Abstract
Cancer is a disease which causes a considerate amount of pain and suffering, and is topped only by cardiovascular disease as cause of mortality in first world countries1. The necessity for new therapies along with the potent tumorigenic potential of the immune system highlights tumour immunotherapies as viable to pursue for further research and development. Here we have investigated a format of bi-specific T cell engager (BiTE) which utilises the tumour-associated antigen tissue factor, covalently linked to a single 41BBL protomer for co-stimulation of T cells, with the aim of assessing its ability to trimerise in solution and activate T cells at the site of antigen expression. We postulated that differences in linker domain structure between the α-tissue factor and 41BBL subunits may alter trimerisation properties and BiTE activity, leading us to investigate different linker constr ucts. We found that the α-tissue factor-41BBL BiTE with either a glycine serine or a leucine zipper linker was able to bind specifically at each end to its target protein, however neither of the two BiTE constructs investigated were able to elicit effector functions from T Cells in vitro when provided as co-stimulation alongside CD3 agonist. Investigating other design formats such as covalently linking multiple 41BB protomers may be required in the future to promote greater trimerisation.