COI Project 2: Modulating immune responses in cancer and inflammation by rewiring receptor signalling via phosphatase recruitment

About the Project

Signalling through immunoreceptor tyrosine activation motifs (ITAMs) underpins a broad range of immune activation processes, from pathogen defence to allergic inflammation. Aberrant ITAM signalling drives hyperactivation in diseases such as asthma and atopic dermatitis, yet current biologics, including omalizumab, primarily act by neutralising ligands rather than directly regulating receptor phosphorylation. We have developed a new technology, Receptor Inhibition by Phosphatase Recruitment (RIPR), which uses bispecific molecules to recruit surface phosphatases like CD45 in cis to target receptors, effectively silencing tyrosine phosphorylation and rewiring signalling pathways. This project will extend the RIPR concept to ITAM-containing receptors, such as FcεRI, with the goal of directly suppressing activation without blocking receptor-ligand interactions. The work will define how targeted phosphatase recruitment can dampen activation, test the concept in physiologically relevant primary cells, and build translational models to predict the therapeutic potential of RIPR molecules compared with current standards of care.

Background and Rationale

Our group recently demonstrated that RIPR can be used to shut down signalling by inhibitory checkpoint receptors such as PD-1, CTLA-4, and SIRPα, which contain ITIM and ITSM motifs that recruit intracellular phosphatases to attenuate immune responses (Fernandes et al., Nature 2020). By enforcing phosphatase proximity in cis, RIPR bypasses the need for antibody blockade and efficiently silences receptor signalling. This strategy has proven effective in reversing T-cell inhibition and shows promise for therapeutic development across multiple immune checkpoints.

However, while inhibitory receptor signalling can be silenced through ITIM/ITSM dephosphorylation, there are currently no effective strategies to shut down signalling by activatory receptors that signal through ITAM motifs. A prime example is the high-affinity IgE receptor FcεRI, which is expressed on mast cells, basophils, and eosinophils. Engagement of FcεRI by IgE-antigen complexes triggers a powerful activation cascade, driving degranulation and release of histamine, prostaglandins, and cytokines that mediate allergic inflammation and anaphylaxis. Despite its central role in allergy, directly modulating FcεRI signalling has been remarkably difficult. The receptor’s multimeric nature, high surface density, and constitutive association with signalling kinases make it resistant to standard inhibitory approaches. Existing drugs such as omalizumab target free IgE to prevent receptor crosslinking, but do not influence receptor phosphorylation once complexes are formed.

RIPR offers a conceptually new way to tackle this problem by recruiting a phosphatase directly to the activated FcεRI complex, allowing targeted dephosphorylation of ITAM tyrosines and termination of downstream signalling, even after receptor engagement. Extending RIPR to this context would establish a new mechanism to silence activatory receptor pathways and potentially transform the treatment landscape for allergic and inflammatory diseases.

Research Objectives

1. Develop and determine the ability of RIPR molecules to disrupt ITAM-mediated signalling, independent of ligand binding, in engineered cell lines.
2. Confirm that phosphatase recruitment maintains its inhibitory function in primary cells expressing endogenous FcεRI and CD45.
3. Develop translational models to compare the predicted efficacy of RIPR molecules with standard therapies such as omalizumab.

Experimental Approach

The project will begin with the molecular reconstruction of known monoclonal antibodies targeting FcεRI and CD45, using published and patented sequences as templates. These will serve as the building blocks for bispecific RIPR molecules designed to recruit CD45 to the FcεRI complex. The student will then establish engineered reporter cell lines expressing the complete FcεRI complex, allowing quantitative measurement of ITAM phosphorylation and downstream signalling responses using phospho-flow cytometry, calcium imaging, super-resolution imaging and transcriptional reporters.

Once validated in engineered cells, the project will move to primary systems that naturally express FcεRI, for instance, human basophils or mast-cell-like cells derived from haematopoietic progenitors. These assays will provide a physiologically relevant setting to test whether RIPR molecules can suppress, or even reset, the activation triggered by IgE-antigen complexes by targeting FcεRI directly.

Parallel experiments will evaluate how RIPR-mediated inhibition compares mechanistically and quantitatively with omalizumab, which acts by blocking free IgE. By combining experimental data with computational models of receptor occupancy and signal propagation, the project will derive quantitative predictions of efficacy that bridge in vitro biology with translational potential. This integrated approach, spanning molecular design, cell engineering, and modelling, will yield a rich framework to understand and exploit receptor rewiring as a therapeutic principle.