A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells.

Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease.

Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like particles and development of virus-specific monoclonal antibodies.

Human metapneumovirus (hMPV) is a member of the Pneumovirinea subfamily within the Paramyxoviridea family. Since its discovery in 2001, hMPV has been isolated in several continents, which suggests its prevalence worldwide. hMPV resembles human respiratory syncytial virus with regard to disease symptoms and its ability to infect and cause disease in young infants as well as individuals of all ages. The aim of the current study was to construct an efficient high-level yeast expression system for the generation of hMPV nucleocapsid (N) protein and to develop monoclonal antibodies (MAbs) suitable for hMPV detection. The genome of hMPV was isolated from oral fluid of an infected patient by using specific primers and reverse transcriptase polymerase chain reaction (RT-PCR). DNA sequence corresponding to the N protein gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast Saccharomyces cerevisiae harbouring recombinant plasmid revealed the presence of a protein band of approximately 43 kDa corresponding to the molecular weight of hMPV N protein. Electron microscopy analysis of purified N protein revealed nucleocapsid-like structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Recombinant hMPV N protein was reactive with human serum specimens collected from patients with confirmed hMPV infection. After immunization of mice with recombinant hMPV N protein, a panel of MAbs was generated. The specificity of newly generated MAbs was proven by immunofluorescence analysis of hMPV-infected cells. Epitope mapping using truncated variants of hMPV N revealed localization of linear MAb epitopes at the N-terminus of hMPV N protein, between amino acid residues 1 and 90. The MAbs directed against conformational epitopes did not recognize hMPV N protein variants containing either N- or C-terminal truncations. The reactivity of recombinant hMPV N protein with hMPV-positive serum specimens and the ability of MAbs to recognize virus-infected cells confirms the antigenic similarity between yeast-expressed hMPV N protein and native viral nucleocapsids. In conclusion, recombinant hMPV N protein and hMPV-specific MAbs provide new diagnostic reagents for hMPV infection.