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

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 immunoconjugates 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.

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.

Molecular studies on DNA sequences coding for factor VII and factor XII of human coagulation.

In this paper are described the immunological and molecular procedures that have allowed the identification and the nucleotide sequence characterization of recombinant cDNA coding for factor XII of human coagulation and have suggested the possible identification of other cDNA clones as coding for factor VII of human coagulation.

Expression of single-chain antibodies in transgenic plants.

There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-alpha HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana. Single-chain antibodies (ScFvs) extracted and purified from E. coli and plant tissues were tested for functionality and specificity by flow cytometry analysis on several cell lines and showed positive results when used on breast cancer slides coming from human frozen tissues. As a result, scFv-alpha HER2 represents a good opportunity for application and use in diagnosis and therapy.

The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing.

Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA.

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development.

'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 immunohistochemical 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.