Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action

Abstract

Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly constructed from veltuzumab (humanized anti-CD20) and epratuzumab (humanized anti-CD22) and evaluated in vitro and in vivo. While none of the parental mAbs alone or mixed had notable antiproliferative activity against Burkitt lymphoma cells when not crosslinked, the bsAbs [eg, anti-CD20 IgG-anti-CD22 (scFv) 2 ] were inhibitory without cross-linking and synergistic with B-cell antigen (BCR)-mediated inhibition. The bsAbs demonstrated higher antibodydependent cellulary cytoxicity (ADCC) activity than the parental mAbs, but not complement-dependent cytoxicity (CDC) of the parental CD20 mAb. Cross-linking both CD20 and CD22 with the bsAbs resulted in the prominent redistribution of not only CD20 but also CD22 and BCR into lipid rafts. Surprisingly, appreciable translocation of CD22 into lipid rafts was also observed after treatment with epratuzumab. Finally, the bsAbs inhibited Daudi lymphoma transplant growth, but showed a significant advantage over the parental anti-CD20 mAb only at the highest dose tested. These results suggest that recombinantly fused, complementary, bispecific, anti-CD20/22 antibodies exhibit functional features distinct from their parental antibodies, perhaps representing new candidate therapeutic molecules. IntroductionCD20 is the principal target in the immunotherapy of B-cell lymphomas, 1,2 while a mAb against CD22, epratuzumab, also has promising activity as a single agent. [3][4][5] Despite encouraging results with these mAbs, there are ongoing efforts to improve immunotherapy, because durable responses are only achieved in a portion of patients. 5 One emerging approach is combination therapy with different biologic agents, 6,7 such as 2 mAbs directed against distinct cell-surface antigens. Ideally, the 2 mAbs would have distinct mechanisms of action so that the therapeutic outcome would be additive or synergistic, and the combined antitumor effects would mitigate resistance to either of the mAbs.In vitro cell-based studies have shown that the combination of epratuzumab and rituximab has a greater antiproliferative effect than either mAb alone. 8 The results of 3 phase 2 clinical trials showed that epratuzumab combined with rituximab was well tolerated and suggested an improved activity in non-Hodgkin lymphoma (NHL), compared with historical reports of rituximab in similar patients. 9-11 However, combination mAb therapy involves sequential administration, thus requiring lengthy infusion times and potentially higher costs. The objectives of this study were to generate a single agent, namely a bsAb that targets both CD20 and CD22 antigens, and to evaluate its properties.A bsAb can be prepared by chemical conjugation, and this was used to prepare a bsAb Fab'x Fab' from hA20 (veltuzumab), a humanized anti-CD20 mAb, 12 and hLL2 (epratuzumab), a humanized anti-CD22 mAb, 13 in earlier studies. While chemical linkage to prepare bsAbs ...

Extensive crosslinking of CD22 by epratuzumab triggers BCR signaling and caspase-dependent apoptosis in human lymphoma cells

“…As shown in These results confirm the ability of soluble epratuzumab (the Wet-I format) to stabilize the localization of CD22 in lipid rafts, 38 and suggest that the cytotoxicity of epratuzumab requires the concurrent translocation of CD22, CD79a, and CD79b to lipid rafts.…”

“…33 Pre-incubation with F(ab 0 ) 2 of epratuzumab was recently reported to inhibit calcium mobilization and phosphorylation of Syk and PLCg2 in normal human B cells after BCR stimulation, 34 and the ability of epratuzumab-based agents to effectively mediate Fc-dependent trogocytosis of multiple B-cell surface markers by FcgR-bearing cells, was established. 35,36 In cell lines and xenografts of human Burkitt lymphoma, soluble epratuzumab, although capable of phosphorylating CD22 37 and translocating CD22 to lipid rafts, 38 as demonstrated in vitro, was not cytotoxic or cytostatic, 31,38 and displayed only minimal toxicity even when crosslinked by goat anti-human IgG Fcg (GAH). 31 On the other hand, in vitro cytotoxicity of epratuzumab comparable to that achievable with anti-IgM (10 mg/mL) could be consistently demonstrated in Ramos and D1-1, a subclone of Daudi selected for a high expression of membrane IgM (mIgM), when the antibody was immobilized to plastic plates, or added in combination with suboptimal amounts of anti-IgM (for example, 1 mg/mL or less) along with GAH.…”

Investigational Immunotherapeutics for B-Cell Malignancies

“…88,89 These bispecific mAbs have shown higher ADCC activity than the single parental mAbs, but not CDC. 88,89 Therapy with anti-CD20/anti-CD22 bispecific mAbs prolonged the survival of SCID mice in a Burkitt's lymphoma model over that seen with parental anti-CD20 mAb.…”

“…88,89 These bispecific mAbs have shown higher ADCC activity than the single parental mAbs, but not CDC. 88,89 Therapy with anti-CD20/anti-CD22 bispecific mAbs prolonged the survival of SCID mice in a Burkitt's lymphoma model over that seen with parental anti-CD20 mAb. 88 The antitumor efficacy of this bispecific mAb was abolished when ADCC potential was diminished by depletion of neutrophils and NK cells, thus suggesting that ADCC plays a critical role in the killing of malignant cells in these murine models.…”

Anticorps bispécifiques : quel avenir ?

“…Un AcBs anti-EGFR/anti-IGFR est capable de bloquer la fixation de ces 2 ligands et d'inhiber in vitro et in vivo la prolifération de cellules tumorales avec la même efficacité qu'un cocktail des anticorps parentaux [17]. Un AcBs anti-CD20/anti-CD22 inhibe la prolifération de certaines lignées de lymphome B plus efficacement que les anticorps parentaux et, a priori, avec un mécanisme d'action différent [18].…”