Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the ...in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies.
A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows ...the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 μM and 500 μM, and 100% for 1000 μM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.
The major impediment to the development of murine monoclonal antibodies (mAbs) for therapy in humans has been the difficulty in reducing their potential immunogenicity. XenoMouse™ mice obviate this ...problem while retaining the relative ease of generating mAbs from a mouse. XenoMouse strains include germline-configured, megabase-sized YACs carrying portions of the human IgH and Igκ loci, including the majority of the variable region repertoire, the genes for Cμ, Cδ and either Cγ1, Cγ2, or Cγ4, as well as the
cis elements required for their function. The IgH and Igκ transgenes were bred onto a genetic background deficient in production of murine immunoglobulin. The large and complex human variable region repertoire encoded on the Ig transgenes in XenoMouse strains support the development of large peripheral B cell compartments and the generation of a diverse primary immune repertoire similar to that from adult humans. Immunization of XenoMouse mice with human antigens routinely results in a robust secondary immune response, which can ultimately be captured as a large panel of antigen-specific fully human IgGκ mAbs of sub-nanomolar affinities. Monoclonal antibodies from XenoMouse animals have been shown to have therapeutic potential both in vitro and in vivo, and appear to have the pharmacokinetics of normal human antibodies based on human clinical trials. The utility of XenoMouse strains for the generation of large panels of high-affinity, fully human mAbs can be made available to researchers in the academic and private sectors, and should accelerate the development and application of mAbs as therapeutics for human disease.
Technical advances made in the 1980s and early 1990s resulted in monoclonal antibodies that are now approved for human therapy. Novel transgenic mouse strains provide a powerful technology platform ...for creating fully human monoclonal antibodies as therapeutics; ten such antibodies have entered clinical trials since 1998 and more are in preclinical testing. Improved transgenic mouse strains provide a powerful technology platform for creating human therapeutics in the future.
After more than a decade of research and breakthroughs in genetic engineering, transgenic mice are producing fully human monoclonal antibodies suitable as human therapeutics, some of which are now entering pivotal clinical trials.
Abstract The objective of this study was to determine the impact of a bout of ruminal acidosis on energy metabolism of cattle unadapted to a high concentrate diet. Eleven ruminally cannulated steers ...body weight (BW) = 352 kg ± 27 were blocked into 3 groups based on initial BW. Before the start of the experiment, animals were consuming a forage-based diet as well as adapted to the headbox style respiration chambers. Additionally, before the experiment, gas emission data were collected over a 24-h period when cattle received an ad libitum forage-based diet for use as a covariate in the statistical analysis. For the experiment, steers were moved into headboxes at the conclusion of a 24-h fast and subsequently received 1 of 2 treatment diets: control (CON), forage-based diet or acidosis (AC), concentrate-based diet. Steers remained in the headboxes for 48 h. Gas concentrations from each headbox were collected hourly and analyzed with an infrared photoacoustic gas analyzer. Ruminal pH, fecal pH, volatile fatty acids, and lactate were also measured during the acidosis challenge. Data were analyzed with the MIXED procedure of SAS 9.4. A treatment × day effect (P = 0.03) was observed for dry matter intake with intake being similar for both CON and AC steers on d 1 but AC steers consuming 2.02 kg less on d 2. Treatment affected ruminal pH (P < 0.01) as CON steers had a greater ruminal pH than AC steers. Greater total volatile fatty acids (P < 0.01) were observed for steers on AC treatment compared with CON. A treatment × time interaction (P < 0.01) was observed for ruminal lactate concentration with AC steers having greater concentrations from h 16 to 36. Fecal pH was affected by a treatment × time interaction (P = 0.05) as AC steers had lower fecal pH from h 24 to h 32. Over the 48-h observation period, there was a treatment × time interaction (P = 0.04) for carbon dioxide production as AC steers produced less CO2 after h 24. No treatment × time interaction was detected for oxygen consumption. However, there was a time effect (P < 0.01). Respiratory quotient was not affected by treatment, day, or their interaction (P ≥ 0.19). Heat production tended (P = 0.06) to be different between treatments, with AC having less heat production than CON. Thus, the effects of ruminal acidosis on gas emissions and energetics may be more transient during the time course of an acidotic bout or have a longer duration even after ruminal pH has recovered.
Abstract
The objective of this study was to determine the impact of a bout of ruminal acidosis on gas emissions of cattle unadapted to a high concentrate diet. Eleven ruminally cannulated steers ...(body weight = 352 kg ± 27) were blocked into 3 groups based on initial body weight. Prior to the start of the experiment, animals were consuming a forage-based diet as well as adapted to the headbox style respiration chambers. Additionally, prior to the experiment, gas emission data were collected over a 24-hour period when cattle received an ad libitum forage-based diet for use as a covariate in the statistical analysis. For the experiment, steers were moved into headboxes at the conclusion of a 24 hour fast and subsequently received 1 of 2 treatment diets: control (CON), forage-based diet or acidosis (ACID), concentrate-based diet. Steers remained in the headboxes for 48 hours. Gas concentrations from each headbox were collected hourly and analyzed with an infrared photoacoustic gas analyzer. Data were analyzed with the MIXED procedure of SAS 9.4. There was a tendency for a treatment × day effect (P = 0.09) with steers on the ACID treatment consuming 1.95 kg less on day 2 than CON steers. Dry matter intake was affected by day (P < 0.01) with steers consuming 4.35 kg less on day 2. There was an effect of treatment (P < 0.01) with CON steers having a greater ruminal pH than ACID steers. Steers on the ACID treatment had less CO2 emissions (P < 0.01) than CON steers, but there were no observed differences (P ≥ 0.19) in O2 emissions or respiratory quotient. Acidosis decreased dry matter intake and CO2 emissions but not O2 emissions for cattle unadapted to a high concentrate diet.
The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. ...Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.
Summary
Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop new ...agents with improved efficacy. Towards this end, using XenoMouse® technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity
in vitro,
as compared to rituximab
,
in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcγRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an
in vitro
whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.
We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, ...complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex® multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity.
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