Patrizia Galeffi

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Site‐Specific Conjugation of ScFvs Antibodies to Nanoparticles by Bioorthogonal Strain‐Promoted Alkyne–Nitrone Cycloaddition

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

Fully "recombinant enzyme-linked immunosorbent assays" using genetically engineered single-chain antibody fusion proteins for detection of Citrus tristeza virus.

ABSTRACT Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.

Structure and dynamics of the anti-AMCV scFv(F8): effects of selected mutations on the antigen combining site.

The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the V(H) chain, playing a crucial role in the antigen binding recognition. Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen-binding site. Structural models of the wild type scFv(F8) and of its H47 and H58 mutants were built by homology modelling and assessed by multiple 15.5ns of molecular dynamics simulations. Computational results indicate that the 47H substitution strongly affects the CDR-H(2) conformation, destabilizes the V(H)/V(L) interface and confers high conformational flexibility to the antigen-binding site, leading the mutant to functional loss. The mutation at position H58 strenghtens the binding site, bestowing a high antigen specificity on the mutant. The essential dynamics and the analysis of the protein-solvent interface further corroborate the correspondence between the extent of the structurally-determined flexibility of the binding site with the different functional behaviours proved by the wild-type and its mutants. These results may have useful implications for structure-based design of antibody combining site.

Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells.

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface.

Understanding structural/functional properties of immunoconjugates for cancer therapy by computational approaches.

Abstract Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immuno- conjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti- HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies.

Dependence of nanoparticle-cell recognition efficiency on the surface orientation of scFv targeting ligands

The surface activation of multifunctional nanoparticles (MNPs) with peptide ligands directing their targeting to cancer cells is an emerging research area in nanobiotechnology. In this paper, water-soluble MNPs have been synthesized and functionalized with an scFv antibody variant specific toward the HER2 receptor overexpressed in several breast cancer cell lines. The scFv was genetically engineered to introduce a cysteine residue inside the loop sequence bridging the VH and VL lobes of the molecule and a histidine tag at the C-terminus in the VL fragment. The Cys and 6 × His functionalities were exploited as orthogonal reactive groups driving the scFv conjugation to MNPs. In this way, scFv positioning on the MNP surface was forced into two different orientations depending on the molecular binding site used for conjugation. The resulting scFv-functionalized MNP1 and MNP2, respectively, were assessed as to their labeling efficiency and selectivity to HER2-positive MCF7 cells. We demonstrate that, while both MNP1 and MNP2 were selective for HER2, there is a remarkable preference for scFv presentation with VH and VL lobes concurrently available for receptor recognition (MNP1) in terms of cell binding efficiency, suggesting that ligand orientation may strongly affect cell binding from MNPs.

Engineering of a Single Chain Variable Fragment Antibody Specific for the Citrus tristeza virus and its Expression in Escherichia coli and Nicotiana tabacum

Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed.

Variability and expression profile of the DRF1 gene in four cultivars of durum wheat and one triticale under moderate water stress conditions

The dehydration responsive element binding (DREB) proteins are important transcription factors that contribute to stress endurance in plants triggering the expression of a set of abiotic stress-related genes. A DREB2-related gene, previously referred to as dehydration responsive factor 1 (DRF1) was originally isolated and characterized in durum wheat. The aim of this study was to monitor the expression profiles of three alternatively spliced TdDRF1 transcripts during dehydration experiments and to evaluate the effects of genetic diversity on the molecular response, using experimental conditions reflecting as closely as possible water stress perceived by cereals in open field. To investigate the effect of moderate water stress conditions, time-course dehydration experiments were carried out under controlled conditions in the greenhouse on four durum wheat and one triticale genotypes. Differences were observed in molecular patterns, thus, suggesting a genotype dependency of the DRF1 gene expression in response to the stress induced. The biodiversity of the transcripts of the DRF1 gene was explored in order to assess the level of polymorphism and its possible effects on structure and function of putative proteins. A total of nine haplotypes were identified in the sequences cloned, seven of which encompassing polymorphisms in exon 4, including the region codifying for the DNA binding Apetala2 (AP2) domain. The 3D structural models of the AP2 domain were generated by homology modelling using the variability observed. The polymorphisms analysed did not significantly affect the structural arrangement of the DNA binding domains, thus resulting compatible with the putative functionality.

Genomic characterization of Phenylalanine ammonia lyase gene in buckwheat

Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.