The CD40 Receptor - Target,Tool and Technology

Sammanfattning: CD40 is a cell surface receptor of pivotal importance that is expressed on several of the cells in the immune system. It is critical for many important events, such as T cell dependent B cell activation, isotype switching, somatic mutation and generation of B cell memory. The central role of CD40 in the immune system makes it an ideal target for antibody based immunotherapy. This led us to characterise a panel of monoclonal anti-CD40 antibodies. In PAPER I, we investigated their cellular activation potential and analysed to what extent this correlates with their affinity, epitope specificity and domain recognition profile. The antibody profiles we obtained in this first study may be valuable for understanding of the mechanisms that influence the therapeutic capacity of these antibodies. In fact, one of the antibodies that we investigated is currently in phase I/II trials. However, all of the antibodies that we characterised in PAPER I are of mouse origin, which probably limits their clinical efficiency, due to the human anti-mouse response that most patients develop against such antibodies. Therefore we selected a set of human anti-CD40 antibodies, which are described in PAPER II, from a recombinant antibody gene library. These antibodies display a wide variety of distinct properties, which may make them a valuable source when evaluating therapeutic candidates for in vivo trials. In PAPER III, we have used some of the anti-CD40 antibodies described in PAPER II to create an antibody library that was utilised to investigate antibody evolution in vitro. The results from this study showed that events, which resembles receptor revision, i.e. secondary rearrangements of antibody genes in the periphery, may provide an evolving antibody with competitive advantages during a selection process that is similar to the affinity maturation process in vivo. Our data reinforce the suggestion that receptor revision is an important complement to point mutations and insertions and deletions in the somatic hypermutation process that occur in germinal centres. It has been suggested that members of the TNFR family pre-associate in the membrane via one of the extracellular domains, the pre-ligand assembly domain (PLAD). Therefore, in PAPER IV, we investigated the functional role of the different domains of CD40, in a B cell model system. The results from this study showed that neither of the extracellular domains is essential for signal transduction and, furthermore, implies that conformational changes play no critical role for the CD40 signalling pathway. Based on the findings that all of the extracellular part of CD40 can be replaced with retained signalling capacity, we developed a novel selection method, named Selection of Protein Interactions by Receptor Engagement (SPIRE). In PAPER V, we demonstrated that this selection system can be used for clonal enrichment of cells that display a mock-CD40 receptor, used as prey, on the surface by interaction with a certain bait protein. Thus, SPIRE allows for clonal selection of interacting protein pairs in a mammalian environment. SPIRE may have several different applications such as identification of tumour antigens or for molecular evolution of complex proteins.

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