Daniel John Lightwood

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength

The development of bone‐rebuilding anabolic agents for treating bone‐related conditions has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin‐neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin‐neutralizing monoclonal antibody (Scl‐AbIV) to gonad‐intact female cynomolgus monkeys. Two once‐monthly subcutaneous injections of Scl‐AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl‐AbIV treatment had clear anabolic effects, with marked dose‐dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl‐AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle‐treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest‐dose group. Taken together, the marked bone‐building effects achieved in this short‐term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.

Blockade of colony stimulating factor‐1 (CSF‐1) leads to inhibition of DSS‐induced colitis

Background Intestinal inflammation associated with inflammatory bowel disease (IBD) is typically characterized by an inflammatory cell infiltrate and pro‐inflammatory cytokine production. Of particular interest, the frequency of colony stimulating factor‐1 (CSF‐1)‐expressing cells is increased in active lesions. In this study, we have investigated the role of CSF‐1 in mucosal inflammation, using a murine model of colitis induced by dextran sulfate sodium (DSS). Methods A neutralizing anti‐CSF‐1 antibody was administered to Balb/c mice that received DSS in their drinking water. Signs of colitis, such as clinical disease score, cellular infiltrate, and cytokine production, were assessed. Results Administration of a neutralizing anti‐CSF‐1 antibody significantly inhibited DSS‐induced colitis. Clinical symptoms, such as weight loss and the appearance of diarrhea or fecal blood, were reduced by CSF‐1 blockade; histologic scores were also improved. The cellular infiltrate of macrophages and T cells was inhibited and a trend toward reduced production of pro‐inflammatory cytokines was noted. Conclusions This is the first study to demonstrate that CSF‐1 plays an important role in mediating intestinal mucosal inflammation and therefore may prove to be an attractive therapeutic target for intestinal diseases such as inflammatory bowel disease. (Inflamm Bowel Dis 2006)

A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels of In Vitro Potency Shows In Vivo Protection in a Hamster Infection Model

ABSTRACT Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts 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 vitro binding 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.

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.

Development of a therapeutic monoclonal antibody that targets secreted fatty acid-binding protein aP2 to treat type 2 diabetes.

A monoclonal antibody to fatty acid–binding protein aP2 has antidiabetic effects on glucose output and utilization. Kill the messenger A variety of metabolic messengers—many from adipose tissue itself—controls the energy state of organs and organisms. Recently, researchers showed that the fatty acid binding protein aP2, once thought to live and work only in the cytoplasm, is also secreted by adipose tissue and spurs metabolic changes in other organs. Now, Burak and colleagues test whether secreted aP2 can serve as a therapeutic target for type 2 diabetes. In mice, the secreted form of aP2 regulates glucose production in liver, systemic glucose homeostasis, and insulin resistance. Serum levels of aP2 were shown to be elevated in obese mice and humans and to correlate with metabolic complications. The authors identified a monoclonal antibody to aP2 that lowered fasting blood glucose, increased insulin sensitivity, and lowered both fat mass and fatty liver (steatosis) in obese mouse models, relative to a control antibody, but not in aP2-deficient mice. The antidiabetic effects of the therapeutic antibody were linked to the regulation of hepatic glucose output and peripheral glucose utilization. Together, these findings suggest that an aP2-targeted antibody that kills the messenger is a viable approach for diabetes treatment. The lipid chaperone aP2/FABP4 has been implicated in the pathology of many immunometabolic diseases, including diabetes in humans, but aP2 has not yet been targeted for therapeutic applications. aP2 is not only an intracellular protein but also an active adipokine that contributes to hyperglycemia by promoting hepatic gluconeogenesis and interfering with peripheral insulin action. Serum aP2 levels are markedly elevated in mouse and human obesity and strongly correlate with metabolic complications. These observations raise the possibility of a new strategy to treat metabolic disease by targeting serum aP2 with a monoclonal antibody (mAb) to aP2. We evaluated mAbs to aP2 and identified one, CA33, that lowered fasting blood glucose, improved systemic glucose metabolism, increased systemic insulin sensitivity, and reduced fat mass and liver steatosis in obese mouse models. We examined the structure of the aP2-CA33 complex and resolved the target epitope by crystallographic studies in comparison to another mAb that lacked efficacy in vivo. In hyperinsulinemic-euglycemic clamp studies, we found that the antidiabetic effect of CA33 was predominantly linked to the regulation of hepatic glucose output and peripheral glucose utilization. The antibody had no effect in aP2-deficient mice, demonstrating its target specificity. We conclude that an aP2 mAb–mediated therapeutic constitutes a feasible approach for the treatment of diabetes.

The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation.

We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.

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.

A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

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.

Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.

A2.24 Plasma cell derived monoclonal anti-citrulline antibodies from ra synovial fluid are multireactive

Background and objectives Human recombinant monoclonal antibodies have previously been generated at our laboratory from isolated patient-derived memory B cells to assess the repertoire and specificity of human anti-citrulline antibodies. The aim of the current study was to access joint-derived antibody secreting plasma cells and explore the proportion of citrulline reactivity, the immunoglobulin mutation patterns and antigen reactivity patterns. Clearly these antibodies can be utilised as tools and can help dissect the contribution antibodies make towards disease manifestations. Materials and methods Spontaneously IgG antibody-secreting cells (referred to as plasma cells) from synovial fluid of the inflamed joints of RA patients were isolated by a fluorescent foci method. The cells were identified and selected utilising a micromanipulator-equipped microscope. Recombinant monoclonal antibodies were generated from the isolated cells by amplification of the heavy and light chain variable antibody regions and were subsequently cloned into expression vectors. The monoclonal antibodies were screened for citrulline-reactivity by in-house ELISA or by a multiplex peptide array of RA associated antigens. Results We generated 375 IgG sequences and 101 recombinant antibodies from both ACPA+ (n = 3) and ACPA- (n = 1) patients. Four of the antibodies from ACPA+ synovial fluid displayed strong reactivity towards a citrullinated RA antigen peptide, but not to the cognate native peptide. The antibodies were multireactive to a diverse backbone with only the citrulline residue as common denominator, however with different reactivity patterns. The majority of the anti-citrulline clones are from the VH4 gene family. We found clonality within the plasma cells (including within the anti-citrulline antibodies), but also between different B cell populations. Conclusions Only a minority of the local plasma cells in the synovial fluid were autoreactive as measured by citrulline reactivity. However, the citrulline reactive plasma cells in the inflamed joint of RA patients where shown to be multireactive to several different citrulline peptides, with unique reactivity patterns. Further studies are needed to distinguish whether these plasma cells have differentiated locally in the joint. Still, it is tempting to speculate that the antibody characteristics that lead to multireactivity are driven by interaction towards several modified antigens, as has been suggested in the setting of influenza.