SummaryHuman CD32B (FccRIIB), the low-affinity inhibitory Fcc receptor (FccR), is highly homologous in its extracellular domain to CD32A (FccRIIA), an activating FccR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no crossreactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcc receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when coligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FceRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FccR-mediated responses to the treatment of allergy and autoimmunity.

Human CD32B (FcRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential im-munotherapeutic target for B-cell malig-nancies. A high-affinity monoclonal anti-body (mAb 2B6), from a novel panel of anti-human CD32B-specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5 were capable of directing cytotoxicity by peripheral blood mononuclear cells and monocyte-derived macrophages against B-lymphoma lines in vitro. In a human B-cell lymphoma mouse xenograft model, administration of ch2B6 or hu2B6-3.5 reduced tumor growth rate and improved tumor-free survival. Both the in vitro and in vivo activities of 2B6 required an intact Fc, suggesting an FcR-mediated mechanism of action. These data support the hypothesis that CD32B is a viable target for mAb treatment of B-cell lymphoprolif-erative disorders. (Blood. 2006;108: 2384-2391) Introduction Non-Hodgkin lymphoma (NHL) is the fifth most common form of cancer in the United States, with approximately 55 000 new cases diagnosed annually. 1 B-cell malignancies comprise more than 85% of diagnosed lymphomas, with diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) being the most common (30% and 22%, respectively) of all diagnosed NHLs. 2 The anti-CD20 chimeric antibody, rituximab (Rituxan) has been used successfully to improve the outcome of certain groups of patients with NHL. 3-5 The improvements in treatment gained with ritux-imab have been remarkable; however, the limitations of this therapy in certain B-cell malignancies, including those that show limited or no expression of CD20, make a strong case for identifying additional monoclonal antibody (mAb) therapeutics. The inhibitory Fc-receptor IIB (FcRIIB, CD32B), a type I transmembrane receptor with low affinity for monomeric IgG (K d 10 6 M 1), is a critical regulatory element in B-cell homeostasis. CD32B controls the threshold and the extent of cell activation by counterbalancing the stimulatory activity of a variety of receptors, including the B-cell antigen receptor. 6,7 Consistent with its regulatory function, CD32B is found on several hematopoietic cell types, which also express activating FcRs, and represents the predominant FcR expressed by the B-cell lineage. 6,8 The generation of mAbs specific for the extracellular region of human CD32B has been hindered by its homology to CD32A (FcRIIA), with 96% identity to CD32B within the extracellular region. 9 CD32A is highly expressed by myeloid cells and is absent in B cells. 8,10 Currently available anti-human CD32 mAbs show variable degrees of cross-reactivity with both receptors or react selectively with CD32A. 11 A therapeutic mAb that engages only CD32B, and spares CD32A-expressing cells, may have utility in the treatment of B-cell malignancies. By immunizing CD32A transgenic mice, we have succeeded in generating a set of...

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.

No abstract

Objective. To exploit the physiologic Fc␥ receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications.Methods. DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the ␤-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. Conclusion. This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.Activation-inhibition coupling, the pairing of a positive signal with an inhibitory loop, controls the magnitude and duration of many biologic processes (1-3). In B lymphocytes, recognition of an antigen by the clonotypic B cell receptor (BCR) induces a signal that can direct clonal expansion, differentiation, the release of cytokines, and, ultimately, Ig production. Uncontrolled activation is prevented by exhaustion of the activating stimulus as well as by the triggering of a negative feedback loop that involves the engagement of an inhibitory Fc␥ receptor (Fc␥R), Fc␥RIIb (CD32B) (4). The latter mechanism is triggered when the BCR recognizes immune-complexed antigen, resulting in the concomitant engagement of CD32B by the Fc domain of the complex-bound IgG, thus preventing the expansion of B cell clones that share the same specificity as that recognized by the soluble IgG. Attesting to its critical role in immune regulation, CD32B-knockout mice on the Th1-prone C57BL/6 background develop a lupuslike glomerulonephritis (5,6). We have focused on CD32B inhibitory signaling in B lymphocytes as a model system for the development of an alternative class of biologics that exploit activation-inhibition coupling for the control of immune activation. In addition to Ig