Antibody-dependent Mechanisms Research Articles

Neutralization and enhancement of trans infection by erythrocyte-bound HIV with antibodies and complement

Background Antibodies to HIV envelope glycoproteins, with or without neutralization when assayed in standard neutralization assays, are reported to have potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Although most of the standard neutralization assays use free virus rather than carrier-bound HIV as the infectious agent; a cell line as the target for infection; and no complement (C), these conditions may not reflect in vivo events that would include antibody-dependent innate effector mechanisms. Methods We investigated the Fc-receptor-mediated and the complement-mediated antibody-dependent enhancement in trans infection neutralization assays using two broadly neutralizing monoclonal antibodies, 4E10 and b12; primary HIV; PBMC target cells naturally expressing Fc receptors and complement receptors; and with or without complement. Results In the absence of complement, the 4E10 mAb did not neutralize erythrocyte-bound HIV, and the b12 mAb neutralized erythrocyte-bound HIV less effective than the free virus. At low concentrations, 4E10 caused enhancement of infection. In contrast, in the presence of complement, 4E10 neutralized erythrocyte-bound HIV, or caused enhancement of trans infection of erythrocytebound HIV, depending on the mechanism of binding of HIV to erythrocytes. Conclusion Our results are consistent with the concept that the binding of HIV to erythrocytes represents a mechanism of establishing a “safe harbor” for HIV from the adverse effects of antibodies and complement that might otherwise be detrimental to free circulating HIV. Our data shows that broadly neutralizing antibody 4E10 can cause both C-mediated neutralization and enhancement of trans infection. This suggests that erythrocyte-bound HIV-1 serves as a sort of battleground between neutralization and enhancement by antibodies in the presence of C. Because of this, we propose that induction of neutralizing vs. enhancing antibodies can only be differentiated by utilization of a trans infection neutralization assay that might yield more relevant results for in vivo conditions.

Licensing of killer dendritic cells in mouse and humans: Functional similarities between IKDC and human blood γδ T-lymphocytes

Dendritic cells are characterized by the ability to induce primary antigen-specific immune responses in both major histocompatibility complex (MHC) Class I-restricted CD8 cells and MHC Class II-restricted CD4 cells. This professional antigen presentation function is associated with the up-regulation of co-stimulatory molecules and Class II MHC. While it has been recognized that several types of innate lymphocytes in mouse and humans can express co-stimulatory molecules and present antigen, the property of antigen presentation to elicit responses in naïve cells has been considered the exclusive domain of the dendritic cell. This concept has been challenged through the description of innate lymphocytes, capable of killing using NK receptors, but also up-regulating co-stimulatory molecules and driving the antigen-specific proliferation of naïve lymphocytes to the same extent as dendritic cells. Interferon (IFN)-γ secreting killer dendritic cells (IKDC) have been described in mice and share immunophenotypic properties of both dendritic cells and natural killer cells. Human blood γδ T-lymphocytes have innate tumor cell killing properties by both antibody-dependent and natural killer receptor-dependent mechanisms. This article reviews data from the authors’ own laboratory showing a particular feature in common between the mouse IKDC and human blood γδ T-lymphocytes; namely their requirement for interaction with a target cell for specific licensing for professional antigen presentation.

Functional identification of pathogenic autoantibody responses in patients with multiple sclerosis

Pathological and clinical studies implicate antibody-dependent mechanisms in the immunopathogenesis of multiple sclerosis. We tested this hypothesis directly by investigating the ability of patient-derived immunoglobulins to mediate demyelination and axonal injury in vitro. Using a myelinating culture system, we developed a sensitive and reproducible bioassay to detect and quantify these effects and applied this to investigate the pathogenic potential of immunoglobulin G preparations obtained from patients with multiple sclerosis (n = 37), other neurological diseases (n = 10) and healthy control donors (n = 13). This identified complement-dependent demyelinating immunoglobulin G responses in approximately 30% of patients with multiple sclerosis, which in two cases was accompanied by significant complement-dependent antibody mediated axonal loss. No pathogenic immunoglobulin G responses were detected in patients with other neurological disease or healthy controls, indicating that the presence of these demyelinating/axopathic autoantibodies is specific for a subset of patients with multiple sclerosis. Immunofluorescence microscopy revealed immunoglobulin G preparations with demyelinating activity contained antibodies that specifically decorated the surface of myelinating oligodendrocytes and their contiguous myelin sheaths. No other binding was observed indicating that the response is restricted to autoantigens expressed by terminally differentiated myelinating oligodendrocytes. In conclusion, our study identifies axopathic and/or demyelinating autoantibody responses in a subset of patients with multiple sclerosis. This observation underlines the mechanistic heterogeneity of multiple sclerosis and provides a rational explanation why some patients benefit from antibody depleting treatments.

Treatment targets in systemic lupus erythematosus: biology and clinical perspective

Systemic lupus erythematosus (SLE) is a complex disease characterized by numerous autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset and progression of clinical manifestations are also complex and multi-step, including breach of tolerance in the adaptive immune system, amplification of autoimmunity through innate and adaptive immune system dysregulation, and end-organ damage. Studies of murine genetic manipulations and human risk variants have provided important clues to the cellular and molecular pathogenesis of SLE, operating at multiple of these steps. The breakdown of B-cell tolerance is probably a defining and early event in the disease process and may occur by multiple pathways, including alterations in factors that affect B-cell activation thresholds, B-cell longevity, and apoptotic cell processing. Examples of amplification of autoimmunity on the adaptive immune system side include disturbances in B-cell/T-cell collaboration. B cells can also amplify innate immune cell activation via antibody-dependent and antibody-independent mechanisms. Indeed, one of the key amplification loops in SLE is the activation of plasmacytoid dendritic cells via autoantibodies and RNA-containing and DNA-containing immune complexes, which act as Toll-like receptor ligands, stimulating the secretion of large quantities of IFNα. A more recent link between the innate and adaptive immune system in SLE includes the neutrophil, which can be primed by interferon and autoantibodies to release neutrophil extracellular traps as an additional source of immunogenic DNA, histones, and neutrophil proteins. The innate immune system activation then feeds back, driving autoreactive B-cell and T-cell survival and maturation. This self-perpetuating disease cycle creates the opportunity for targeted treatment inventions at multiple steps.

B cells contribute to MS pathogenesis through antibody-dependent and antibody-independent mechanisms

For many years, central dogma defined multiple sclerosis (MS) as a T cell-driven autoimmune disorder; however, over the past decade there has been a burgeoning recognition that B cells contribute to the pathogenesis of certain MS disease subtypes. B cells may contribute to MS pathogenesis through production of autoantibodies (or antibodies directed at foreign bodies, which unfortunately cross-react with self-antigens), through promotion of T cell activation via antigen presentation, or through production of cytokines. This review highlights evidence for antibody-dependent and antibody-independent B cell involvement in MS pathogenesis.

Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice

Current therapies for non-Hodgkin lymphoma commonly include CD20 mAb to deplete tumor cells. However, the response is not durable in a substantial proportion of patients. Herein, we report our studies in mice testing the hypothesis that heterogeneity in endogenous tissue CD20+ B cell depletion influences in vivo lymphoma therapy. Using highly effective CD20 mAbs that efficiently deplete endogenous mature B cells and homologous CD20+ primary lymphoma cells through monocyte- and antibody-dependent mechanisms, we found that lymphoma depletion and survival were reduced when endogenous host B cells were not depleted, particularly a rare IL-10-producing B cell subset (B10 cells) known to regulate inflammation and autoimmunity. Even small numbers of adoptively transferred B10 cells dramatically suppressed CD20 mAb-mediated lymphoma depletion by inhibiting mAb-mediated monocyte activation and effector function through IL-10-dependent mechanisms. However, the activation of innate effector cells using a TLR3 agonist that did not activate B10 cells overcame the negative regulatory effects of endogenous B10 cells and enhanced lymphoma depletion during CD20 immunotherapy in vivo. Thus, we conclude that endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells able to inhibit lymphoma depletion by CD20 mAbs. Consequently, B10 cell removal could provide a way to optimize CD20 mAb-mediated clearance of malignant B cells in patients with non-Hodgkin lymphoma.

A CD8α− subpopulation of macaque circulatory natural killer cells can mediate both antibody-dependent and antibody-independent cytotoxic activities

Natural killer (NK) cells are important components of the innate immune system that mediate effector and regulatory functions. As effector cells, NK cells help control virus-infected cells through cell-mediated antibody-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). Although macaques are an important and reliable animal model for the study of retrovirus-induced human diseases, and despite the crucial role played by NK cells in innate and adaptive immune responses against simian immunodeficiency virus (SIV), only a few studies have attempted to characterize different macaque NK cell subpopulations. In the present study, we identified a subpopulation of circulatory CD8α(-) macaque NK cells that express NK lineage markers and exhibit cytotoxic potential. CD8α(-) NK cells were phenotypically characterized as CD3(-) CD14(-) CD20(-) CD8α(-) cells that express NK cell markers including CD16, CD56, granzyme B, perforin, NKG2D and KIR2D. Based on their CD56/CD16 expression patterns, cells within the CD8α(-) gate can be divided into four subpopulations: CD56(dim) CD16(bright) , CD56(dim) CD16(-) , CD56(bright) CD16(-) , and CD56(-) CD16(-) cells. In contrast, CD8α(+) NK cells are 95% CD56(dim) CD16(bright) , which correlates with their high cytotoxic potential. Upon interleukin-15 activation, CD8α(-) cells up-regulated CD69 expression and produced low levels of interferon-γ and tumour necrosis factor-α. Sorted CD8α(-) NK cells were capable of killing MHC-I-devoid target cells and mediated ADCC responses against SIV gp120-coated target cells in the presence of macaque anti-gp120 antibodies. Taking into account CD8α(-) myeloid dendritic cells, we show that about 35% of macaque CD8α(-) cells represent a novel, functional population of circulatory NK cells that possesses cytotoxic potential and is capable of mediating anti-viral immune responses.

Immunization with the recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice

Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis.

Dengue virus isolation relying on antibody-dependent enhancement mechanism using FcγR-expressing BHK cells and a monoclonal antibody with infection-enhancing capacity

Background Virus isolation is the most reliable evidence of dengue virus (DENV) infection. However, conventional virus isolation methods generally posses lower sensitivity and are time consuming as compared to other diagnostic methods such as detection of viral genome by RT-PCR, and determination of NS1 antigen and anti-DENV antibody by ELISA. Objectives A virus isolation method relying on the antibody-dependent enhancement mechanism was established and the assay's efficacy in DENV isolation was confirmed. Study design FcγR-expressing BHK cells were used for DENV isolation from patient serum samples in the presence of a flavivirus-group reactive monoclonal antibody, mAb4G2, which possesses DENV infection-enhancement activity. DENV genome copy numbers in the culture supernatant fluids of FcγR-expressing BHK cells were assessed and compared to those of parent BHK cells and C6/36 mosquito cells, a cell line commonly used for DENV isolation. Results The virus titer levels were higher in the culture supernatant fluid of FcγR-expressing BHK cells in the presence of enhancing antibody in comparison with other cell lines using laboratory-established strains and some clinical samples. DENV was isolated from 7 of 16 serum samples by using FcγR-expressing BHK cells in the presence of mAb4G2, but not by using cell lines commonly employed in conventional isolation assays, the FcγR-negative BHK cells and C6/36 cell lines. Conclusions The results demonstrate that FcγR-expressing BHK cell line in the presence of antibodies, which possess antibody dependent enhancement (ADE) activity, is a useful tool for DENV isolation.

Cytomegalovirus-based cancer vaccines expressing TRP2 induce potent antitumor effect against melanoma in mice (156.20)

Abstract Cytomegalovirus (CMV) induces strong and long-lasting immune responses, which make it an attractive candidate for a cancer vaccine vector. In this study, we tested whether a tumor antigen expressed in CMV can induce an anti-tumor effect. We expressed the melanoma tumor antigen, tyrosinase-related protein 2 (TRP2), in murine cytomegalovirus (MCMV). A single prophylactic dose of MCMV-TRP2 induced complete rejection of B16 melanoma cells. In a therapeutic setting, MCMV-TRP2 significantly inhibited the growth of melanoma. In addition, the growth of B16 cells inoculated five months after a single vaccination with MCMV-TRP2 was still inhibited, which indicates the vaccine induced a long-term protection. MCMV-TRP2 induced the production of both TRP2-specific CD8 T cells and antibodies. Depletion of CD4 and CD8 T cells did not compromise the antitumor effect, but MCMV-TRP2-vaccinated B cell deficient (µMT) mice did not reject B16 cells. This indicated that antibody rather than T cells was the main mediator of the antitumor effect induced by MCMV-TRP2. We also made a spread-deficient MCMV-TRP2 vaccine, which showed a similar antitumor effect. In conclusion, our study indicates that TRP2 expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody dependent mechanism. Our findings demonstrated for the first time that CMV is a promising vector for cancer vaccines.

Synthesis of a Single-Molecule l-Rhamnose-Containing Three-Component Vaccine and Evaluation of Antigenicity in the Presence of Anti-l-Rhamnose Antibodies

Carbohydrates are generally considered to be poorly immunogenic. Therefore, new approaches for enhancing their immunogenicity are important for the development of carbohydrates as vaccine components. We hypothesized that conjugation of an l-rhamnose (Rha) moiety to a carbohydrate antigen would enhance the antigenicity of the antigen in mice possessing anti-Rha antibodies via an antibody-dependent antigen uptake mechanism. To explore this hypothesis, we synthesized a single-molecule three-component vaccine containing the GalNAc-O-Thr (Tn) tumor-specific antigen, a 20 amino acid helper T-cell epitope (YAF) derived from an outer-membrane protein of Neisseria meningitides, and a Rha moiety. The vaccine was synthesized by automated Fmoc-based solid-phase peptide synthesis and deacetylated by brief treatment with NaOMe. Groups of female BALB/c mice were immunized and boosted with Rha-ovalbumin (Rha-OVA) formulated with either TiterMax Gold or Sigma Adjuvant System for a period of 35 days in order to determine optimal conditions for generating anti-Rha titers in mice. Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than in the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups initially immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a 2-fold increase in anti-Tn titer at 1/100 serum dilution relative to mice not immunized with Rha-OVA. An in vitro T-cell proliferation study using cells primed with either Rha-YAF-Tn or YAF-Tn was done to examine possible differences in antigen uptake and presentation due to anti-Rha antibody and chemical modification. Proliferation of T cells was stimulated by a 10-fold lower antigen concentration in the presence of Rha antibodies. The results strongly suggest that T cells present in the spleen were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.

Significance of B cells and B cell clonality in Sjögren's syndrome

Sjogren’s syndrome (SS) is a chronic autoimmune disease in which the exocrine glands become sites of intense immunologic activity, leading to tissue damage that is manifested as mucosal dryness (1). This disorder can occur either alone, as primary SS, or on a background of another connective tissue disease, such as rheumatoid arthritis (RA), systemic sclerosis (SSc; scleroderma), or systemic lupus erythematosus (SLE), as secondary SS. One theory of the disease immunopathogenesis proposes that the syndrome is initiated by epitheliitis and is sustained by the ensuing influx of lymphocytes (2). Hence, the disease was referred to by those investigators as autoimmune epitheliitis. All affected organs, especially the salivary glands, exhibit a lymphoproliferative sialadenitis. Lymphoproliferative sialadenitis in SS is associated with lymphocyte infiltration, epithelial cell proliferation, and apoptosis (3). Compared with healthy individuals, patients with SS are at greater risk of developing non-Hodgkin’s lymphoma (4). Lymphocytes are therefore central to the pathophysiology of this autoimmune condition. However, there is controversy about which set of lymphocytes directs the immunopathologic process. Examination of the available evidence at a glance suggests that T lymphocytes play the principal role in the immunopathogenesis of these diseases. This proposition is based on the numerical predominance of T lymphocytes in the cellular composition of the inflamed tissues and on the restriction of the T cell receptor (5). One potential contradiction of this proposition, however, is that if the T lymphocytes were indeed the principal driving cells, then no increases in monoclonal Ig and B cell lymphoma would be seen in primary SS. Therefore, because the numerical predominance of T lymphocytes is not consistent with the clonal expansion of B cells in SS patients, attention has recently focused on the possibility that B lymphocytes play the leading role. Furthermore, there is good evidence to indicate that B lymphocytes promote the immune responses to self and non-self antigens through antibody-dependent and antibodyindependent mechanisms (6). Accumulation of B cells in exocrine glands results in their clustering into aggregates (7). There is nonetheless evidence from phenotype analyses suggesting that these aggregates are benign (8). Such findings raise questions as to why lymphomas frequently develop on a background of polyclonal proliferation of B cells in patients with SS (9). The frequent leukemic transformation in these patients with primary SS was first noted by Bunim and Talal (10). Subsequent studies by these investigators indicated that the two B cell aberrations were associated (11). They went on further to show a whole range of benign-to-malignant B cell proliferations in patients with primary SS, but not in those with secondary SS (12). Lymphoma in these patients has thus changed from an all-or-nothing phenomenon to one of a continuous spectrum of disease. This concept fits in with, but does not explain, the high frequency of lymphoma in patients with primary SS. In this review, we venture into the area of the ambiguous relationship between primary SS and B cell lymphoma. This analysis is focused in part on highlighting the centrality of B lymphocytes to the pathogenesis of primary SS and on exploring the likely processes that underpin the polyclonality or monoclonality of B cells in the disease. Our paradigm does not exclude a key role of Supported by the Association Francaise du Gougerot-Sjogren et des Syndromes Secs, the French Ministry for Education and Research, and the Institut Francais pour la Recherche Odontologique. Pierre Youinou, MD, DSc, Valerie Devauchelle-Pensec, MD, PhD, Jacques-Olivier Pers, DDS, PhD: EA2216 Immunology and Pathology, IFR 148 ScInBioS, European University of Brittany, and Brest University Medical School Hospital, Brest, France. Address correspondence and reprint requests to Pierre Youinou, MD, DSc, Laboratory of Immunology, Brest University Medical School Hospital, BP824, F29609 Brest, France. E-mail: youinou@univbrest.fr. Submitted for publication March 19, 2010; accepted May 11, 2010.

The autophilic anti-CD20 antibody DXL625 displays enhanced potency due to lipid raft-dependent induction of apoptosis

Despite widespread use of anti-CD20 antibodies as therapeutic agents for oncologic and autoimmune indications, precise descriptions of killing mechanisms remain incomplete. Complement-dependent cytolysis and antibody-dependent cell-mediated cytotoxicity are indicated as modes of target cell depletion; however, the importance of apoptosis induction is controversial. Studies showing that the therapeutic anti-CD20 antibody rituximab (Rituxan) mediates apoptosis of tumor cell targets in vitro after cross-linking by anti-Fc reagents suggest that enhancement strategies applied to Fc-independent activities for anti-CD20 antibodies could improve therapeutic efficacy. An anti-CD20 antibody designated DXL625, with autophilic properties such as increased binding avidity, is shown here to independently induce caspase-mediated apoptosis of an established B-cell lymphoma line in vitro. Depletion of membrane cholesterol or chelation of extracellular calcium abrogated the pro-apoptotic activity of DXL625, indicating that intact lipid rafts and calcium are required for this activity. The Fc-mediated complement-dependent and antibody-dependent cellular killing mechanisms are maintained by DXL625 despite conjugation of the parental Rituxan antibody to the autophilic DXL peptide sequence. This study shows a strategy for improving anti-CD20 immunotherapy by endowing therapeutic antibodies with self-interacting properties.

Antiplatelet antibodies in WASP(−) mice correlate with evidence of increased in vivo platelet consumption

To study the role of antiplatelet antibodies in the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). A flow cytometric method was developed for detection of serum antiplatelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with 5-chloromethylfluorescein diacetate (CMFDA) in order to track their clearance from the circulation. WASP(-)muMT(-/-) mice were generated by standard breeding methods. Serum antiplatelet antibodies were detected in approximately 40% of WASP(-) males. The mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(-) males show an approximately 50% reduction in platelet counts, 5% to 10% show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies. In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily immunoglobulin G, and are also detected in >30% of CD47(-/-) males. WASP(-)muMT(-/-) males, which demonstrate no serum- or platelet-associated antibodies, show a degree of thrombocytopenia similar to that of WASP(-) males. Their platelet clearance rates remain accelerated--more so in WASP(-)muMT(-/-) than WASP(+)muMT(-/-) recipients. These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms: an antibody-independent mechanism that largely requires WASP deficiency in trans, and an antibody-dependent mechanism that does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody-dependent clearance of WASP(-) platelets.

Physiological and pathophysiological functions of intestinal mast cells

The normal gastrointestinal (GI) mucosa is equipped with mast cells that account for 2-3% of lamina propria cells under normal conditions. Mast cells are generally associated with allergic disease, and indeed, food allergy that manifests in the GI tract is usually mast cell dependent. On the other hand, mast cells have a number of physiological functions in the GI tract, namely regulatory functions such as control of blood flow and coagulation, smooth muscle contraction and peristalsis, and secretion of acid, electrolytes, and mucus by epithelial cells. One of the most intriguing functions of intestinal mast cells is their role in host defense against microbes like bacteria, viruses, or parasites. Mast cells recognize microbes by antibody-dependent mechanisms and through pattern-recognition receptors. They direct the subsequent immune response by attracting both granulocytes and lymphocytes to the site of challenge via paracrine cytokine release. Moreover, mast cells initiate, by releasing proinflammatory mediators, innate defense mechanisms such as enhanced epithelial secretion, peristalsis, and alarm programs of the enteric nervous This initiation can occur in response to a primary contact to the microbe or other danger signals, but becomes much more effective if the triggering antigen reappears and antibodies of the IgE or IgG type have been generated in the meantime by the specific immune system. Thus, mast cells operate at the interface between innate and adaptive immune responses to enhance the defense against pathogens and, most likely, the commensal flora. In this respect, it is important to note that mast cells are directly involved in controlling the function of the intestinal barrier that turned out to be a crucial site for the development of infectious and immune-mediated diseases. Hence, intestinal mast cells perform regulatory functions to maintain tissue homeostasis, they are involved in host defense mechanisms against pathogens, and they can induce allergy once they are sensitized against foreign antigens. The broad spectrum of functions makes mast cells a fascinating target for future pharmacological or nutritional interventions.

Phosphoethanolamine Substitution of Lipid A and Resistance ofNeisseria gonorrhoeaeto Cationic Antimicrobial Peptides and Complement-Mediated Killing by Normal Human Serum

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

Adverse Consequences of Immunostimulation

The therapeutic uses of immunostimulatory agents are generally in the treatments of infections or cancer. The traditional example of vaccination is one form of immunostimulation used in the prevention of pathogenic infections or cancer (e.g., human papillomavirus vaccine). Recombinant cytokines are increasingly used to stimulate immune system function. For example, interferon-α (IFNα) and interleukin (IL)-2 have been used to treat chronic hepatitis C virus infection and metastatic melanoma, respectively. In contrast, monoclonal antibodies are used to target malignant cells for elimination via antibody-dependent cytotoxicity mechanisms or apoptosis, including the anti-CD20 monoclonal antibody rituximab and the anti-CD56 monoclonal antibody alemtuzumab used in the treatment of B-cell malignancies, and the anti-erb2 receptor antibody trastuzumab used in the treatment of breast cancer. Finally, immunostimulation may develop via modulation of pathways involved in immune system regulation. For example, the anti-CD28 monoclonal antibody TGN1412 was developed as an agonist of regulatory T-cells for treatment of T-cell-mediated chronic inflammatory diseases or leukemias. A panel was convened to discuss potential toxicities associated with immunostimulation. At the Immunotoxicology IV meeting in 2006, a panel, moderated by Dr. Robert House (Dynport Vaccine Co., Frederick, MD), included Drs. Gary Burleson (Burleson Research Technologies, Inc., Raleigh, NC), Kenneth Hastings (US FDA, Center for Drug Evaluation and Research [CDER], Rockville, MD), Barbara Mounho (Amgen, Thousand Oaks, CA), Rafael Ponce (ZymoGenetics, Inc., Seattle, WA), Mark Wing (Huntington Life Sciences, Cambridgeshire, United Kingdom), Lauren Black (Navigators Consulting, Sparks, NV) and Anne Pilaro (US FDA, CDER, Rockville, MD). This paper reviews the major identified toxicities associated with immunostimulation, including the acute phase response, cell and tissue abnormalities/injury, cytokine release/cytokine storm, tumor lysis syndrome, vascular leak, and autoimmunity that were discussed by this panel.

Enhancement of Antibody-Dependent Mechanisms of Tumor Cell Lysis by a Targeted Activator of Complement

Complement inhibitors expressed on tumor cells provide a hindrance to the therapeutic efficacy of some monoclonal antibodies (mAb). We investigated a novel strategy to overwhelm complement inhibitor activity and amplify complement activation on tumor cells. The C3-binding domain of human complement receptor 2 (CR2; CD21) was linked to the complement-activating Fc region of human IgG1 (CR2-Fc), and the ability of the construct to target and amplify complement deposition on tumor cells was investigated. CR2 binds C3 activation fragments, and CR2-Fc targeted tumor cells by binding to C3 initially deposited by a tumor-specific antibody. Complement deposition on Du145 cells (human prostate cancer cell line) and anti-MUC1 mAb-mediated complement-dependent lysis of Du145 cells were significantly enhanced by CR2-Fc. Anti-MUC1 antibody-dependent cell-mediated cytotoxicity of Du145 by human peripheral blood mononuclear cells was also significantly enhanced by CR2-Fc in both the presence and the absence of complement. Radiolabeled CR2-Fc targeted to s.c. Du145 tumors in nude mice treated with anti-MUC1 mAb, validating the targeting strategy in vivo. A metastatic model was used to investigate the effect of CR2-Fc in a therapeutic paradigm. Administration of CR2-Fc together with mAb therapy significantly improved long-term survival of nude mice challenged with an i.v. injection of EL4 cells. The data show that CR2-Fc enhances the therapeutic efficacy of antibody therapy, and the construct may provide particular benefits under conditions of limiting antibody concentration or low tumor antigen density.

A phase I study of MORAb-003, a fully humanized monoclonal antibody against folate receptor alpha, in advanced epithelial ovarian cancer

5553 Background: Folate receptor alpha (FRA) is over-expressed in the majority of epithelial ovarian cancers (EOC) but is largely absent from normal tissue. MORAb-003 is a humanized monoclonal antibody (MAb) to FRA. Binding of MORAb-003 to FRA can prevent phosphorylation of substrates specific for Lyn kinase, suppress proliferation of cells over-expressing FRA, mediate FRA-positive tumor cell killing via antibody-dependent cellular and complement-dependent mechanisms, and suppress tumor growth in vivo of FRA-expressing tumors in rodent xenograft models. Toxicology studies in non-human primates found no evidence of toxicity with MORAb-003 at supra- pharmacological doses. This open-label, multiple-infusion, first-in-human, dose-escalation trial evaluates the safety, pharmacokinetics (PK), and anti-tumor activity of MORAb-003 in patients with platinum-resistant EOC. Methods: Sequential cohorts of patients received four weekly infusions at escalating dose levels of MORAb-003 from 12.5 mg/m2 to 400 mg/m2. Eligible patients had platinum-resistant EOC, acceptable organ function, KPS = 70%, and measurable disease by GOG-RECIST criteria. A subset of subjects received a tracer dose of 111In-labelled MORAb-003 and SPECT-CT and planar imaging. Human anti-human antibody (HAHA) and PK analyses were performed. Results: To date, 18 subjects have been dosed with up to 200 mg/m2. No dose limiting toxicity or significant related adverse events have been observed. Four subjects had rigors (2 grade 1) or fevers (3 grade 1, 1 grade 2) following the first infusion. Nine subjects have had radiologically stable disease, and 7 subjects have had stable or declining CA125. Two subjects have received extended therapy for an apparent clinical benefit. Radiolabelled tracer studies have demonstrated significant tumor uptake of labeled MORAb-003. The 400 mg/m2 dose cohort is currently enrolling. Conclusions: The FRA-specific MAb MORAb-003 appears to be well tolerated in patients with EOC and may have activity in platinum-resistant patients. These results support further evaluation of the efficacy of MORAb-003 in a phase 2 study which is ongoing. [Table: see text]

The role of B cells in animal models of rheumatoid arthritis

Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease that affects approximately 1% of the population. Recent studies demonstrate a significant improvement in clinical symptoms in patients treated with Rituximab, an anti-CD20 monoclonal antibody that depletes pro-B cells and mature B cells but not plasma cells. These findings indicate that B cells are an important contributor to the pathogenesis of RA. In this review we will examine the role of B cells in several different murine models of RA. There are a number of antibody-dependent mechanisms by which B cells support inflammatory processes in the joint. However, there are also antibody-independent mechanisms that involve B cell/T cell collaboration where B cells may modulate autoreactive T cell responses. In addition, B cells may be an important source of cytokines that either stimulate or inhibit autoimmune responses. Understanding the role of B cells in RA will provide new and directed therapeutic approaches to the treatment of disease.