C-Terminal Selective Ligation to Access Homogeneous Antibody Conjugates

Antibody-Drug Conjugates (ADCs) represent one of the most exciting classes of new targeted anti-cancer therapeutics, with 4 ADCs achieving clinical approval over the last 8 years. They consist of an antibody, which is a protein macromolecule that specifically binds to an antigen overexpressed on a cancer cell surface, attached to a cytotoxic chemical 'warhead'. They aim to overcome the limitations of existing chemotherapeutics by delivering the cytotoxic drug specifically to the cancer cells, and thus reducing the side-effects associated with damaging healthy tissue. To maximise the chances for ADCs achieving their therapeutic potential, their design and chemical construction must be improved. Critically, current approaches for the attachment of the drug to the antibody have relied on unselective chemistries, which generate highly complex mixtures of conjugates with variable pharmacological profiles. Instead, it is now widely accepted that the attachment method for the next generation of optimised ADCs must be site-selective.
In this project we are proposing to pioneer a new approach for the generation of ADCs, which attaches the drugs at specific locations on the antibody by extending the protein chain, generating superior homogeneous conjugates. Our strategy, crucially, will not require genetic engineering of the antibodies to incorporate reactive handles, and is thus applicable directly to native 'off-the-shelf' antibodies. This will maximise the accessibility of homogenous ADCs to researchers across the world and ensure that the production yields are maintained as high as possible, ultimately reducing the cost of these relatively complex biopharmaceuticals. Moreover, it will form ADCs with a controlled loading of 2 drugs, which is timely as many of the new ultra-potent warheads can only be tolerated at such loadings as they are so hydrophobic.
We will achieve this goal by targeting the C-terminal cysteines present on the vast majority of clinically validated antibodies. This is a challenging aim as whilst a wide variety of methods have been established for the selective modification of various amino acids on peptides/proteins, there is an absence of a reliable strategy for modification at the C-terminus. As such, the development of a C-terminal modification strategy would also provide a fundamental advance in the broader field of bioconjugation. Our strategy exploits cysteine reactive reagents, which will be designed to transfer from Cys-to-C-terminus via highly favourable 6-membered ring intermediates. The reactions will result in the formation of amide linkages, which are already extremely well characterised and known to be robustly stable in vivo. Overall this C-terminal modification strategy will represent an optimum approach for producing next generation ADCs and facilitate the wider success of these exciting targeted therapies.

This project aims to increase our capability to produce targeted cancer therapies, and thus improved patient healthcare is the main, long-term, prospective impact. By overcoming key limitations in the design and synthesis of ADCs this work will contribute to the broader success of these therapeutics going forward. By developing a homogeneous conjugation strategy that can be employed directly on native antibodies to create ADCs with a loading of two is timely; as this is considered the ideal loading for the new wave of highly potent toxins suitable for ADCs. Moreover, forming clinically validated, robustly stable amide linkages maximises the chances of this approach being taken-up into clinical products. It also ensures the costs are kept as low as possible, and will even allow existing clinical antibodies to be re-purposed as antibody conjugates.
The high economic value associated with antibody conjugates, particularly for therapy and imaging applications, also reveals that our approach could have very significant economic impact. For example the ADC field alone is expected to be worth $10 billion by 2025. We will work closely with UCL Business to obtain IP protection at key moments in the project, to establish a proprietary platform which will incentivize the future investment required to pursue final healthcare products.
The technology development described in this proposal will be carried out using Herceptin, which is a clinically validated antibody alone and is used in the treatment of breast, oesophageal and stomach cancers. This will ensure the products we generate are of direct potential for future clinical development in these areas.
Throughout this project we will disseminate our findings widely, through publications, posters and seminars, with the aim of facilitating impact throughout the various scientific communities in which antibody conjugates play diverse roles.