Characterising the molecular interactions in the CTLA-4 and PD-1 pathways as a basis for novel strategies in immune checkpoint blockade

Immunotherapy has revolutionised the treatment of cancer. The 2018 Nobel Prize in medicine to Allison and Honjo recognised this groundbreaking shift in our approach to cancer treatment whereby instead of cytotoxic drugs targeting cancer tissue, antibodies are used to remove the "brakes" on the immune system. This targeting of the immune system and not the cancer represents a fundamental change in thinking, recognising that cancer can be targeted by the immune system and therefore manipulating the immune response can be highly beneficial.

The two main checkpoints targeted to date have been the CTLA-4 and PD-1 pathways, which have been targeted both separately and increasingly together. Despite over two decades of research into these pathways we still understand remarkably little about how these pathways actually work. In addition, it is now emerging that there are clear biophysical interactions between these pathways at the molecular level which have yet to be properly characterised.

The Sansom lab has made significant progress in this area by identifying that CTLA-4 has the remarkable ability to capture and destroy its ligands (CD80 and CD86) using a cell biological process known as Transendocytosis(TE) providing a platform for our studies. Because of the unusual cell biology of CTLA-4 involving intracellular delivery to a confined immune synapse, rapid internalisation, recycling and degradation the best strategies for antibody targeting of CTLA-4 have yet to be determined.

Recent data has also revealed that one of the CTLA-4 ligands (CD80) also directly interacts with a ligand from the PD-1 pathway (PD-L1). This produces numerous additional scenarios when considering antibody modulation. For example, it is likely that the level of CTLA-4 expression, by controlling the availability of CD80, directly regulates the PD-L1- PD-1 pathway.

The proposed project will therefore explore different strategies for manipulating these checkpoints including the impact of multiple formulations of antibodies (high affinity vs low affinity, blocking vs non-blocking, different Fc regions and different sizes (Fab2, Fab', Fv, etc) used alone alone and in combination within the CTLA-4 and PD-1 pathways. The project will evaluate impacts at both the molecular level (binding, dissociation, trafficking, degradation effects) and cellular levels (immune modulation, T cell activity, ADCC etc.) in order to better understand, manipulate and predict their use in treatment scenarios. Overall the project will provide a focused training on the biology of immune checkpoints as well as direct experience of the pharmaceutical industry relating to antibody engineering.

In this project we therefore aim to:

1). Provide training in the study of the relevant partners involved, including measurement of their biophysical characteristics using purified proteins and functional impacts genetically manipulated cell lines and primary human cells.

2). Explore the design of new proteins (antibody variants, soluble mimics, ligand mutants) that can act to modulate the above interactions and determine their mode of action.

3). Determine the influence of transendocytosis process on the regulation of the PD-1 pathway via CD80 interaction with PD-L1.

4). Characterise the impact of clinically available anti-CTLA-4 and anti-PD-L1 antibodies for their ability to influence the above processes and compare them with novel variants produced in this programme.


Since both the CTLA-4 and PD-1 pathways affect cancer biology and are key control points for common autoimmune diseases, the understanding of these pathways in molecular detail and how to best manipulate them therapeutically is highly relevant to the fundamental mechanisms of disease theme.