For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets.
Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, highcontent imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins β1 and β6 or αV and β1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.
Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.
Induction of tumour-specific CD8+ T cell responses in melanoma patients is the major aim of antigen-specific immunotherapy. This study evaluated a prime-boost immunotherapeutic vaccine (Hi-8™ MEL) in stage III/IV melanoma patients using vectors encoding a polyepitope string of seven CD8+ T cell epitopes from five melanoma antigens. T cell responses were evaluated in cryopreserved PBMC samples taken before and during treatment. CD8+ T cells specific for the immunodominant Melan-A epitope were detected by ex vivo tetramer assay in 24/36 (67%) of all patients and in 10/11 (91%) of patients that received the highest treatment dose. Eleven of 36 (31%) patients mounted an ex vivo IFN-γ ELISPOT response: 9/36 (25%) to Melan-A and 3/36 (8%) to other epitopes in the string (NY-ESO-1 and MAGE-3). Notably, the Melan-A-tetramer+ CD8+ T cells in one patient reached 7.25% and acquired a CD45RA−CCR7− effector/memory phenotype. This patient also exhibited an ELISPOT response and a partial clinical response (>12 months). Across the study, tetramer responders showed a median survival of 86 weeks versus 37 weeks for non-responders (p<0.001), indicating that Hi-8™ MEL immunotherapy can elicit melanoma-specific CD8+ T cell responses that are associated with tumour control and increased survival. Additional immune assays are underway to further elucidate the nature of these efficacious T cell responses.
Therapeutic targeting of PC populations has potential value in various auto-immune, allergic and neoplastic cell states, but at present few options exist for selective targeting. However, the unique physiology and niche dependence of PCs suggests that such targeting may be achievable. To this end we have developed a sensitive screening platform capable of detecting changes in PC biology. Starting from our published methods for in vitro PC generation we optimised conditions to allow maintenance of PCs in polarised niche environments reflective of homeostatic and inflammatory conditions in microtitre plates. Using this assay format we probed PC biology via small molecule inhibitors (SMIs) targeting BCL2 family members, proteasome, and kinase signalling pathways. We were able to identify SMIs with broad and selective toxicity, as well as SMIs with no effect on PCs. Under all conditions analysed we found consistent divergence in the impact of targeting aspects of B cell receptor (BCR)-associated signalling pathways. Targeting SYK via two distinct SMIs consistently resulted in selective killing of subsets of PCs. By contrast BTK inhibition had no significant effect on PC viability. Whilst the importance of SYK signalling has been well documented at the B cell stage its importance to PCs has to our knowledge not been extensively documented. Retention of functional BCRs has been recently described in BM IgA+ and IgM+ PCs, and examination of expression data indicates selective retention of components of the BCR signalling complex in our assay system. The data presented here demonstrate the potential utility of an in vitro screening platform to evaluate the selective dependency of subsets of PCs on therapeutically targetable pathways.
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