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ABSTRACTMicrotubule-associated protein tau is inextricably linked to a group of clinically diverse neurodegenerative diseases termed tauopathies. The ratio balance of the major tau splicing isoform groups (3 R- and 4 R-tau) is critical in maintaining healthy neurons. An imbalance causing excess 4 R tau is associated with diseases such as progressive supranuclear palsy and frontotemporal dementia. The mechanisms by which increased 4 R results in neuronal dysfunction and neurodegeneration are not fully understood, and progress has been limited partly by a lack of suitable tools to investigate tau isoform imbalance. This work generated novel 3 R- and 4 R-specific antibody tools and 4 R-tau degrading intracellular antibody fragment “degrabodies”. These were used to probe the molecular mechanisms of excess 4 R-tau in disease-mutant induced pluripotent stem cell-derived neurons. For the first time, we demonstrate a causative link between excess 4 R-tau and mitochondrial membrane hyperpolarization with wide-ranging potential for elucidating novel therapeutic approaches to treat neurodegenerative disease.

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.

ABSTRACTRozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

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.

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.

ABSTRACTClostridium difficileinfections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficileinfection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficileexerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitrobinding and neutralization assays (~10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

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 109B 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 (KD) in the sub 10 pM range against a range of therapeutic targets are routinely recovered using this process.

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 109B 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 (KD) in the sub 10 pM range against a range of therapeutic targets are routinely recovered using this process.

We describe therapeutic monoclonal antibodies isolated from human volunteers vaccinated with recombinant adenovirus expressing Ebola virus glycoprotein (EBOV GP) and boosted with modified vaccinia virus Ankara. Among 82 antibodies isolated from peripheral blood B cells, almost half neutralized GP pseudotyped influenza virus. The antibody response was diverse in gene usage and epitope recognition. Although close to germline in sequence, neutralizing antibodies with binding affinities in the nano- to pico-molar range, similar to “affinity matured” antibodies from convalescent donors, were found. They recognized the mucin-like domain, glycan cap, receptor binding region, and the base of the glycoprotein. A cross-reactive cocktail of four antibodies, targeting the latter three non-overlapping epitopes, given on day 3 of EBOV infection, completely protected guinea pigs. This study highlights the value of experimental vaccine trials as a rich source of therapeutic human monoclonal antibodies.

A monoclonal antibody to fatty acid–binding protein aP2 has antidiabetic effects on glucose output and utilization.