Vladka Čurin Šerbec

Exploring the Binding Sites of Anti-Infliximab Antibodies in Pediatric Patients With Rheumatic Diseases Treated With Infliximab

Over the past decade, the treatment of a variety of immune-mediated diseases has improved greatly due to the introduction of biologics for therapies in cases that are nonresponsive to traditional treatments. However, a side effect not encountered in traditional treatments is the immunogenicity of the biologics themselves. Our aim was to investigate the anti-infliximab-antibody response in pediatric patients receiving infliximab for juvenile idiopathic arthritis and other pediatric rheumatic diseases, with a focus on an analysis of the binding sites of these antibodies. We show that anti-infliximab antibodies developed in 43% of patients receiving infliximab therapy. Neutralization studies showed that in all these patients, the antibodies were directed toward the variable domains of infliximab, as they inhibited binding of infliximab to TNF. A more precise determination of the antibody epitopes using synthetic peptides was not achieved, indicating that all the antibody binding sites were composed of discontinuous segments of infliximab.

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.

Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori

Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrPSc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: Identification of a novel C-terminally truncated prion fragment

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP(C)) and the disease-associated (PrP(Sc)) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP(Sc)-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP(C) and PrP(Sc). Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP(Sc) that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.

Mutations in the hemochromatosis gene and the clinical outcome of multiple sclerosis.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for its variability in disease outcome. Given a possible role for dysregulation of iron metabolism in MS disease pathogenesis, we investigated whether or not mutations in the HFE gene influence the prognosis of the disease. A cohort of sporadic MS cases, taken from opposite extremes of the putative distribution of long-term outcome using the most stringent clinical criteria to date, was used to determine the role of HFE on MS disease severity. This approach increases the effective sample size by some 40-fold. Genotyping the two sets of MS patients (112 benign and 51 malignant) provided no evidence to suggest that mutations in HFE have any outcome modifying activity, although small effects cannot be ruled out. The frequency of HFE mutations was not different in MS compared to the general population.

Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrPSc-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.

A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies.

The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrP(Sc) humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrP(Sc)-specific mAbs being the most abundant.

Antibody variable-region sequencing as a method for hybridoma cell-line authentication.

Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories.