Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells

Abstract

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3ɛ-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid–hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab′)2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases. © 2001 Cancer Research Campaign http://www.bjcancer.com

Bi20 (fBTA05), a novel trifunctional bispecific antibody (anti‐CD20 × anti‐CD3), mediates efficient killing of B‐cell lymphoma cells even with very low CD20 expression levels

“…For example, the bispecific mouse antibody Bis20 (mouse IgG1 CD20 3 mouse IgG2b CD3) induced nearly 60% B-cell depletion at the same E:T ratio used in our experiments but with a 10-fold higher antibody concentration, preactivated T cells and a shorter incubation time. 37 In another study using an anti-CD20 3 anti-CD3 diabody, lysis of tumor cells (Raji) could only be achieved when PBLs were prestimulated with IL-2. Even at a diabody concentration of 1,000 ng/ml and an E:T ratio of 20:1, only about 35% of cells were lysed.…”

The Role of B Cell-Mediated T Cell Costimulation in the Efficacy of the T Cell Retargeting Bispecific Antibody BIS20x3

“…BIS20x3 was produced and purified by IQ Products (Groningen, The Netherlands) as previously described (16). The immunoreactivity of both binding moieties within BIS20x3 was characterized using flow cytometric analysis, using secondary Abs against the murine IgG1 (CD20) and IgG2b (CD3) chains within the BsAb that were purchased from IQ Products.…”

“…T cell activation was determined using the Jurkat T cell subline Jurkat AM/T as previously described (16,21). Briefly, in each experiment 1.0 ϫ 10 6 Jurkat AM/T cells were activated in 1.0 ml of medium at 37°C, using the stimuli described, in the presence or absence of 0.2 ϫ 10 6 B cells (Ramos, Ramos-IkB␣ ND , Ramos LZRS , Raji, Daudi, or Jy).…”

“…In addition to this, BIS20x3-mediated cross-linking of CD20 at the cell surface of B cells induced significant apoptosis in the B cell lymphoma cell line Ramos (16). In the present study we investigated the mechanism of T cell activation mediated by BIS20x3 and B cells.…”

“…BIS20x3 combines the Ag specificity for CD20 on B cells with the Ag specificity for CD3⑀ on T cells, within one Ab molecule (16). In contrast to rituximab, this BsAb uses the patients T cell compartment for its antitumor effect by activating and retargeting T cells to CD20 ϩ B cells, independent of the TCR and MHC specificity of the T cells.…”