Search

T cells are a cornerstone of the mammalian adaptive immune system. A range of T-cell subsets exist that can orchestrate the overall immune response to pathogens or cancers, either by directly killing infected cells or licensing other cells to do so. Dysregulation of this important process can result in immunodeficiency or autoimmunity. Although T cells have been studied extensively over many decades, the detailed mechanisms underlying T-cell activation remain to be fully resolved. This thesis describes the development of new optogenetic approaches for the modulation of T-cell signalling dynamics and the interrogation of key events in T-cell activation to help investigate this question. Optogenetics is a rapidly emerging technique whereby light can be used to control the spatial and temporal activation, or inactivation of signalling pathways at unprecedented resolution. The methods described in this work utilise the blue light-responsive LOV2 photo-domain from the common oat A. Sativa, which is the foundation of the both the ‘LOVTRAP’ and ‘TULIPs’ optogenetic toolkits. T-cell antigen receptor (TCR) microclusters arise early during the interaction between T cells and antigen presenting cells (APCs). These TCR signalling platforms contain the proteins necessary for sustained T-cell activation, yet the processes underlying their formation and dissociation are still not fully characterised as they have been difficult to investigate with current chemical and genetic manipulations of T cells. Using two optogenetics systems combining either LOVTRAP or TULIPs and the microcluster- scaffolding protein LAT (Linker for the Activation of T cells), it was possible to modulate early T-cell signalling events and measure functional outputs in real-time. Unfortunately, the biological limitations of these LAT-based systems meant that they could not be used to quantitatively investigate microcluster formation. However, in an alternative approach, a drug-inducible, light-controllable chimeric antigen receptor was successfully developed that yielded important new insights into the rapid rate of signal decay within the TCR signalling pathway and the temporal dynamics of T-cell activation over several timescales. T cell-dependent bispecific antibodies (TDBs) are a new class of immuno-therapeutics that can specifically direct a T-cell response towards tumours, by crosslinking the TCR complex to a surface- expressed target on the cancerous cells. However, their mechanism of action has not been studied in detail. The close apposition of the T cell and target cells driven by the TDB interaction can result in the steric exclusion of phosphatases, such as CD45, away from the TCR at the TDB-generated cell-cell interface due to their large, rigid extracellular domains. Using the myeloma-expressed antigen, FcRH5, it was found that membrane-proximal epitopes of FcRH5 drive more robust TCR clustering and increased CD45 exclusion than membrane distal epitopes, which strongly correlated with effective killing of the target cell. These findings have important implications for therapeutic design and implementation of TDBs.

Introduction: Immune checkpoint inhibitors and adoptive T-cell therapy based on chimeric antigen receptors are the spearhead strategies to exploit the immune system to fight cancer. To take advantage of the full potential of the immune system, cancer immunotherapy must incorporate new biotechnologies such as mRNA technology that may synergize with already approved immunotherapies and act more effectively on immune targets. Areas covered: This review describes the basics of mRNA biotechnology and provides insight into the recent advances in the use of mRNA for the local and systemic delivery of immunostimulatory antibodies, proinflammatory cytokines or for optimizing adoptive T-cell therapy. Expert opinion: mRNA-based nanomedicines have great potential to expand the arsenal of immunotherapy tools due to their ability to simplify and accelerate drug development and their suitability for transient and local expression of immunostimulatory molecules, whose systemic and sustained expression would be toxic. The success of mRNA-based COVID-19 vaccines has highlighted the feasibility of this approach. Continuous advances in the delivery and construction of RNA-based vectors hold promise for improvements in clinical efficacy.

Therapeutic antibodies are among the fastest growing class of biopharmaceuticals. This advance has been enabled by the development of various antibody formats through protein engineering, which alter the traditional structure of the IgG molecule. One such format is bispecific antibodies, which harbour two different antigen-binding specificities at either end of the Fab arms. Among the many novel uses for this format, is the capability to target two growth factor receptors simultaneously or to target a tumour specific receptor whilst simultaneously recruiting a T cell response to the site. Unfortunately, current methods used for producing bispecific antibodies are suboptimal and yield non-functional antibodies due to misassembled chains. Here we investigate strategies to overcome chain misassembly by a combination of phage display selection, mutagenesis, x-ray crystallography and mass spectrometry. We initially demonstrate a selection method using a VH phage display library, to identify substitutions that confer optimal VL-VH pairing, and from which a complementarity determining region (CDR) mutational hot spot was discovered. Consistently, the identified hot spot mutations were shown to enhance the affinity between cognate VH and VL domains. This affinity improvement could then be transferred to the full-length IgG molecule, producing superior heavy-light chain pairing when expressed in HEK293 cells, as judged by MS analysis. Crystal structures of this antibody were determined, suggesting that the identified CDR mutations enhance pairing through a “knobs into holes” mechanism, thereby increasing the shape complementarity of the VH-VL interface. To determine if our identified mutations could be more broadly applied to improve cognate heavy chain- light chain pairing, we analysed the VH-VL interface of other commercial antibodies for which structural data was available. These analyses suggested that other antibodies could be amenable to, and benefit from, our “knobs into hole” CDR mutational approach. Hence we produced a bispecific antibody under this rationale, and in combination with our mutational and structural strategy, were able to improve the cognate heavy-light chain pairing in this particular bispecific antibody. Collectively, this study suggests that CDR mutations that improve VH-VL affinity, can be directly transferred to the full-length IgG modality to enhance cognate heavy chain-light chain pairing and future work using our methodology may characterise additional CDR hotspots in other antibodies, to produce novel bispecific combinations