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Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

The gene encoding green fluorescent protein (GFP) from Aequorea victoria was resynthesized to adapt its codon usage for expression in plants by increasing the frequency of codons with a C or a G in the third position from 32 to 60%. The strategy for constructing the synthetic gfp gene was based on the overlap extension PCR method using 12 long oligonucleotides as the starting material and as primers. The new gene contains 101 silent nucleotide changes compared to its wild-type counterpart used in this study. Several transgenic tobacco lines containing the wild-type gfp gene contained minute amounts of a smaller protein cross-reacting with GFP antiserum, whereas only one protein of the expected size was found in transgenics with the synthetic gfp gene. The smaller protein was probably encoded by a truncated gfp mRNA created by splicing of a 84 bp cryptic intron as detected by a reverse transcription-PCR technique. A comparison of GFP production in transgenics with the wild-type and the synthetic gfp gene under the control of the enhanced CaMV 35S promoter showed that the large-scale alterations in the gfp gene increased the frequency of high expressors in the transgenic population but hardly changed the maximum GFP concentrations.The latter phenomenon may be attributed to a reduced regeneration capacity of transformed cells with higher GFP concentrations.

An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700–4200 J/m2 and those of 60Co gamma radiation over the range 0.15–1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m2 UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human β-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1

A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable.