Meryn Griffiths

Structure and Interactions of the Human Programmed Cell Death 1 Receptor

Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2. Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1. Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected. Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors. PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

High-Throughput Screening for High Affinity Antibodies

UCB Selected Lymphocyte Antibody Method (SLAM) is a rapid and efficient process for the generation of high-quality monoclonal antibodies, in which variable region gene sequences are recovered directly from specific, single B cells. Monoclonal antibody generation has been limited in the past by the relatively low efficiency of the hybridoma process. UCB SLAM process is well suited to high-throughput screening and has been extensively automated at UCB. If necessary, in excess of 1 times 10 9 B cells can be screened in a campaign, to discover a rare therapeutic antibody candidate, which meets the stringent selection criteria. Primary screening for antigen binders, on purified or cell expressed antigen, is performed using a homogeneous fluorescence assay format. Supernatants from positive wells are consolidated to allow further secondary screening and selection of antibodies with desired characteristics. Individual, specific B cells are identified using a fluorescence based method and isolated using a micromanipulator. The antibody variable region genes are cloned from DNA extracted from the single B cell. The genes are sequenced then prepared for transient expression to confirm activity. Antibodies with affinities (K D) in the sub 10 pM range against a range of therapeutic targets are routinely recovered using this process.

The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method

Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.

Dual IL-17A and IL-17F neutralisation by bimekizumab in psoriatic arthritis: evidence from preclinical experiments and a randomised placebo-controlled clinical trial that IL-17F contributes to human chronic tissue inflammation

Objective Interleukin (IL)-17A has emerged as pivotal in driving tissue pathology in immune-mediated inflammatory diseases. The role of IL-17F, sharing 50% sequence homology and overlapping biological function, remains less clear. We hypothesised that IL-17F, together with IL-17A, contributes to chronic tissue inflammation, and that dual neutralisation may lead to more profound suppression of inflammation than inhibition of IL-17A alone. Methods Preclinical experiments assessed the role of IL-17A and IL-17F in tissue inflammation using disease-relevant human cells. A placebo-controlled proof-of-concept (PoC) clinical trial randomised patients with psoriatic arthritis (PsA) to bimekizumab (n=39) or placebo (n=14). Safety, pharmacokinetics and clinical efficacy of multiple doses (weeks 0, 3, 6 (240 mg/160 mg/160 mg; 80 mg/40 mg/40 mg; 160 mg/80 mg/80 mg and 560 mg/320 mg/320 mg)) of bimekizumab, a humanised monoclonal IgG1 antibody neutralising both IL-17A and IL-17F, were investigated. Results IL-17F induced qualitatively similar inflammatory responses to IL-17A in skin and joint cells. Neutralisation of IL-17A and IL-17F with bimekizumab more effectively suppressed in vitro cytokine responses and neutrophil chemotaxis than inhibition of IL-17A or IL-17F alone. The PoC trial met both prespecified efficacy success criteria and showed rapid, profound responses in both joint and skin (pooled top three doses vs placebo at week 8: American College of Rheumatology 20% response criteria 80.0% vs 16.7% (posterior probability >99%); Psoriasis Area and Severity Index 100% response criteria 86.7% vs 0%), sustained to week 20, without unexpected safety signals. Conclusions These data support IL-17F as a key driver of human chronic tissue inflammation and the rationale for dual neutralisation of IL-17A and IL-17F in PsA and related conditions. Trial registration number NCT02141763; Results.

THU0038 Bimekizumab dual inhibition of IL-17A and IL-17F provides evidence of IL-17F contribution to chronic inflammation in disease-relevant cells

Background IL-17A and IL-17F share structural homology and have similar biological function1. Although the contribution of IL-17A to immune-mediated inflammatory diseases has been widely reported1–3, the role of IL-17F is less well characterised in human tissue inflammation. Bimekizumab, a humanised monoclonal IgG1 antibody, was developed to neutralise both IL-17A and IL-17F potently and selectively, and is under clinical development as a treatment for psoriatic arthritis (PsA) and other immune-mediated conditions such as psoriasis. Objectives To assess the involvement of IL-17F in chronic inflammation in tissue from patients with PsA and disease-relevant cells, and to determine the effect of dual neutralisation of IL-17A and IL-17F in suppressing inflammation, compared with blockade of IL-17A. Methods Synovial and lesional skin tissue from patients with PsA was probed by immunostaining for expression of IL-17F protein. Normal dermal fibroblasts and synoviocytes, in the presence of TNFα, were stimulated with recombinant IL-17A and IL-17F to assess the inflammatory response. Using cytokine-specific blocking antibodies, the individual and combined effects of IL-17A and IL-17F were explored with: pro-inflammatory cytokine expression in a complex in vitro model (synoviocytes from patients with PsA and normal dermal fibroblasts were treated with pro-inflammatory mediators from supernatant [SN] of sorted Th17 cells), microarray and cell migration studies. Results IL-17F expression was observed in tissue biopsies from patients with PsA. In normal dermal fibroblasts, normal synoviocytes and synoviocytes from patients with PsA, stimulation with recombinant IL-17F promoted production of pro-inflammatory mediators, such as IL-6 and IL-8, though to a lesser extent than with recombinant IL-17A. Treatment of Th17 SN-stimulated synoviocytes from patients with PsA with bimekizumab (neutralising IL-17A and IL-17F) led to greater reductions of IL-6 (42% lower p<0.05) and IL-8 (35% lower p<0.05) production than IL-17A inhibition. Bimekizumab treatment of Th17 SN-stimulated normal dermal fibroblasts also reduced production of IL-6 (35% lower p<0.0001) and IL-8 (57% lower p<0.0001) more than IL-17A alone. Combining IL-17A + IL-17F monoclonal antibodies produced similar results to bimekizumab. Levels of expression of 27 inflammation-linked genes, including CXCL1, CXCL2, CXCL3 and IL-15RA, were lower with dual neutralisation of IL-17A and IL-17F by bimekizumab versus inhibition of IL-17A. Suppression of migration of neutrophils (Fig.) and monocytes, both involved in tissue destruction in immune-mediated diseases, was substantially greater with bimekizumab treatment than with single blockade of IL-17A. Conclusions Dual neutralisation of IL-17A and IL-17F provides evidence for the contribution of IL-17F to inflammation in joints and skin beyond IL-17A alone. As a result, dual inhibition of IL-17A and IL-17F by bimekizumab may provide an effective treatment for immune-mediated inflammatory diseases such as PsA. References C Johansen et al. Br J Dermatol 2009:160:319–24. AL Lima et al. Br J Dermatol 2016:174:514–21. C Doe et al. Chest 2010:138:1140–7. Disclosure of Interest A. Maroof Employee of: UCB Pharma, R. Okoye Employee of: UCB Pharma, T. Smallie Employee of: UCB Pharma, D. Baeten Grant/research support from: UCB Pharma, Consultant for: AbbVie, Pfizer, MSD, Roche, BMS, Novartis, Eli Lilly, Boehringer Ingelhaim and Glenmark, S. Archer Employee of: UCB Pharma, C. Simpson Employee of: UCB Pharma, M. Griffiths Consultant for: UCB Pharma, Employee of: UCB Pharma, S. Shaw Employee of: UCB Pharma

02.13 Il-17f contributes to human chronic inflammation in synovial tissue: Preclinical evidence with dual il-17a and il-17f inhibition with bimekizumab in psoriatic arthritis

Background IL-17A is an established target in several chronic immune-mediated inflammatory diseases; IL-17F, sharing significant structural homology and overlapping biological function with IL-17A,1 is a relatively understudied cytokine. Bimekizumab is a humanised monoclonal IgG1 antibody that potently and selectively neutralises the biological function of both IL-17A and IL-17F. Using preclinical human in vitro models we studied the contribution of IL-17F to inflammation, via stimulation and blocking assays, in joint cells from patients with psoriatic arthritis (PsA). Materials and methods Immunostaining was performed on synovial tissue from patients with PsA to probe for IL-17F. Recombinant IL-17A and IL-17F, with/without TNFα, were added to synoviocytes from patients with PsA, to examine their effect on inflammatory cytokine production. A complex in vitro assay was developed, using pro-inflammatory mediators from sorted Th17 cells, to explore the mechanism of action of bimekizumab and relative contributions of IL-17A and IL-17F to chronic disease. The Th17-cell supernatant profile had a complex mix of disease-relevant pro-inflammatory cytokines akin to those found elevated in joint tissue biopsies from patients with PsA. IL-17-isoform-specific blocking antibodies provided an overview of the individual and collective influence of IL-17A and IL-17F in regulating key disease-pathology-relevant cytokine and chemokine production. Results IL-17F protein was detected in inflamed synovium from patients with PsA. Stimulation of normal synoviocytes with recombinant IL-17A or IL-17F promoted qualitatively similar responses, notably induction of key pro-inflammatory mediators (eg, IL-8 and IL-6), albeit to a lesser extent with IL-17F than IL-17A. Similar results were obtained in synoviocytes from patients with PsA, stimulated in the presence of TNFα. When synoviocytes from patients with PsA were stimulated with Th17-cell supernatant, dual IL-17A and IL-17F inhibition with bimekizumab resulted in a greater downregulation of IL-6 (42% lower; p<0.05) and IL-8 (35.4% lower; p<0.05) expression than inhibition of IL-17A alone. Similar results were obtained using monospecific anti-IL-17A and anti-IL-17F antibodies. Conclusions Data support hypotheses that IL-17F contributes to human tissue inflammation in joints, beyond IL-17A alone, and that dual inhibition of IL-17A and IL-17F may be therapeutically beneficial in immune-mediated inflammatory diseases. Acknowledgements The authors thank the subjects and their caregivers in addition to the investigators and their teams who contributed to this study. This study was funded by UCB Pharma. The authors acknowledge the contributions of Tim Smallie, of UCB, Louise Healy, formerly of UCB, for their work on functional characterisation of bimekizumab. The authors also acknowledge the contribution of Catherine Simpson, of UCB, for flow cytometry cell sorting support, Remi Okoye, of UCB, for the immunohistochemistry work, and Iris Blijdorp of the Academic Medical Centre, Amsterdam, for work on blockade experiments in synoviocytes. The authors would like to acknowledge Ailsa Dermody, PhD, of iMed Comms, an Ashfield Company, part of UDG Healthcare plc, for editorial assistance that was funded by UCB Pharma. Reference Johansen C, Usher PA, Kjellerup RB, et al,.. Characterisation of the interleukin-17 isoforms and receptors in lesional psoriatic skin. Br J Dermatol 2009; 160(2): 319-24.