Improving the functional affinity of human anti-glycan monoclonal antibodies (mabs) for cancer therapy

Over the last decade, antibodies have become one of the major growth areas in the Pharmaceutical industry. They combine a high level of specificity for their target (the 'antigen') with the ability to recruit powerful immune system-mediated effects. Indeed, antibodies are one of the main mechanisms by which the immune system normally eliminates infectious agents.. We have a proven track record in the production of clinically valuable anti-cancer antibodies (two of our antibodies are currently in clinical trials) and we are striving to produce more. However, we have observed that during the molecular procedures necessary to allow an antibody to be used in patients, they often lose a substantial fraction of their potency. We believe that we now have an explanation for why this might be occurring, and we are seeking the funding to make the changes necessary to avoid this loss of potency.
Antibodies are typically produced by immunising mice with a target 'antigen'. Antibody-producing cells are isolated from responding mice and the cells immortalised. The antibodies being produced are then screened for reactivity and clinical applicability, and the genes encoding the antibodies are cloned. The areas of the antibody that are not responsible for the target specificity (the 'constant regions') are then replaced with their human equivalent, in a process referred to as the 'humanisation' of the antibody. This is essential to create a clinically useful reagent; if the majority of the mouse areas remain, an immune response can develop in patients that negates the effectiveness of the antibody. We have observed that after this 'humanisation' process, the antibodies bind less strongly to their target and that many of the clinically beneficial effects of the antibodies are lost. We believe that this reduced potency is due to the replacement of the original mouse 'subclass' of the antibody, called mouse IgG3, as we do not observe it when other mouse subclasses are involved.
Previous investigators have suggested that mouse IgG3 antibodies might combine together once bound to their target, in a way that other antibody subclasses do not. This stabilises binding and amplifies the clinical potency of the antibody. Our proposal is to confirm the areas of mouse IgG3s that are having this effect, and to transfer these areas to human IgG1, the most common human subclass used clinically. This is feasible because, although the antibodies are very similar in sequence, they nevertheless have unique differences. Rather than investigate these numerous differences individually, we propose to alter whole patches of the surface of human IgG1 to resemble mouse IgG3. In this way, we will cover the differences in a more time- and cost-efficient way, and also pick up any changes where multiple simultaneous differences are necessary for the effect. Once we have isolated which overall area is responsible for the effect, we will investigate the area change by change to identify exactly which changes are important. We have two candidate antibodies in which we have shown a hundred-fold and ten-fold reduction in potency, respectively, introduced by the change from mouse IgG3 to human IgG1. These make ideal candidates to test which alterations in the human IgG1 re-introduce this lost potency.
We believe that this work will result in a number of very exciting prospects. It will permit the use of antibodies against clinically useful targets that would otherwise have failed at the 'humanisation' stage; it will allow the use of lower doses of antibody, with a lower risk of side-effects; and it will increase the potency of currently available antibodies, increasing their clinical effectiveness. We therefore consider that the proposed project is an excellent use of resources with patient and economic benefits encompassing our antibodies and many other therapeutic antibodies.

Mabs targeting tumours show variable efficacy and improving this is a key goal to this $billion industry. Glycolipids are excellent targets for mabs as they are over-expressed in tumours and are co-accessory molecules for key survival pathways. Blocking these pathways can lead to direct cell killing. Despite the attractiveness of the targets, there are very few mabs recognising glycolipids as they fail to provide T cell help and induce low affinity IgM responses. We have overcome this limitation and produced high functional affinity mouse IgG3 (mIgG3) mabs that show potent in vitro and in vivo anti-tumour responses. These mabs show proinflammatory direct tumour killing without the need for immune effector cells or complement and can reinitiate immune responses in the tumour microenvironment. mIgG3 has the unique ability to non-covalently multimerise on the cell surface through intermolecular cooperativity, resulting in increased functional affinity and enhanced tumour cell killing. This only occurs at high antigen density (tumour) thus reducing off target (normal) toxicity. We have chimerised the mouse mabs to human IgG1 (hIgG1) but they have lost direct cell killing and have reduced functional affinity as intermolecular cooperativity is not present in human IgGs. By crosslinking the chimeric mabs with anti-human Ig anti-serum or by changing one key amino acid residue we have shown that direct killing is directly related to functional affinity. The next stage in development of these novel mabs is to recapitulate the functional affinity of the mouse mabs by introducing intermolecular cooperativity in the chimeric constructs. No one has mapped the mIgG3 cooperativity and the key amino acids that differ between hIgG1 and mIgG3 are not patented giving us a unique position. This project will not only instigate the development of our series of anti-glycolipid mabs but could be used by other groups to improve the therapeutic index of any mab.

Our research benefits patients, commerce and the public with an overall goal of improving health and quality of life. Our research will provide a novel therapy for a wide range of cancer patients that should improve quality of life and achieve long term cure from this debilitating disease. The impact of our research spans both economic impact and patient benefit.

Commercial and economic impact
Nottingham University Therapeutic Antibody centre (NUTAC) has been established to develop and licence monoclonal antibodies (mAbs). Professor Durrant has had success with this process through her spin out company Scancell Ltd using external investment. The current business model is that NUTAC would develop and licence new mAbs using University (£500k)/grant (£500k) income so that all future income flows to the University will enable development of further mAbs. We have produced four mouse mAbs which show selective in vitro and in vivo tumour killing. Chimerisation of those mAbs to human IgG1 mAbs coincided with a reduction in potency. The current grant is designed to confirm the key residues in the mouse mAbs that are required for this increased potency and to transfer it to the human mAbs. This will not only allow us to produce 3 novel human mAbs for licensing to the Biotechnology/Pharmaceutical industry but could form a platform to improve the efficacy of all human mAbs.The top two lead mAbs, FG27 and FG88, could potentially treat 214,500 and 421,500 patients per annum based upon the % of tumours that express the target antigen. These are patients who currently have failed all other therapies and die of their disease. If we assume market penetration of 50% and a treatment price of £10,000 per patient this is a potential market of £1-2 billion.

Patient Benefit
Our mAbs can be used in the treatment of a wide range of cancer patients. As they not only directly kill tumours but initiative immune responses they should confer similar survival benefits to the new checkpoint inhibitors which are currently curing 20-50% of advanced melanoma patients. If our mAbs are used to treat over 300,000 patients per annum and a conservative 20% are cured this could result in saving over 60,000 lives per annum.

Developing an engagement and impact culture
Our strategy is to translate our developmental findings into an investable product, through attracting innovation funding. Professor Durrant, Dr Spendlove and Dr Ramage run an MSc course in Cancer Immunotherpy and Biotechnology to train young scientists to enter the Biotechnology industry. New clinical translation, protecting IP and business plan writing modules have been introduced for Masters students.
The School's Research Committee (est. 2009) drives strategy for impact. Its membership includes senior clinical and basic scientist co-chairs, a Business Development Executive (BDE) and experts in industrial liaison, clinical translation and engagement. It organises workshops led by impact champions, promotes knowledge exchange events, encourages new collaborations and closer engagement with the NHS. Key features include the review and facilitation of impact development and highlighting successful impact approaches. Workshops include sessions on: maximising research impact, industrial collaboration, translational research, intellectual property (IP), patient involvement and impact surgeries. Annual research days highlight University support for knowledge exchange, through Business Engagement & Innovation Services (BEIS), and knowledge exchange funding schemes.