Determination of insecticidal Cry1Ab protein in soil collected in the final growing seasons of a nine-year field trial of Bt-maize MON810.

Cultivation of genetically modified maize (Bt-maize; event MON810) producing recombinant δ-endotoxin Cry1Ab, leads to introduction of the insecticidal toxin into soil by way of root exudates and plant residues. This study investigated the fate of Cry1Ab in soil under long-term Bt-maize cultivation in an experimental field trial performed over nine growing seasons on four South German field sites cultivated with MON810 and its near isogenic non Bt-maize variety. Cry1Ab protein was quantified in soil (<2 mm size) using an in-house validated ELISA method. The assay was validated according to the criteria specified in European Commission Decision 2002/657/EC. The assay enabled quantification of Cry1Ab protein at a decision limit (CCα) of 2.0 ng Cry1Ab protein g(-1) soil with analytical recovery in the range 49.1-88.9%, which was strongly correlated with clay content. Cry1Ab protein was only detected on one field site at concentrations higher than the CCα, with 2.91 and 2.57 ng Cry1Ab protein g(-1) soil in top and lower soil samples collected 6 weeks after the eighth growing season. Cry1Ab protein was never detected in soil sampled in the spring before the next farming season at any of the four experimental sites. No experimental evidence for accumulation or persistence of Cry1Ab protein in different soils under long-term Bt-maize cultivation can be drawn from this field study.

Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment.

The objective of the study was to track the fate of recombinant Cry1Ab protein in a liquid manure field trial when feeding GM maize MON810 to dairy cows. A validated ELISA was applied for quantification of Cry1Ab in the agricultural chain from GM maize plants, feed, liquid manure and soil to crops grown on manured fields. Starting with 23.7 µg of Cry1Ab g(-1) dry weight GM maize material, a rapid decline of Cry1Ab levels was observed as 2.6% and 0.9% of Cry1Ab from the GM plant were detected in feed and liquid manure, respectively. Half of this residual Cry1Ab persisted during slurry storage for 25 weeks. After application to experimental fields, final degradation of Cry1Ab to below detectable levels in soil was reported. Cry1Ab exhibited a higher rate of degradation compared to total protein in the agricultural processes. Immunoblotting revealed a degradation of the 65 kDa Cry1Ab into immunoreactive fragments of lower size in all analyzed materials.

Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach.

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.