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Comparative Study
. 2016 Feb;24(2):298-305.
doi: 10.1038/mt.2015.209. Epub 2015 Nov 19.

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

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Free PMC article
Comparative Study

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

Kristina Nalivaiko et al. Mol Ther. .
Free PMC article

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 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.

Figures

Figure 1
Figure 1
The three CD20-antibodies compared in this paper. (a) a chimeric antibody with variable regions derived from the 2H7 antibody and human constant domains, (b) the chimeric molecule with an optimized Fc-part, (c) a bispecific molecule in the Fabsc-format with an N-terminal CD95-specificity and a C-terminal single chain binding CD20. To prevent homodimerization and binding to Fc-receptors cysteins (x) and amino acid residues mediating FcR-binding (o), respectively, were replaced as described in the Materials and Methods section. As a control a bispecific Fabsc-antibody was produced that is directed to the melanoma associated antigen CSPG4 (BS95Mel).
Figure 2
Figure 2
Antibody dependent cellular cytotoxicity against various lymphoma cell lines. Peripheral blood mononuclear cells (PBMCs) and various target cells were incubated with the indicated antibodies at PBMC:target ratios of 5:1 or 10:1 and pulsed after 24 hours with 3H-thymidine. Mean values and standard deviations of triplicate samples are indicated.
Figure 3
Figure 3
Depletion of lymphoma cells in the presence of peripheral blood mononuclear cells (PBMC). (a) SKW 6.4 and (b) Raji cells were incubated with the indicated antibodies at PBMC:target ratios of 10:1 and analyzed after 48 hours by flow cytometry as described in the Materials and Methods section. The mean values of technical duplicates are indicated. Representative results with one (of three) different healthy donors are shown.
Figure 4
Figure 4
Antitumor activity and serum elimination of the various anti-CD20 antibodies. In (a) groups of eight C.B-17 SCID mice were inoculated with 1 × 107 SKW6.4 lymphoma cells and treated i.p. after 24 hours with 1 × 60 µg (day 1) and 3 × 20 µg (days 1, 2, and 3) of mono- and bispecific antibodies, respectively. Mice were killed when signs of lymphoma growth were present and tumor growth was confirmed by necroscopy. All long term surviving animals were killed at day 120 and were subjected to a histological examination. In one animal of the BS9520 group, local tumor growth in the thymus was detected. In (b) C57BL/6 mice were injected with 50 µg of the respective antibody i.v. and serum concentrations were determined at the indicated time points as described in the Materials and Methods section. Mean values and standard deviations obtained with three mice per time point are indicated.
Figure 5
Figure 5
Depletion of B- and T-cells in resting and pokeweed mitogen (PWM)-activated peripheral blood mononuclear cell (PBMC) cultures. PBMC, either untreated (resting) or activated with 1 µg/ml PWM for 6 days, were incubated for 2 days with the indicated antibodies (0.1 µg/ml) and were then analyzed by flow cytometry as described in the Materials and Methods section. In (a) and (b) fluorescence-activated cell sorting analysis of B-cell depletion within PBMC cultures of one donor, marked with an open square in (c–f), is shown. In (c–f) results obtained with four resting (c,e) and five activated (d,f) PBMC cultures from different healthy donors are summarized. Symbols indicate the mean values of technical duplicates.
Figure 6
Figure 6
Annexin V staining of CD19+ and CD4+ cells in activated peripheral blood mononuclear cell cultures of one donor (marked with filled circles in Figure 4) were incubated with the respective antibodies (1 µg/ml) and stained with Annexin V. After 4 hours, cells were analyzed by flow cytometry as described in the Methods section. One representative experiment of three is shown.
Figure 7
Figure 7
Suppression of panclonal and specific IgG production in peripheral blood mononuclear cell (PBMC) cultures by various antibodies. PBMC of different healthy donors were stimulated for 6 days with pokeweed mitogen (a) and tetanus toxoid (b). Cells were then washed and incubated for 2 days with the indicated antibodies. Antibody production in the culture supernatants was estimated by enzyme-linked immunosorbent assay as described in the Materials and Methods section.

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