SummaryPublic concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection / regeneration strategies.

Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man 5 GlcNAc 2 -PP-Dol a1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Dalg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response.

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 ␤-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic corebound 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.biopharmaceutical ͉ glycosylation ͉ immunogenicity

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Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HCABD/LCABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LCABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.

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 antiproliferative 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.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reverse-phase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

Degenerate oligonucleotides to highly conserved regions of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14), the key enzyme in ethylene biosynthesis, were used to prime the synthesis and amplification of fragments of about 1,180 bp by polymerase chain reaction (PCR) in samples of cDNA to total RNA isolated from senescing carnation (Dianthus caryophyllus) flowers. Two putative ACC synthase PCR clones were isolated one of which was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (Plant Mol Biol 18 (1992) 377-386). The other clone (CARAS1) was ca. 66% homologous at the amino acid level to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized and, using PCR, we were able to distinguish between the two ACC synthase transcripts in samples of total RNA isolated from different carnation flower parts and leaves. DNA blots of PCR fragments revealed that, in flowers, both ageing and ethylene stimulated the occurrence of these transcripts in an organ-specific way. CARACC3 was more abundant in RNA from the petals whereas CARAS1 was more abundant in RNA from the styles. Despite a high ethylene production observed in ovaries, the level of both transcripts was low, suggesting the existence of a third ACC synthase gene that is specifically expressed in the ovary. Transcript levels in leaves were low irrespective of treatment.