Moreover, it has been reported that the small subpopulation of peripheral blood B-cells in immunized human subjects, capable of producing specific antibody, is sensitive to CD95-mediated cell death.27 Such cells might be killed by BS9520-induced bystander lysis18 even if they have lost CD20 expression during differentiation into antibody producing cells. Physique S5: Inhibition of proliferation of different lymphoma cells in the absence of effector cells. mt2015209x5.eps (337K) GUID:?37B829DB-D585-449C-8C2C-3BD24B736AF8 Abstract 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 CD20CD95-antibody in a newly developed Bithionol 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 and 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 Bithionol was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production application of the reagent. An additional explanation for the superior suppressive effect of BS9520 on antibody production might be, that this susceptibility of B-cells toward CD95-mediated killing may change during the process of B-cell activation that continues 6 days in the experiments described here. In this regard, we have noticed in preliminary experiments that this sensitivity of B-cells toward CD95-mediated cell death is steadily increasing in PWM-activated PBMC cultures from day 3 to day 6. Moreover, it has been reported that the small subpopulation of peripheral blood B-cells in immunized human subjects, capable of producing specific antibody, is sensitive to CD95-mediated cell death.27 Such cells might be killed by BS9520-induced bystander lysis18 even if they have lost CD20 expression during differentiation into antibody producing cells. In any case, the superior suppressive effects of BS9520 on antibody production imply that this reagent may be particularly suitable for the treatment of B-cell-mediated autoimmune disease. Materials and Methods PBMCs, isolated from heparinized blood of healthy donors by density-gradient centrifugation (Biocoll separating answer, Biochrom, Berlin, Germany), SKW6.4- Daudi-, Jurkat-, C1R-, JY-, and Raji-cell lines (ATCC, Manassas) were kept in RPMI 1640 (Life Technologies, Darmstadt, Germany), mouse Sp2/0-Ag14 cells (ATCC) in IMDM (Lonza, Basel, Switzerland). All media were supplemented with 10% heat-inactivated fetal calf serum (Biochrom), 100?U/ml penicillin, 100 g/ml streptomycin (Sigma-Aldrich, Hamburg, Germany), 1 mmol/l sodium-pyruvate (Biochrom), nonessential amino-acids (Biochrom), 2 mmol/l L-glutamine (Lonza) and 50 mol/l -mercaptoethanol (Merck, Darmstadt, Germany). Human cells lines were cultured at 37 C and 5% CO2, the mouse myeloma cell line Sp2/0-Ag14 and transfected Sp2/0 cells Bithionol were propagated at 7.5% CO2. Clinical grade material (Roche, Basel, Switzerland) diluted in phosphate-buffered saline was used in all experiments utilizing the Rituximab antibody. The variable domains of the 2H7 antibody (GenBank no.: “type”:”entrez-nucleotide”,”attrs”:”text”:”M17954″,”term_id”:”197015″,”term_text”:”M17954″M17954 and “type”:”entrez-nucleotide”,”attrs”:”text”:”M17953″,”term_id”:”196223″,”term_text”:”M17953″M17953) were synthesized using the ligase chain reaction with overlapping oligonucleotides. For the generation of chimerized and Fc-optimized antibodies (amino-acid exchanges at S239D and I332E), the VJ and VDJ elements were amplified and cloned into a eukaryotic expression vector made up of regulatory elements of the IgG locus, a human constant heavy- and light chain as described previously.28 Heavy and light chain plasmids of the chimeric and optimized antibody constructs were linearized with AhdI and SfiI, respectively, and transfected into Sp2/0-Ag14 cells by electroporation. Antibodies were purified from culture supernatants of transfected Sp2/0 cells using protein A affinity chromatography (GE Healthcare, Munich, Germany). For construction of bispecific antibodies, the variable domains of the antibodies APO-1 (anti-CD95) and 9.2.27 Rabbit Polyclonal to GAK (anti-chondroitin sulfate proteoglycan, CSPG4) were cloned from the respective hybridoma cells as previously described.18,28 At the C-terminus of the Fab fragment of the APO-1 antibody, a modified CH2 domain of human Ig1 and the respective scFv-fragments of 2H7 or 9.2.27 were added. To abrogate FcR-binding, glycosylation sites and the formation of disulfide bonds the following modifications were introduced into the hinge region and the CH2 domain name (EU-index): C226S; C229S; E233P; L234V; L235A; G236; D265G;.