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Showing papers on "Antibody published in 2009"


Journal ArticleDOI
02 Apr 2009-Nature
TL;DR: The IgG memory B-cell compartment in the selected group of patients with broad serumneutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.
Abstract: Serologic memory is an important factor in long-term vaccine efficacy, but there is little understanding of the antibodies produced by memory B cells in individuals infected with important human pathogens such as HIV To examine the memory antibody response to HIV, Scheid et al cloned more than 500 antibodies from HIV-specific memory B cells from six HIV-infected patients with high serum titres of broadly neutralizing antibodies The B-cell memory response to HIV in these patients was composed of up to 50 independent expanded B clones expressing a heterogeneous collection of antibodies to different viral epitopes, several of which may be important for broad HIV neutralization and effective vaccination This study clones and characterizes antibodies present in six HIV-infected subjects with low-to-intermediate viral loads Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum However, little is known about the specificity and activity of these antibodies1,2,3 To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120

896 citations


Journal ArticleDOI
12 Mar 2009-Nature
TL;DR: In this article, the authors investigated the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques.
Abstract: Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here we investigate the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T-cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Blockade was effective during the early (week 10) as well as late ( approximately week 90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for control of human immunodeficiency virus infections.

731 citations


Journal ArticleDOI
TL;DR: IgG4 by itself is unlikely to be a cause of allergic symptoms, but the presence of allergen‐specific IgG4 indicates that anti‐inflammatory, tolerance‐inducing mechanisms have been activated.
Abstract: Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also referred to as 'Fab-arm exchange', results usually in asymmetric antibodies with two different antigen-combining sites. While these antibodies are hetero- bivalent, they will behave as monovalent antibodies in most situations. Another aspect of IgG4, still poorly understood, is its tendency to mimic IgG rheumatoid factor (RF) activity by interacting with IgG on a solid support. In contrast to conventional RF, which binds via its variable domains, the activity of IgG4 is located in its constant domains. This is potentially a source of false positives in IgG4 antibody assay results. Because regulation of IgG4 production is dependent on help by T-helper type 2 (Th2) cells, the IgG4 response is largely restricted to non-microbial antigens. This Th2-dependency associates the IgG4 and IgE responses. Another typical feature in the immune regulation of IgG4 is its tendency to appear only after prolonged immunization. In the context of IgE-mediated allergy, the appearance of IgG4 antibodies is usually associated with a decrease in symptoms. This is likely to be due, at least in part, to an allergen-blocking effect at the mast cell level and/or at the level of the antigen-presenting cell (preventing IgE-facilitated activation of T cells). In addition, the favourable association reflects the enhanced production of IL-10 and other anti-inflammatory cytokines, which drive the production of IgG4. While in general, IgG4 is being associated with non-activating characteristics, in some situations IgG4 antibodies have an association with pathology. Two striking examples are pemphigoid diseases and sclerosing diseases such as autoimmune pancreatitis. The mechanistic basis for the association of IgG4 with these diseases is still enigmatic. However, the association with sclerosing diseases may reflect an excessive production of anti-inflammatory cytokines triggering an overwhelming expansion of IgG4-producing plasma cells. The bottom line for allergy diagnosis: IgG4 by itself is unlikely to be a cause of allergic symptoms. In general, the presence of allergen-specific IgG4 indicates that anti-inflammatory, tolerance-inducing mechanisms have been activated. The existence of the IgG4 subclass, its up-regulation by anti-inflammatory factors and its own anti-inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions.

697 citations


Journal ArticleDOI
TL;DR: The principle of a new class of bispecific antibodies called BiTE (for "bispecific T-cell engager") antibodies is reviewed, suggesting that this therapeutic paradigm is finally showing promise for treatment of both bulky and minimal residual disease.
Abstract: There is increasing evidence that T cells are able to control tumor growth and survival in cancer patients, both in early and late stages of the disease. However, tumor-specific T-cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells selected during immunoediting. An alternative approach to engage T cells for cancer therapy are antibodies, which are bispecific for a surface target antigen on cancer cells, and for CD3 on T cells. These are capable of connecting any kind of cytotoxic T cell to a cancer cell, independently of T-cell receptor specificity, costimulation, or peptide antigen presentation. Here, we review the principle of a new class of bispecific antibodies called BiTE (for "bispecific T-cell engager") antibodies. Recent results from clinical studies with a CD19/CD3-bispecific BiTE antibody suggest that this therapeutic paradigm is finally showing promise for treatment of both bulky and minimal residual disease.

622 citations


Patent
08 Dec 2009
TL;DR: In this paper, anti-PD-L1 antibodies are used to enhance T-cell function to upregulate cell-mediated immune responses and for the treatment of T cell dysfunctional disorders, including infection (e.g., acute and chronic) and tumor immunity.
Abstract: The present application relates to anti-PD-L1 antibodies, nucleic acid encoding the same, therapeutic compositions thereof, and their use enhance T-cell function to upregulate cell-mediated immune responses and for the treatment of T cell dysfunctional disorders, including infection (e.g., acute and chronic) and tumor immunity.

571 citations


Journal ArticleDOI
TL;DR: A new mouse model of memory B cell labeling dependent on the cytidine deaminase AID is used, which shows that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M–positive and IgG1+ B cells in germinal center–like structures that persisted up to 8 months after immunized.
Abstract: Memory B cells are at the center of longstanding controversies regarding the presence of antigen for their survival and their re-engagement in germinal centers after secondary challenge. Using a new mouse model of memory B cell labeling dependent on the cytidine deaminase AID, we show that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M-positive (IgM(+)) and IgG1(+) B cells in germinal center-like structures that persisted up to 8 months after immunization, as well as IgM(+) and IgG1(+) B cells with a memory phenotype outside of B cell follicles. After challenge, the IgG subset differentiated into plasmocytes, whereas the IgM subset reinitiated a germinal center reaction. This model, in which B cell memory appears in several layers with different functions, reconciles previous conflicting propositions.

556 citations


Journal ArticleDOI
TL;DR: Results indicate substantial plasticity of Th17 commitment toward a Th1-like profile and suggest a major role of Th1 cells in the induction of disease in this model of type 1 diabetes.
Abstract: Th17 cells are involved in the pathogenesis of many autoimmune diseases, but it is not clear whether they play a pathogenic role in type 1 diabetes. Here we investigated whether mouse Th17 cells with specificity for an islet antigen can induce diabetes upon transfer into NOD/SCID recipient mice. Induction of diabetes in NOD/SCID mice via adoptive transfer of Th1 cells from BDC2.5 transgenic mice was prevented by treatment of the recipient mice with a neutralizing IFN-γ–specific antibody. This result suggested a major role of Th1 cells in the induction of disease in this model of type 1 diabetes. Nevertheless, transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients with similar rates of onset as in transfer of Th1 cells. However, treatment with neutralizing IL-17–specific antibodies did not prevent disease. Instead, the transferred Th17 cells, completely devoid of IFN-γ at the time of transfer, rapidly converted to secrete IFN-γ in the NOD/SCID recipients. Purified Th17 cells also upregulated Tbet and secreted IFN-γ upon exposure to IL-12 in vitro and in vivo in NOD/SCID recipients. These results indicate substantial plasticity of Th17 commitment toward a Th1-like profile.

555 citations


Journal ArticleDOI
TL;DR: It is indicated that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.
Abstract: The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naive subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.

533 citations


Journal ArticleDOI
TL;DR: The serum of most neuromyelitis optica patients contains autoantibodies directed against the aquaporin‐4 water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain, and their role in disease pathogenesis was determined.
Abstract: Objective The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. Methods Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). Results Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. Interpretation Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease. Ann Neurol 2009;66:617–629

517 citations


Journal ArticleDOI
TL;DR: It is shown that when incubated with IL-3 and antigen or complexes of antigen and immunoglobulin E (IgE), basophils internalized, processed and presented antigen as complexes of peptide and major histocompatibility complex class II and produced IL-4.
Abstract: Basophils act as effector cells in immunoglobulin E–mediated hypersensitivity responses. Artis, Nakanishi and Medzhitov and their colleagues report that basophils present antigen and induce T helper type 2 responses to helminths, allergens and immunoglobulin E immune complexes. Basophils express major histocompatibility complex class II, CD80 and CD86 and produce interleukin 4 (IL-4) in various conditions. Here we show that when incubated with IL-3 and antigen or complexes of antigen and immunoglobulin E (IgE), basophils internalized, processed and presented antigen as complexes of peptide and major histocompatibility complex class II and produced IL-4. Intravenous administration of ovalbumin-pulsed basophils into naive mice 'preferentially' induced the development of naive ovalbumin-specific CD4+ T cells into T helper type 2 (TH2) cells. Mice immunized in this way, when challenged by intravenous administration of ovalbumin, promptly produced ovalbumin-specific IgG1 and IgE. Finally, intravenous administration of IgE complexes rapidly induced TH2 cells only in the presence of endogenous basophils, which suggests that basophils are potent antigen-presenting cells that 'preferentially' augment TH2-IgE responses by capturing IgE complex.

504 citations


Journal ArticleDOI
TL;DR: Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD.
Abstract: Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti–CADM-140 antibody–positive prototype serum sample was used to screen a HeLa cell–derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro–transcribed and in vitro–translated products using anti–CADM-140 antibody–positive and anti-CADM-140 antibody–negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti–CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro–transcribed and in vitro–translated products, we obtained a cDNA clone encoding melanoma differentiation–associated gene 5 (MDA-5). The anti–CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the “gold standard” immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti–CADM-140 antibody routinely available.

Journal ArticleDOI
TL;DR: The approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time and can be completed with as little as 20 ml of human blood and in as much as 28 d when optimal.
Abstract: We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

Journal ArticleDOI
TL;DR: A crucial role is identified for RORgamma-expressing Th17 cells in chronic intestinal inflammation, which controls IL-17A andIL-17F production and these cytokines have a redundant but highly pathogenic role in gut inflammation.

Journal ArticleDOI
TL;DR: SLE flare might be linked to the expansion of the Th17 cell population and the depletion of natural Treg cell subpopulations and Antagonism of Th17 cells by IL-17A-blocking antibodies should be explored as a treatment of SLE.
Abstract: Objective To investigate the relative abundance and activities of Th17 cells and natural Treg cells in systemic lupus erythematosus (SLE). Methods Blood samples were collected from 50 adult patients with SLE. Samples were processed to detect Th17 cells and natural Treg cells by flow cytometry, and related gene expression was assessed by real-time reverse transcription–polymerase chain reaction. Skin biopsy specimens were collected for histologic assessment. The function of Th17 cells in relation to human umbilical vein endothelial cells (HUVECs) was studied in vitro. Th17 cells were also examined in lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice. Results We demonstrated the presence of Th17 cells among the peripheral blood mononuclear cells (PBMCs) and in the involved organs of patients with active SLE. Both the percentage of circulating Th17 cells and the ability to produce interleukin-17A (IL-17A) were increased in samples derived from patients with active SLE. The number of Th17 cells increased during SLE flare, especially in patients with vasculitis, and decreased following certain treatments. We observed that IL-17A from patients with SLE could induce adhesion molecule messenger RNA expression in HUVECs and adhesion of T cells to HUVECs. An increase in the percentage of Th17 cells was correlated with natural Treg cell depletion during disease flare. Finally, expansion of the Th17 cell population was detected in MRL/lpr mice. Conclusion SLE flare might be linked to the expansion of the Th17 cell population and the depletion of natural Treg cell subpopulations. Expansion of the Th17 cell population might be related to a distinct cytokine environment in active SLE. Th17 cells and microenvironmental IL-17A are involved in vascular inflammation in SLE. Antagonism of Th17 cells by IL-17A–blocking antibodies should be explored as a treatment of SLE.

01 Jan 2009
TL;DR: In this paper, the authors evaluated the role of interferon (IFN)-producing T helper (TH) 1 and interleukin (IL)-17-expressing TH17 lymphocytes in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE).
Abstract: Objective: There is substantial evidence supporting the role of interferon (IFN)-–producing T helper (TH) 1 and interleukin (IL)-17–expressing TH17 lymphocytes in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). However, to date little is known about the potential cooperative interplay between these 2 cytokines. In the current study, we sought to evaluate the frequency of IFN-–expressing TH17 lymphocytes in MS and EAE, and study their recruitment into the central nervous system (CNS). Methods: Human TH17 lymphocytes were expanded in vitro from the blood of healthy controls and relapsing MS patients using IL-23. Immune cell migration to the CNS was assessed in vitro with primary cultures of human blood–brain barrier (BBB)-derived endothelial cells, and in vivo in EAE mice. Results: We demonstrate that in response to IL-23, human memory lymphocytes expand into a TH17 phenotype, with a subpopulation of cells simultaneously expressing IFN- and IL-17. We note that lymphocytes obtained from the blood of relapsing MS patients have an increased propensity to expand into IFN-–producing TH17 cells and identify numerous T lymphocytes coexpressing IL-17 and IFN- in brain tissue of MS patients. We also find lymphocytes expressing both the TH1and the TH17-associated transcription factors RORt and T-bet, in situ and in vitro. We further provide in vitro and in vivo evidence that IFN- TH17 lymphocytes preferentially cross the human BBB and accumulate in the CNS of mice during the effector phase of EAE. Interpretation: Our data underscore the involvement of IFN- TH17 lymphocytes in the pathology of MS and EAE and their preferential recruitment into the CNS during inflammatory events. Ann Neurol 2009;66:390 – 402 Multiple sclerosis (MS) is an immune-mediated disorder of the central nervous system (CNS) characterized by multifocal areas of leukocyte infiltration, demyelination, and axonal damage. Abundance of immune cells and their products (cytokines, chemokines, and immunoglobulins) in MS plaques, and their accumulation in the cerebrospinal fluid of affected individuals, support the notion that MS is a CNS-targeted inflammatory disorder. 1,2 Typically, demyelination is associated with an infiltration of memory CD4CD45RO T lymphocytes, effector memory CD8 T lymphocytes, macrophages, and dendritic cells that arise from migration of peripheral blood immune cells across CNS microvascular endothelial cells (ECs) forming the blood– brain barrier (BBB). 3–9

Journal ArticleDOI
TL;DR: Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non‐inflammatory antibody shielding of internal body surfaces – mediated principally by secretory immunoglobulin A (SIgA).
Abstract: Prevention of infections by vaccination remains a compelling goal to improve public health. Most infections involve the mucosae, but the development of vaccines against many of these pathogens has yet to be successful. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces – mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA) – produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). In 1974, a common SC-dependent transport of pIgA and pentameric IgM was proposed. From the basolateral surface, pIg-SC complexes are taken up by endocytosis and finally extruded into the lumen. Membrane SC is now referred to as polymeric Ig receptor (pIgR). In 1980, it was shown to be synthesized as a larger transmembrane protein – first cloned from rabbit and then from human. Mice deficient for pIgR showed that this is the only receptor responsible for epithelial transport of IgA and IgM. In the gut, induction of B cells occurs in gut-associated lymphoid tissue, particularly the Peyer’s patches, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the memory/effector cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue – but by different homing receptors. Such compartmentalization is a challenge for development of mucosal vaccines.

Journal ArticleDOI
TL;DR: A mouse monoclonal antibody targeting the IDH1 R132H mutation is developed and shown the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens.
Abstract: IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.

Journal ArticleDOI
TL;DR: Findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.
Abstract: IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.

Journal ArticleDOI
20 Mar 2009-Science
TL;DR: This work isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF).
Abstract: The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such "two-in-one" antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Journal ArticleDOI
TL;DR: A panel of JFH1‐based cell culture systems of all seven major HCV genotypes and important subtypes were completed and used in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies.

Journal ArticleDOI
TL;DR: SCS macrophage development depended on lymphotoxin-α1β2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface, characterized as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.
Abstract: Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

Journal ArticleDOI
26 Mar 2009-Blood
TL;DR: It is shown that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer cells displaying anti-leukemia activity were generated and maintained even late after transplantation, and this may have important clinical implications for the selection of optimal donors for haplo-HSCT.

Journal ArticleDOI
TL;DR: The breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV are described.
Abstract: Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27hi CD38hi plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27+ and surface IgG+. These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.

Patent
11 Aug 2009
TL;DR: In this article, isolated monoclonal antibodies that specifically bind to LAG-3 with high affinity, particularly human immunoglobulin-III (LAG-III)-specific antibodies, are described.
Abstract: The present disclosure provides isolated monoclonal antibodies that specifically bind to LAG-3 with high affinity, particularly human monoclonal antibodies. Preferably, the antibodies bind human LAG-3. In certain embodiments, the antibodies bind both human and monkey LAG-3 but do not bind mouse LAG-3. The invention provides anti-LAG-3 antibodies that can inhibit the binding of LAG-3 to MHC Class II molecules and that can stimulate antigen- specific T cell responses. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. This disclosure also provides methods for detecting LAG-3, as well as methods for treating stimulating immune responses using an anti-LAG-3 antibody of the invention. Combination therapy, in which an anti- LAG-3 antibody is co-administered with at least one additional immuno stimulatory antibody, is also provided.

Journal ArticleDOI
TL;DR: The conservation of a large fraction of T-cell epitopes suggests that the severity of an S-OIV infection, as far as it is determined by susceptibility of the virus to immune attack, would not differ much from that of seasonal flu.
Abstract: A major concern about the ongoing swine-origin H1N1 influenza virus (S-OIV) outbreak is that the virus may be so different from seasonal H1N1 that little immune protection exists in the human population. In this study, we examined the molecular basis for pre-existing immunity against S-OIV, namely the recognition of viral immune epitopes by T cells or B cells/antibodies that have been previously primed by circulating influenza strains. Using data from the Immune Epitope Database, we found that only 31% (8/26) of B-cell epitopes present in recently circulating H1N1 strains are conserved in the S-OIV, with only 17% (1/6) conserved in the hemagglutinin (HA) and neuraminidase (NA) surface proteins. In contrast, 69% (54/78) of the epitopes recognized by CD8+ T cells are completely invariant. We further demonstrate experimentally that some memory T-cell immunity against S-OIV is present in the adult population and that such memory is of similar magnitude as the pre-existing memory against seasonal H1N1 influenza. Because protection from infection is antibody mediated, a new vaccine based on the specific S-OIV HA and NA proteins is likely to be required to prevent infection. However, T cells are known to blunt disease severity. Therefore, the conservation of a large fraction of T-cell epitopes suggests that the severity of an S-OIV infection, as far as it is determined by susceptibility of the virus to immune attack, would not differ much from that of seasonal flu. These results are consistent with reports about disease incidence, severity, and mortality rates associated with human S-OIV.

Journal ArticleDOI
TL;DR: The antibody that was identified was detected in most patients with autoimmune pancreatitis but also in some patients with pancreatic cancer, making it an imperfect test to distinguish between these two conditions.
Abstract: BackgroundAutoimmune pancreatitis is characterized by an inflammatory process that leads to organ dysfunction. The cause of the disease is unknown. Its autoimmune origin has been suggested but never proved, and little is known about the pathogenesis of this condition. MethodsTo identify pathogenetically relevant autoantigen targets, we screened a random peptide library with pooled IgG obtained from 20 patients with autoimmune pancreatitis. Peptide-specific antibodies were detected in serum specimens obtained from the patients. ResultsAmong the detected peptides, peptide AIP1-7 was recognized by the serum specimens from 18 of 20 patients with autoimmune pancreatitis and by serum specimens from 4 of 40 patients with pancreatic cancer, but not by serum specimens from healthy controls. The peptide showed homology with an amino acid sequence of plasminogen-binding protein (PBP) of Helicobacter pylori and with ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells ...

Journal ArticleDOI
15 May 2009-AIDS
TL;DR: There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers, and for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.
Abstract: OBJECTIVE: To determine the spectrum of antiviral antibodies in HIV-1-infected individuals in whom viral replication is spontaneously undetectable, termed HIV controllers (HICs). DESIGN: Multicenter French trial ANRS EP36 studying the viral control in HICs. METHODS: Neutralizing Antibody (nAb) activities (neutralization assay, competition with broadly reactive monoclonal antibodies, and reactivity against the viral MPER gp41 region), FcgammaR-mediated antiviral activities, antibody-dependent cell cytotoxicity (ADCC), as well as autoantibody levels, were quantified in plasma from 22 controllers and from viremic individuals. The levels of these different antibody responses and HIV-specific CD8 T cell responses quantified by enzyme-linked immunosorbent spot (ELISPOT) IFNgamma assay were compared in each controller. RESULTS: The levels of antibody against the gp120 CD4 binding site, gp41, as well as Env epitopes near to the sites bound by broadly nAbs 2F5 and 1b12 were not different between HICs and viremic individuals. We did not find significant autoantibody levels in HICs. The magnitude and breadth of nAbs were heterogeneous in HICs but lower than in viremic individuals. The levels of nAbs using FcgammaR-mediated assay inhibition were similar in both groups. Regardless of the type of antibody tested, there was no correlation with HIV-specific CD8 T cell responses. ADCC was detectable in all controllers tested and was significantly higher than in viremic individuals (P < 0.0002). CONCLUSION: There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers. Rather, for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.

Journal ArticleDOI
TL;DR: It is demonstrated that, although accounting for only 2-3% of antibody mass, glycosylation of the IgG-Fc is essential to the activation of downstream biologic mechanisms (effector functions) and the precise structure of the attached oligosaccharide can influence biologic efficacy.

Journal ArticleDOI
TL;DR: The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.
Abstract: Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B-cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross-reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab-treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr-1 or asialo-GM-1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.

Journal ArticleDOI
TL;DR: Questions are raised regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.
Abstract: Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody's reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.