Comparative phenotype of circulating versus tissue immune cells in human lung and blood compartments during health and disease.

The lung is a dynamic mucosal surface constantly exposed to a variety of immunological challenges including harmless environmental antigens, pollutants, and potentially invasive microorganisms. Dysregulation of the immune system at this crucial site is associated with a range of chronic inflammatory conditions including asthma and Chronic Pulmonary Obstructive Disease (COPD). However, due to its relative inaccessibility, our fundamental understanding of the human lung immune compartment is limited. To address this, we performed flow cytometric immune phenotyping of human lung tissue and matched blood samples that were isolated from 115 donors undergoing lung tissue resection. We provide detailed characterization of the lung mononuclear phagocyte and T cell compartments, demonstrating clear phenotypic differences between lung tissue cells and those in peripheral circulation. Additionally, we show that CD103 expression demarcates pulmonary T cells that have undergone recent TCR and IL-7R signalling. Unexpectedly, we discovered that the immune landscape from asthmatic or COPD donors was broadly comparable to controls. Our data provide a much-needed expansion of our understanding of the pulmonary immune compartment in both health and disease.

Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients.

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.

Novel Selection Approaches to Identify Antibodies Targeting Neoepitopes on the C5b6 Intermediate Complex to Inhibit Membrane Attack Complex Formation.

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein-protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.