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

Abstract

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.

Angiogenin Mutants as Novel Effector Molecules For the Generation of Fusion Proteins With Increased Cytotoxic Potential

“…They comprise a toxic effector domain fused to a tumor cell-specific binding component, which is usually an antibody or a derivative thereof. [3][4][5] To circumvent these limitations, the murine antibody components have been replaced by humanized or fully human counterparts and the bacterial and plant toxins have been replaced by human proapoptotic enzymes such as granzymes or ribonucleases (RNases). The major advantage of ITs compared with traditional chemotherapy is their exceptional target cell specificity, but their disadvantages include side effects caused by potential immunogenicity.…”

Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein

“…Antibodies should be a powerful tool to solve this question. Despite the insufficient biochemical characterization of PrP Sc , many attempts have been made to establish antibodies with fine specificity using mice immunized with partially purified PrP Sc from prion-infected cells or brain extracts (6,10,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). However, most antibodies were specific to PrP C or cross-reactive to both PrP C and PrP Sc but not monospecific to PrP Sc , although more recently a PrP Sc -specific murine IgG1 antibody, W261, has been established (10).…”

Single-Chain Fragment Variable Passive Immunotherapies for Neurodegenerative Diseases

“…The humanized V5B2 scFv was engineered with the aid of computer modeling. The resulting scFv had human amino acid residues and 13 mutations introduced at key positions compared with the original murine mAb, yet retained V5B2 mAb’s stability, specificity and affinity [42,45]. However, the effectiveness of V5B2 scFv has not been evaluated.…”

“…scFvs can be engineered into multimers such as diabodies or triabodies to increase antigen binding [22,95]. scFvs sequence optimization including the replacement of unpaired cysteine residue in the scFv fragment [64], coding region mutagenesis, computer-guided codon humanization [42,45], and addition of the PEST domain to the scFvs [80,96] has helped to increase scFv stability and decrease immune response against the scFvs. In certain cases, changes in scFvs may lead to reduced affinity against cognate antigens.…”