted with various BsAbs prior to microscopy. EDVTMnanocells are represented in green (AF488) whilst DyLight650-labelled EGFR-His is represented in purple (color substitution from red has been applied to improve co-localisation imaging). Overlap of the two fluorophores (co-localisation) results in near-white imaging. Panels represent the filter showing EGFR (DL650) alone, EDV (AF488) alone and the merged overlay image of the two. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. 3.4. Discussion and Conclusion A variety of BsAb formats were investigated for their ability to bind EDVTMnanocells and recombinant or native EGFR. Initial proof of principle studies were performed using rigorously tested approved therapeutic antibodies, which have ideal properties such as high affinity and stability. The modular nature of BsAb constructs allows alternative antibody specificities to be substituted depending on the receptor target. In order to create a cancer cell-targeting domain we selected EGFR, as several marketed anti-cancer therapeutics target this molecule.12, 14, 16, 17

Panitumumab (Vectibix or ABX-EGF) is a FDA approved commercial anti-EGFR mAb developed via transgenic humanised mouse technology by Abgenix Inc and Amgen.24, 67, 83 The mAb has picomolar affinity for EGFR and is the first fully human mAb created through transgenic humanised mouse technology to be approved for therapeutic use. Its properties, including affinity, specificity and immunogenicity, have been extensively evaluated making Panitumumab an ideal candidate to be incorporated in the EnGeneIC drug delivery system.129

The tandem scFv BsAb was designed to include a 5 amino acid glycine-serine linker (G4S) between the Vectibix- and 1H10-scFv. The shorter linker reduces flexibility and therefore prevents misfolding from occurring between the two scFv components. Administered in isolation, tandem scFv BsAbs exhibit low avidity, and due to their small size have a low serum half life.156, 193 However pharmacokinetic properties of tandem scFv BsAbs bound to the large EDVTMnanocell (400 nm in diameter), will be enhanced. Furthermore, coating of the EDVTMnanocell with tandem scFv BsAbs imparts multivalency and avidity to the NP, and provides multiple binding sites for the cancer-targeting domain.129

To produce more complex BsAbs compared to the tandem scFv BsAb, manipulation of the standard IgG mAb format has produced a plethora of Fc-containing BsAbs, whereby designs generally rely on the principle that Fc-containing BsAbs will dimerise through the Fc, similar to native, whole mAbs.118, 154, 156, 169, 193 Features of these antibodies include engineering Fc components to improve or remove various related functions, forced heterodimerisation, removal of redundant domains and addition of other binding motifs to the IgG molecule.84, 154-156, 259 We used an IgG1 Fc-domain to link and separate the two scFvs spatially (Vectibix-scFv at the N-terminus of human IgG1 Fc domain, and an 1H10- scFv at the C-terminus) to create the Vectibix-Fc-1H10 homodimeric Fc-containing BsAb, 94 similar to the scFv-Fc-scFv BsAb dimer described in Jendreyko et al. 2003 and the Emergent BiosolutionsTM product termed ADAPTIRTM Multi-Specific.151, 266

A benefit of the Fc-containing BsAbs over the tandem scFv format is the presence of two scFvs to bind each target – promoting high avidity binding to both the EDVTMnanocell and the EGFR on the cancer cell surface; however a potential drawback could result if the Fc- domain stimulates an unwanted immune response and complement cascade.144, 239 Although it is well known that Fc-related immune responses can be avoided through various mutations 84 throughout the domain – our work thus far has focussed on the production of a stable drug delivery system rather than potential immunogenicity. EnGeneIC’s original BsAb format also retained fully functional Fc domains without significant immunogenicity being noted.103, 129, 239

Presence of aggregation is undesirable in therapeutic products and therefore necessitated the need to attempt stability engineering of the Fc-containing format.136, 181, 267 Previous studies have found that inclusion of a number of design modifications, including variations in CH3-scFv linker length and type, and also scFv stability engineering via addition of disulfide bonds, improved construct stability.134, 138, 141, 181 However, the stability appears to be largely related to the nature of the scFv at the CH3 domain – an observation reiterated in our results (Table 3.3.K – N).136, 138 A concern with the Fc-linked dimeric design was whether the presence of two anti-LPS scFvs would result in EDVTMnanocell aggregation via binding of multiple nanocells rather than high avidity binding of a single nanocell.

An alternative Fc-linked BsAb was engineered consisting of two EGFR targeting scFvs at the N-terminus (high avidity) and a single anti-LPS scFv at the C-terminus of the final product. Heterodimeric BsAb formats were based on the common Knob-in-hole constructs138, 155 and partly on the TandAb BsAb designed by Affimed where variable linker lengths are used to determine chain association for functional BsAb formation.140, 190 The single anti-LPS site is formed through forced heterodimerisation of the CH3 domain via

Knob-in-Hole (KIH) engineering, where the VH and VΚ domains of the scFv are expressed on separate polypeptides in a single cell. To ensure that the VH- and VΚ-chains associate with one another to form a functional 1H10-scFv, a longer linker between CH3 and the 1H10-scFv component was designed to allow extra flexibility in the area for functional association of the nanocell-targeting scFv components.138

95 In summary we engineered several BsAb constructs and found differences in format design affected BsAb production and function. These are important findings as they can influence potential application of BsAbs as targeting reagents for NP drug delivery vehicles and also have implications for scale-up production thereof. Based on co-localisation images of the various BsAb formats binding to EDVTMnanocells and to EGFR, four of the BsAbs are of particular interest for further development: tandem scFv, Fc-containing (Cys), Fc-containing (Cys Longlink) and KIH (Cys). The other formats resulted in clumping of the EDVTMnanocells. All of these constructs can be routinely expressed in CHO-S and purified through simple purification protocols.

Preliminary results suggest that although the disulphide engineered Fc-linked BsAbs are poorly expressed in CHO-S cultures it is possible to increase product yield through optimisation of the transfection protocol. The differences in anti-EGFR avidity between constructs would likely not impact the BsAb-EDVTMnanocell function significantly due to the presence of multiple BsAbs bound to the surface of the EDVTMnanocell. Where avidity binding will likely be of importance though is during the targeting process of BsAbs binding to EDVTMnanocells – multiple anti-LPS scFvs can provide a stronger interaction between BsAb and EDVTMnanocell resulting in more EDVTMnanocells retaining targeting capabilities. However as there are expected to be multiple binding sites present on the EDVTMnanocell surface, if a tandem scFv does dissociate from the EDV it will re-associate at a second LPS site.

BsAb format stability plays an important role in the development of the EDVTMnanocell targeting system. The tandem scFv was shown to have superior stability over the Fc- containing variants indicating that individual scFv components were not responsible for any observed stability issues, but that stability was influenced by the linker connecting them. Without the ability to routinely produce predictable amounts of stable BsAb, the quality of the BsAb targeted EDVTMnanocells cannot be maintained and would hinder production of a viable therapeutic drug delivery system.

96 4. Targeting EnGeneIC drug delivery vehicles (EDVTMnanocells)

4.1. Chapter summary Engineered BsAbs are proteins designed to be capable of binding two distinct targets; whereas traditional mAbs or scFvs only bind a single target. One arm of a BsAb binding domain is engineered to bind a disease antigen while the second is used as a targeting moiety. The use of BsAbs as therapeutic products is on the increase – particularly in pre- clinical cancer treatment studies. NPs are also being developed as therapeutic agents. Greater therapeutic efficacy is achieved through loading NPs with cytotoxic drugs, siRNA and proteins, and by actively targeting the NPs reduced systemic toxicity can be achieved. Our work describes how EGFR displaying cancers could potentially be treated by BsAbs that bind the disease target while carrying a therapeutic payload enclosed in a NP, the EnGeneIC delivery vehicle (EDVTMnanocell). We show that stable BsAbs can be bound to EDVTMnanocells to produce a uniform, non-chemically conjugated BsAb-EDVTMnanocell distribution with little product loss, thereby improving scalability. The BsAb-EDVTMnanocell products maintain functional binding in vitro as illustrated through flow cytometry against EGFR-overexpressing MDA-MB-468 breast cancer cells. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDVTMnanocells were actively targeted with tandem scFv BsAb in vivo to MDA-MB-468 derived tumours in mouse xenograft models, and showed enhanced tumour regression by 40% compared to passively targeted EDVTMnanocells. BsAbs therefore provide a functional means to deliver EDVTMnanocells to target cells.

4.2. Introduction Recently a large number of engineered NPs have been developed.218, 226, 233, 234, 268, 269 The concept of NP-mediated drug delivery is attractive as it can lead to improvement in drug safety and efficacy. A myriad of engineered NPs for drug delivery have been developed, and include liposomes, polymeric-based NPs, silica, carbon, metal oxides and other materials. Some are also derived from bacteria, viruses (e.g. virus-like particles) and eukaryotic cells (exosomes). 220, 223, 226, 227, 232-234, 270 Advantages of NP-mediated drug delivery include protection of payloads such as DNA and RNA from degradation, improved drug pharmacokinetics, being able to increase the dose concentration above what would be too toxic for systemic exposure and enabling effective drug dosing where drugs are insoluble.225, 226, 239, 252 Notwithstanding the large number of NPs that have been developed for therapeutic application, at present there are only six nanomedicines approved for 97 clinical use, including liposomal NPs Myocet, Doxil, Daunoxome and Depocyt, Abraxane (albumin-based NP) and Genexol-PM (micelle).235 All these nanomedicines operate through passive targeting, i.e. enhanced extravasation of NPs at the tumour site, facilitated by poorly differentiated tumour vasculature. There are a number of nanomedicines presently in clinical trials, with several of these targeted by antibodies.218, 231

Barriers to successful NP-mediated drug delivery - including the reticular endothelial system, efficient extravasation, crossing the anatomical and physical barriers between endothelial and tumour cells, penetrating the tumour structure, endocytosis uptake and intracellular release of therapeutic agents, can be improved upon through active targeting of NPs using targeting agents. NPs can carry drug payloads that include small molecule drugs, DNA or RNA.225, 226, 252 Targeted NPs are being developed to enable efficient delivery of therapeutic compounds directly to a disease target (active targeting), such as cancer cell surface markers, thereby localising the effects of the therapeutic agent.49, 271 An emerging application of BsAbs is active targeting of drug-loaded NP to tumour sites by cross-linking the NP to tumour cells, leading to endocytosis, fusion with lysosomes and drug release intracellularly.103, 118, 129, 246

Delivery of drugs sequestered within or attached to NPs opens the drug therapeutic window and addresses the low therapeutic index of free-drug administration; i.e. systemic drug delivery at required therapeutic doses results in acute toxicity and can elicit severe patient side effects. Furthermore multiple drug resistance (MDR) can be alleviated by packaging NPs with siRNA or shRNA capable of interfering with cellular mechanisms promoting MDR.103, 239 For active targeting of NPs to tumour cells with antibodies, anti- tumour mAbs can be chemically conjugated to the NP, or alternatively be tethered to the NPs by using BsAbs which simultaneously bind the NP and the tumour target antigen.224, 236

BsAbs have gained popularity in recent years owing to their ability to target multiple antigens.118, 124, 188, 255 EnGeneIC’s EDVTMnanocell technology incorporates both the use of a novel drug delivery vehicle and a means to target cancer cells through BsAbs. EDVTMnanocells or minicells are bacterially derived, anucleate NPs capable of being loaded with high concentrations of various drug classes including chemotherapeutics and siRNA; and are successfully internalised on targeted cell surfaces despite their large size of 400nm.129 The only disadvantage of the EDVTMnanocells is the presence of LPS which 98 can illicit an immune response in patients; however it is possible that when the anti-LPS component of BsAbs bind the O-polysaccharides on the LPS-arms, these sites are shielded from the immune system (MacDiarmid personal communication).129 Coupling EDVTMnanocells to BsAbs with affinity for specific cancer cell surface antigens provides the potential of having high cytotoxic payloads or siRNA delivered directly to where required; thereby limiting the severe toxicity associated with non-targeted drug delivery.103, 129, 226, 239 Once delivered to the cancer cell surface, the BsAb and drug-loaded EDVTMnanocells are internalised by the cell; and on completion of intracellular degradation the cytotoxic payload is released.103, 129 Drug resistance can be circumvented using this technology through a two-step process of firstly delivering siRNA and then cytotoxic drugs to the target cells.103, 272

EnGeneIC’s initial approach to producing a BsAb compatible with the EDVTMnanocell included two full mAbs - one directed towards the O-polysaccharides on the LPS-arms and another towards the target cell surface antigen (EGF-Receptors), linked through the Fc regions of the mAbs by means of purified recombinant protein A/G (Figure 4.1).

Figure 4.1: Diagrammatic representation of a BsAb created through Protein A/G linkage. The diagram shows how a Protein A/G molecule with six potential binding sites for antibody Fc-domains can form a functional BsAb when bound to two different mAbs targeting different antigens.

Using this protein A/G as a linker complicates manufacturing and produces variable construct sizes since the linker is capable of binding variable proportions of up to six different anti-LPS or anti-EGFR mAbs. Initial studies showed that these BsAb- EDVTMnanocells are effectively internalised by targeted cancer cells but to optimise the therapeutic benefits and manufacturing of the technology the BsAb design needs to be improved.129 Three stable BsAb formats, a G4S-linked tandem scFv format and cysteine

99 stabilised homodimeric Fc-containing and heterodimeric Knob-in-Hole (KIH) engineered BsAbs were evaluated as to their ability to simultaneously bind EDVTMnanocells (Figure 4.2) and a cancer target – EGFR; both in vitro and in vivo.

Figure 4.2: BsAb format binding to EnGeneICTMnanocells. Graphical representation of how different types of BsAb formats are expected to bind the EnGeneICTMnanocell with different levels of avidity for the LPS O-polysaccharide on the nanocell surface. A) Shows a Knob-in-Hole BsAb and B) a tandem scFv BsAb binding to a nanocell, both BsAbs bind with lower avidity than C) the homodimeric Fc-containing BsAb format.

100 4.3. Results

4.3.1. Characterisation of stable BsAbs binding to EDVTMnanocells Conjugation of BsAbs to AF488 labelled EDVTMnanocells showed limited aggregation as illustrated in nanosight data (Table 4.1). EDVTMnanocells were resuspended to 10 approximately 4 x 10 EDVs/ml after completion of binding and wash steps. Actual mean counts indicate very little variation from this expected value indicating that little product loss occurred during the conjugation process. If aggregation is high due to the conjugation process then particle counts are significantly lower than pre-conjugation counts and the size distribution also wider than the observed “tight” peaks. Fluorescent images of BsAb- EDVTMnanocells (AF488) samples taken with microscope showed presence of single green or aggregated (green clumps) BsAb-EDVTMnanocells (Figure 4.3). Particle sizes were also distributed within the expected range of at around 400 – 500 nm.

4.3.2. In vitro binding of BsAb-EDVTMnanocells to MDA-MB-468 breast cancer cells The BsAbs which resulted in a uniform distribution in the co-localisation study described in chapter 3 were further investigated by binding to cells using flow cytometry (Figure 4.4) and confocal microscopy (Figure 4.5). For confocal microscopy MDA-MB-468 cells (labelled with AF647) were targeted with AF488-labelled BsAb-EDVTMnanocells. Non- targeted EDVTMnanocells did not localise on MDA-MB-468 cell surfaces nor did non- specific EDVTMnanocells. Confocal microscopy (Figure 4.5) and flow cytometry analyses (Figure 4.4) showed that while all BsAbs localised on the MDA-MB-468 cell surface, the tandem scFv and KIH 1 (Cys) BsAb formats, possessing only a single 1H10-scFv, displayed a lower distribution across the cell surface compared to the distributions of the Fc-containing (Cys) and Fc-containing (Cys Longlink) targeted EDVTMnanocells, with two 1H10-scFvs. Variation in flow cytometry binding shifts therefore reflects effects that can be associated with the degree of binding avidity. The Fc-containing (Cys) and Fc-containing (Cys Longlink) targeted EDVTMnanocells presented as larger shifts compared to the tandem scFv and KIH 1 (Cys) targeted EDVTMnanocells. These results could also indicate that the tandem scFv and KIH 1 (Cys) formats don’t bind EDVTMnanocells as tightly as the other two formats, resulting in more BsAb loss with increased time from initial conjugation to EDVTMnanocells through to final cellular uptake. However significant numbers of nanocells still reach the target cell surface and preliminary observations at EnGeneIC indicate that high numbers of nanocells bound to target cell-surfaces could encumber cellular uptake (personal communication Ilya Sedliarou). 101 Table 4.1: Experimental sample breakdown. BsAb-EDVTMnanocells particle characterisation and counts post- conjugation.

Mean EDV count Mean size Sample ID 10 (x10 EDVs/ml) distribution (nm)

1 Non-targeted EDVTMnanocells 4.83 455

2 Non-specific tandem scFv- EDVTMnanocells 4.62 429

3 Tandem scFv- EDVTMnanocells 4.56 483

4 Fc-containing (Cys)- EDVTMnanocells 3.78 460

Fc-containing (Cys Longlink)- 5 4.73 464 EDVTMnanocells

6 KIH (Cys)- EDVTMnanocells 3.99 474

102 Figure 4.3: Analysis of BsAb-EDVTMnanocell aggregation post-conjugation. Each individual green spot is illustrative of a single AF488-labelled BsAb-EDVTMnanocell. Where clumps are visible as in KIH 1 (Cys)-EDVs in the bottom right panel, particle aggregation has occurred.

Figure 4.4: In vitro binding of BsAb-EDVTMnanocells to EGFR overexpressing MDA-MB-468 breast cancer cells. All specific BsAbs include ABX-EGF-scFvs (anti-EGFR), non-specific formats include a Synagis-scFv (anti-RSV) and all BsAbs include the 1H10 (anti-LPS) scFv. The diagram illustrates degrees of binding shifts associated with AlexaFluor 488 labelled EDVTMnanocells (EDVs) binding to cells via various bound BsAbs. All non-aggregating BsAb formats were used to target EDVs to MDA-MB-468 cells. Positive binding shifts occurred for all formats targeted to EGFR with slightly larger shifts observed for BsAbs, Fc-containing (Cys) and Fc-containing (Cys Longlink), where higher avidity binding to EDVs is possible. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. 103 Figure 4.5: Confocal in vitro imaging of stable BsAb- EDVTMnanocells binding to the surface of MDA-MB-468 breast cancer cells. MDA-MB-468 cells were labelled with anti-EGFR- AF647 (red) to visualise the cell surface while BsAb-EDVs were labelled with AF488 (green). After 3 hours of incubating labelled BsAb-EDVs with EGFR overexpressing MDA-MB-468 cells, the samples were washed, fixed and labelled with the AF647.

104 Cover slips were mounted and cells visualised on a Confocal LSM Zeiss 710 using a Plan- Apochromat 63x/1.40 Oil DIC M27 objective. Analyses were performed using ZEN 2008 software. Scale bars in white indicate a length of 10 µm. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. 4.3.3. EDVTMnanocell-mediated tumour regression in a mouse xenograft model The tandem scFv BsAb can be produced as uniform, predictable and stable amounts and therefore it was the only recombinant BsAb format tested in a mouse xenograft model for tumour regression. To evaluate the efficacy of EDVTMnanocells targeted using the ABX- EGF tandem scFv (Tandem scFv EDV) compared to those targeted with EnGeneIC’s original, Protein A cross-linked anti-LPS / anti-EGFR BsAb (Protein A/G EDV),129 in vivo experiments were carried out utilising a MDA-MB-468 mouse xenograft model, with dosing commencing 14 days after subcutaneous injection of MDA-MB-468 cells (tumour volume approx. 80-100 mm3) (Figure 4.6). EDVTMnanocells targeted with the tandem scFv and Tandem scFv loaded with Doxorubicin ( EDV Dox) effectively suppressed tumour growth over a Tandem scFv 38 day period post xenograft compared to controls. Active targeting with EDV Dox showed tumour regression was similar to that achieved with EDVTMnanocells targeted by Protein A/G protein A/G cross-linked BsAbs ( EDV Dox). Importantly, active targeting of Doxorubicin-loaded EDVTMnanocells by the tandem scFv BsAb resulted in a 40% reduction in tumour volume compared to that of loaded passively targeted EDVTMnanocells 3 3 (EDV Dox) 38 days post xenograft (i.e. 140 mm and 240 mm , respectively). Targeted EDVTMnanocells not loaded with Doxorubicin (Tandem scFv EDV) had a minor tumour TM suppressing effect, whereas non-targeted, Doxorubicin-loaded EDV nanocells (EDV Dox) showed significant tumour regression compared to saline controls; the latter being most likely due to passive targeting through leaky tumour vasculature of tumour xenografts (pore sizes 0.2–1.2 µm).222, 231, 273

105 Figure 4.6: Mouse MDA-MB- 468 xenograft results following EDVTMnanocell (EDV) treatment. Tandem scFv refers to the EGFR targeting ABX-EGF BsAb format in the BsAb- EDVTMnanocell configuration whereas protein A/G refers to EnGeneIC’s original BsAb format composed of anti- EGFR and 1H10 mAbs connected through a protein A/G molecule. Dox refers to

106 the chemotherapeutic agent Doxorubicin. At various time points, indicated with a blue triangle, mice were treated with 1x109 EDVs. Error bars represent standard error of the mean (SEM) where n = 7. Copyright 2015 The Authors. Published with license by Taylor & Francis Group, LLC. 4.4. Discussion and Conclusion The Protein A/G linked BsAb format originally included as EnGeneIC’s active targeting moiety in the EDVTMnanocell system presented quality results as a proof of principle concept in the ability of BsAbs to act as facilitators of targeted drug delivery.103, 129, 239, 245 However BsAb production proved to be cumbersome and non-uniform owing to the presence of six potential binding sites on the Protein A/G molecule, and limited control over the ratio of each mAb component incorporated into the BsAb.129 We initially evaluated various engineered BsAb formats with respect to their product yield, stability and target binding post-purification from mammalian cell culture. From these results we were able to select improved formats for use in the EDVTMnanocell system and further analyse their ability in respect to being suited to their end purpose, which is to facilitate active drug delivery to targeted cancer cells.

The modular nature of the designed BsAbs means that clinically approved as well as new targeting scFv moieties can easily be substituted into the BsAb to target various disease phenotypes once the optimal BsAb-EDVTMnanocell system is fully evaluated. Our formats incorporated a scFv derived from a clinically approved mAb (Vectibix, Panitumumab or ABX-EGF)24, 67, 83 which targets EGFR on cancer cells, thereby circumventing the need for complete validation of the BsAbs specificity when bound to the cancer cell as this information is readily available in published results and patent information.

Given that the biggest requirement for the BsAb-EDVTMnanocell system associated with our work is the facilitation of cytotoxic payload delivery to the intended target, BsAb mode of action as a therapeutic in isolation by itself and the resulting variation thereof between formats (and variation from the standard mAb format) wasn’t a priority factor in the initial product development. All proposed formats utilising the commercial model (Panitumumab) were expected to be effective in the system albeit that there would be higher avidity associated with the Fc-containing (Cys) and Fc-containing (Cys Longlink) formats due to the presence of two scFvs targeting each of EGFR (cancer) and LPS (EDVTMnanocell). The added avidity would improve NP binding but further research would be required to evaluate the higher binding avidity’s influence on tumour penetration compared with the tandem scFv format. Another possible difference might be in the immunogenicity associated with presence of an Fc linker between scFvs rather than a simple G4S linker. However, our work thus far has focussed on the production of a stable drug delivery system rather than potential immunogenicity. EnGeneIC’s original BsAb format also 107 retained fully functional Fc domains without significant immunogenicity being noted.103, 129, 239

Based on co-localisation images of the various BsAb formats binding to EDVTMnanocells and to EGFR, four of the BsAbs were of particular interest for further development: tandem scFv, Fc-containing (Cys), Fc-containing (Cys Longlink) and KIH 1 (Cys). The other formats resulted in clumping of the EDVTMnanocells (Figure 3.14). Confocal images (Figure 4.5) showed that there was a lower distribution of KIH 1 (Cys) BsAb- EDVTMnanocells present on MDA-MB-468 cell surfaces. A possible explanation for this observation is that the association of the Vh and Vk domains of the anti-LPS scFv from different polypeptides in the KIH format might not be very stable, in addition to the lower avidity effects associated with the presence of only a single anti-LPS scFv on the construct. The sum of which results in less affinity of the BsAb to the EDVTMnanocells and consequently less reaching the MDA-MB-468 cell surfaces compared with the IgG-like (Cys) and IgG-like (CysLonglink) BsAb formats. Additional stability engineering in the anti- LPS scFv components might benefit the construct to improve functional heterodimer formation.

As the expression yields of the disulphide-engineered, Fc-linked BsAbs were 6-9 fold lower than the tandem scFv, the tandem scFv was chosen for further investigation. The tandem scFv BsAb was designed to include a 5 amino acid glycine-serine linker (G4S) between the ABX-EGF- and 1H10-scFv. The shorter linker reduces flexibility and therefore prevents misfolding from occurring between the two scFv components. Administered in isolation, tandem scFv BsAbs exhibit low avidity, and due to their small size have a low serum half life.156, 193 However pharmacokinetic properties of tandem scFv BsAbs bound to the large EDVTMnanocell (400 nm in diameter), will be enhanced. Furthermore, coating of the EDVTMnanocell with tandem scFv BsAbs imparts multivalency and avidity to the NP, and provides multiple binding sites for the cancer-targeting domain.129

Tumour regression in MDA-MB-468 breast cancer cell xenografts showed that BsAb- mediated targeting with anti-EGFR antibody is essential for optimal inhibition of tumour growth. Although EDVTMnanocells have previously been successfully targeted by cross- linking two IgG antibodies with protein A, utilising the tandem scFv, whereby one arm binds the EDVTMnanocell and the other targets the tumour, requires the production of one new molecular entity (NME). A biologic NME can be characterised for binding activity and 108 manufactured according to cGMP. Fc-containing BsAbs produced during this project have not as yet been produced as stable single NMEs at predictable yields and are therefore currently unsuitable for cGMP production. Although the 1H10-scFv is of murine origin the scFv is expected to be shielded between the nanocell and the BsAb, thereby limiting the possibility of a HAMA response.103, 129, 239 The coating of BsAb on the surface of the EDVTMnanocell also imparts other properties to the nanocell. Depending on BsAb density on the nanocell surface, the imparted avidity would enhance apparent affinity of the EDVTMnanocell for its target, compared to that of the target scFv alone. Furthermore, coating with a BsAb, where the cancer targeting scFv is of human origin, alters the physicochemical properties of the EDVTMnanocell in vivo, and would impact parameters such as pharmacokinetics and immunogenicity.

109

5. Concluding remarks and future work

During this project we successfully engineered and expressed a stable BsAb format best suited to targeting drug-laden EnGeneICTMnanocells to cancer cells presenting the EGFR. In contrast to EnGeneIC’s original BsAb format, the selected tandem scFv BsAb can be produced as a uniform and reproducible product with consistent characteristics relating to physical structure (one binding arm per target), yield, stability, binding affinity and function.129, 240 EnGeneIC has subsequently initiated a number of clinical trials both in the U.S. and in Australia wherein they have incorporated the tandem scFv as their targeting moiety (personal communication Ilya Sedliarou). A stable cell line has been developed for this BsAb format and they have since also progressed to having the product manufactured at cGMP scale. BsAb stability and release specifications were also recently finalised on pre-IND with the FDA.

A Phase 1 clinical trial was completed in Australia during June 2014 using the EGFR- targeting EDVTMnanocells for the treatment of recurrent glioma. Results from these trials have informed EnGeneIC’s ability to progress to a Phase 2a clinical trial in the U.S. using the same nanocell technology loaded with doxorubicin, in the treatment of recurrent glioblastoma – an aggressive form of brain tumour. The results from a Phase 1 clinical trial in paclitaxel-loaded EDVTMnanocells showed limited toxicity associated with the treatment; a result then reiterated in a phase 1 trial of glioblastoma patients treated with doxorubicin- loaded nanocells. Another Phase 1 clinical trial (collaboration between EnGeneIC and the Asbestos Disease Research Institute) seeks to evaluate the potential of the EGFR- targeting nanocell technology when loaded with microRNA in the treatment of malignant pleural mesothelia (MPM) (www.prnewswire.com).

The collaborative efforts between our group at the Australian Institute for Bioengineering and Nanotechnology (AIBN) and EnGeneIC in selecting the optimal BsAb targeting moiety has subsequently provided the opportunity for further research and development of the novel EDVTMnanocell platform, specifically in regards to new and novel targets. EnGeneIC have been evaluating the potential of targeting cancers relating to mesothelioma, non- small cell lung cancer and melanoma.

Currently AIBN researchers are working on identifying and producing tandem scFv BsAbs to be used as an alternative to the EGFR-targeting product. The modular nature of the 110 engineered tandem scFv BsAb makes this possible by using basic molecular techniques where newly identified targeting sequences can be inserted into the BsAb framework through restriction enzyme digestion and subsequent cloning to produce new vectors for mammalian protein expression. Currently the work involves incorporating a mesothelin targeting scFv derived from into the technology to target human mesothelioma and identifying novel binding moieties against canine c-kit (CD117) through phage display. EnGeneIC are currently performing in vivo studies on the mesothelin- targeting BsAb-EDVTMnanocells (personal communication Mohamed Alfaleh). Furthermore, the mechanism that facilitates EDVTMnanocell uptake is currently unknown and the subject of a recently submitted ARC-Linkage grant application.

In addition to the work being completed through collaboration between EnGeneIC and AIBN, our engineered tandem scFv BsAb has informed other researchers in the field of diagnostic and therapeutic NPs as to the potential of using BsAbs to facilitate targeted particle delivery. Current collaborative work includes the development of a BsAb targeting Polyethylene glycol (PEG) on a NP surface and EGFR simultaneously using the described tandem scFv BsAb format. This work is currently confidential pending the investigators pursuing the potential to patent the developed product.

Although the tandem scFv format has shown great promise as a facilitator of EDVTMnanocell delivery to target cell surfaces, it is worth noting that this may not be the case for all applications. The tandem scFv does not possess high avidity binding due to the BsAb being monovalent for each of its two targets – if a different particle was used that had low levels of target cell surface density the targeting moiety may be lost if the binding affinity isn’t strong enough or the dissociation rate too high. In the case of the EDVTMnanocell however, LPS on the nanocell surface is readily available which endows the product with the potential of high avidity – the tandem scFv to reattach should it become dissociated from one LPS site.

In conclusion, the work described in this thesis has been able to inform a wide audience spanning fields such as molecular, chemical and cell biology in the potential use and production of BsAbs. We have shown that although BsAbs are most commonly used as a therapeutic where the mode of action is to redirect an immune response, a large amount of potential is evident in using BsAbs as facilitators of targeted NP delivery.29, 118, 123, 124 The work brings together NP and antibody engineering fields to improve on the current 111 knowledge base to develop next generation novel therapeutics capable of improving the availability and quality of disease treatment options. Nanomedicine is poised to play an increasingly important role in cancer therapy in the future, with at present seven passively targeted nanomedicines approved for cancer therapy.218, 231 Actively targeting NPs to tumours with antibodies, as demonstrated in this study, can have significant advantages and improve efficacy compared to passive targeting alone. Designing new BsAbs that are able to efficiently target NPs opens a new dimension in active NP targeting.

112 6. List of References

1. Tiwari AK, Roy HK. Progress against cancer (1971–2011): how far have we come? Journal of Internal Medicine 2012; 271:392-9. 2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: A Cancer Journal for Clinicians 2011; 61:69-90. 3. Allen TM. Ligand-targeted therapeutics in anticancer therapy. Nature Reviews Cancer 2002; 2:750-63. 4. Umar A, Dunn BK, Greenwald P. Future directions in cancer prevention. Nature Reviews Cancer 2012; 12:835-48. 5. White CS. National Lung Screening Trial A Breakthrough in Lung Cancer Screening? J Thorac Imaging 2011; 26:86-7. 6. Thompson PA, Gerner EW. Current concepts in colorectal cancer prevention. Expert review of gastroenterology & hepatology 2009; 3:369-82. 7. Smith L, MacNeil S. State of the art in non-invasive imaging of cutaneous melanoma. Skin Res Technol 2011; 17:257-69. 8. Muller JM, Erasmi H, Stelzner M, Zieren U, Pichlmaier H. Surgical therapy of esophageal-carcinoma. Br J Surg 1990; 77:845-57. 9. Chabner BA, Roberts TG. Timeline - Chemotherapy and the war on cancer. Nature Reviews Cancer 2005; 5:65-72. 10. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nature Reviews Immunology 2008; 8:59-73. 11. Shanbhag S, Burtness B. Antibody-Based Therapies for Solid Tumors Cancer Management in Man: Chemotherapy, Biological Therapy, Hyperthermia and Supporting Measures. In: Minev BR, ed. Cancer Management In Man: Chemotherapy, Biological Therapy, Hyperthermia and Supporting Measures: Springer Netherlands, 2011:245-56. 12. Reichert JM, Dhimolea E. The future of antibodies as cancer drugs. Drug Discovery Today 2012; 17:954-63. 13. Kaspar AA, Reichert JM. Future directions for peptide therapeutics development. Drug Discovery Today 2013; 18:807-17. 14. Reichert JM. Antibodies to watch in 2013: Mid-year update. mAbs 2013; 5:513-7. 15. Grunwald V, Hidalgo M. The epidermal growth factor receptor: A new target for anticancer therapy. Current Problems in Cancer 2002; 26:109-64.

113 16. Mendelsohn J, Baselga J. Epidermal growth factor receptor targeting in cancer. Semin Oncol 2006; 33:369-85. 17. Elvin JG, Couston RG, van der Walle CF. Therapeutic antibodies: Market considerations, disease targets and bioprocessing. International Journal of Pharmaceutics 2013; 440:83-98. 18. Boonstra J, Rijken P, Humbel B, Cremers F, Verkleij A, Henegouwen PVE. The epidermal growth-factor. Cell Biol Int 1995; 19:413-30. 19. Wang X, Wang B, Zhang Q. Anti-tumor targeted drug delivery systems mediated by aminopeptidase N/CD13. Acta Pharmaceutica Sinica B 2011; 1:80-3. 20. Moore HE, Davenport EL, Smith EM, Muralikrishnan S, Dunlop AS, Walker BA, Krige D, Drummond AH, Hooftman L, Morgan GJ, et al. Aminopeptidase inhibition as a targeted treatment strategy in myeloma. Molecular Cancer Therapeutics 2009; 8:762- 70. 21. Gabizon A, Horowitz AT, Goren D, Tzemach D, Mandelbaum-Shavit F, Qazen MM, Zalipsky S. Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: In vitro studies. Bioconjugate Chem 1999; 10:289-98. 22. Schroeder JE, Shweky I, Shmeeda H, Banin U, Gabizon A. Folate-mediated tumor cell uptake of quantum dots entrapped in lipid nanoparticles. Journal of Controlled Release 2007; 124:28-34. 23. Campbell IG, Jones TA, Foulkes WD, Trowsdale J. Folate-binding protein is a marker for ovarian-cancer. Cancer Research 1991; 51:5329-38. 24. Cohenuram M, Saif MW. Panitumumab the first fully human monoclonal antibody: from the bench to the clinic. Anti-Cancer Drugs 2007; 18:7-15. 25. Friedman M, Lindström S, Ekerljung L, Andersson-Svahn H, Carlsson J, Brismar H, Gedda L, Frejd FY, Ståhl S. Engineering and characterization of a bispecific HER2 × EGFR-binding . Biotechnology and Applied Biochemistry 2009; 54:121-31. 26. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. New England Journal of Medicine 2005; 353:1673-84. 27. Zahnd C, Wyler E, Schwenk JM, Steiner D, Lawrence MC, McKern NM, Pecorari F, Ward CW, Joos TO, Pluckthun A. A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of Molecular Biology 2007; 369:1015-28.

114 28. Ekerljung L, Bass T, Wållberg H, Sohrabian A, Anderson K, Friedman M, Frejd F, Ståhl S, Gedda L. Bispecific Affibody Molecules for Targeting of EGFR and HER2. mAbs 2012; 4:14-6. 29. Brack S, Attinger-Toller I, Schade B, Mourlane F, Klupsch K, Woods R, Hachemi H, von der Bey U, Koenig-Friedrich S, Bertschinger J, et al. A Bispecific HER2-Targeting FynomAb with Superior Antitumor Activity and Novel Mode of Action. Molecular Cancer Therapeutics 2014; 13:2030-39. 30. Yazaki PJ, Lee B, Channappa D, Cheung CW, Crow D, Chea J, Poku E, Li L, Andersen JT, Sandlie I, et al. A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance. Protein Eng Des Sel 2013; 26:187-93. 31. Waldron NN, Kaufman DS, Oh S, Inde Z, Hexum MK, Ohlfest JR, Vallera DA. Targeting Tumor-Initiating Cancer Cells with dCD133KDEL Shows Impressive Tumor Reductions in a Xenotransplant Model of Human Head and Neck Cancer. Molecular Cancer Therapeutics 2011; 10:1829-38. 32. Chames P, Baty D. Bispecific antibodies for cancer therapy The light at the end of the tunnel? mAbs 2009; 1:539-47. 33. Vanosdol W, Fujimori K, Weinstein JN. An analysis of monoclonal-antibody distribution in microscopic tumor nodules - consequences of a binding site barrier. Cancer Research 1991; 51:4776- 84. 34. Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK, Marks JD, Weiner LM. High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules. Cancer Research 2001; 61:4750-5. 35. Tabrizi M. Considerations in Establishing Affinity Design Goals for Development of Antibody-Based Therapeutics. In: Tabrizi MA, Bornstein GG, Klakamp SL, eds. Development of Antibody-Based Therapeutics: Springer New York, 2012:141-51. 36. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. European Journal of Cancer 2001; 37:S9-S15. 37. Burgess AW, Cho HS, Eigenbrot C, Ferguson KM, Garrett TPJ, Leahy DJ, Lemmon MA, Sliwkowski MX, Ward CW, Yokoyama S. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Molecular Cell 2003; 12:541-52. 38. Rae JM, Lippman ME. Evaluation of novel epidermal growth factor receptor tyrosine kinase inhibitors. Breast Cancer Res Treat 2004; 83:99-107.

115 39. Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, Greene MI. ErbB receptors: from oncogenes to targeted cancer therapies. Journal of Clinical Investigation 2007; 117:2051-8. 40. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000; 103:211-25. 41. Citri A, Skaria KB, Yarden Y. The deaf and the dumb: the biology of ErbB-2 and ErbB- 3. Experimental Cell Research 2003; 284:54-65. 42. Falls DL. Neuregulins: functions, forms, and signaling strategies. Experimental Cell Research 2003; 284:14-30. 43. Hackel BJ, Neil JR, White FM, Wittrup KD. Epidermal growth factor receptor downregulation by small heterodimeric binding proteins. Protein Engineering Design and Selection 2012; 25:47-57. 44. Fan Z, Baselga J, Masui H, Mendelsohn J. Antitumor effect of antiepidermal growth-factor receptor monoclonal-antibodies plus cis- diamminedichloroplatinum on well established A431 cell xenografts. Cancer Research 1993; 53:4637-42. 45. Goldstein NI, Prewett M, Zuklys K, Rockwell P, Mendelsohn J. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clinical Cancer Research 1995; 1:1311-8. 46. Yang XD, Jia XC, Corvalan JRF, Wang P, Davis CG, Jakobovits A. Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Research 1999; 59:1236- 43. 47. Johns TG, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK, Scott AM, Furnari FB. The Efficacy of Epidermal Growth Factor Receptor-Specific Antibodies against Glioma Xenografts Is Influenced by Receptor Levels, Activation Status, and Heterodimerization. Clinical Cancer Research 2007; 13:1911-25. 48. Adams TE, Koziolek EJ, Hoyne PH, Bentley JD, Lu L, Lovrecz G, Ward CW, Lee FT, Scott AM, Nash AD, et al. A truncated soluble epidermal growth factor receptor-Fc fusion ligand trap displays anti-tumour activity in vivo. Growth Factors 2009; 27:141- 54. 49. Yang LL, Mao H, Wang YA, Cao ZH, Peng XH, Wang XX, Duan HW, Ni CC, Yuan QG, Adams G, et al. Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging. Small 2009; 5:235-43.

116 50. Spangler JB, Neil JR, Abramovitch S, Yarden Y, White FM, Lauffenburger DA, Wittrup KD. Combination antibody treatment down-regulates epidermal growth factor receptor by inhibiting endosomal recycling. Proceedings of the National Academy of Sciences 2010; 107:13252-7. 51. Dong J, Sereno A, Aivazian D, Langley E, Miller BR, Snyder WB, Chan E, Cantele M, Morena R, Joseph IBJK, et al. A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity. mAbs 2011; 3:273-88. 52. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: Targeting the epidermal growth factor receptor. Clinical Cancer Research 2001; 7:2958-70. 53. Cohen RB. Current challenges and clinical investigations of epidermal growth factor receptor (EGFR)- and ErbB family-targeted agents in the treatment of head and neck squamous cell carcinoma (HNSCC). Cancer Treatment Reviews 2014; 40:567-77. 54. Pirker R. EGFR-directed monoclonal antibodies in non-small cell lung cancer. Targeted Oncology 2013; 8:47-53. 55. Strohl WR. Therapeutic Monoclonal Antibodies: Past, Present, and Future. Therapeutic Monoclonal Antibodies: John Wiley & Sons, Inc., 2009:1-50. 56. Goldstein G. Overview of the development of Orthoclone-OKT3-monoclonal antibody for therapeutic use in transplantation. Transplant Proc 1987; 19:1-6. 57. Hooks MA, Wade CS, Millikan WJ. Muromonab CD-3 - A review of its pharmacology, pharmacokinetics, and clinical use in transplantation. Pharmacotherapy 1991; 11:26-37. 58. Brekke OH, Sandlie I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nature Reviews Drug Discovery 2003; 2:52-62. 59. Elgert KD. Antibody Structure and Function. Immunology : understanding the immune system. New York: Wiley-Liss, 1996:58-78. 60. Neuberger MS, Williams GT, Mitchell EB, Jouhal SS, Flanagan JG, Rabbitts TH. A hapten-specific IgE antibody with human physiological effector function. Nature 1985; 314:268-70. 61. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human-antibody with those from a mouse. Nature 1986; 321:522-5.

117 62. Lonberg N. Fully human antibodies from transgenic mouse and phage display platforms. Current Opinion in Immunology 2008; 20:450-9. 63. Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. Journal of Molecular Biology 1997; 270:26-35. 64. Hoogenboom HR, Chames P. Natural and designer binding sites made by phage display technology. Immunol Today 2000; 21:371-8. 65. Saggy I, Wine Y, Shefet-Carasso L, Nahary L, Georgiou G, Benhar I. Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining. Protein Engineering Design and Selection 2012; 25:539-49. 66. Luginbuhl B, Kanyo Z, Jones RM, Fletterick RJ, Prusiner SB, Cohen FE, Williamson RA, Burton DR, Pluckthun A. Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation. Journal of Molecular Biology 2006; 363:75-97. 67. Jakobovits A, Amado RG, Yang X, Roskos L, Schwab G. From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nature Biotechnology 2007; 25:1134-43. 68. Reichert JM. Monoclonal Antibodies as Innovative Therapeutics. Curr Pharm Biotechnol 2008; 9:423-30. 69. Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nature Reviews Drug Discovery 2010; 9:767-74. 70. Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nature Biotechnology 2005; 23:1147-57. 71. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nature Reviews Cancer 2012; 12:278-87. 72. Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market. mAbs 2014; 7:9-14. 73. Dimitrov DS. Therapeutic proteins. Methods in molecular biology (Clifton, NJ) 2012; 899:1-26. 74. Reichert JM. Antibodies to watch in 2014. mAbs 2014; 6:799-802. 75. Reichert JM. Antibodies to watch in 2015. mAbs 2014; 7:1-8. 76. Russell JS, Colevas AD. The use of epidermal growth factor receptor monoclonal antibodies in squamous cell carcinoma of the head and neck. Chemotherapy research and practice 2012; 2012:761518. 118 77. Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, Valtorta E, Schiavo R, Buscarino M, Siravegna G, et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486:532-U131. 78. Roskoski R. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res 2014; 79:34-74. 79. Bardelli A, Siena S. Molecular Mechanisms of Resistance to Cetuximab and Panitumumab in Colorectal Cancer. Journal of Clinical Oncology 2010; 28:1254-61. 80. Saxena B, Sundaram ST, Walton W, Patel I, Kuo P, Khan S, Matathia A, Purohit A, Crowley R, Zhou Q. Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: In vitro biological and binding activities. Journal of Clinical Oncology 2011; 29:e13030. 81. Fauvel B, Yasri A. Antibodies directed against receptor tyrosine kinases. mAbs 2014; 6:838-51. 82. Argiles G, Dienstmann R, Elez E, Tabernero J. Panitumumab: a summary of clinical development in colorectal cancer and future directions. Future Oncology 2012; 8:373- 89. 83. Gemmete JJ, Mukherji SK. Panitumumab (Vectibix). American Journal of Neuroradiology 2011; 32:1002-3. 84. Labrijn AF, Aalberse RC, Schuurman J. When binding is enough: nonactivating antibody formats. Current Opinion in Immunology 2008; 20:479-85. 85. Casi G, Neri D. Antibody–drug conjugates: Basic concepts, examples and future perspectives. Journal of Controlled Release 2012; 161:422-8. 86. Govindan SV, Goldenberg DM. Designing immunoconjugates for cancer therapy. Expert Opin Biol Ther 2012; 12:873-90. 87. Vincent KJ, Zurini M. Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates. Biotechnol J 2012; 7:1444-50. 88. Sassoon I, Blanc V. Antibody-Drug Conjugate (ADC) Clinical Pipeline: A Review. In: Ducry L, ed. Antibody-Drug Conjugates. Totowa: Humana Press Inc, 2013:1-27. 89. Sievers EL, Senter PD. Antibody-Drug Conjugates in Cancer Therapy. Annual Review of Medicine, Vol 64 2013; 64:15-29. 90. Linenberger ML, Hong T, Flowers D, Sievers EL, Gooley TA, Bennett JM, Berger MS, Leopold LH, Appelbaum FR, Bernstein ID. Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 2001; 98:988-94.

119 91. Sievers EL, Larson RA, Stadtmauer EA, Estey E, Lowenberg B, Dombret H, Karanes C, Theobald M, Bennett JM, Sherman ML, et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. Journal of Clinical Oncology 2001; 19:3244-54. 92. Sievers EL, Linenberger M. Mylotarg: antibody-targeted chemotherapy comes of age. Current Opinion in Oncology 2001; 13:522-7. 93. Trail PA, Bianchi AB. Monoclonal antibody drug conjugates in the treatment of cancer. Current Opinion in Immunology 1999; 11:584-8. 94. Beck A, Reichert JM. Antibody-drug conjugates Present and future. mAbs 2014; 6:15- 7. 95. Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. mAbs 2014; 6:34-45. 96. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, Borzilleri RM. Antibody-drug conjugates: current status and future directions. Drug Discovery Today 2014; 19:869-81. 97. Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, Cuong T, Uter NT, Yin G, Rivers PJ, et al. Production of Site-Specific Antibody-Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System. Bioconjugate Chem 2014; 25:351-61. 98. Human Monoclonal Antibodies: Methods and Protocols. In: Steinitz M, ed. Human Monoclonal Antibodies: Methods and Protocols: Humana Press Inc, 999 Riverview Dr, Ste 208, Totowa, Nj 07512-1165 USA, 2014. 99. Falconer RJ, Chan C, Hughes K, Munro TP. Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients. J Chem Technol Biotechnol 2011; 86:942-8. 100. Zider A, Drakeman DL. The future of monoclonal antibody technology. mAbs 2010; 2:361-4. 101. Dinnis DM, James DC. Engineering mammalian cell factories for improved recombinant monoclonal antibody production: Lessons from nature? Biotechnology and Bioengineering 2005; 91:180-9. 102. Chan AC, Carter PJ. Therapeutic antibodies for autoimmunity and inflammation. Nature Reviews Immunology 2010; 10:301-16. 103. MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss J, Sedliarou I, Wetzel S, Kochar K, Brahmbhatt VN, Phillips L, Pattison ST, Petti C, et al. Sequential treatment

120 of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Nature Biotechnology 2009; 27:643-51. 104. Garber K. Bispecific antibodies rise again. Nature reviews Drug discovery 2014; 13:799-801. 105. Byrne H, Conroy PJ, Whisstock JC, O'Kennedy RJ. A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends in Biotechnology 2013; 31:621-32. 106. Riethmuller G. Symmetry breaking: bispecific antibodies, the beginnings, and 50 years on. Cancer immunity 2012; 12:12. 107. Kontermann RE. Bispecific Antibodies: Developments and Current Perspectives. In: Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011:1-28. 108. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of pre- defined specificity . Nature 1975; 256:495-7. 109. Williams G. Novel antibody reagents - production and potential. Trends in Biotechnology 1988; 6:36 110. Nisonoff A, Rivers MM. Recombination of a mixture of univalent antibody fragments of different specificity. Arch Biochem Biophys. 1961; 93:460. 111. Nisonoff A, Mandy WJ. Quantitative estimation of hybridization of rabbit antibodies. Nature 1962; 194:355. 112. Milstein C, Cuello AC. Hybrid hybridomas and their use in immunohisto- chemistry. Nature 1983; 305:537-40. 113. Suresh MR, Cuello AC, Milstein C. Bispecific monoclonal antibodies from hybrid hybridomas. Method Enzymol 1986; 121:210-28. 114. Glennie MJ, McBride HM, Worth AT, Stevenson GT. Preparation and performance of bispecific F(ab'-gamma)2 antibody containing thioether-linked Fab'-gamma fragments. Journal of Immunology 1987; 139:2367-75. 115. Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011: DOI 10.1007/978-3-642-20910-9 116. Fanger MW, Segal DM, Rometlemonne JL. Bispecific antibodies and targeted cellular cytotoxicity. Immunol Today 1991; 12:51-4. 117. Byrne H, Conroy PJ, Whisstock JC, O'Kennedy RJ. A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends in Biotechnology 2013; 31:621-32.

121 118. Kontermann R. Dual targeting strategies with bispecific antibodies. mAbs 2012; 4:182-97. 119. Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nature Reviews Immunology 2010; 10:345-52. 120. Brennan FR, Cauvin A, Tibbitts J, Wolfreys A. Optimized Nonclinical Safety Assessment Strategies Supporting Clinical Development of Therapeutic Monoclonal Antibodies Targeting Inflammatory Diseases. Drug Development Research 2014; 75:115-61. 121. Stubenrauch K, Wessels U, Essig U, Vogel R, Waltenberger H, Hansbauer A, Koehler A, Heinrich J. An immunodepletion procedure advances free angiopoietin-2 determination in human plasma samples during anti-cancer therapy with bispecific anti-Ang2/VEGF CrossMab. Journal of Pharmaceutical and Biomedical Analysis 2015; 102:459-67. 122. Miossec P, Kolls JK. Targeting IL-17and T(H)17 cells in chronic inflammation. Nature Reviews Drug Discovery 2012; 11:763-76. 123. Lutterbuese R, Raum T, Kischel R, Hoffmann P, Mangold S, Rattel B, Friedrich M, Thomas O, Lorenczewski G, Rau D, et al. T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells. Proceedings of the National Academy of Sciences of the United States of America 2010; 107:12605-10. 124. Brischwein K, Schlereth B, Guller B, Steiger C, Wolf A, Lutterbuese R, Offner S, Locher M, Urbig T, Raum T, et al. MT110: A novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Molecular Immunology 2006; 43:1129-43. 125. Zhu Z. Dual-Targeting Bispecific Antibodies as New Therapeutic Modalities for Cancer. In: Wood CR, ed. Antibody Drug Discovery. Germany: Imperial College Press, 2011:373-408. 126. Johnson S, Burke S, Huang L, Gorlatov S, Li H, Wang W, Zhang W, Tuaillon N, Rainey J, Barat B, et al. Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion. Journal of Molecular Biology 2010; 399:436-49. 127. Silacci M, Baenziger-Tobler N, Lembke W, Zha WJ, Batey S, Bertschinger J, Grabulovski D. Linker Length Matters, Fynomer-Fc Fusion with an Optimized Linker Displaying Picomolar IL-17A Inhibition Potency. Journal of Biological Chemistry 2014; 289:14392-8. 122 128. Metz S, Haas AK, Daub K, Croasdale R, Stracke J, Lau W, Georges G, Josel H-P, Dziadek S, Hopfner K-P, et al. Bispecific digoxigenin-binding antibodies for targeted payload delivery. Proceedings of the National Academy of Sciences 2011; 108:8194-9. 129. MacDiarmid JA, Mugridge NB, Weiss JC, Phillips L, Burn AL, Paulin RP, Haasdyk JE, Dickson KA, Brahmbhatt VN, Pattison ST, et al. Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics. Cancer Cell 2007; 11:431-45. 130. Moldenhauer G. Bispecific Antibodies from Hybrid Hybridoma. In: Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011:29-46. 131. Ellerman D, Scheer JM. Generation of Bispecific Antibodies by Chemical Conjugation. In: Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011:47-63. 132. Pluckthun A, Pack P. New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 1997; 3:83-105. 133. Carter P. Bispecific human IgG by design. Journal of Immunological Methods 2001; 248:7-15. 134. Mabry R, Lewis KE, Moore M, McKernan PA, Bukowski TR, Bontadelli K, Brender T, Okada S, Lum K, West J, et al. Engineering of stable bispecific antibodies targeting IL-17A and IL-23. Protein Engineering Design and Selection 2009; 23:115-27. 135. Gunasekaran K, Pentony M, Shen M, Garrett L, Forte C, Woodward A, Ng SB, Born T, Retter M, Manchulenko K, et al. Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: Applications to bispecific molecules and monovalent IgG. Journal of Biological Chemistry 2010; 285:19637-46. 136. Miller BR, Demarest SJ, Lugovskoy A, Huang F, Wu XF, Snyder WB, Croner LJ, Wang N, Amatucci A, Michaelson JS, et al. Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-57. 137. Jin P, Zhu ZP. The Design and Engineering of IgG-Like Bispecific Antibodies. In: Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011:151-69. 138. Moore GL, Bautista C, Pong E, Nguyen D-HT, Jacinto J, Eivazi A, Muchhal US, Karki S, Chu SY, Lazar GA. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. mAbs 2011; 3:546-57. 139. Müller D, Kontermann RE. Recombinant Bispecific Antibodies for Cancer Therapy. 2011; 7:235-49. 123 140. Rajkovic E, Hucke C, Knackmuss S, Molkenthin V, Reusch U, Legall F, Little M. Recruit-TandAbs: Engaging immune cells to kill cancer cells AFM13: A bispecific tetravalent TandAb for treating Hodgkin Lymphoma. 11th International Conference on Malignant Lymphoma 15-18 June 2011. Lugano (Switzerland): Annals of Oncology, 2011:240-1. 141. Schanzer J, Jekle A, Nezu J, Lochner A, Croasdale R, Dioszegi M, Zhang J, Hoffmann E, Dormeyer W, Stracke J, et al. Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains. Antimicrobial Agents and Chemotherapy 2011; 55:2369-78. 142. Kuo S-R, Wong L, Liu J-S. Engineering a CD123xCD3 bispecific scFv immunofusion for the treatment of leukemia and elimination of leukemia stem cells. Protein Engineering Design and Selection 2012; 25: 561-70. 143. Wang L, He YR, Zhang G, Ma J, Liu CZ, He W, Wang W, Han HM, Boruah BM, Gao B. Retargeting T Cells for HER2-Positive Tumor Killing by a Bispecific Fv-Fc Antibody. PLoS ONE 2013; 8: e75589. 144. Chelius D, Ruf P, Gruber P, Plöscher M, Liedtke R, Gansberger E, Hess J, Wasiliu M, Lindhofer H. Structural and functional characterization of the catumaxomab. mAbs 2010; 2:309-19. 145. Linke R, Klein A, Seimetz D. Catumaxomab: Clinical development and future directions. mAbs 2010; 2:129-36. 146. Atanackovic D, Reinhard H, Meyer S, Spöck S, Grob T, Luetkens T, Yousef S, Cao Y, Hildebrandt Y, Templin J, et al. The trifunctional antibody catumaxomab amplifies and shapes tumor-specific immunity when applied to gastric cancer patients in the adjuvant setting. Human Vaccines & Immunotherapeutics 2013; 9:2533-42. 147. Weidle UH, Kontermann RE, Brinkmann U. Tumor-Antigen–Binding Bispecific Antibodies for Cancer Treatment. Semin Oncol 2014; 41:653-60. 148. Roberts JP. Bispecific biologics rush. Nature Biotechnology 2011; 29:959. 149. Laventie BJ, Rademaker HJ, Saleh M, de Boer E, Janssens R, Bourcier T, Subilia A, Marcellin L, van Haperen R, Lebbink JHG, et al. Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins. Proceedings of the National Academy of Sciences 2011; 108:16404-9. 150. Schaefer W, Regula JT, Bahner M, Schanzer J, Croasdale R, Durr H, Gassner C, Georges G, Kettenberger H, Imhof-Jung S, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proceedings of the National Academy of Sciences of the United States of America 2011; 108:11187-92. 124 151. Jendreyko N. Intradiabodies, Bispecific, Tetravalent Antibodies for the Simultaneous Functional Knockout of Two Cell Surface Receptors. Journal of Biological Chemistry 2003; 278:47812-9. 152. Gramer MJ, van den Bremer ETJ, van Kampen MD, Kundu A, Kopfmann P, Etter E, Stinehelfer D, Long J, Lannom T, Noordergraaf EH, et al. Production of stable bispecific IgG1 by controlled Fab-arm exchange Scalability from bench to large-scale manufacturing by application of standard approaches. mAbs 2013; 5:961-72. 153. Kufer P, Lutterbuse R, Baeuerle PA. A revival of bispecific antibodies. Trends in Biotechnology 2004; 22:238-44. 154. Merchant AM, Zhu ZP, Yuan JQ, Goddard A, Adams CW, Presta LG, Carter P. An efficient route to human bispecific IgG. Nature Biotechnology 1998; 16:677-81. 155. Ridgway JBB, Presta LG, Carter P. 'Knobs-into-holes' engineering of antibody C(H)3 domains for heavy chain heterodimerization. Protein Engineering 1996; 9:617- 21. 156. Presta LG. Molecular engineering and design of therapeutic antibodies. Current Opinion in Immunology 2008; 20:460-70. 157. Beckman RA, Weiner LM, Davis HM. Antibody constructs in cancer therapy - Protein engineering strategies to improve exposure in solid tumors. Cancer 2007; 109:170-9. 158. Wozniak-Knopp G, Bartl S, Bauer A, Mostageer M, Woisetschlager M, Antes B, Ettl K, Kainer M, Weberhofer G, Wiederkum S, et al. Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties. Protein Eng Des Sel 2010; 23:289- 97. 159. Schaefer G, Haber L, Crocker Lisa M, Shia S, Shao L, Dowbenko D, Totpal K, Wong A, Lee Chingwei V, Stawicki S, et al. A Two-in-One Antibody against HER3 and EGFR Has Superior Inhibitory Activity Compared with Monospecific Antibodies. Cancer Cell 2011; 20:472-86. 160. Lee CV, Koenig P, Fuh G. A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions. mAbs 2014; 6:622-7. 161. Zhang H, Yun S, Batuwangala TD, Steward M, Holmes SD, Pan L, Tighiouart M, Shin HJC, Koenig L, Park W, et al. A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment. International Journal of Cancer 2012; 131:956-69. 125

162. DiGiammarino E, Ghayur T, Liu J. Design and generation of DVD-Ig molecules for dual-specific targeting. Methods in molecular biology (Clifton, NJ) 2012; 899:145-56. 163. Gu JJ, Ghayur T. Generation of dual-variable domain immunoglobulin molecules for dual-specific targeting. In: Wittrup KD, Verdine GL, eds. Methods in Enzymology, Vol 502: Protein Engineering for Therapeutics, Pt A. San Diego: Elsevier Academic Press Inc, 2012:25-41. 164. Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, Guntas G, Leaver-Fay A, Smith EM, Ho C, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat Biotech 2014; 32:191-8. 165. Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, Guntas G, Leaver-Fay A, Smith EM, Ho C, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nature Biotechnology 2014; 32:191-8. 166. Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, Wittrup KD. A modular IgG-scFv bispecific antibody topology. Protein Engineering Design and Selection 2010; 23:221-8. 167. Michaelson JS, Demarest SJ, Miller B, Amatucci A, Snyder WB, Wu X, Huang F, Phan S, Gao S, Doern A, et al. Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR. mAbs 2009; 1:128-41. 168. Dong J, Sereno A, Snyder WB, Miller BR, Tamraz S, Doern A, Favis M, Wu X, Tran H, Langley E, et al. Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity. Journal of Biological Chemistry 2011; 286:4703-17. 169. Mabry R, Gilbertson DG, Frank A, Vu T, Ardourel D, Ostrander C, Stevens B, Julien S, Franke S, Meengs B, et al. A dual-targeting PDGFR beta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo. mAbs 2010; 2:20-34. 170. Kanakaraj P, Puffer BA, Yao XT, Kankanala S, Boyd E, Shah RR, Wang GP, Patel D, Krishnamurthy R, Kaithamana S, et al. Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis. mAbs 2012; 4:600-13. 171. LaFleur DW, Abramyan D, Kanakaraj P, Smith RG, Shah RR, Wang GP, Yao XT, Kankanala S, Boyd E, Zaritskaya L, et al. Monoclonal antibody therapeutics with up to five specificities Functional enhancement through fusion of target-specific peptides. mAbs 2013; 5:208-18.

126 172. Buck PM, Kumar S, Singh SK. Consequences of glycan truncation on Fc structural integrity. mAbs 2013; 5:904-16. 173. Rose RJ, van Berkel PHC, van den Bremer ETJ, Labrijn AF, Vink T, Schuurman J, Heck AJR, Parren PWHI. Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG. mAbs 2013; 5:219-28. 174. Monnet C, Jorieux S, Souyris N, Zaki O, Jacquet A, Fournier N, Crozet F, de Romeuf C, Bouayadi K, Urbain R, et al. Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody. mAbs 2014; 6:422-36. 175. Lazar GA, Dang W, Karki S, Vafa O, Peng JS, Hyun L, Chan C, Chung HS, Eivazi A, Yoder SC, et al. Engineered antibody Fc variants with enhanced effector function. Proceedings of the National Academy of Sciences of the United States of America 2006; 103:4005-10. 176. Desjarlais JR, Lazar GA, Zhukovsky EA, Chu SY. Optimizing engagement of the immune system by anti-timor antibodies: an engineer's perspective. Drug Discovery Today 2007; 12:898-910. 177. Dhodapkar MV, Steinman RM. Antigen-bearing immature dendritic cells induce peptide-specific CD8(+) regulatory T cells in vivo in humans. Blood 2002; 100:174-7. 178. Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N. An engineered human IgG1 antibody with longer serum half-life. Journal of Immunology 2006; 176:346-56. 179. Datta-Mannan A, Witcher DR, Tang Y, Watkins J, Wroblewski VJ. Monoclonal antibody clearance - Impact of modulating the interaction of IgG with the neonatal . Journal of Biological Chemistry 2007; 282:1709-17. 180. Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nature Biotechnology 1997; 15:159-63. 181. Demarest SJ, Glaser SM. Antibody therapeutics, antibody engineering, and the merits of protein stability. Curr Opin Drug Discov Dev 2008; 11:675-87. 182. Croasdale R, Wartha K, Schanzer JM, Kuenkele KP, Ries C, Mayer K, Gassner C, Wagner M, Dimoudis N, Herter S, et al. Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition. Arch Biochem Biophys 2012; 526:206-18. 183. Mouquet H, Warncke M, Scheid JF, Seaman MS, Nussenzweig MC. Enhanced HIV-1 neutralization by antibody heteroligation. Proceedings of the National Academy of Sciences 2012; 109:875-80. 127 184. Fenn S, Schiller CB, Griese JJ, Duerr H, Imhof-Jung S, Gassner C, Moelleken J, Regula JT, Schaefer W, Thomas M, et al. Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain. PLoS ONE 2013; 8: e61953. 185. Wurch T, Larbouret C, Robert B. Keystone Symposium on Antibodies as Drugs: March 27-April 1, 2009, Whistler, BC CA. mAbs 2009; 1:318-25. 186. Labrijn AF, Meesters JI, de Goeij B, van den Bremer ETJ, Neijssen J, van Kampen MD, Strumane K, Verploegen S, Kundu A, Gramer MJ, et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proceedings of the National Academy of Sciences of the United States of America 2013; 110:5145-50. 187. Holliger P, Prospero T, Winter G. Diabodies - small bivalent and bispecific antibody fragments. Proceedings of the National Academy of Sciences of the United States of America 1993; 90:6444-8. 188. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005; 23:1126-36. 189. Muller D, Karle A, Meibburger B, Hofig I, Stork R, Kontermann RE. Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. Journal of Biological Chemistry 2007; 282:12650-60. 190. McAleese F, Eser M. RECRUIT-TandAbs (R): harnessing the immune system to kill cancer cells. Future Oncology 2012; 8:687-95. 191. Zhao XB, Lapalombella R, Joshi T, Cheney C, Gowda A, Hayden-Ledbetter MS, Baurn PR, Lin TS, Jarjoura D, Lehman A, et al. Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 2007; 110:2569-77. 192. Cheney CM, Stephens DM, Mo XK, Rafiq S, Butchar J, Flynn JM, Jones JA, Maddocks K, O'Reilly A, Ramachandran A, et al. Ocaratuzumab, an Fc- engineered antibody demonstrates enhanced antibody- dependent cell- mediated cytotoxicity in chronic lymphocytic leukemia. mAbs 2014; 6:748-54. 193. Enever C, Batuwangala T, Plummer C, Sepp A. Next generation immunotherapeutics—honing the magic bullet. Current Opinion in Biotechnology 2009; 20:405-11. 194. Kasaian MT, Marquette K, Fish S, DeClercq C, Agostinelli R, Cook TA, Brennan A, Lee J, Fitz L, Brooks J, et al. An IL-4/IL-13 Dual Antagonist Reduces Lung Inflammation, Airway Hyperresponsiveness, and IgE Production in Mice. American Journal of Respiratory Cell and Molecular Biology 2013; 49:37-46. 128 195. Chames P, Baty D. Bispecific Single Domain Antibodies. In: Kontermann RE, ed. Bispecific Antibodies. New York: Springer, 2011:101-114 196. Rozan C, Cornillon A, Petiard C, Chartier M, Behar G, Boix C, Kerfelec B, Robert B, Pelegrin A, Chames P, et al. Single-Domain Antibody-Based and Linker-Free Bispecific Antibodies Targeting Fc gamma RIII Induce Potent Antitumor Activity without Recruiting Regulatory T Cells. Molecular Cancer Therapeutics 2013; 12:1481- 91. 197. Saerens D, Ghassabeh GH, Muyldermans S. Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 2008; 8:600-8. 198. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY. Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett; 584:2670-80. 199. Muller D, Kontermann RE. Diabodies, Single-Chain Diabodies, and Their Derivatives. Kontermann RE, ed. Bispecific Antibodies. New York:Springer,2011:83-100. 200. Moore PA, Zhang WJ, Rainey GJ, Burke S, Li H, Huang L, Gorlatov S, Veri MC, Aggarwal S, Yang YH, et al. Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma. Blood 2011; 117:4542-51. 201. Wolf E, Hofmeister R, Kufer P, Schlereth B, Baeuerle PA. BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discovery Today 2005; 10:1237-44. 202. Emanuel SL, Engle LJ, Cao C, Chao G, Lin Z, Zhu R-R, Yamniuk AP, Hosbach J, Brown J, Fitzpatrick E, et al. Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors. Proceedings of the American Association for Cancer Research Annual Meeting 2010; 51:627. 203. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY. Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 2010; 584:2670-80. 204. Stumpp MT, Binz HK, Amstutz P. DARPins: A new generation of protein therapeutics. Drug Discovery Today 2008; 13:695-701. 205. Binz HK, Amstutz P, Plückthun A. Engineering novel binding proteins from nonimmunoglobulin domains. Nature Biotechnology 2005; 23:1257-68. 206. Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, Nagy-Davidescu G, Dreier B, Schibli R, Binz HK, Waibel R, et al. Efficient Tumor Targeting with High- Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size. Cancer Research 2010; 70:1595-605. 129 207. Richter A, Eggenstein E, Skerra A. Anticalins: Exploiting a non-Ig scaffold with hypervariable loops for the engineering of binding proteins. FEBS Lett 2014; 588:213- 8. 208. Baeuerle PA, Reinhardt C. Bispecific T-Cell Engaging Antibodies for Cancer Therapy. Cancer Research 2009; 69:4941-4. 209. Baeuerle PA, Itin C. Clinical Experience with Gene Therapy and Bispecific Antibodies for T Cell-based Therapy of Cancer. Curr Pharm Biotechnol 2012; 13:1399- 408. 210. Frankel SR, Baeuerle PA. Targeting T cells to tumor cells using bispecific antibodies. Current Opinion in Chemical Biology 2013; 17:385-92. 211. Nagorsen D, Kufer P, Baeuerle PA, Bargou R. Blinatumomab: A historical perspective. Pharmacology & Therapeutics 2012; 136:334-42. 212. Walter RB. Biting back: BiTE antibodies as a promising therapy for acute myeloid leukemia. Expert Rev Hematol 2014; 7:317-9. 213. Skerra A. Alternative non-antibody scaffolds for molecular recognition. Current Opinion in Biotechnology 2007; 18:295-304. 214. Binz HK, Stumpp MT, Forrer P, Amstutz P, Pluckthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. Journal of Molecular Biology 2003; 332:489-503. 215. Forrer P, Stumpp MT, Binz HK, Pluckthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett 2003; 539:2-6. 216. Boder ET, Jiang W. Engineering Antibodies for Cancer Therapy. In: Prausnitz JM, ed. Annual Review of Chemical and Biomolecular Engineering, Vol 2. Palo Alto: Annual Reviews, 2011:53-75. 217. Akbulut M, D'Addio SM, Gindy ME, Prud'homme RK. Novel methods of targeted drug delivery: the potential of multifunctional nanoparticles. Expert review of clinical pharmacology 2009; 2:265-82. 218. Sanna V, Pala N, Sechi M. Targeted therapy using nanotechnology: focus on cancer. Int J Nanomed 2014; 9:467-83. 219. Goodall S, Jones ML, Mahler S. Monoclonal antibody-targeted polymeric nanoparticles for cancer therapy – future prospects. Journal of Chemical Technology & Biotechnology 2014; DOI: 10.1002/jctb.4555. 220. Couvreur P. Nanoparticles in drug delivery: Past, present and future. Advanced Drug Delivery Reviews 2013; 65:21-3.

130 221. Livney YD, Assaraf YG. Rationally designed nanovehicles to overcome cancer chemoresistance. Advanced Drug Delivery Reviews 2013; 65:1716-30. 222. Hobbs SK, Monsky WL, Yuan F, Roberts WG, Griffith L, Torchilin VP, Jain RK. Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment. Proceedings of the National Academy of Sciences of the United States of America 1998; 95:4607-12. 223. Steichen SD, Caldorera-Moore M, Peppas NA. A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics. European Journal of Pharmaceutical Sciences 2013; 48:416-27. 224. Bertrand N, Wu J, Xu XY, Kamaly N, Farokhzad OC. Cancer nanotechnology: The impact of passive and active targeting in the era of modern cancer biology. Advanced Drug Delivery Reviews 2014; 66:2-25. 225. Fay F, Scott CJ. Antibody-targeted nanoparticles for cancer therapy. Immunotherapy 2011; 3:381-94. 226. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nano 2007; 2:751-60. 227. Webster DM, Sundaram P, Byrne ME. Injectable nanomaterials for drug delivery: Carriers, targeting moieties, and therapeutics. Eur J Pharm Biopharm 2013; 84:1-20. 228. Kao CH, Wang JY, Chuang KH, Chuang CH, Cheng TC, Hsieh YC, Tseng YL, Chen BM, Roffler SR, Cheng TL. One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles. Biomaterials 2014; 35:9930-40. 229. Leserman LD, Barbet J, Kourilsky F, Weinstein JN. Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A. Nature 1980; 288:602-4. 230. Heath TD, Fraley RT, Papahadjopoulos D. Antibody targeting of liposomes - cell specificity obtained by conjugation of F(Aab')2 to vesicle surface. Science 1980; 210:539-41. 231. Felice B, Prabhakaran MP, Rodriguez AP, Ramakrishna S. Drug delivery vehicles on a nano-engineering perspective. Mater Sci Eng C-Mater Biol Appl 2014; 41:178-95. 232. Battaglia DP. Targeted Drug Delivery. 181-234; in Nanomaterials for medical applications. ed. Aguilar. Amsterdam: Elsevier Science Bv, 2013. 233. Ferrari M. Cancer nanotechnology: Opportunities and challenges. Nature Reviews Cancer 2005; 5:161-71

131 234. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery 2005; 4:145-60. 235. Haley B, Frenkel E. Nanoparticles for drug delivery in cancer treatment. Urol Oncol- Semin Orig Investig 2008; 26:57-64. 236. Goodall S, Howard CB, Jones ML, Munro T, Jia Z, Monteiro MJ, Mahler S. An EGFR Targeting Nanoparticle Self Assembled from a Thermoresponsive Polymer. Journal of Chemical Technology & Biotechnology 2014; DOI: 10.1002/jctb.4509. 237. Peer D, Gianneschi N, Luo D. Themed issue on nanoparticles in biology. J Mat Chem B 2013; 1:5174-6. 238. van der Meel R, Vehmeijer LJC, Kok RJ, Storm G, van Gaal EVB. Ligand-targeted particulate nanomedicines undergoing clinical evaluation: Current status. Advanced Drug Delivery Reviews 2013; 65:1284-98. 239. MacDiarmid JA, Brahmbhatt H. Minicells: Versatile vectors for targeted drug or si/shRNA cancer therapy. Current Opinion in Biotechnology 2011; 22:909-16. 240. Taylor K, Howard CB, Jones ML, Sedliarou I, MacDiarmid J, Brahmbhatt H, Munro TP, Mahler SM. Nanocell targeting using engineered bispecific antibodies. mAbs 2014; 7:53-65. 241. Kaitu'u-Lino TuJ, Pattison S, Ye L, Tuohey L, Sluka P, MacDiarmid J, Brahmbhatt H, Johns T, Horne AW, Brown J, et al. Targeted Nanoparticle Delivery of Doxorubicin Into Placental Tissues to Treat Ectopic Pregnancies. Endocrinology 2013; 154:911-9. 242. Metz S, Panke C, Haas AK, Schanzer J, Lau W, Croasdale R, Hoffmann E, Schneider B, Auer J, Gassner C, et al. Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing. Protein Engineering Design and Selection 2012; 25:571-80. 243. Schneider B, Grote M, John M, Haas A, Bramlage B, Lckenstein LM, Jahn- Hofmann K, Bauss F, Cheng WJ, Croasdale R, et al. Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies. Mol Ther- Nucl Acids 2012; 1:e46. 244. Girgis MD, Federman N, Rochefort MM, McCabe KE, Wu AM, Nagy JO, Denny C, Tomlinson JS. An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles. J Surg Res 2013; 185:45-55.

132 245. Solomon B, Desai J, Scott A, Rosenthal M, McArthur G, Rischin D, Segelov E, MacDiarmid J, Brambhatt H. 585 First-in-man, Multicenter, Phase I Trial Evaluating the Safety of First-in-class Therapeutic, EGFR-targeted, Paclitaxel-Packaged Minicells. European Journal of Cancer 2012; 48, Supplement 6:179. 246. Burden RE, Caswell J, Fay F, Scott CJ. Recent advances in the application of antibodies as therapeutics. Future Medicinal Chemistry 2012; 4:73-86. 247. Morrison SL. Cloning, Expression, and Modification of Antibody V Regions. Ch. 2; in. Current Protocols in Immunology. ed. Coligan et. al. John Wiley & Sons, Inc., 2001. 248. Xiao Y-H, Pei Y. Asymmetric Overlap Extension PCR Method for Site-Directed Mutagenesis. In: Park DJ, ed. PCR Protocols: Methods in Molecular Biology: Springer Science and Business Media, 2011:277-82. 249. Jones ML, Seldone T, Smede M, Linville A, Chin DY, Barnard R, Mahler SM, Munster D, Hart D, Gray PP, et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. Journal of Immunological Methods 2010; 354:85-90. 250. Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 2004; 22:1393-8. 251. Codamo J, Hou JJC, Hughes BS, Gray PP, Munro TP. Efficient mAb production in CHO cells incorporating PEI-mediated transfection, mild hypothermia and the co- expression of XBP-1. J Chem Technol Biotechnol 2011;86:923-34. 252. Yoo J-W, Irvine DJ, Discher DE, Mitragotri S. Bio-inspired, bioengineered and biomimetic drug delivery carriers. Nature Reviews Drug Discovery 2011; 10:521-35. 253. Perchiacca JM, Ladiwala ARA, Bhattacharya M, Tessier PM. Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions. Protein Engineering Design and Selection 2012; 25:591-602. 254. Teillaud J-L. From whole monoclonal antibodies to single domain antibodies: think small. Methods in molecular biology (Clifton, NJ) 2012; 911:3-13. 255. Pasquetto MV, Vecchia L, Covini D, Digilio R, Scotti C. Targeted Drug Delivery Using Immunoconjugates: Principles and Applications. Journal of Immunotherapy 2011; 34:611-28. 256. May C, Sapra P, Gerber H-P. Advances in bispecific biotherapeutics for the treatment of cancer. Biochem Pharmacol 2012; 84:1105-12.

133 257. Zhu JW. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 2012; 30:1158-70. 258. Ruigrok Vincent JB, Levisson M, Eppink Michel HM, Smidt H, van der Oost J. Alternative affinity tools: more attractive than antibodies? Biochemical Journal 2011; 436:1-13. 259. Zhu ZP, Presta LG, Zapata G, Carter P. Remodeling domain interfaces to enhance heterodimer formation. Protein Science 1997; 6:781-8. 260. Feige MJ, Groscurth S, Marcinowski M, Shimizu Y, Kessler H, Hendershot LM, Buchner J. An Unfolded C(H)1 Domain Controls the Assembly and Secretion of IgG Antibodies. Molecular Cell 2009; 34:569-79. 261. Kolhe P, Amend E, Singh SK. Impact of Freezing on pH of Buffered Solutions and Consequences for Monoclonal Antibody Aggregation. Biotechnol Prog 2010; 26:727- 33. 262. Sharma VK, Chih H, Mrsny RJ, Daugherty AL. The Formulation and Delivery of Monoclonal Antibodies. In: An Z, ed. Therapeutic Monoclonal Antibodies: From Bench to Clinic. New Jersey: John Wiley & Sons, Inc., 2009:675-709. 263. Baynes BM, Wang DIC, Trout BL. Role of Arginine in the Stabilization of Proteins against Aggregation†. Biochemistry 2005; 44:4919-25. 264. Roberts CJ, Nesta DP, Kim N. Effects of Temperature and Osmolytes on Competing Degradation Routes for an IgG1 Antibody. J Pharm Sci 2013; 102:3556-66. 265. Mendez MJ, Green LL, Corvalan JRF, Jia XC, MaynardCurrie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL, et al. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nature Genet 1997; 15:146-56. 266. Abou-Nassar K, Brown JR. Novel agents for the treatment of chronic lymphocytic leukemia. Clinical advances in hematology & oncology : H&O 2010; 8:886-95. 267. Lee CC, Perchiacca JM, Tessier PM. Toward aggregation-resistant antibodies by design. Trends in Biotechnology 2013; 31:612-20. 268. Serda RE, Godin B, Blanco E, Chiappini C, Ferrari M. Multi-stage delivery nano- particle systems for therapeutic applications. Biochim Biophys Acta-Gen Subj 2011; 1810:317-29. 269. Greco F, Vicent MJ. Combination therapy: Opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Advanced Drug Delivery Reviews 2009; 61:1203-13.

134 270. Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress. Journal of Controlled Release 2012; 161:175-87. 271. Geers B, De Wever O, Demeester J, Bracke M, De Smedt SC, Lentacker I. Targeted Liposome-Loaded Microbubbles for Cell-Specific Ultrasound-Triggered Drug Delivery. Small 2013; 9:4027-35. 272. Karagiannis ED, Anderson DG. Minicells overcome tumor drug-resistance. Nat Biotech 2009; 27:620-1. 273. Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D, Jain RK. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Research 1994; 54:3352-6.

135 7. Appendices

7.1. mAbs paper published online December 2014 REPORT mAbs 7:1, 53--65; January/February 2015; Published with license by Taylor & Francis Group, LLC

Nanocell targeting using engineered bispecific antibodies Karin Taylor1,*, Christopher B Howard1, Martina L Jones1, Ilya Sedliarou2, Jennifer MacDiarmid2, 1 1,3 Himanshu Brahmbhatt2, Trent P Munro , and Stephen M Mahler

1Australian Institute for Bioengineering and Nanotechnology (AIBN); University of Queensland, St Lucia; Queensland, Australia; 2EnGeneIC Ltd; New South Wales, Australia; 3School of Chemical Engineering; University of Queensland, St Lucia; Queensland, Australia Keywords: bispecific antibody, disulfide bridge, mammalian expression, nanoparticle, single chain Fv, surface plasmon resonance, tumor regression Abbreviations: BsAb, bispecific antibody; EDVTM, EnGeneIC Delivery Vehicle; scFv, single chain variable fragment; EGFR, epidermal growth factor receptor; LPS, lipopolysaccharide; IgG, ; mAb, monoclonal antibody; NP, nanoparticle

There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or TM TM EDV nanocell) to the epidermal growth factor receptor (EGFR). EDV nanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. TM However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV nanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and TM expression yield. Doxorubicin-loaded EDV nanocells actively targeted with tandem scFv BsAb in vivo to MDA- MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively TM TM targeted EDV nanocells. BsAbs therefore provide a functional means to deliver EDV nanocells to target cells.

Introduction The most prominent therapeutic utility for BsAbs in cancer thera- peutics is the cross-linking of cell-surface antigens or receptors, so Therapeutic monoclonal antibodies (mAbs) approved for that immune cells can be tethered to cancer cells through a bispe- Ò clin-ical use, such as (Avastin ), adalimumab 9,11,12,16,17 Ò Ò Ò cific antibody. Catumaxomab (Removab) was (Humira ), trastuzumab (Herceptin ), cetuximab (Erbitux ) approved for therapeutic use in Europe in 2009, and remains the Ò and panitumumab (Vectibix ), comprise a significant portion only approved therapeutic BsAb, although the number of BsAb 1,2 of the global pharmaceutical market. Of the total mAb sales formats entering clinical trials is increasing steadily.7, 13 in 2010, half were attributed to cancer-related therapies.3 In Catumaxomab is a T-cell engager, with specificity for both CD3 2012, half of the antibody-based oncology products under on cytotoxic T cells and the EpCAM antigen on ovarian cancer clinical evaluation were full-length IgG mAbs, while the 18-20 cells, for the treatment of malignant ascites. Another remaining candidates included drug-conjugated or radio-labeled emerging application of BsAbs is active targeting of drug-loaded mAbs, protein- and glyco-engineered mAbs and fragment or nanoparticles (NP) to tumor sites by cross-linking the NP to domain antibodies.4–6 Also under evaluation are bispecific tumor cells, leading to endocytosis, fusion with lysosomes and 7 antibodies (BsAbs) , which are subject to increasing interest in drug release intracellularly.1,11,21,22 recent years owing to their ability to target multiple The concept of NP-mediated drug delivery is attractive as it 8-11 antigens. can lead to improvement in drug safety and efficacy. A myriad BsAbs are able to crosslink antigenic determinants and so have of engineered NPs for drug delivery have been developed, and 1,11-15 value beyond that of single antigen-specific mAbs (Fig.1). include liposomes, polymeric-based NPs, silica, carbon, metal © Karin Taylor, Christopher B Howard, Martina L Jones, Ilya Sedliarou, Jennifer MacDiarmid, Himanshu Brahmbhatt, Trent P Munro, and Stephen M Mahler * Correspondence to: Karin Taylor; Email: [email protected]

Submitted: 09/29/2014; Revised: 10/29/2014; Accepted: 11/04/2014 http://dx.doi.org/10.4161/19420862.2014.985952

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by- nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. www.tandfonline.com mAbs 53 136 alternatively be tethered to the NPs by using BsAbs that simulta- neously bind the NP and the tumor target antigen.37, 38 BsAbs can be produced by chemical conjugation of 2 mAbs or frag- ments with different specificities (e.g., via bis-maleimide cross- linking through Thiomab technology)39 or alternatively via an affinity ligand such as Protein A or Protein G.21 However, these methods can be associated with antibody inactivation, low yields and product heterogeneity; for example, yields of 10 - 40% dimeric BsAb from hetero-bifunctional reagents and 65 - 75% from homo-bifunctional reagents.39 These methods rely on het- erodimeric association of 2 mAbs. However homodimers can also be produced, limiting the yield of functional product.40, 41 Another method to construct BsAbs is to combine binding moieties such as scFvs or single domain antibodies into a single recombinant polypeptide chain using linkers such as the flexible glycine-serine (G4S) motif9,10,17 or Fc (Hinge-CH2-CH3) domain. Improved scFv stability can be achieved through engi- neering additional cysteine residues within framework regions.41 The modular design of recombinant BsAbs has resulted in the creation of a variety of novel BsAbs.14 Formats range from non- IgG like varieties containing chemical or protein linkers, through to recombinant Fc-containing, Dock-and-lock and knobs-into- holes (KIH) BsAbs.42 Some of the more well-known formats include Triomabs, dual affinity retargeting (DART) molecules, 2TM Figure 1. Applications of bispecific antibodies. Therapeutic modalities bispecific T-cell engagers (BiTEs), and the F-star mAb 9,20,43-45 include: (A) crosslinking separate antigens on the cell surface; (B) T cell BsAbs. Design features may endow the BsAb with spe- engagement by cross-linking CD3 on cytotoxic T cells to tumor cells; (C) cific properties, such as enhanced serum half-life or complement targeting drugs or radiolabels to cell surface; (D) targeted delivery of activation, by fusion with PEG, BSA or Fc domains.46, 47 drug-loaded nanoparticles to tumor cells (image not to scale). In this study, we designed several BsAbs based on 3 basic design formats for active targeting of the EnGeneIC delivery oxides and other materials. Some are also derived from bacteria, vehicle (EDVTMnanocell) to the epidermal growth factor recep- viruses (e.g., virus-like particles) and eukaryotic cells (exosomes). tor (EGFR), to replace the currently used protein A/G linked 23-30 Advantages of NP-mediated drug delivery include protec- mAbs.48-50 The EDVTMnanocell (a 400 nm particle of bacterial tion of payloads such as DNA and RNA from degradation, origin capable of carrying a drug payload) was originally targeted improved drug pharmacokinetics and the enabling of effective using a BsAb moiety produced by incubating 2 different mAbs 2, 31 drug dosing where drugs are insoluble. Notwithstanding the with protein A/G (Pierce, USA).21 Although functional BsAbs large number of NPs that have been developed for therapeutic capable of delivering the EDVTMnanocell were prepared, it was application, at present there are only 7 nanomedicines approved noted that multimer formation and the inability to control the for cancer therapy. All these nanomedicines operate through pas- ratio of mAbs bound to the protein A/G, which has 6 potential sive targeting, i.e., enhanced extravasation of NPs at the tumor Fc-binding sites, rendered the targeting entity unsuitable for site, facilitated by poorly differentiated tumor vasculature. There commercial manufacture and downstream clinical trials.21, 32 are a number of nanomedicines presently in clinical trials, with Recombinant BsAbs offer an advantage of producing one anti- 32, 33 several of these targeted by antibodies. body with dual target binding capability. NPs can carry drug payloads that include small molecule drugs, The EDVTMnanocell is coated with lipopolysaccharide (LPS),21 2,30,34 DNA or RNA. Accumulation of the NPs at the tumor site andthusBsAbdesignsincorporatedscFvsderivedfromananti-LPS by passive targeting decreases biodistribution, ideally localizing the antibody (1H10) with the anti-EGFR antibody ABX-EGF.51-53 35, 36 therapeutic effects of the drug payload at the tumor site(s). ABX-EGF (panitumumab) was developed via transgenic humanized Delivery of drugs sequestered within or attached to NPs opens the mouse technology by Abgenix Inc.,51-53 and is approved for clinical drug therapeutic window and addresses the low therapeutic index use in the treatment of colorectal cancer. ABX-EGF has picomolar of free-drug administration, i.e., systemic drug delivery at required affinity for EGFR, and its properties, including affinity, specificity therapeutic doses results in acute toxicity and can elicit severe and immunogenicity, have been extensively evaluated and is an ideal patient side effects. Furthermore, multiple drug resistance (MDR) model antibody to incorporate into the design and testing of bispe- can be alleviated by packaging NPs with siRNA or shRNA capable cific formats.21 Three BsAb formats were evaluated for EDVTMna- 22, 31 of interfering with cellular mechanisms promoting MDR. nocell targeting; a G4S-linked tandem scFv format, homodimeric For active targeting of NPs to tumor cells with antibodies, Fc-containing BsAbs and KIH-engineered formats to promote anti-tumor mAbs can be chemically conjugated to the NP, or forced heterodimerisation of the EDV-targeting variable fragments.

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137 Table 1. BsAb format and sequence design parameters. (A) Tandem scFv, (B) Fc-containing and (C) Knobs-into-Holes (KIH) BsAbs. (A) The tandem scFv, incorporated a G4S linker as well as N-terminal His and C-terminal myc tags. Homodimeric Fc-containing variants (B) incorporated an IgG1-Fc linker; variants of (B) included either G4S or a longer (Longlink) linker to connect 1H10 scFvs to the CH3 domain. Variants also included or excluded engineered cysteine residues in scFvs for enhanced stability. KIH constructs (C) are heterodimers with 1H10 heavy and light variable regions on separate chains. Format design included a longer (Longlink) linker connecting CH3 to 1H10 variable fragments and again either included or excluded engineered cysteine residues in scFvs

CH3-aEDV Linker Additional Structural BsAb ID Sequence information disulfide bridges G4S Longlink Illustration

A Tandem scFv His-(ABX-EGF scFv)-G4S-(1H10 scFv)-myc NA NA

B Fc-containing (ABX-EGF scFv)-G4S-Fc-G4S-(1H10 scFv)

Fc-containing (ABX-EGF scFv)-G4S-Fc-Longlink-(1H10 (Longlink) scFv)

Fc-containing (ABX-EGF scFv) (cys)-G4S-Fc-G4S-(1H10 (Cys) scFv) (cys)

Fc-containing (ABX-EGF scFv) (cys)-G4S-Fc-Longlink-(1H10 (Cys Longlink) scFv) (cys)

C KIH 1 (Cys) (ABX-EGF scFv) (cys)-G4S-Fc(Knob)- Longlink-VH(1H10)(cys) C

(ABX-EGF scFv) (cys)-G4S-Fc(Hole)-Longlink- VL(1H10)(cys) KIH 2 (ABX-EGF scFv)-G4S-Fc(Knob)-Longlink-VH (1H10) C (ABX-EGF scFv)-G4S-Fc(Hole)-Longlink-VL (1H10)

Additionally, modifications of the latter 2 formats were investigated, increased 2-3 fold compared to that of the tandem scFv BsAb. incorporating additional disulfide bridges in the scFv domains and Engineering disulfide bridges into Fc-containing and KIH vari- longer linkers between the Fc-CH3 and anti-LPS scFv interface ant scFvs dramatically decreased BsAb yields 6-9 fold compared (Table 1). We evaluated these different formats with regard to to those of non-stabilised formats. A qualitative Western blot expression yields, their ability to bind respective targets and their confirmed that BsAbs were efficiently secreted and that the lower effect on EDVTMnanocell clumping. The tandem scFv design was product yields were not due to retention in the endoplasmic further evaluated to show its ability to target the EDVTMnanocell to reticulum via cellular mechanisms such as the unfolded protein MDA-MB-468 breast cancer cells overexpressing EGFR in vitro, response (results not shown). 54-57 and to target the drug-loaded EDVTMnanocell to mouse xenografts in vivo, resulting in tumor regression. Targeted binding Binding activity was maintained for both EGFR and LPS tar- gets for all engineered BsAb formats (Table 2; Fig. 2). Surface Results plasmon resonance (SPR) analysis of recombinant EGFR binding using Biacore T-200 (GE) showed that BsAb association con- Production and yield stants (ka) were 5 to 10-fold lower than that of the published Various BsAbs of different designs were expressed in Chinese ABX-EGF (using purified native EGFR).58, 59 Dissociation con- hamster ovary (CHO)-S transient cell culture and purified from stants (kd) of the ABX-EGF-scFvs incorporated within the tan- culture supernatant. We observed that BsAb yields from transient dem scFv, Fc-containing and Fc-containing (Longlink) BsAbs CHO-S cultures were lower compared to that of full length, have values similar to the published kd for ABX-EGF-IgG2 mAb, ABX-EGF-IgG1 mAb (52.6 mg/L); tandem scFv BsAb expres- whereas all remaining EGFR-targeting formats have kd values up sion routinely yielded around 5 mg/L in transient cell culture to 10-fold slower. A control, non-specific tandem scFv BsAb tar- supernatant, whereas yields of Fc-containing and KIH BsAbs geting RSV and LPS, showed no binding to EGFR.

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138 Table 2. Surface plasmon resonance binding affinities for recombinant EGFR-mFc collected from a Biacore T-200. Surface Plasmon Resonance (Biacore) anal- ysis of BsAb binding to EGFR – ka (association constant), kd (dissociation constant), and KD (binding affinity). High performance (Multi-cycle) Kinetic Assay (HPKA) data is shown for all BsAb constructs as well as for a reassembled, complete IgG1 ABX-EGF mAb. Standard error values corresponding to binding val- 6 ¡5 ues of recombinant EGFR is shown as § x £ 10 for ka and § y £ 10 for kd

Binding to recombinant EGFR

6 –5 Antibody ID ka (1/Ms) (£10 )(§SE ) kd (1/s) (£10 )(§SE ) KD (nM)

ABX-EGF IgG2 mAb (published) 1.97 11.3 0.05 ABX-EGF IgG1 mAb (reformatted) 2.18 § 0.0025 15.5 § 0.047 0.07 Non-specific Tandem scFv No binding No binding No binding Tandem scFv 0.26 § 0.0004 13.6 § 0.021 0.52 Fc-containing 0.36 § 0.0023 9.42 § 0.028 0.26 Fc-containing (Longlink) 0.19 § 0.0005 21.6 § 0.084 1.13 Fc-containing (Cys) 0.48 § 0.0002 4.89 § 0.019 0.10 Fc-containing (Cys Longlink) 0.70 § 0.0004 2.47 § 0.025 0.04 KIH 1 (Cys) 0.82 § 0.0005 2.67 § 0.026 0.03 KIH 2 0.50 § 0.0021 6.75 § 0.021 0.13

Reformatting the ABX-EGF-scFv to an IgG1 mAb resulted in BsAb showed greater binding affinity than that of the KIH 2 similar ka, kd and binding affinity (KD) values as those reported BsAb. Although the reasons for the effect of cysteine stabilization for the published ABX-EGF IgG2 mAb (Fig. 2). KD for the of scFvs within a specific BsAb format were not investigated, it is EGFR-targeting BsAbs were all in the nanomolar (nM) range. hypothesized that the additional disulfide bonds offer a further The Fc-containing (Longlink) BsAb showed a 4 to 5-fold degree of stabilization and rigidity within the scFv binding enti- decrease in KD compared to that of the Fc-containing BsAb. ties, which enhances binding affinity. Flexibility imparted by the long linker may have a destabilizing Binding of all BsAbs to LPS was confirmed by ELISA (results effect on the scFvs within the BsAb. Interestingly, cysteine stabili- not shown). As LPS is naturally adhesive and binds strongly to zation restores the high affinity of Fc-containing (Longlink) BsAb surfaces (e.g., glass, plastic), performing binding studies by SPR for EGFR (0.04 nM). Similarly, cysteine-stabilised KIH 1 (Cys) was not compatible with the microfluidics system of the Biacore. The kinetics of 1H10-scFv component of the BsAbs binding to LPS were therefore determined by the Octet system (Forte- Bio), which circumvents the problems of a fluidics-based system, and provided bind- ing data for the original, hybridoma- derived 1H10-mAb [KD D 0.2 nM, ka D 8.20EC05 (§1 .25EC04), kd D 1.36E-04 (§1 .72E-05)] and 1H10-scFv of the tan- dem scFv format [KD D 10.0 nM, ka D 2.20EC04 (§1 .76EC03), kd D 5.64E-04 (§2.39E-05)] (Fig. 2C). The 60-fold dif- ference in KD between the 2 formats is indicative of differences in avidity. Flow cytometry analyses showed that all EGFR-targeting formats (mAb and BsAb) were able to bind to EGFR-over- expressing MDA-MB-468 cells, while non-specific formats showed no binding (Fig. 3A-C). A decreased shift for the tandem scFv BsAb compared to the Fc- linked formats can be attributed to the higher avidity of the EGFR-bivalent, Fc- Figure 2. ABX-EGF and 1H10 representative binding curves. SPR sensorgrams illustrative of (A) ABX- EGF tandem scFv (1 nM, 3 nM, 10 nM, 30 nM and 100 nM BsAb) and (B) ABX-EGF-IgG1 mAb (1 nM, 3 linked BsAbs. The lower fluorescence nM, 10 nM and 30 nM BsAb) binding to immobilised recombinant EGFR. (C) Biolayer interferometry intensity of the tandem scFv could also kinetic curve of ABX-EGF tandem scFv the 1H10-mAb and ABX-EGF IgG1 mAb at 100 nM binding to be due to the different secondary anti- captured LPS. body used for detection.

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139 Figure 3. In vitro binding of IgG1 mAbs, BsAbs and BsAb-EDVs to EGFR overexpressing MDA-MB-468 breast cancer cells. All specific BsAbs include ABX- EGF-scFvs (anti-EGFR), non-specific formats include a palivizumab-scFv (anti-RSV) and all BsAbs include the 1H10 (anti-LPS) scFv. (A) Whole mAbs, non- specific (anti-RSV) and ABX-EGF-IgG1 (anti-EGFR) binding to cells detected by APC-conjugated anti-human IgG; (B) Tandem scFv BsAbs (anti-RSV and anti-EGFR) binding to cells detected by FITC-conjugated anti-c-myc; (C) Various Fc-containing BsAbs binding to cells detected by APC-conjugated anti- human IgG; and (D) AlexaFluor 488 labeled EDVTMnanocells (EDVs) binding to cells via various bound BsAbs.

It was of interest to investigate the ability of BsAbs to crosslink 468 cell surfaces nor did non-specific EDVTMnanocells. Confo- the 2 antigens LPS and EGFR. A co-localization experiment cal microscopy (Fig. 5) and flow cytometry analyses (Fig. 3D) using fluorescently labeled recombinant-EGFR (DL650) and showed that while all BsAbs localized on the MDA-MB-468 cell BsAb-targeted EDVTMnanocells (AF488) provided visual confir- surface, the tandem scFv and KIH 1 (Cys) BsAb formats, possess- mation that BsAbs could simultaneously bind their 2 targets ing only one 1H10-scFv, displayed a lower distribution across the (Fig. 4). Results show that non-targeted and non-specifically tar- cell surface compared to the distributions of the Fc-containing geted EDVTMnanocells do not co-localize in the presence of (Cys) and Fc-containing (CysLonglink) targeted EDVTMnano- recombinant EGFR (green fluorescence only). We included all 7 cells, with 2 1H10-scFvs. of the BsAb formats, and observed their effect on nanocell clumping. The ABX-EGF tandem scFv, KIH 1 (Cys), Fc-con- taining (Cys) and Fc-containing (CysLonglink) BsAbs caused Stability less EDVTMnanocell aggregation, as illustrated by a uniform dis- tribution of co-localized EGFR and targeted EDVTMnanocells, Various buffer formulations were tested to improve the stor- whereas nanocells targeted with KIH 2, Fc-containing and Fc- age stability of the BsAbs. Formulations tested thus far have not containing (Longlink) BsAbs produced large, aggregated clumps shown significantly improved stability of Fc-containing and KIH of co-localized recombinant EGFR and EDVTMnanocells. This variants, and there is evidence of concentration-dependent aggre- illustrates how different recombinant BsAbs affect the EDVTM- gation above 0.1 mg/ml (results not shown). Size exclusion chro- nanocells during and post targeting, and the final product stabil- matography (SEC) HPLC analyses over a 1 month period ity. These findings highlight how BsAb design and properties in indicated that tandem scFv stability, when stored at a concentra- solution may influence the performance of the BsAb-EDVTMna- tion of 0.12 mg/ml, is affected by buffer formulation. Product nocell formulated product. storage in a formulation of PBS/trehalose resulted in slightly The BsAbs that resulted in a uniform distribution in the co- increased aggregation levels when stored at -20C compared with localization study were further investigated by binding to cells 4C. However, stability was maintained at both 4C and -20C using flow cytometry (Fig. 3D) and confocal microscopy in a HEPES/Trehalose buffer formulation (Fig. 6A). Dynamic (Fig. 5). For confocal microscopy, MDA-MB-468 cells (labeled light scattering (DLS) data confirmed the tandem scFv to be a with AF647) were targeted with AF488-labeled BsAb-EDVs. monomer of 55 kDa in size, with a hydrodynamic radius of Non-targeted EDVTMnanocells did not localize on MDA-MB- 2.5 nm (results not shown). Thermostability determined by

www.tandfonline.com mAbs 57 140 Figure 4. Co-localization imaging by confocal microscopy of recombinant EGFR-His (DL650) and EDVTMnanocells (AF488) with or without specific BsAb. Recombinant EGFR was co-incubated with EnGeneIC Delivery Vehicles (EDVTMnanocells or EDVs) pre-targeted with various BsAbs prior to microscopy. EDVTMnanocells are represented in green (AF488) while DyLight650-labeled EGFR-His is represented in purple (color substitution from red has been applied to improve co-localization imaging). Overlap of the 2 fluorophores (co-localization) results in near-white imaging. Panels represent the filter showing EGFR (DL650) alone, EDV (AF488) alone and the merged overlay image of the 2.

Protein A/G differential scanning calorimetry (DSC) indicated that the tan- protein A/G cross-linked BsAbs ( EDV Dox). Impor- dem scFv has a melting temperature (Tm) of 57C(Fig. 6B). tantly, active targeting of doxorubicin-loaded EDVTMnanocells Additionally, co-incubating the tandem scFv with human serum by the tandem scFv BsAb resulted in a 40% reduction in tumor (HS) over a 48-hour period showed no protease-mediated degra- volume compared to that of loaded passively targeted EDVTMna- 3 dation of the BsAb (results not shown). nocells (EDV Dox) 38 d post xenograft (i.e., 140 mm and 240 mm3, respectively). Targeted EDVTMnanocells not loaded Tandem scFv EDVTMnanocell-mediated tumor regression in a mouse with doxorubicin ( EDV) had a minor tumor suppress- TM xenograft model ing effect, whereas non-targeted, doxorubicin-loaded EDV na- Due to the superior properties of the tandem scFv BsAb, it nocells (EDV Dox) showed significant tumor regression was the only recombinant BsAb format tested in a mouse xeno- compared to saline controls; the latter being most likely due to graft model for tumor regression. To evaluate the efficacy passive targeting through leaky tumor vasculature of tumor xeno- 32,60,61 of EDVTMnanocells targeted using the ABX-EGF tandem scFv grafts (pore sizes 0.2–1.2 mm). (Tandem scFv EDV) compared to those targeted with EnGeneIC’s original, Protein A cross-linked anti-LPS / anti-EGFR BsAb (Protein A/G EDV),21 in vivo experiments were carried out utilizing Discussion a MDA-MB-468 mouse xenograft model, with dosing com- mencing 14 d after subcutaneous injection of MDA-MB-468 A variety of BsAb formats were investigated for their ability to cells (tumor volume approx. 80–100 mm3)(Fig. 7). EDVTMna- bind EDVTMnanocells and recombinant or native EGFR. This nocells targeted with the tandem scFv and loaded with doxorubi- initial proof-of-principle study was performed using BsAbs con- Tandem scFv cin ( EDV Dox) effectively suppressed tumor growth taining the scFv binding sequence derived from ABX-EGF (pani- over a 38 day period post xenograft compared to controls. Active tumumab), a rigorously tested, approved therapeutic antibody, Tandem scFv targeting with EDV Dox showed tumor regression was which has ideal properties such as high affinity and stability. similar to that achieved with EDVTMnanocells targeted by ABX-EGF targets EGFR, which is commonly over-expressed on

58 mAbs Volume 7 Issue 1 141 Figure 5. Confocal in vitro imaging of stable BsAb-EDVTMnanocells binding to the surface of MDA-MB-468 breast cancer cells. MDA-MB-468 cells were labeled with anti-EGFR-AF647 (red) to visualize the cell surface while BsAb-EDVs were labeled with AF488 (green). After 3 hours of incubating labeled BsAb-EDVs with EGFR overexpressing MDA-MB-468 cells, the samples were washed, fixed and labeled with the AF647. Cover slips were mounted and cells visualised on a Confocal LSM Zeiss 710 using a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Analyses were performed using ZEN 2008 software. Scale bars in white indicate a length of 10 mm.

a variety of cancer cells and the target for several marketed anti- To produce more complex BsAbs compared to the tandem cancer therapeutics.3,7,50,62 The modular nature of BsAb con- scFv BsAb, manipulation of the standard IgG mAb format has structs allows alternative antibody specificities to be substituted produced a plethora of Fc-containing BsAbs, whereby designs depending on the receptor target. generally rely on the principle that Fc-containing BsAbs will The tandem scFv BsAb was designed to include a 5 amino dimerize through the Fc, similar to native, whole acid glycine-serine linker (G4S) between the ABX-EGF- and mAbs.11,46,47,63,64 Features of these antibodies include engineer- 1H10-scFv. The shorter linker reduces flexibility and therefore ing Fc components to improve or remove various related func- prevents misfolding from occurring between the 2 scFv compo- tions, forced heterodimerisation, removal of redundant domains nents. Administered in isolation, tandem scFv BsAbs exhibit low and addition of other binding motifs to the IgG mole- avidity and, due to their small size, a low serum half-life;46, 47 cule.47,63,65-67 We used an IgG1 Fc-domain to link and separate however, pharmacokinetic properties of tandem scFv BsAbs the 2 scFvs spatially (ABX-EGF scFv at the N-terminus of bound to the large EDVTMnanocell (400 nm in diameter) will human IgG1 Fc domain, and an 1H10 scFv at the C-terminus) be enhanced. Furthermore, coating of the EDVTMnanocell with to create the ABX-EGF-Fc-1H10 homodimeric Fc-containing tandem scFv BsAbs imparts multivalency and avidity to the NP, BsAb, similar to the scFv-Fc-scFv BsAb dimer described in Jen- and provides multiple binding sites for the cancer-targeting dreyko et al. and the Emergent BiosolutionsTM product termed domain.21 ADAPTIRTM Multi-Specific.68, 69 A benefit of the Fc-containing

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142 BsAbs over the tandem scFv format is the presence of 2 scFvs to bind each target – promoting high avidity binding to both the EDVTMnanocell and the EGFR on the cancer cell surface; whereas a potential drawback could result if the Fc-domain stim- ulates an unwanted immune response and complement cas- cade.20,31 Although it is well known that Fc-related immune responses can be avoided through various mutations 67 through- out the domain, our work thus far has focused on the production of a stable drug delivery system rather than on potential immu- nogenicity. EnGeneIC’s original BsAb format also retained fully functional Fc domains without significant immunogenicity being noted.21,22,31 Previous studies have found that inclusion of a number of design modifications, including variations in CH3-scFv linker length and type, and also scFv stability engineering via addition of disulfide bonds, improved construct stability. 70-75 A concern with the Fc-linked dimeric design was whether the presence of 2 anti-LPS scFvs would result in EDVTMnanocell aggregation via binding of multiple nanocells rather than high avidity binding of a single nanocell. An alternative Fc-linked BsAb was engineered consisting of 2 EGFR targeting scFvs at the N-terminus (high avidity) and a single anti-LPS scFv at the C-terminus of the final product. Heterodimeric BsAb formats were based on the com- mon KIH constructs65, 75 and partly on the TandAb BsAb designed by Affimed where variable linker lengths are used to determine chain association for functional BsAb formation.76, 77 Figure 6. Tandem scFv stability data. (A) Tandem scFv samples were The single anti-LPS site is formed through forced heterodimer- stored in various buffers at different temperatures to evaluate stability isation of the CH3 domain via KIH engineering, where the VH during storage over a 3 month period. The representative trace is indica- and VK domains of the scFv are expressed on separate polypepti- tive of stable product stored in Hepes/trehalose buffer for 3 months. (B) des in a single cell. To ensure that the VH- and VK-chains associ- Thermostability of tandem scFv BsAb in PBS was determined by differen- tial scanning calorimetry and indicates unfolding and subsequent aggre- ate with one another to form a functional 1H10-scFv, a longer gation of the tandem scFv BsAb at 57C. linker between CH3 and the 1H10-scFv component was designed to allow extra flexibility in the area for functional associ- ation of the nanocell-targeting scFv components.75 Based on co-localization images of the various BsAb formats binding to EDVTMnanocells and to EGFR, 4 of the BsAbs are of particular interest for further development: tandem scFv, Fc-con- taining (Cys), Fc-containing (Cys Longlink) and KIH (Cys). The other formats resulted in clumping of the EDVs. However, as the expression yields of the disulfide-engineered, Fc-linked BsAbs were 6-9 fold lower than the tandem scFv, the tandem scFv was chosen for further investigation. Tumor regression in MDA-MB-468 breast cancer cell xeno- grafts showed that BsAb-mediated targeting with anti-EGFR antibody is essential for optimal inhibition of tumor growth. Although EDVTMnanocells have previously been successfully tar- geted by cross-linking 2 IgG antibodies with protein A, utilizing the tandem scFv, whereby one arm binds the EDVTMnanocell and the other targets the tumor, requires the production of a new molecular entity (NME). A biologic NME can be characterized Figure 7. Mouse MDA-MB-468 xenograft results following EDVTMnano- for binding activity and manufactured according to cGMP. The cell (EDV) treatment. Tandem scFv refers to the EGFR targeting ABX-EGF TM BsAb format in the BsAb-EDVTMnanocell configuration whereas protein coating of BsAb on the surface of the EDV nanocell also A/G refers to EnGeneIC’s original BsAb format composed of anti-EGFR imparts other properties to the nanocell. Depending on BsAb and 1H10 mAbs connected through a protein A/G molecule. Dox refers density on the nanocell surface, the imparted avidity would to the chemotherapeutic agent doxorubicin. At various time points, indi- enhance apparent affinity of the EDVTMnanocell for its target, cated with a blue triangle, mice were treated with 1£109 EDVs. compared to that of the target scFv alone. Furthermore, coating

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143 with a BsAb, where the targeting scFvs are of human origin, alters Mammalian expression and purification the physicochemical properties of the EDVTMnanocell in vivo, Transient expression of all BsAbs was performed using PEI- and would affect parameters such as pharmacokinetics and mediated transfection of suspension adapted CHO cells as previ- immunogenicity. ously described.82 KIH chains were co-transfected at a 1:1 DNA Nanomedicine is poised to play an increasingly important role ratio (1H10-Vh : 1H10-Vk). Culture supernatants were har- in cancer therapy in the future, with at present 7 passively tar- vested 7 - 10 d post-transfection by centrifugation, then filtered geted nanomedicines approved for cancer therapy.32, 33 Actively and stored frozen at -80C until purification could be completed. targeting nanoparticles to tumors with antibodies, as demon- For the tandem scFv purification, 2 sequential IMAC chroma- strated in this study, can have significant advantages and improve tography steps were utilised, first with HisTrap Excel (GE efficacy compared to passive targeting alone. Designing new Healthcare), which tolerates nickel-chelating agents present in BsAbs that are able to efficiently target nanoparticles opens a new mammalian cell culture media, followed by HisTrap FF (GE dimension in active nanoparticle targeting. Healthcare), which further removes impurities. Manufacturer- recommended buffers were used for equilibration and loading, with 500 mM Imidazole added for elution. The eluted product was buffer exchanged into PBS using a HiPrep desalting column after each IMAC step. Tandem scFv BsAb monomer was isolated using a GE gel filtration column (HiPrep 26/60 Sephacryl S-200 Materials and Methods HR) and the monomer subsequently used for kinetic experiments and long-term stability studies. Sequence design For constructs containing an Fc domain, purification was per- The DNA sequence of the extracellular domain (amino acid formed in one step using a MabSelect Sure Protein A HP col- residues 25 - 645) of wild type human EGFR (Swiss-Prot umn. Following purification the BsAbs were desalted using the P00533) was synthesized by Geneart, incorporating a mamma- HiPrep desalting column into Dulbecco’s PBS buffer for storage lian leader sequence from IgK and a C-terminal His tag, with at 4C unless specified otherwise. codon optimisation for expression in CHO cells. Further con- structs consisting of amino acid residues 25 - 525 followed by Dynamic Light Scattering either human or mouse Fc tags were created, similar to that Analysis was completed by CSL, Melbourne, Australia. Molar described by Adams, 2009.78 The heavy and light chain variable mass and hydrodynamic radius (Rh) was determined using SEC- region sequences of the anti-LPS monoclonal antibody were MALS. A Wyatt WTC-030-N5 4.6 column was used with buffer determined by isolating and sequencing cDNA isolated from the composed of 100 mM phosphate/200 mM sodium chloride, pH hybridoma 1H10, using established protocols.79 The sequence 6.8. The column was equilibrated in the buffer and flow rate set information for the heavy and light chain variable regions for to 0.2 ml/min with 100 ml sample injections. A DAWN Heleos panitumumab (ABX-EGF) was obtained from US Patent 6 235 MALS detector was normalized using BSA and used in series 883. The sequence for an unrelated control antibody (anti-RSV) with an Agilent 1200 series UV diode array detector and an Opti- palivizumab (SynagisÒ), was obtained from the RCSB Protein lab T-rEx RI detector. Wyatt’s Astra VI software was used to cal- Data Bank file, 2 HWZ. culate the weight-averaged molar mass and hydrodynamic radius. The DNA templates for all BsAb formats were synthesized by Geneart with codon optimisation for CHO expression, Analysis of tandem scFv by differential scanning calorimetry then cloned into the mammalian expression vector pcDNA3.1 The tandem scFv BsAb was concentrated to 1 mg/ml in PBS (Invitrogen). Cysteine stabilization substitutions were made at using membrane based centrifugal concentrators with nominal Kabat position VH44 and VL100 of the scFvs.72, 73 Variants molecular weight cutoff of 10 Kda (Millipore). 500 ml of 1 mg/ included either a short (G4S) linker or a longer linker ml tandem scFv was vacuum degassed and loaded into the sample (SSDKTHTSPPSPGGGGSGGGGSGGGGSGGGG), as des- chamber of the VP differential scanning calorimeter (VP-DSC, cribed in Moore et al., connecting the CH3 domain to the Microcal). The same volume of PBS was loaded into the refer- 1H10-scFv.75 A “knob” was produced by amino acid substitu- ence chamber. The sample and reference chambers were scanned tion for a longer side chain (T366’W), whereas a "hole" was pro- once from 25Cto90C for 1 h to determine the melting profile duced by amino acid substitutions for shorter side chains of the tandem scFv. The data was analyzed using Origin 7.0 (T366’S:L368’A:Y407’V); the sequence positions are as indicated software and graphed as time (min) (x axis) versus heat capacity by Kabat et al.80 The tandem scFv included 6xHis and c-myc (Cp) (y axis). tags for purification and detection, while the other constructs uti- lised the Fc domain for purification and detection. For the KIH Size exclusion chromatography HPLC constructs, 2 DNA templates were required, one containing the A mobile phase of 0.1 M sodium phosphate buffer, 0.2 M 1H10 Vh region and one containing the 1H10 Vk region. Whole sodium chloride, pH 6.8 was prepared and filtered using a mAbs for panitumumab (ABX-EGF) and palivizumab were cre- 0.22 mm filter. A gel filtration standard was included on each ated by reformatting the BsAb sequences using a previously run of the HPLC to calculate molecular weights by their reten- described method.81 tion times. Samples were not concentrated prior to performing

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144 SEC so as to avoid the potential that concentration-based aggre- used as the secondary antibody for detecting binding shifts when gation may occur. The guard column and SEC-HPLC column the ABX-EGF tandem scFv and non-specific tandem scFv were were connected to the HPLC system, flushed and equilibrated in incubated with MDA-MB-468 breast cancer cells; whereas APC- m mobile phase. 100 l sample injections were used in all cases conjugated F(ab)’2 fragment goat anti-human IgG (Jackson with a flow rate of 0.8 ml/min. Columns were stored in 20% eth- ImmunoResearch) was used for detection of all mAb, Fc-contain- anol pre- and post-run completion. ing and KIH constructs. Fluorescence shift was then analyzed on Tandem scFv stability in buffer formulations of PBS/trehalose the Accuri C6 flow cytometer. Cells were gated using forward and HEPES/trehalose was completed by The Australian Protein and side scatter, and the cell sample incubated with secondary Analysis Facility. SEC was performed using a Zorbax BioSeries antibody only was used to determine the cut-off fluorescence for GF-250 column (Agilent). Flow rate was set to 0.5 ml/min and a non-specific binding. Events were limited to 100 ml or 100,000 mobile phase of 100 mM sodium phosphate, pH 7, 250 mM events. NaCl used as a buffer. Sample injections were set to 25 ml. Preparation of labeled BsAb-EDVTMnanocells Surface plasmon resonance One £ 1012 Non-targeted EDVTMnanocells were incubated To evaluate binding affinities of the EGFR-targeting ABX- with 1 mg of Invitrogen’s NH2-reactive AlexaFluor488 (internal EGF-scFvs on all constructs, kinetic data was collected from protocol) and excess dye was removed by 4 spin wash cycles in either Single Cycle Kinetic (SCK) Assays or High Performance sterile PBS. The fluorescently labeled EDVTMnanocells were (Multi-cycle) Kinetic Assays (HPKA) performed on a Biacore T- counted using a NanoSight and found to be 5 £ 1010 EDVs/ml 200. A CM5 chip was coated with an anti-mouse IgG antibody at EnGeneIC Pty Ltd. Doses were prepared as 2.5 £ 1010 EDV on flow cells 1 and 2 as described in the Mouse Antibody Cap- (AF488) particles in 0.5 ml. 2.5 £ 1010 EDV (AF488) particles ture Kit guidelines (GE Healthcare). Recombinant EGFR-mFc were incubated with 6 mg of various BsAb constructs at room was captured on flow cell 2 at 10 mg/ml for 8 - 15 seconds and temperature for 30 min while shaking at 300 rpm. Samples then variable concentrations of BsAb (1 – 100 nM) flowed over both underwent 3 PBS spin wash cycles at 9,000 g for 8 min each to flow cells 1 and 2. Recommended flow rates (30 mL/min) and remove any excess BsAb. BsAb-EDVTMnanocells were resus- regeneration conditions (10 mM glycine pH 1.7; 10 mL/min for pended to concentrations as required for specific experiments. 180 s) were used. The dissociation phase was 1800 s. Reference subtractions of flow cells 2 - 1 were incorporated in the analysis Flow cytometry analysis of EDVTMnanocell binding to as were blank and buffer only sample runs. Kinetic analyses were MDA-MB-468 cells performed using a 1:1 fit of binding curves using BiaEvaluation One £ 105 MDA-MB-468 cells grown on coverslips in software (GE Healthcare). 400 ml RPMI C 5% FCS C 1% PC-SM were treated with 1 £ 109 pre-targeted and AF488-labeled EDVTMnanocells and  Biolayer interferometry kinetic characterization of LPS returned to 37 C for 3 h. This was repeated for the non-targeted binding EDVTMnanocell and each of the 5 BsAb-EDVTMnanocells – The binding of mAbs and BsAbs to LPS molecules was tested non-specific and ABX-EGF tandem-scFv, KIH 1 (Cys), Fc-con- utilizing Biolayer Interferometry and the ForteBio Octet mod- taining (Cys) and Fc-containing (CysLonglink), with only the ule. Aminopropylsilane (APS) biosensors (ForteBio) were briefly control remaining untreated. On completion of the 3 h incuba- hydrated in PBS and coated with 1 mg/ml LPS (Salmonella enter- tion period, coverslips were washed 3 times with sterile DPBS ica, serotype typhimurium - Sigma L7261) diluted in PBS. A and cells scraped for flow analyses on a Beckman FC500 flow concentration range of the 1H10-mAb (anti-LPS) or tandem cytometer. Flow rate was set to high and cells were again gated scFv BsAb was set up using 2-fold dilutions starting at 100 nM using forward and side scatter. The “cells only” sample was used and subsequently tested for binding to immobilised LPS. PBS to determine the cut-off fluorescence for non-specific binding. was added to LPS coated sensors as a reference. Events were detected over a 250 sec period. Final analyses were completed using CXP and VenturiOne software. Flow cytometry of BsAb and mAb samples against MDA- MB-468 cells Co-localization  All BsAb and mAb samples had been stored at 4 C prior to AF488-Labeled, non-targeted and targeted EDVTMnanocells commencement of the experiment. The Indirect Flow Cytometry were prepared as previously mentioned and observations made (FACS) Protocol described by Abcam was used (available online regarding clumping of the BsAb-EDVTMnanocells during each at http:\\www.abcam.com). All components were added to a cell resuspension. BsAb-EDVTMnanocells were resuspended to an 5 suspension of 2 £ 10 MDA-MB-468 cells in 100 ml of 10% approximate concentration of 4 £ 1010 EDVs/ml in 500 ml.  FCS/DPBS and kept at 4 C to prevent receptor-mediated endo- Counts and aggregation analysis were again performed using cytosis from occurring. Primary BsAb and mAb stock dilutions established techniques on the nanosight. EGFR-His (1 mg) label- to 10 mg/ml were made in ice cold 5% BSA C Dulbecco’s PBS ing was completed using a DyLight650 amine-reactive labeling kit buffer as were secondary antibody dilutions. Final BsAb and (Thermo Scientific) as per the manufacturer’s instructions after mAb concentrations were at 1 mg/ml in the final cell suspension having been concentrated to 1.6 mg/ml using a 10 kDa spin volume. Mouse anti-c-myc:FITC (AbD Serotec; Bio-Rad) was column.

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145 To set up samples for co-localization imaging, 2 mg EGFR- MDA-MB-468 cells were obtained from ATCC and grown in His (DyLight650) was added to 5 £ 109 particles of each of the RPMI with 10% fetal calf serum and penicillin/streptomycin. conjugated BsAb-EDVTMnanocells. The resulting suspension Each mouse was injected subcutaneously on the left flank with 1 was then incubated at RT for 30 min on a shaker at 300 rpm to £ 107 cells in 100 ml of media together with 100 ml of growth allow sufficient time for recombinant EGFR binding to occur. factor reduced matrigel (BD Biosciences). Tumor volume was Samples underwent 3 spin wash cycles as before to remove excess determined by measuring length (l) and width (w) and calculat- unbound labeled EGFR-His and checked for evidence of clump- ing volume (V D l £ w2 £ 0.5). Once the tumors reached 80 – ing resulting from BsAb-nanocell interaction with recombinant 100 mm3 the mice were randomized into treatment groups EGFR. EGFR-BsAb-nanocell suspensions were resuspended to a which included 7 mice per group. The treatment groups were as concentration of 0.4 £ 1010 EDVs/ml. For analysis on an Olym- follows: pus IX81 confocal microscope using Xcellence RT software, slides were prepared by pipetting 10 ml sample onto a clean slide  Group 1 – Saline that is then spread out by addition of a coverslip.  Group 2 – Tandem scFv EDV (ABX-EGF tandem scFv BsAb tar- geted EDVTMnanocell not loaded with Doxorubicin)  TM Confocal microscopy of in vitro binding of BsAb- Group 3 – EDV Dox (Non-targeted EDV nanocell loaded EDVTMnanocells to MDA-MB-468 cells with Doxorubicin)  Protein A/G MDA-MB-468 cells were incubated on coverslips in the pres- Group 4 – EDV Dox (Protein A/G linked BsAb tar- ence or absence of targeted [non-specific and ABX-EGF tandem geted EDVTMnanocell loaded with Doxorubicin)21,22  Tandem scFv scFv, KIH 1 (Cys), Fc-containing (Cys) and Fc-containing Group 5 – EDV Dox (ABX-EGF tandem scFv BsAb (CysLonglink)] and non-targeted EDVTMnanocells – 7 slides targeted EDVTMnanocell loaded with Doxorubicin) total. EDVTMnanocells (labeled with AF488) were added to each of 6 samples at a ratio of 10,000 EDVs per cell and the seventh The amount of Doxorubicin was measured by HPLC for all was left as a "cells only" control. Plates were returned to 37C for loaded EDVTMnanocells and determined to be 1 § 0.1 mg per 1 3 hours which was followed by 3 washes in sterile DPBS. £ 109 EDVTMnanocells (one intravenous dose); method com- Fixative volume of 500 ml, or enough to cover each coverslip, pleted as previously described.21,22 All treatments were injected of 4% PFA was added to each of the 7 samples and left for 10 intravenously via the tail vein and administered 3 times a week minutes for cells to become fixed. This was again followed by 3 for 3 weeks. Tumors were measured using a caliper 3 times a DPBS washes. After the final wash, 500 ml DPBS was added to week. The mice were weighed twice a week for 3 weeks. each well to cover the coverslips completely. An anti-EGFR mAb, 528 mAb, was labeled with AF647 to be used as a cell membrane stain; 4 mg was added directly to each sample. Wells Disclosure of Potential Conflicts of Interest were mixed gently and left for 10 min to allow cells to stain suffi- No potential conflicts of interest were disclosed. ciently. All coverslips were again washed 3 times with DPBS fol- lowed by a final wash in Milli-Q water. Coverslips were removed from the water and placed on tissue Acknowledgments paper cell side up to allow complete drying to occur, at which Acknowledgment is given to the National Biologics Facility point they were mounted on clean slides using Fluka Eukitt (AIBN) for providing protein expression facilities. EnGeneIC (Sigma-Aldrich) quick-hardening mounting medium. Dry slides Ltd kindly provided access to microscopy facilities and EDVTM-  were stored at 4 C until confocal microscopy could be com- nanocells for binding assays, in addition to completing in vivo pleted. Microscopy was completed on a LSM Zeiss 710 confocal mouse studies using the selected BsAb format. This work was using a plan-apochromat 63£/1.40 Oil DIC M27 objective and performed in part at the Queensland node of the Australian ZEN 2008 software for image formatting. National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to pro- Effect of ABX-EGF tandem scFv-EDVTMnanocell on tumor vide nano- and micro-fabrication facilities for Australia’s regression researchers. Additional thanks are due to CSL for the completion The experiment was performed in compliance with the Aus- of DLS and The Australian Protein Analysis Facility for evaluat- tralian National Health and Medical Research Council guidelines ing BsAb stability in buffer formulations. for the care and use of laboratory animals and with the approval of the EnGeneIC Animal Ethics Committee. Five-to-6 week old female Balb/C athymic nude mice were obtained from the Labo- Funding ratory Animal Services at the University of Adelaide, South Aus- This work was supported by an ARC Linkage Project Grant tralia. The animals were housed at the EnGeneIC Animal funded by the Australian Government. Karin Taylor is supported Facility under specific pathogen-free conditions. by an Australian Postgraduate Award scholarship.

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146 References 2010; 399:436-49; PMID:20382161; http://dx.doi. 34. Fay F, Scott CJ. Antibody-targeted nanoparticles for 1. Burden RE, Caswell J, Fay F, Scott CJ. Recent advances org/10.1016/j.jmb.2010.04.001 cancer therapy. Immunotherapy 2011; 3:381-94; € in the application of antibodies as therapeutics. Future 18. Atanackovic D, Reinhard H, Meyer S, Spock S, Grob PMID:21395380; http://dx.doi.org/10.2217/imt.11.5 Med Chem 2012; 4:73-86; PMID:22168165; http:// T, Luetkens T, Yousef S, Cao Y, Hildebrandt Y, Tem- 35. Geers B, De Wever O, Demeester J, Bracke M, De dx.doi.org/10.4155/fmc.11.165 plin J, et al. The trifunctional antibody catumaxomab Smedt SC, Lentacker I. Targeted Liposome-Loaded 2. Yoo J-W, Irvine DJ, Discher DE, Mitragotri S. Bio- amplifies and shapes tumor-specific immunity when Microbubbles for Cell-Specific Ultrasound-Trig- inspired, bioengineered and biomimetic drug delivery applied to gastric cancer patients in the adjuvant set- gered Drug Delivery. Small 2013; 9:4027-35; carriers. Nat Rev Drug Discov 2011; 10:521-35; ting. Hum Vaccin Immunother 2013; 9:5-4; http://dx. PMID:23737360; http://dx.doi.org/10.1002/smll. PMID:21720407; http://dx.doi.org/10.1038/nrd3499 doi.org/10.4161/hv.26065 201300161 3. Elvin JG, Couston RG, van der Walle CF. Therapeutic 19. Linke R, Klein A, Seimetz D. Catumaxomab: Clinical 36. YangLL,MaoH,WangYA,CaoZH,PengXH,Wang antibodies: Market considerations, disease targets and development and future directions. MAbs 2010; 2:129- XX,DuanHW,NiCC,YuanQG,AdamsG,etal.Single bioprocessing. Int J Pharm 2013; 440:83-98; 36; PMID:20190561; http://dx.doi.org/10.4161/ Chain Epidermal Growth Factor Receptor Antibody Con- PMID:22227342; http://dx.doi.org/10.1016/j.ijpharm. mabs.2.2.11221 jugated Nanoparticles for in vivo Tumor Targeting and € 2011.12.039 20. Chelius D, Ruf P, Gruber P, Ploscher M, Liedtke R, Imaging. Small 2009; 5:235-43; PMID:19089838; http:// 4. Perchiacca JM, Ladiwala ARA, Bhattacharya M, Tessier Gansberger E, Hess J, Wasiliu M, Lindhofer H. Struc- dx.doi.org/10.1002/smll.200800714 PM. Aggregation-resistant domain antibodies engi- tural and functional characterization of the trifunc- 37. Goodall S, Howard CB, Jones ML, Munro T, Jia Z, neered with charged mutations near the edges of the tional antibody catumaxomab. MAbs 2010; 2:309-19; Monteiro MJ, Mahler S. An EGFR Targeting Nano- complementarity-determining regions. Protein Eng PMID:20418662; http://dx.doi.org/10.4161/mabs.2.3. particle Self Assembled from a Thermoresponsive Poly- Des Sel 2012; 25:591-602; PMID:22843678; http:// 11791 mer. J Chem Technol Biotechnol 2014; 10.1002/ dx.doi.org/10.1093/protein/gzs042 21. MacDiarmid JA, Mugridge NB, Weiss JC, Phillips L, jctb.4509 5. Chames P, Baty D. Bispecific Single Domain Antibod- Burn AL, Paulin RP, Haasdyk JE, Dickson KA, 38. Bertrand N, Wu J, Xu XY, Kamaly N, Farokhzad OC. ies. New York: Springer, 2011. Brahmbhatt VN, Pattison ST, et al. Bacterially derived Cancer nanotechnology: The impact of passive and 6. Teillaud J-L. From whole monoclonal antibodies to 400 nm particles for encapsulation and cancer cell tar- active targeting in the era of modern cancer biology. single domain antibodies: think small. Methods Mol geting of chemotherapeutics. Cancer Cell 2007; Adv Drug Deliv Rev 2014; 66:2-25; PMID:24270007; Biol 2012; 911:3-13. 11:431-45; PMID:17482133; http://dx.doi.org/ http://dx.doi.org/10.1016/j.addr.2013.11.009 7. Reichert JM, Dhimolea E. The future of antibodies as 10.1016/j.ccr.2007.03.012 39. Ellerman D, Scheer JM. Generation of Bispecific Anti- cancer drugs. Drug Discov Today 2012; 17:954-63; 22. MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss bodies by Chemical Conjugation. New York: Springer, PMID:22561895; http://dx.doi.org/10.1016/j. J, Sedliarou I, Wetzel S, Kochar K, Brahmbhatt VN, 2011 drudis.2012.04.006 Phillips L, Pattison ST, Petti C, et al. Sequential treat- 40. Holliger P, Prospero T, Winter G. DIABODIES - 8. Pasquetto MV, Vecchia L, Covini D, Digilio R, Scotti ment of drug-resistant tumors with targeted minicells SMALL BIVALENT AND BISPECIFIC ANTIBODY C. Targeted Drug Delivery Using Immunoconjugates: containing siRNA or a cytotoxic drug. Nat Biotechnol FRAGMENTS. Proc Nati Acad Sci U S A 1993; Principles and Applications. J Immunother 2011; 2009; 27:643-51; PMID:19561595; http://dx.doi.org/ 90:6444-8; PMID:8341653; http://dx.doi.org/ 34:611-28; PMID:21989410; http://dx.doi.org/ 10.1038/nbt.1547 10.1073/pnas.90.14.6444 10.1097/CJI.0b013e318234ecf5 23. Torchilin VP. Recent advances with liposomes as phar- 41. Muller D, Kontermann RE. Diabodies, Single-Chain 9. Brischwein K, Schlereth B, Guller B, Steiger C, Wolf A, maceutical carriers. Nat Rev Drug Discov 2005; 4:145- Diabodies, and Their Derivatives. New York: Springer, Lutterbuese R, Offner S, Locher M, Urbig T, Raum T, 60; PMID:15688077; http://dx.doi.org/10.1038/ 2011. et al. MT110: A novel bispecific single-chain antibody nrd1632 42. Kontermann RE. Bispecific Antibodies. New York: construct with high efficacy in eradicating established 24. Ferrari M. Cancer nanotechnology: Opportunities and Springer, 2011, 1-373. tumors. Mol Immunol 2006; 43:1129-43; challenges. Nat Rev Cancer 2005; 5:161-71; 43. Moore PA, Zhang WJ, Rainey GJ, Burke S, Li H, PMID:16139892; http://dx.doi.org/10.1016/j.molimm. PMID:15738981; http://dx.doi.org/10.1038/nrc1566 Huang L, Gorlatov S, Veri MC, Aggarwal S, Yang YH, 2005.07.034 25. Webster DM, Sundaram P, Byrne ME. Injectable et al. Application of dual affinity retargeting molecules 10. Holliger P, Hudson PJ. Engineered antibody fragments nanomaterials for drug delivery: Carriers, targeting to achieve optimal redirected T-cell killing of B-cell and the rise of single domains. Nat Biotechnol 2005; moieties, and therapeutics. Eur J Pharm Biopharm lymphoma. Blood 2011; 117:4542-51; 23:1126-36; PMID:16151406; http://dx.doi.org/ 2013; 84:1-20; PMID:23313176; http://dx.doi.org/ PMID:21300981; http://dx.doi.org/10.1182/blood- 10.1038/nbt1142 10.1016/j.ejpb.2012.12.009 2010-09-306449 11. Kontermann R. Dual targeting strategies with bispecific 26. Couvreur P. Nanoparticles in drug delivery: Past, pres- 44. Wozniak-Knopp G, Bartl S, Bauer A, Mostageer M, antibodies. mAbs 2012; 4:182-97; PMID:22453100; ent and future. Adv Drug Deliv Rev 2013; 65:21-3; Woisetschlager M, Antes B, Ettl K, Kainer M, http://dx.doi.org/10.4161/mabs.4.2.19000 PMID:22580334; http://dx.doi.org/10.1016/j.addr. Weberhofer G, Wiederkum S, et al. Introducing anti- 12. Zhu Z. Dual-Targeting Bispecific Antibodies as New 2012.04.010 gen-binding sites in structural loops of immunoglobu- Therapeutic Modalities for Cancer. In: Wood CR, ed. 27. Lammers T, Kiessling F, Hennink WE, Storm G. Drug lin constant domains: Fc fragments with engineered Antibody Drug Discovery. London, UK: Imperial Col- targeting to tumors: Principles, pitfalls and (pre-) clini- HER2/neu-binding sites and antibody properties. Pro- lege Press, 2011:373-408 cal progress. J Control Release 2012; 161:175-87; tein Eng Des Sel 2010; 23:289-97; PMID:20150180; 13. May C, Sapra P, Gerber H-P. Advances in bispecific PMID:21945285; http://dx.doi.org/10.1016/j.jconrel. http://dx.doi.org/10.1093/protein/gzq005 biotherapeutics for the treatment of cancer. Biochem 2011.09.063 45. Wang L, He YR, Zhang G, Ma J, Liu CZ, He W, Pharmacol 2012; 84:1105-12; PMID:22858161; 28. Steichen SD, Caldorera-Moore M, Peppas NA. A Wang W, Han HM, Boruah BM, Gao B. Retargeting http://dx.doi.org/10.1016/j.bcp.2012.07.011 review of current nanoparticle and targeting moieties T Cells for HER2-Positive Tumor Killing by a Bispe- 14. Chames P, Baty D. Bispecific antibodies for cancer for the delivery of cancer therapeutics. Eur J Pharm Sci cific Fv-Fc Antibody. PLoS ONE 2013; 8(9):e75589; therapy The light at the end of the tunnel? MAbs 2009; 2013; 48:416-27; PMID:23262059; http://dx.doi.org/ PMID:24086580; doi:10.1371/journal.pone.0075589 1:539-47; PMID:20073127; http://dx.doi.org/ 10.1016/j.ejps.2012.12.006 46. Enever C, Batuwangala T, Plummer C, Sepp A. Next 10.4161/mabs.1.6.10015 29. Battaglia DP. Targeted Drug Delivery. Amsterdam, generation immunotherapeutics—honing the magic 15. Byrne H, Conroy PJ, Whisstock JC, O’Kennedy RJ. A Netherlands: Elsevier Science Bv, 2013. bullet. Curr Opin Biotechnol 2009; 20:405-11; tale of two specificities: bispecific antibodies for thera- 30. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, PMID:19709876; http://dx.doi.org/10.1016/j.copbio. peutic and diagnostic applications. Trends Biotechnol Langer R. Nanocarriers as an emerging platform for 2009.07.002 2013; 31:621-32; PMID:24094861; http://dx.doi.org/ cancer therapy. Nat Nano 2007; 2:751-60; http://dx. 47. Presta LG. Molecular engineering and design of thera- 10.1016/j.tibtech.2013.08.007 doi.org/10.1038/nnano.2007.387. peutic antibodies. Curr Opin Immunol 2008; 20:460- 16. Lutterbuese R, Raum T, Kischel R, Hoffmann P, Man- 31. MacDiarmid JA, Brahmbhatt H. Minicells: Versatile 70; PMID:18656541; http://dx.doi.org/10.1016/j. gold S, Rattel B, Friedrich M, Thomas O, Lorenczew- vectors for targeted drug or si/shRNA cancer therapy. coi.2008.06.012 ski G, Rau D, et al. T cell-engaging BiTE antibodies Curr Opin Biotechnol 2011; 22:909-16; 48. Boonstra J, Rijken P, Humbel B, Cremers F, Verkleij specific for EGFR potently eliminate KRAS- and PMID:21550793; http://dx.doi.org/10.1016/j.copbio. A, Henegouwen PVE. THE EPIDERMAL BRAF-mutated colorectal cancer cells. Proc Nati Acad 2011.04.008 GROWTH-FACTOR. Cell Biol Int 1995; 19:413-30; Sci U S A 2010; 107:12605-10; PMID:20616015; 32. Felice B, Prabhakaran MP, Rodriguez AP, Ramak- PMID:7640657; http://dx.doi.org/10.1006/cbir.1995. http://dx.doi.org/10.1073/pnas.1000976107 rishna S. Drug delivery vehicles on a nano-engineering 1086 17. Johnson S, Burke S, Huang L, Gorlatov S, Li H, Wang perspective. Mater Sci Eng C-Mater Biol Appl 2014; 49. Grunwald V, Hidalgo M. The epidermal growth factor W, Zhang W, Tuaillon N, Rainey J, Barat B, et al. 41:178-95; PMID:24907751; http://dx.doi.org/ receptor: A new target for anticancer therapy. Curr Prob- Effector Cell Recruitment with Novel Fv-based Dual- 10.1016/j.msec.2014.04.049. lems in Cancer 2002; 26:109-64; PMID:12085086; http:// affinity Re-targeting Protein Leads to Potent Tumor 33. Sanna V, Pala N, Sechi M. Targeted therapy using dx.doi.org/10.1067/mcn.2002.125874 Cytolysis and in Vivo B-cell Depletion. J Mol Biol nanotechnology: focus on cancer. Int J Nanomed 50. Mendelsohn J, Baselga J. Epidermal growth factor 2014; 9:467-83 receptor targeting in cancer. Semin Oncol 2006;

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147 33:369-85; PMID:16890793; http://dx.doi.org/ interstitial penetration of sterically stabilized (stealth) CCR5 Monoclonal Antibody-Resistant HIV-1 Strains. 10.1053/j.seminoncol.2006.04.003 liposomes in a human tumor xenograft. Cancer Res Antimicrob Agents Chemother 2011; 55:2369-78; 51. Cohenuram M, Saif MW. Panitumumab the first fully 1994; 54:3352-6; PMID:8012948 PMID:21300827; http://dx.doi.org/10.1128/AAC. human monoclonal antibody: from the bench to the 62. Reichert JM. Antibodies to watch in 2013: Mid-year 00215-10 clinic. Anti-Cancer Drugs 2007; 18:7-15; update. MAbs 2013; 5:513-7; PMID:23727858; 74. Mabry R, Lewis KE, Moore M, McKernan PA, Bukow- PMID:17159497; http://dx.doi.org/10.1097/CAD. http://dx.doi.org/10.4161/mabs.24990 ski TR, Bontadelli K, Brender T, Okada S, Lum K, 0b013e32800feecb 63. Merchant AM, Zhu ZP, Yuan JQ, Goddard A, Adams West J, et al. Engineering of stable bispecific antibodies 52. Gemmete JJ, Mukherji SK. Panitumumab (Vectibix). CW, Presta LG, Carter P. An efficient route to human targeting IL-17A and IL-23. Protein Eng Design Sel Am J Neuroradiol 2011; 32:1002-3; PMID:21596817; bispecific IgG. Nat Biotechnol 1998; 16:677-81; 2009; 23:115-27; http://dx.doi.org/10.1093/protein/ http://dx.doi.org/10.3174/ajnr.A2601 PMID:9661204; http://dx.doi.org/10.1038/nbt0798- gzp073 53. Jakobovits A, Amado RG, Yang X, Roskos L, Schwab 677 75. Moore GL, Bautista C, Pong E, Nguyen D-HT, G. From XenoMouse technology to panitumumab, the 64. Mabry R, Gilbertson DG, Frank A, Vu T, Ardourel D, Jacinto J, Eivazi A, Muchhal US, Karki S, Chu SY, first fully human antibody product from transgenic Ostrander C, Stevens B, Julien S, Franke S, Meengs B, Lazar GA. A novel bispecific antibody format enables mice. Nat Biotechnol 2007; 25:1134-43; et al. A dual-targeting PDGFR beta/VEGF-A molecule simultaneous bivalent and monovalent co-engagement PMID:17921999; http://dx.doi.org/10.1038/nbt1337 assembled from stable antibody fragments demonstrates of distinct target antigens. mAbs 2011; 3:546-57; 54. Zhu JW. Mammalian cell protein expression for bio- anti-angiogenic activity in vitro and in vivo. mAbs PMID:22123055; http://dx.doi.org/10.4161/mabs.3.6. pharmaceutical production. Biotechnol Adv 2012; 2010; 2:20-34; PMID:20065654; http://dx.doi.org/ 18123 30:1158-70; PMID:21968146; http://dx.doi.org/ 10.4161/mabs.2.1.10498 76. McAleese F, Eser M. RECRUIT-TandAbs (R): har- 10.1016/j.biotechadv.2011.08.022 65. Ridgway JBB, Presta LG, Carter P. ’Knobs-into-holes’ nessing the immune system to kill cancer cells. Future 55. Dinnis DM, James DC. Engineering mammalian cell engineering of antibody C(H)3 domains for heavy Oncol 2012; 8:687-95; PMID:22764766; http://dx. factories for improved recombinant monoclonal anti- chain heterodimerization. Protein Eng 1996; 9:617-21; doi.org/10.2217/fon.12.54 body production: Lessons from nature? Biotechnol Bio- PMID:8844834; http://dx.doi.org/10.1093/protein/ 77. Rajkovic E, Hucke C, Knackmuss S, Molkenthin V, eng 2005; 91:180-9; PMID:15880827; http://dx.doi. 9.7.617 Reusch U, Legall F, Little M. Recruit-TandAbs: Engag- org/10.1002/bit.20499 66. Zhu ZP, Presta LG, Zapata G, Carter P. Remodeling ing immune cells to kill cancer cells AFM13: A bispe- 56. Metz S, Panke C, Haas AK, Schanzer J, Lau W, Croas- domain interfaces to enhance heterodimer formation. cific tetravalent TandAb for treating Hodgkin dale R, Hoffmann E, Schneider B, Auer J, Gassner C, Protein Sci 1997; 6:781-8; PMID:9098887; http://dx. Lymphoma. 11th International Conference on Malig- et al. Bispecific antibody derivatives with restricted doi.org/10.1002/pro.5560060404 nant Lymphoma 15-18 June 2011. Lugano (Switzer- binding functionalities that are activated by proteolytic 67. Labrijn AF, Aalberse RC, Schuurman J. When binding land): Ann Oncol 2011:240-1 processing. Protein Eng Des Sel 2012; 25:571-80; is enough: nonactivating antibody formats. Curr Opin 78. Adams TE, Koziolek EJ, Hoyne PH, Bentley JD, http://dx.doi.org/10.1093/protein/gzs064 Immunol 2008; 20:479-85; PMID:18577454; http:// Lu L, Lovrecz G, Ward CW, Lee FT, Scott AM, 57. Feige MJ, Groscurth S, Marcinowski M, Shimizu Y, dx.doi.org/10.1016/j.coi.2008.05.010 Nash AD, et al. A truncated soluble epidermal Kessler H, Hendershot LM, Buchner J. An Unfolded C 68. Jendreyko N. Intradiabodies, Bispecific, Tetravalent growth factor receptor-Fc fusion ligand trap displays (H)1 Domain Controls the Assembly and Secretion of Antibodies for the Simultaneous Functional Knockout anti-tumour activity in vivo. Growth Factors 2009; IgG Antibodies. Molecular Cell 2009; 34:569-79; of Two Cell Surface Receptors. J Biol Chem 2003; 27:141-54; PMID:19333814; http://dx.doi.org/ PMID:19524537; http://dx.doi.org/10.1016/j.molcel. 278:47812-9; PMID:12947084; http://dx.doi.org/ 10.1080/08977190902843565 2009.04.028 10.1074/jbc.M307002200 79. Morrison SL. Cloning, expression, and modification of 58. Yang XD, Jia XC, Corvalan JRF, Wang P, Davis CG, 69. Abou-Nassar K, Brown JR. Novel agents for the treat- antibody V regions. Curr Proto Immunol 2002; Chap- Jakobovits A. Eradication of established tumors by a ment of chronic lymphocytic leukemia. Clin Adv Hem- ter 2:Unit 2.12; PMID:18432877; doi:10.1002/ fully human monoclonal antibody to the epidermal atol Oncol : H&O 2010; 8:886-95. 0471142735.im0212s47 growth factor receptor without concomitant chemo- 70. Lee CC, Perchiacca JM, Tessier PM. Toward aggrega- 80. Atwell S, Ridgway JB, Wells JA, Carter P. Stable heter- therapy. Cancer Res 1999; 59:1236-43; tion-resistant antibodies by design. Trends Biotechnol odimers from remodeling the domain interface of a PMID:10096554 2013; 31:612-20; PMID:23932102; http://dx.doi.org/ homodimer using a phage display library. J Mol Biol 59. Mendez MJ, Green LL, Corvalan JRF, Jia XC, May- 10.1016/j.tibtech.2013.07.002 1997; 270:26-35; PMID:9231898; http://dx.doi.org/ nardCurrie CE, Yang XD, Gallo ML, Louie DM, Lee 71. Miller BR, Demarest SJ, Lugovskoy A, Huang F, Wu 10.1006/jmbi.1997.1116 DV, Erickson KL, et al. Functional transplant of mega- XF, Snyder WB, Croner LJ, Wang N, Amatucci A, 81. Jones ML, Seldone T, Smede M, Linville A, Chin DY, base human immunoglobulin loci recapitulates human Michaelson JS, et al. Stability engineering of scFvs for Barnard R, Mahler SM, Munster D, Hart D, Gray PP, antibody response in mice. Nat Genet 1997; 15:146- the development of bispecific and multivalent antibod- et al. A method for rapid, ligation-independent refor- 56; PMID:9020839; http://dx.doi.org/10.1038/ ies. Protein Eng Des Sel 2010; 23:549-57; matting of recombinant monoclonal antibodies. J ng0297-146 PMID:20457695; http://dx.doi.org/10.1093/protein/ Immunol Methods 2010; 354:85-90; 60. Hobbs SK, Monsky WL, Yuan F, Roberts WG, Grif- gzq028 PMID:20153332; http://dx.doi.org/10.1016/j.jim.2010. fith L, Torchilin VP, Jain RK. Regulation of transport 72. Demarest SJ, Glaser SM. Antibody therapeutics, anti- 02.001 pathways in tumor vessels: Role of tumor type and body engineering, and the merits of protein stability. 82. Codamo J, Hou JJC, Hughes BS, Gray PP, Munro TP. microenvironment. Proc Nati Acad Sci U S A 1998; Curr Opin Drug Discov Dev 2008; 11:675-87. Efficient mAb production in CHO cells incorporating 95:4607-12; PMID:9539785; http://dx.doi.org/ 73. Schanzer J, Jekle A, Nezu J, Lochner A, Croasdale R, PEI-mediated transfection, mild hypothermia and the 10.1073/pnas.95.8.4607 Dioszegi M, Zhang J, Hoffmann E, Dormeyer W, co-expression of XBP-1. J Chem Technol Biotechnol 61. Yuan F, Leunig M, Huang SK, Berk DA, Papahadjo- Stracke J, et al. Development of Tetravalent, Bispecific 2011; 86:923-34; http://dx.doi.org/10.1002/jctb.2572 poulos D, Jain RK. Microvascular permeability and CCR5 Antibodies with Antiviral Activity against

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