Engineering bispecific antibodies 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 cancer 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. Chemotherapy 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 antigen that has been extensively evaluated in the literature. EGFR is associated with a number of different cancers including breast and colon, and anti-EGFR mAbs are approved for therapeutic use (e.g. panitumumab and cetuximab). 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 antibody 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 Protein 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