Nanette Mittereder

B cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

A glycoengineered anti-CD19 antibody with potent antibody-dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo

Human cluster of differentiation (CD) antigen 19 is a B cell‐specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI‐551 is an affinity‐optimized and afucosylated CD19 mAb with enhanced antibody‐dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI‐551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI‐551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI‐551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI‐551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC‐enhanced CD19 mAb with an anti‐CD20 mAb could be a novel approach for the treatment of B cell lymphomas.

Follicular Dendritic Cell Activation by TLR Ligands Promotes Autoreactive B Cell Responses.

&NA; A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self‐reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement‐opsonized immune complexes. Here, we examined whether FDCs retain self‐antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self‐immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) &agr; via an IRF5‐dependent pathway. Blocking of FDC secretion of IFN‐&agr; restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN‐&agr; driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus. Graphical Abstract Figure. No caption available. HighlightsInternalization of RNP complexes via CD21 triggers TLR7 and IFN‐&agr; in mouse and human FDCsGC maintenance and &agr; anti‐nuclear antibody production are dependent on TLR7 pathway in FDCsLoss of B cell tolerance in RNP‐specific lupus mice is IFNAR dependentFDCs are an essential source of type I IFN in lupus mice &NA; Follicular dendritic cells (FDCs) maintain germinal centers through the uptake and cycling of complement‐opsonized immune complexes. Das et al. show that in a murine model of lupus, uptake of self‐immune complexes by FDCs activates TLR7 and that these stromal cells are a critical source of the IFN‐&agr; driving autoimmunity.

Loss of Immune Tolerance Is Controlled by ICOS in Sle1 Mice

ICOS, a member of the CD28 family, represents a key molecule that regulates adaptive responses to foreign Ags. ICOS is prominently expressed on T follicular helper (TFH) cells, a specialized CD4+ T cell subset that orchestrates B cell differentiation within the germinal centers and humoral response. However, the contribution of ICOS and TFH cells to autoantibody profiles under pathological conditions has not been thoroughly investigated. We used the Sle1 lupus-prone mouse model to examine the role of ICOS in the expansion and function of pathogenic TFH cells. Genetic deletion of ICOS impacted the expansion of TFH cells in B6.Sle1 mice and inhibited the differentiation of B lymphocytes into plasma cells. The phenotypic changes observed in B6.Sle1-ICOS–knockout mice were also associated with a significant reduction in class-switched IgG, and anti-nucleosomal IgG-secreting B cells compared with B6.Sle1 animals. The level of vascular cell adhesion protein 1, a molecule that was shown to be elevated in patients with SLE and in lupus models, was also increased in an ICOS-dependent manner in Sle1 mice and correlated with autoantibody levels. The elimination of ICOS-expressing CD4+ T cells in B6.Sle1 mice, using a glyco-engineered anti-ICOS–depleting Ab, resulted in a significant reduction in anti-nucleosomal autoantibodies. Our results indicate that ICOS regulates the ontogeny and homeostasis of B6.Sle1 TFH cells and influences the function of TFH cells during aberrant germinal center B cell responses. Therapies targeting the ICOS signaling pathway may offer new opportunities for the treatment of lupus and other autoimmune diseases.

Characterization of the Hypercitrullination Reaction in Human Neutrophils and Other Leukocytes

Autoantibodies against citrullinated proteins are diagnostic for rheumatoid arthritis. However, the molecular mechanisms driving protein citrullination in patients with rheumatoid arthritis remain poorly understood. Using two independent western blotting methods, we report that agents that trigger a sufficiently large influx of extracellular calcium ions induced a marked citrullination of multiple proteins in human neutrophils, monocytes, and, to a lesser extent, T lymphocytes and natural killer cells, but not B lymphocytes or dendritic cells. This response required 250–1,000 μM extracellular calcium and was prevented by EDTA. Other neutrophil activating stimuli, such as formyl-peptides, GM-CSF, IL-6, IL8, TNFα, or phorbol ester, did not induce any detectable increase in protein citrullination, suggesting that receptor-induced calcium mobilization is insufficient to trigger hypercitrullination. We conclude that loss of membrane integrity and subsequent influx of high levels of calcium, which can be triggered by perforin released from cytotoxic cells or complement mediated formation of membrane attack complexes in the joints of rheumatoid arthritis patients, are sufficient to induce extensive protein citrullination in immune cells, notably neutrophils. This mechanism may provide the citrullinated autoantigens that drive autoimmunity in this devastating disease.

T follicular helper–like cells contribute to skin fibrosis

T follicular helper–like cells and IL-21 are drivers of skin fibrosis in systemic sclerosis. Scleroderma’s little helpers Scleroderma, also known as systemic sclerosis, is a devastating disease involving multi-organ fibrosis. Taylor et al. examined immune cells from patients and in the skin of a graft-versus-host disease–based mouse model to elucidate the key players in this disease. They observed that a subset of T follicular helper–like cells expressing inducible costimulator (ICOS) correlated with disease scores. Blocking these cells with anti-ICOS or anti–IL-21, an important T follicular helper cell cytokine, ameliorated disease in the mouse model. Targeting these not-so-helpful T cells could dampen dermal fibrosis and bring relief to scleroderma patients. Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS+ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4+ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS+ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)–SSc mice and contributed to dermal fibrosis via an interleukin-21– and matrix metalloproteinase 12–dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS+ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS+ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

Spontaneous secretion of the citrullination enzyme PAD2 and cell surface exposure of PAD4 by neutrophils

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Perspective on Protein Arginine Deiminase Activity—Bicarbonate Is a pH-Independent Regulator of Citrullination

Protein citrullination catalyzed by peptidyl arginine deiminase (PADs) is involved in autoimmune disease pathogenesis, especially in rheumatoid arthritis. Calcium is a key regulator of PAD activity, but under normal physiological conditions it remains uncertain how intracellular calcium levels can be raised to sufficiently high levels to activate these enzymes. In pursuit of trying to identify other factors that influence PAD activity, we identified bicarbonate as a potential regulator of PAD activity. We demonstrate that physiological levels of bicarbonate upregulate citrullination by recombinant PAD2/4 and endogenous PADs in neutrophils. The impact of bicarbonate is independent of calcium and pH. Adding bicarbonate to commercial PAD activity kits could increase assay performance and biological relevance. These results suggest that citrullination activity is regulated by multiple factors including calcium and bicarbonate. We also provide commentary on the current understanding of PAD regulation and future perspective of research in this area.

Abstract C186: A glycoengineered anti‐CD19 antibody (MEDI‐551) is effective as monotherapy or in combination treatment in mouse models of B‐cell malignancies

Human cluster of differentiation (CD) antigen 19 is a B cell‐specific surface antigen that is expressed by early pre‐B cells and throughout B cell differentiation until it is down‐regulated during terminal differentiation of plasma cells. Expression of CD19 is maintained following malignant transformation and therefore CD19 is expressed on the majority of B cell‐derived leukemias and lymphomas. MEDI‐551 is a humanized immunoglobulin G1 kappa (IgG1) monoclonal antibody (MAb) directed against CD19. The Fc portion of MEDI‐551 does not contain a fucose sugar moiety due to its expression in BioWa9s Potelligent® cells. This afucosylated form of MEDI‐551 exhibits enhanced CD16 binding and antibody‐dependent cellular cytotoxicity (ADCC). Rituximab, another B‐cell targeted therapy, is directed against CD20 and is widely used for the treatment of B cell malignancies. However, a subset of patients is unresponsive to rituximab therapy and the majority of patients will relapse following treatment. We have previously shown that the glycoengineered, ADCC‐enhanced anti‐CD19 MAb is effective in in vitro and in vivo models of B cell leukemia and lymphoma. The purpose of the present study was to explore the anti‐tumor activity of the anti‐CD19 MAb in SCID mouse models of human lymphoma as single agent and in combination with rituximab. In several models the combination of anti‐CD19 with anti‐CD20 resulted in prolonged suppression of tumor growth with no untoward adverse events observed. The results suggest that combination therapy targeting both CD19 and CD20 could be an effective approach in the clinic for the treatment of B cell lymphomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C186.