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Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy- and light-chain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells.

In a randomized, double-blind study, we administered placebo and flumazenil to 40 healthy Chinese boys, aged 3-12 yr, undergoing circumcision. The children received midazolam 0.5 mg kg-1 orally for premedication and 0.5 mg kg-1 i.v. during induction. After operation the patients were given 0.1 ml kg-1 of a blinded solution followed by 0.05 ml kg-1 min-1 until either they awoke or the 10-ml ampoule of solution was empty. Efficacy of antagonism of midazolam was assessed by times to eye opening and self identification, modified Steward coma scale, a post-box toy completion-time ratio and qualitatively by an independent observer. The difference between flumazenil and placebo was both clinically and statistically different in the first 2 h. Children receiving flumazenil awoke approximately four times faster and identified themselves nearly three times sooner; 65% of this group could complete the post-box toy at 10 min, compared with none of the placebo group. There were no cases of resedation, but one child did not awaken for 30 min after i.v. administration of flumazenil 1.0 mg. The mean total dose of flumazenil administered was 0.024 (SD 0.019) mg kg-1. Flumazenil rapidly antagonized midazolam-induced hypnosis in children and was associated with minimal change in cardiorespiratory variables.