Complex membrane proteins, multi-spanning membrane proteins, represent a sizeable therapeutic class that include GPCR’s, ion channels, transporters, and to a lesser extent, membrane-bound enzymes. They are involved in many physiological processes, playing a broad range of roles in both normal and disease states. Currently, there are 127 antibodies products available across the United States and Europe, with 77 of these targeting membrane proteins. Among the most well-known include Mogamulizumab (Potilegeo) for treating leukaemia and Erenumab (Aimovig) to help treat migraines. The global market for therapeutic antibodies is projected to be worth 179.56 billion USD by 2025. Key players in the field include AstraZeneca, Amgen, Bionomics, Bristol Myers Squibb, Integral Molecular, Merus, Novartis, and Novo Nordisk.
Antibodies provide a therapeutic opportunity for biologics against multi-spanning membrane proteins. In particular, they hold the promise of exquisite specificity by targeting certain structures of a membrane protein. However, due to various challenges, membrane proteins remain an understudied and underutilised opportunity for the application of therapeutic antibodies. In this article, we delve into some of the solution technologies available to optimise the targeting of complex membrane proteins and hear from an industry leading pharmaceutical representative, about the future of antibody discovery.
Monoclonal Antibodies Structure and Function:
Monoclonal antibodies are complex molecules containing a heavy and light chain. They consist of a variable domain and the Fc constant domain. The variable domain houses complementarity determining regions (CDRs) and engages with the target protein of interest. Here, epitope selection is important as it impacts the function of the antibody. According to our unnamed source, “a lot of care must be taken over what epitopes are selected as the properties can be engineered to target specific residues in the CDRs”.
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They continued, “this, in turn, has the potential to improve affinity, potency, modulate cross-reactivity, and have an impact on the specificity of the antibody against a particular target”. The Fc constant domain involves binding to FC receptors and engaging effector functions that provide the antibody-dependent with cellular cytotoxicity. The senior representative described these features as “very useful and accountable opportunities”. The Fc domain also indicates where clinicians may want to deplete target cells in a patient for a desired therapeutic effect. In addition to this, the modular nature of antibodies allows for the insertion of specified features such as introducing toxins to target ion channels, engineer glycosylation status, and modify isotype choice.
Targeting Complex Membrane Proteins with Monoclonal Antibodies: Overcoming the Challenges
Complex membrane proteins are difficult to express at high levels and require purification using detergents. Currently, this remains an empirical and costly process, limiting the broader use of these therapeutics. As dynamic molecules with multiple conformations, the large size of antibodies can limit tumour penetration. Furthermore, their long serum half-life lacks applicability for imaging and radioimmunotherapy treatments. Nevertheless, advances in engineering mean there are several solution technologies and approaches available to optimise the targeting of complex membrane proteins with monoclonal antibodies.
1.) Immunisation Solutions: Immunisation of animals with DNA raises immune responses specific to the selected protein. Our source stated, “you can avoid purifying the protein entirely by going down the route of DNA immunisation”. It involves introducing a target protein into an animal via a DNA vector and is an important source of antibodies. “Immunisation uses stable cell lines over expressing the target of interest” they explained. This is a tried and tested method where various animal models have been used to isolate antibodies, including wild-type mice, and velocimmune mice.
2.) Protein Reconstitution: There are also various ways of reconstituting proteins such as nanodisc technologies and virus-like particles to preserve the native conformation of a purified membrane protein for antigenic purposes. It is also possible to reconstitute proteins into proteoliposomes, systems that mimic the lipid membranes liposome to which a protein has been incorporated or inserted. Strategies for expressing proteins in different hosts, such as using tetrahymena as a host, help determine the functioning and reconstructing of proteins.
Target knowledge significantly impacts the ability to target complex membrane proteins. Despite the challenges to the development of therapeutic antibodies targeting complex membrane proteins, applications of engineering, selection, and screening techniques will facilitate and enable the next generation of targeting. In the future, we can expect to see more clearly defined guidelines and regulatory advice pertaining to the optimal antibody-generation platforms and antigen format for a given target. Targeting complex membrane proteins is an understudied but exciting therapeutic opportunity, and at Oxford Global, we can hardly wait to see the impact this creates for the field of biologics.
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