Showing papers on "Antibody published in 2005"

Immunology of multiple sclerosis

Abstract: Multiple sclerosis (MS) develops in young adults with a complex predisposing genetic trait and probably requires an inciting environmental insult such as a viral infection to trigger the disease. The activation of CD4+ autoreactive T cells and their differentiation into a Th1 phenotype are a crucial events in the initial steps, and these cells are probably also important players in the long-term evolution of the disease. Damage of the target tissue, the central nervous system, is, however, most likely mediated by other components of the immune system, such as antibodies, complement, CD8+ T cells, and factors produced by innate immune cells. Perturbations in immunomodulatory networks that include Th2 cells, regulatory CD4+ T cells, NK cells, and others may in part be responsible for the relapsing-remitting or chronic progressive nature of the disease. However, an important paradigmatic shift in the study of MS has occurred in the past decade. It is now clear that MS is not just a disease of the immune system, but that factors contributed by the central nervous system are equally important and must be considered in the future.

Mast cells in the development of adaptive immune responses

Abstract: Mast cells are so widely recognized as critical effector cells in allergic disorders and other immunoglobulin E-associated acquired immune responses that it can be difficult to think of them in any other context. However, mast cells also can be important as initiators and effectors of innate immunity. In addition, mast cells that are activated during innate immune responses to pathogens, or in other contexts, can secrete products and have cellular functions with the potential to facilitate the development, amplify the magnitude or regulate the kinetics of adaptive immune responses. Thus, mast cells may influence the development, intensity and duration of adaptive immune responses that contribute to host defense, allergy and autoimmunity, rather than simply functioning as effector cells in these settings.

Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

Abstract: Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

Abstract: Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Divergent Immunoglobulin G Subclass Activity Through Selective Fc Receptor Binding

Abstract: Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity. We suggest that these highly predictable functions assigned by Fc binding will be an important consideration in the design of therapeutic antibodies and vaccines.

Regulation of plasma-cell development

Abstract: Plasma cells are the terminally differentiated, non-dividing effector cells of the B-cell lineage. They are cellular factories devoted to the task of synthesizing and secreting thousands of molecules of clonospecific antibody each second. To respond to microbial pathogens with the necessary specificity and rapidity, B cells are exquisitely regulated with respect to both development in the bone marrow and activation in the periphery. This review focuses on the terminal differentiation of B cells into plasma cells, including the different subsets of B cells that become plasma cells, the mechanism of regulation of this transition, the transcription factors that control each developmental stage and the characteristics of long-lived plasma cells.

Adaptive immune responses in acute and chronic hepatitis C virus infection

Abstract: The hepatitis C virus (HCV) persists in the majority of infected individuals and is a significant cause of human illness and death globally Recent studies have yielded important insights into immunity to HCV, in particular revealing the central role of T cells in viral control and clearance Other key features of adaptive immune responses remain obscure, including mechanisms by which T cells control HCV replication, the role of antibodies in conferring protection and how cellular and humoral immunity are subverted in persistent infection

Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells.

Abstract: The majority of breast cancer patients who achieve an initial therapeutic response to the human epidermal growth factor receptor 2 (HER-2)-targeted antibody trastuzumab will show disease progression within 1 year. We previously reported the characterization of SKBR3-derived trastuzumab-resistant pools. In the current study, we show that HER-2 interacts with insulin-like growth factor-I receptor (IGF-IR) uniquely in these resistant cells and not in the parental trastuzumab-sensitive cells. The occurrence of cross talk between IGF-IR and HER-2 exclusively in resistant cells is evidenced by the IGF-I stimulation resulting in increased phosphorylation of HER-2 in resistant cells, but not in parental cells, and by the inhibition of IGF-IR tyrosine kinase activity leading to decreased HER-2 phosphorylation only in resistant cells. In addition, inhibition of IGF-IR tyrosine kinase activity by I-OMe-AG538 increased sensitivity of resistant cells to trastuzumab. HER-2/IGF-IR interaction was disrupted on exposure of resistant cells to the anti-IGF-IR antibody alpha-IR3 and, to a lesser extent, when exposed to the anti-HER-2 antibody pertuzumab. Heterodimer disruption by alpha-IR3 dramatically restored sensitivity to trastuzumab and resistant cells showed a slightly increased sensitivity to pertuzumab versus parental cells. Neither alpha-IR3 nor pertuzumab decreased HER-2 phosphorylation, suggesting that additional sources of phosphorylation other than IGF-IR exist when HER-2 and IGF-IR are not physically bound. Our data support a unique interaction between HER-2 and IGF-IR in trastuzumab-resistant cells such that cross talk occurs between IGF-IR and HER-2. These data suggest that the IGF-IR/HER-2 heterodimer contributes to trastuzumab resistance and justify the need for further studies examining this complex as a potential therapeutic target in breast cancers that have progressed while on trastuzumab.

Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma

Abstract: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis and one for which immunotherapy remains a viable option. Experimental tumor models have shown that regulatory T cells, a functionally unique subset of T cells, can suppress effective antitumor immune responses. This suppression might explain the poor outcome of some of the immunotherapy protocols currently being used. A better understanding of the role of regulatory T cells in HCC is important for design of future immunotherapy-based clinical protocols. We have studied regulatory T cells from 84 patients with HCC and 74 controls, including healthy donors, patients with chronic hepatitis B virus and hepatitis C virus infection and nonviral liver cirrhosis. Regulatory T cells were identified by fluorescence-activated cell sorting using a panel of antibodies and by real-time PCR analysis for Foxp3 expression. Functional studies were done to analyze their inhibitory role. Finally, regulatory T cells were analyzed in tumors and ascites from patients with HCC. Patients with HCC have increased numbers of CD4+CD25+ regulatory T cells in their peripheral blood, which express high levels of HLA-DR, GITR, and low or no CD45RA. These cells were anergic toward T-cell receptor stimulation and, when cocultured with activated CD4+CD25- cells, potently suppressed their proliferation and cytokine secretion. There were also high numbers of regulatory T cells in tumor-infiltrating lymphocytes of HCC patients comparable with the increase in their peripheral blood. Our data suggest that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against HCC and could be important in design of immunotherapeutic approaches.

Suppression of allergic airway inflammation by helminth-induced regulatory T cells

Abstract: Allergic diseases mediated by T helper type (Th) 2 cell immune responses are rising dramatically in most developed countries. Exaggerated Th2 cell reactivity could result, for example, from diminished exposure to Th1 cell‐inducing microbial infections. Epidemiological studies, however, indicate that Th2 cell‐stimulating helminth parasites may also counteract allergies, possibly by generating regulatory T cells which suppress both Th1 and Th2 arms of immunity. We therefore tested the ability of the Th2 cell‐inducing gastrointestinal nematode Heligmosomoides polygyrus to influence experimentally induced airway allergy to ovalbumin and the house dust mite allergen Der p 1. Inflammatory cell infiltrates in the lung were suppressed in infected mice compared with uninfected controls. Suppression was reversed in mice treated with antibodies to CD25. Most notably, suppression was transferable with mesenteric lymph node cells (MLNC) from infected animals to uninfected sensitized mice, demonstrating that the effector phase was targeted. MLNC from infected animals contained elevated numbers of CD4 � CD25 � Foxp3 � T cells, higher TGF- � expression, and produced strong interleukin (IL)-10 responses to parasite antigen. However, MLNC from IL-10‐deficient animals transferred suppression to sensitized hosts, indicating that IL-10 is not the primary modulator of the allergic response. Suppression was associated with CD4 � T cells from MLNC, with the CD4 � CD25 � marker defining the most active population. These data support the contention

Antitumor Activity in Melanoma and Anti-Self Responses in a Phase I Trial With the Anti-Cytotoxic T Lymphocyte–Associated Antigen 4 Monoclonal Antibody CP-675,206

Abstract: Purpose Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) blockade with CP-675,206, a fully human anti-CTLA4 monoclonal antibody, may break peripheral immunologic tolerance leading to effective immune responses to cancer in humans. A phase I trial was conducted to test the safety of CP-675,206. Patients and Methods Thirty-nine patients with solid malignancies (melanoma, n = 34; renal cell, n = 4; colon, n = 1) received an intravenous (IV) infusion of CP-675,206 at seven dose levels. The primary objective was to determine the maximum-tolerated dose and the recommended phase II dose. Results Dose-limiting toxicities and autoimmune phenomena included diarrhea, dermatitis, vitiligo, panhypopituitarism and hyperthyroidism. Two patients experienced complete responses (maintained for 34+ and 25+ months), and there were two partial responses (26+ and 25+ months) among 29 patients with measurable melanoma. There have been no relapses thus far after objective response to therapy. Four other patients had stable di...

Antibody composition-producing cell

Abstract: The present invention relates to a cell for the production of an antibody molecule such as an antibody useful for various diseases having high antibody-dependent cell-mediated cytotoxic activity, a fragment of the antibody and a fusion protein having the Fc region of the antibody or the like, a method for producing an antibody composition using the cell, the antibody composition and use thereof.

Maintenance of serum antibody levels

Abstract: In vertebrates, serum antibodies are an essential component of innate and adaptive immunity and immunological memory They also can contribute significantly to immunopathology Their composition is the result of tightly regulated differentiation of B lymphocytes into antibody-secreting plasma blasts and plasma cells The survival of antibody-secreting cells determines their contribution to the immune response in which they were generated and to long-lasting immunity, as provided by stable serum antibody levels Short-lived plasma blasts and/or plasma cells secrete antibodies for a reactive immune response Short-lived plasma blasts can become long-lived plasma cells, probably by competition with preexisting plasma cells for occupation of a limited number of survival niches in the body, in a process not yet fully understood Limitation of the number of long-lived plasma cells allows the immune system to maintain a stable humoral immunological memory over long periods, to react to new pathogenic challenges, and to adapt the humoral memory in response to these antigens

Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion

Abstract: Natural antibodies are produced at tightly regulated levels in the complete absence of external antigenic stimulation. They provide immediate, early and broad protection against pathogens, making them a crucial non-redundant component of the humoral immune system. These antibodies are produced mainly, if not exclusively, by a subset of long-lived, self-replenishing B cells termed B-1 cells. We argue here that the unique developmental pattern of these B-1 cells, which rests on positive selection by self antigens, ensures production of natural antibodies expressing evolutionarily important specificities that are required for the initial defense against invading pathogens. Positive selection for reactivity with self antigens could also result in the production of detrimental anti-self antibodies. However, B-1 cells have evolved a unique response pattern that minimizes the risk of autoimmunity. Although these cells respond rapidly and strongly to host-derived innate signals, such as cytokines, and to pathogen-encoded signals, such as lipopolysaccharide and phosphorylcholine, they respond very poorly to receptor-mediated activation. In addition, they rarely enter germinal centers and undergo affinity maturation. Thus, their potential for producing high-affinity antibodies with harmful anti-self specificity is highly restricted. The positive selection of B-1 cells occurs during the neonatal period, during which the long-lived self-renewing B-1 population is constituted. Many of these cells (B-1a) express CD5, although a smaller subset (B-1b) does not express this surface marker. Importantly, B-1a cells should not be confused with short-lived anergic B-2 cells, which originate in the bone marrow in adults and initiate CD5 expression and programmed cell death following self-antigen recognition. In summary, we argue here that the mechanisms that enable natural antibody production by B-1 cells reflect the humoral immune system, which has evolved in layers whose distinct developmental mechanisms generate complementary repertoires that collectively operate to maximize flexibility in responses to invading pathogens. B-2 cells, present in what may be the most highly evolved layer(s), express a repertoire that is explicitly selected against self recognition and directed towards the generation of high-affinity antibody response to external antigenic stimuli. B-1 cells, whose repertoire is selected by recognition of self antigen, belong to what may be earlier layer(s) and inherently maintain production of evolutionarily important antibody specificities that respond to pathogen-related, rather then antigen-specific signals.

Terminal Differentiation into Plasma Cells Initiates the Replicative Cycle of Epstein-Barr Virus In Vivo

Abstract: In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10-, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Abstract: Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

Multiplex Human Papillomavirus Serology Based on In Situ-Purified Glutathione S-Transferase Fusion Proteins,

Abstract: Background: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity–purified recombinant HPV proteins. Methods: Twenty-seven HPV proteins were expressed as glutathione S -transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. Results: Glutathione S -transferase fusion proteins bound with high affinity to glutathione-displaying beads ( K d = 6.9 × 10−9 mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1 000 000. Imprecision (median CV) was ≤5.4%, and assay reproducibility was high ( R 2 = 0.97). Results on clinical samples showed high concordance with ELISA (κ = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer ( P <0.001). Conclusion: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.

Interferon‐α/β–mediated innate immune mechanisms in dermatomyositis

Abstract: Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement-mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by interferon-alpha/beta were highly overexpressed, and immunohistochemistry for the interferon-alpha/beta inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 13/14 patients. Of 36 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and 3 of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of interferon-alpha, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and interferon-alpha/beta inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus.

Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I/II study.

Abstract: Background Cytotoxic T lymphocyte–associated antigen (CTLA)-4 can inhibit T-cell responses and is involved in tolerance against self antigens. We previously reported autoimmune manifestations and objective cancer regressions in patients with metastatic melanoma treated with CTLA-4 blockade. The possibility of activating tumor-reactive T cells while removing inhibitory activity with CTLA-4 blockade has stimulated interest in using anti–CTLA-4 antibodies in combination with other cancer immunotherapies to improve clinical outcomes. In this study, we assessed the antitumor activity and autoimmune toxicity of CTLA-4 blockade in combination with an immune-activating stimulus, interleukin (IL)-2, in patients with metastatic melanoma.

Direct access to CD4 + T cells specific for defined antigens according to CD154 expression

Abstract: The direct assessment of T helper (T(H))-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific T(H) cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of T(H) cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific T(H) cells. Moreover, the isolation of antigen-specific CD154(+) T(H) cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific T(H) cell lines. Our approach allows assessment of T(H) cells with a defined specificity for the combined quantitative and qualitative analysis of T(H)-cell immunity as well as for the isolation of specific T(H) cells for targeted cellular immunotherapies.

Antibody-mediated organ-allograft rejection

Abstract: Recent studies show that alloantibodies mediate a substantial proportion of graft-rejection episodes, contributing to both early and late graft loss. Rejection that is caused by antibody is mediated by different mechanisms from rejection that is caused by T cells, thereby requiring other approaches to treatment and prevention. Antibody induces rejection acutely through the fixation of complement, resulting in tissue injury and coagulation. In addition, complement activation recruits macrophages and neutrophils, causing additional endothelial injury. Antibody and complement also induce gene expression by endothelial cells, which is thought to remodel arteries and basement membranes, leading to fixed and irreversible anatomical lesions that permanently compromise graft function.

Autoimmunity in a Phase I Trial of a Fully Human Anti-Cytotoxic T-Lymphocyte Antigen-4 Monoclonal Antibody With Multiple Melanoma Peptides and Montanide ISA 51 for Patients With Resected Stages III and IV Melanoma

Abstract: Purpose Nineteen patients with high-risk resected stage III and IV melanoma were immunized with three tumor antigen epitope peptides from gp100, MART-1, and tyrosinase emulsified with adjuvant Montanide ISA 51 and received a fully human anti-cytotoxic T-lymphocyte antigen-4 (anti–CTLA-4) monoclonal antibody MDX-010. Each of three cohorts received escalating doses of antibody with vaccine primarily to evaluate the toxicities and maximum-tolerated dose (MTD) of MDX-010 with vaccine. MDX-010 pharmacokinetics and immune responses were secondary end points. Patients and Methods Peptide immunizations with MDX-010 were administered every 4 weeks for 6 months and then every 12 weeks for 6 months. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was performed before treatment and after the sixth vaccination. Patients were observed until relapse. Results Grade 3 gastrointestinal (GI) toxicity (diarrhea or abdominal pain) was observed in three patients in the highest dose cohort and o...

Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery

Abstract: Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.

Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.

Abstract: We have engineered the Fc region of a human immunoglobulin G (IgG) to generate a mutated antibody that modulates the concentrations of endogenous IgGs in vivo. This has been achieved by targeting the activity of the Fc receptor, FcRn, which serves through its IgG salvage function to maintain and regulate IgG concentrations in the body. We show that an IgG whose Fc region was engineered to bind with higher affinity and reduced pH dependence to FcRn potently inhibits FcRn-IgG interactions and induces a rapid decrease of IgG levels in mice. Such FcRn blockers (or 'Abdegs,' for antibodies that enhance IgG degradation) may have uses in reducing IgG levels in antibody-mediated diseases and in inducing the rapid clearance of IgG-toxin or IgG-drug complexes.

Circulating osteoblast-lineage cells in humans.

Abstract: Background Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood, we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation, such as during pubertal growth. Methods We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [±SD] age, 14.5±0.7 vs. 37.7±7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. Results Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells ...

Combination Therapy Enhances the Inhibition of Tumor Growth with the Fully Human Anti–Type 1 Insulin-Like Growth Factor Receptor Monoclonal Antibody CP-751,871

Abstract: Purpose: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. Experimental Design: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity ( K d = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. Results: This antibody blocks binding of IGF-1 to its receptor (IC 50 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC 50 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. Conclusion: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.

Correlation between Immunologic Responses to a Recombinant Glycoprotein 120 Vaccine and Incidence of HIV-1 Infection in a Phase 3 HIV-1 Preventive Vaccine Trial

Abstract: Background. An objective of the first efficacy trial of a candidate vaccine containing recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein 120 (rgp120) antigens was to assess correlations between antibody responses to rgp120 and the incidence of HIV-1 infection. Methods. Within the randomized trial (for vaccinees, n = 3598; for placebo recipients, n = 1805), binding and neutralizing antibody responses to rgp120 were quantitated. A case-cohort design was used to study correlations between antibody levels and HIV-1 incidence. Results. Peak antibody levels were significantly inversely correlated with HIV-1 incidence. The relative risk (RR) of infection was 0.63 (95% confidence interval, 0.45-0.89) per log10 higher neutralization titer against HIV-1MN, and the RRs of infection for second-, third-, and fourth-quartile responses of antibody blocking of gp120 binding to soluble CD4 versus first-quartile responses (the lowest responses) were 0.35, 0.28, and 0.22, respectively. Conclusions. Despite inducing a complex, robust immune response, the vaccine was unable to reduce the incidence of HIV-1. Two interpretations of the correlative results are that the levels of antibodies (i) caused both an increased (low responders) and decreased (high responders) risk of HIV-1 acquisition or (ii) represented a correlate of susceptibility to HIV-1 but had no causal effect on susceptibility. Although the data cannot definitively discriminate between these 2 explanations, (ii) appears to be more likely.