Enhancing anti-tumour t cell function by controlled inhibition of checkpoint receptor signalling

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In the past decade, immune checkpoint blockade has emerged as a major therapeutic advance in immunotherapy. However, only a small subset of cancer patients respond to checkpoint blockade, suggesting a fundamental understanding of the basic mechanisms of immune checkpoint receptor signalling is lacking. Novel therapeutic drugs must be developed. This PhD project aims to develop a novel approach to potentiate T cell function and to understand how checkpoint receptors dampen T cell function. Regulation of T-cell signalling by immune checkpoints such as PD-1 and CTLA-4 has been at the centre of recent breakthroughs in cancer immunotherapy. Signalling by PD-1 and CTLA-4 reduces T cell activity and contributes to an "exhausted" phenotype, severely compromising antitumor responses. In the case of PD-1, binding to PD-L1/2 triggers the tyrosine phosphorylation of signalling motifs and results in the recruitment of cytosolic phosphatases such as SHP1/2, which in turn reduces TCR and CD28 signalling. Strikingly, signalling by several immune receptors relies on the Tyr phosphorylation of ITAM/ITIM/ITSM signalling motifs. We hypothesise that tonic receptor phosphorylation and sustained signalling by 'ligand-experienced' receptors impact T cell function and fails to be controlled by extracellular antagonist antibodies. To address this issue, we previously engineered a bi-specific molecule to recruit CD45, an abundant and promiscuous receptor tyrosine phosphatase, within close proximity of PD-1. In this approach, the phosphatase domain of CD45 acts intracellularly, in cis, on the p-Tyr residues of the PD-1 ITIM/ITSM motif, thus inhibiting sustained signalling. This demonstrated that Receptor Inhibition by Phosphatase Recruitment (RIPR) potentiates T cell activity beyond that seen with PD-1/PD-L1 antagonist antibodies, both in the presence and absence of PD-1 ligand-binding in vitro, and reduces tumour growth in mouse models of small cell lung cancer and colon adenocarcinoma (Fernandes et al., Nature, 2020). In this PhD project, we propose to expand this novel approach to shut down signalling by key immune and cancer-specific receptors aimed at generating novel antitumor, RIPR-based, molecules.We propose to develop novel RIPR proteins to shut down signalling by inhibitory checkpoint receptors expressed in cytotoxic T cells, including BTLA, CTLA-4, ILT2 and ILT4. We will systematically test the potency of newly generated RIPR proteins using various anti-CD45 nanobodies. Nanobodies against CD45 isoforms and other phosphatases found in immune cells will also be developed and tested for their ability to shut down inhibitory signalling. Newly developed RIPR molecules will be characterized in biophysical assays. Binding on-rate, off-rate and affinity will be determined by surface plasmon resonance. Next, early readouts for signalling potency will be determined with in vitro activation assays, using T cells, NK cells or macrophages. Candidate RIPR molecules will also be tested for the ability to potentiate T cell cytotoxic functions using co-culture assays with T cells and target cancer cells. Markers of T cell activation will be quantified longitudinally using flow cytometry, western blotting, ELISAs and RNA-Seq. This comprehensive approach is expected to identify determinants of RIPR activity for various checkpoint receptors. This information will guide the design of future antagonists of checkpoint receptor signalling with strong potential for therapeutic applications.