Although treating D1-7 cells with -IgM to ligate BCR induced rapid phosphorylation of CD22 (Figure 5 lane 8), it did not result in colocalization of phospho-CD22 into lipid rafts with BCR

Although treating D1-7 cells with -IgM to ligate BCR induced rapid phosphorylation of CD22 (Figure 5 lane 8), it did not result in colocalization of phospho-CD22 into lipid rafts with BCR. 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 delta-Valerobetaine of CD22 into lipid rafts was also observed after treatment delta-Valerobetaine 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. Introduction CD20 is the principal delta-Valerobetaine target in the immunotherapy of B-cell lymphomas,1,2 CD320 while a mAb against CD22, epratuzumab, also has promising activity as a single agent.3C5 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 2 mAbs would have distinct mechanisms of action so that the therapeutic outcome would be delta-Valerobetaine 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.9C11 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 is convenient, it is a tedious, low-yield process involving multiple steps of modification and purification. Thus, recombinant engineering is preferred, with notable examples including 2 different scFv’s joined with a flexible linker14; minibodies that contain 2 different scFv’s joined at the C-termini via a hinge that is further fused to a helix-turn-helix15 or leucine zipper motif16 to facilitate heterodimerization; and diabodies that form heterodimers with a short linker between the VL and VH domains.17,18 However, these bsAbs have a relatively short half-life in vivo compared with IgG, because of their smaller size and lack of an Fc fragment. To increase the residence time for maximizing therapeutic effects, a variety of fusion bsAbs combining scFvs or diabodies and IgG or Fc fragments have been devised and produced in mammalian cell cultures,19C22 among which the IgG-(scFv)2 format19 is appealing for its divalency for each antigen, easy purification by protein A, a serum half-life comparable to IgG, and the presence of Fc to mediate CDC and ADCC. Veltuzumab contains the same human IgG1/ constant and variable framework regions as in epratuzumab, which has shown rapid and relatively safe infusion properties in clinical studies.3,5,23 CDC, ADCC, and inhibition of cell growth and induction of apoptosis have been shown for veltuzumab in vitro.12 Further, initial clinical studies have confirmed that low doses of veltuzumab can be given in shorter infusion times than rituximab, and it has shown good safety and efficacy results in NHL patients.24 In addition, enhancement of the in vitro and in vivo antitumor effects of veltuzumab was demonstrated experimentally when combined with epratuzumab.8,12 Epratuzumab is a humanized version of LL2, the murine mAb raised against the Raji Burkitt lymphoma cell line, and originally named EPB-2.13,25 LL2 is specifically reactive with B cells and NHL, and recognizes the same extracellular epitope of CD22 as the RFB4 mAb.26,27 In this study, anti-CD20/22 bsAbs composed of veltuzumab IgG and 2 entities of LL2scFv or RFB4scFv were constructed and evaluated for their growth-inhibitory effects in human B-cell lymphomas. Surprisingly,.