Therapeutic control of B cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold.

OBJECTIVE: To exploit the physiologic Fcgamma 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 beta-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. RESULTS: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. 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.

Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors.

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcgammaRIV, a unique mouse activating FcgammaR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcgammaR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158(phe) on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcgammaR present on the surface of tumor-infiltrating effector cell populations.

Endotoxin-induced uveitis in cyclooxygenase-2-deficient mice.

PURPOSE: Endotoxin-induced uveitis (EIU) is a model that mimics human acute anterior uveitis. Cyclooxygenase (COX)-2 is an enzyme that initiates the conversion of arachidonic acid (AA) into prostaglandins (PGs), whereas 5-lipoxygenase (5-LO) generates leukotrienes (LT). The purpose of this study was to delineate the role of COX-2 in acute ocular inflammation. METHODS: EIU was induced in wild-type (WT), heterozygotic (COX-2(+/-)) and COX-2 null (COX-2(-/-)) mice by injection of lipopolysaccharide (LPS). Other mice were coinjected with LPS and IFN gamma. Ocular histology, serum cytokines, and AA products determined by ELISA, and relevant ocular messengers determined by RT-PCR were compared among the different groups. RESULTS: Histology showed that the EIU score was significantly enhanced in COX-2(-/-) mice in comparison to WT and COX-2(+/-). PGE(2) was increased in WT and COX-2(+/-) EIU but not in COX-2(-/-) EIU. LTB(4) in serum and ocular 5-LO transcripts were increased in COX-2(-/-) EIU mice in comparison with WT and COX-2(+/-) EIU mice. IL-6 increased, whereas IFN gamma decreased both in serum and ocular transcripts in COX-2(-/-) EIU mice in comparison with WT and COX-2(+/-). Furthermore, EIU was suppressed in mice treated with recombinant IFN gamma, as shown by the decreased EIU scores, the presence of serum LTB(4) and IL-6 and ocular 5-LO and IL-6 mRNA, and the increases in serum IFN gamma and ocular IFN gamma, particularly in COX-2(-/-) mice. CONCLUSIONS: These data suggest that disturbance of the AA pathway exacerbates EIU in COX-2-deficient mice. IFN gamma moderately reverses this exacerbation and protects against EIU.

MCP-1 expression in endotoxin-induced uveitis.

PURPOSE: Monocyte chemoattractant protein (MCP)-1 (CCL-2) is a chemokine with chemoattractant properties for monocytes, memory T cells, natural killer cells, mast cells, and basophils. To delineate the role played by MCP-1 in acute anterior uveitis, a common ocular inflammation, MCP-1(-/-) mice and wild-type matched control mice were analyzed for the development of endotoxin-induced uveitis (EIU) in response to subcutaneous injection of a sublethal dose of lipopolysaccharide (LPS). METHODS: EIU was induced in MCP-1(-/-) and wild-type control mice by a single subcutaneous injection of Salmonella typhimurium LPS endotoxin at day 0. Alternatively, MCP-1(-/-) mice were injected subcutaneously with LPS plus recombinant MCP-1 at day 0 and with recombinant MCP-1 6 hours later. Mice were killed at day 1 or 3 after injection. Serum levels of IL-1alpha, IL-1beta, IL-6, IFN-gamma, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1alpha, MIP-2, regulated on activation normal T-cell expressed and secreted (RANTES), and MCP-1 were determined by ELISA. Eyes were collected and analyzed histologically and by RT-PCR for MCP-1, IFN-gamma, IL-6, TNF-alpha, beta-actin, MCP-5, RANTES, KC, inflammatory protein (IP)-10, and toll-like receptor (TLR)-4. RESULTS: EIU was strongly reduced in MCP-1(-/-) mice compared with wild-type control mice. The number of ocular inflammatory cells was significantly reduced. Moreover, intraocular IFN-gamma transcription was increased. EIU was induced in MCP-1(-/-) mice by co-administration of recombinant rat MCP-1 and LPS. CONCLUSIONS: Data indicate that MCP-1 plays a crucial role in the induction of EIU. MCP-1 may be a new therapeutic strategy for acute anterior uveitis.

Nonclinical evaluation of GMA161--an antihuman CD16 (FcγRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion.

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

CD32B, the human inhibitory Fc-gamma receptor IIB, as a target for monoclonal antibody therapy of B-cell lymphoma.

Human CD32B (FcgammaRIIB), 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 immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (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 lymphoproliferative disorders.

Expression of chemokine receptors, CXCR4 and CXCR5, and chemokines, BLC and SDF-1, in the eyes of patients with primary intraocular lymphoma.

OBJECTIVE: Chemokines have a range of biologic activities, including regulation of leukocyte trafficking, modulation of hematopoietic cell proliferation, and adhesion to extracellular matrix molecules. Specifically, B-lymphocyte chemoattractant (BLC); BCA-1; CXCL13, SCYB13) and stromal cell-derived factor-1 (SDF-1, CXCL12, SCYB12) are chemotactic for human B cells, and their ligands CXCR4 and CXCR5 are differentially expressed on B cells, including malignant B cells. We investigated the expression of these chemokine/chemokine receptors in eyes with primary intraocular B-cell lymphoma (PIOL). DESIGN: Observational case series (human tissue study). METHODS: Three freshly enucleated eyes with PIOL and a normal autopsied eye were frozen and sectioned. The sections were evaluated using immunohistochemistry (avidin-biotin-complex immunoperoxidase technique) for CXCR4, CXCR5, BLC, and SDF-1 to detect the expression and location. Reverse transcriptase-polymerase chain reaction was used to detect chemokine transcripts of CXCR4, CXCR5, BLC, and SDF-1 in PIOL and retinal pigment epithelium (RPE) cells after microdissection-either by laser capture (Arcturus) or by manual dissection-from frozen sections. MAIN OUTCOME MEASURES AND RESULTS: The three PIOL eyes showed similar pathology, with typical diffuse large B-lymphoma cells subjacent to the RPE. The eyes also demonstrated a similar chemokine profile. High expression levels of CXCR4 and CXCR5 were found limited to the lymphoma cells. In contrast, BLC protein was expressed in the RPE but not located in other ocular resident cells. SDF-1 was barely detected in a few RPE cells. CXCR4 and CXCR5 transcripts were detected abundantly in lymphoma cells, whereas BLC and SDF-1 transcripts were detected only in the RPE and not the malignant cells. No chemokine expression was detected on the RPE cells in the normal control eye. CONCLUSIONS: Chemokines and chemokine receptors selective for B cells were identified in RPE and malignant B cells, respectively. BLC, and possibly SDF-1, attracts both normal and malignant B-cells while promoting migration of only small numbers of T cells and macrophages. We propose that B-cell chemokines may be involved in the pathogenesis of PIOL by selectively attracting lymphoma cells to the RPE from the choroidal circulation. Our data suggest that inhibition of B-cell chemoattractants could be a future strategy for the treatment of PIOL.

Effect of sex hormones on experimental autoimmune uveoretinitis (EAU).

PURPOSE: Sex hormones have been associated with the prevalence, susceptibility, and severity of autoimmune disease. Although the exact mechanism is unknown, sex hormones are reported to influence cytokine production, specifically by affecting the balance of Th1 and Th2 effector cells. We evaluated the effect of estrogen, progesterone, and testosterone in autoimmune uveoretinitis (EAU), a rodent model of human ocular autoimmune disease. METHODS: Lewis rats implanted with either beta-estradiol (estrogen), 5-dihydrotestosterone (5-DHT), norgestrel (progesterone), or estrogen plus progesterone were immunized with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) peptide. Evaluation of EAU was based on histology of the eyes and measurement of peripheral immunological responses of DTH and lymphocyte proliferation to S-antigen. Quantitative RT-PCR was used to measure IFN-gamma and IL-10 mRNA in the eyes. RESULTS: In female rats 5-DHT significantly decreased, estrogen slightly enhanced, but progesterone or estrogen + progesterone did not affect EAU. In contrast, in male rats 5-DHT slightly decreased, estrogen moderately decreased, progesterone did not effect, but, estrogen + progesterone slightly decreased EAU. The results correlated with the ocular levels of Th1 (IFN-gamma) and Th2 (IL-10) cytokine messengers. CONCLUSION: The data support the hypothesis that sex hormones may affect autoimmune diseases by inducing changes in the cytokine balance. This suggests that sex hormone therapy could be considered as an adjunct to anti-inflammatory agents to treat ocular autoimmune diseases in humans.