Open AccessFeature PaperArticle
Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings
Antibodies 2017, 6(4), 15; doi:10.3390/antib6040015 -
Abstract
Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the
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Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323) were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916) were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO)/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes). The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used. Full article
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Open AccessArticle
Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies
Antibodies 2017, 6(4), 14; doi:10.3390/antib6040014 -
Abstract
Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal
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Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections. Full article
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Open AccessArticle
Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly
Antibodies 2017, 6(3), 13; doi:10.3390/antib6030013 -
Abstract
Significant progress in the manufacturing of biopharmaceuticals has been made by increasing the overall titers in the USP (upstream processing) titers without raising the cost of the USP. In addition, the development of platform processes led to a higher process robustness. Despite or
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Significant progress in the manufacturing of biopharmaceuticals has been made by increasing the overall titers in the USP (upstream processing) titers without raising the cost of the USP. In addition, the development of platform processes led to a higher process robustness. Despite or even due to those achievements, novel challenges are in sight. The higher upstream titers created more complex impurity profiles, both in mass and composition, demanding higher separation capacities and selectivity in downstream processing (DSP). This creates a major shift of costs from USP to DSP. In order to solve this issue, USP and DSP integration approaches can be developed and used for overall process optimization. This study focuses on the characterization and classification of host cell proteins (HCPs) in each unit operation of the DSP (i.e., aqueous two-phase extraction, integrated countercurrent chromatography). The results create a data-driven feedback to the USP, which will serve for media and process optimizations in order to reduce, or even eliminate nascent critical HCPs. This will improve separation efficiency and may lead to a quantitative process understanding. Different HCP species were classified by stringent criteria with regard to DSP separation parameters into “The Good, the Bad, and the Ugly” in terms of pI and MW using 2D-PAGE analysis depending on their positions on the gels. Those spots were identified using LC-MS/MS analysis. HCPs, which are especially difficult to remove and persistent throughout the DSP (i.e., “Bad” or “Ugly”), have to be evaluated by their ability to be separated. In this approach, HCPs, considered “Ugly,” represent proteins with a MW larger than 15 kDa and a pI between 7.30 and 9.30. “Bad” HCPs can likewise be classified using MW (>15 kDa) and pI (4.75–7.30 and 9.30–10.00). HCPs with a MW smaller than 15 kDa and a pI lower than 4.75 and higher than 10.00 are classified as “Good” since their physicochemical properties differ significantly from the product. In order to evaluate this classification scheme, it is of utmost importance to use orthogonal analytical methods such as IEX, HIC, and SEC. Full article
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Open AccessArticle
Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality
Antibodies 2017, 6(3), 12; doi:10.3390/antib6030012 -
Abstract
Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant
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Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically “silent”. Full article
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Open AccessArticle
Monoclonal Antibodies against Plasmodium falciparum Circumsporozoite Protein
Antibodies 2017, 6(3), 11; doi:10.3390/antib6030011 -
Abstract
Malaria is a mosquito-borne infectious disease caused by the parasite Plasmodium spp. Malaria continues to have a devastating impact on human health. Sporozoites are the infective forms of the parasite inside mosquito salivary glands. Circumsporozoite protein (CSP) is a major and immunodominant protective
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Malaria is a mosquito-borne infectious disease caused by the parasite Plasmodium spp. Malaria continues to have a devastating impact on human health. Sporozoites are the infective forms of the parasite inside mosquito salivary glands. Circumsporozoite protein (CSP) is a major and immunodominant protective antigen on the surface of Plasmodium sporozoites. Here, we report a generation of specific monoclonal antibodies that recognize the central repeat and C-terminal regions of P. falciparum CSP. The monoclonal antibodies 3C1, 3C2, and 3D3—specific for the central repeat region—have higher titers and protective efficacies against challenge with sporozoites compared with 2A10, a gold standard monoclonal antibody that was generated in early 1980s. Full article
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Open AccessReview
Strategies for Selecting Membrane Protein-Specific Antibodies using Phage Display with Cell-Based Panning
Antibodies 2017, 6(3), 10; doi:10.3390/antib6030010 -
Abstract
Membrane proteins are attractive targets for monoclonal antibody (mAb) discovery and development. Although several approved mAbs against membrane proteins have been isolated from phage antibody libraries, the process is challenging, as it requires the presentation of a correctly folded protein to screen the
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Membrane proteins are attractive targets for monoclonal antibody (mAb) discovery and development. Although several approved mAbs against membrane proteins have been isolated from phage antibody libraries, the process is challenging, as it requires the presentation of a correctly folded protein to screen the antibody library. Cell-based panning could represent the optimal method for antibody discovery against membrane proteins, since it allows for presentation in their natural conformation along with the appropriate post-translational modifications. Nevertheless, screening antibodies against a desired antigen, within a selected cell line, may be difficult due to the abundance of irrelevant organic molecules, which can potentially obscure the antigen of interest. This review will provide a comprehensive overview of the different cell-based phage panning strategies, with an emphasis placed on the optimisation of four critical panning conditions: cell surface antigen presentation, non-specific binding events, incubation time, and temperature and recovery of phage binders. Full article
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Open AccessArticle
Potential Roles of Antiphospholipid Antibodies in Generating Platelet-C4d in Systemic Lupus Erythematosus
Antibodies 2017, 6(3), 9; doi:10.3390/antib6030009 -
Abstract
Premature, accelerated onset of atherothrombotic disease is prevalent in patients with systemic lupus erythematosus (SLE). Most, if not all, atherothrombotic diseases are likely to involve platelets and complement. Previously, we discovered that platelets bearing complement activation product C4d (P-C4d) are present in SLE
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Premature, accelerated onset of atherothrombotic disease is prevalent in patients with systemic lupus erythematosus (SLE). Most, if not all, atherothrombotic diseases are likely to involve platelets and complement. Previously, we discovered that platelets bearing complement activation product C4d (P-C4d) are present in SLE patients, and are significantly associated with antiphospholipid (aPL) antibody positivity and stroke in SLE patients. The goal of the present study was to further elucidate the role of aPL and other platelet-reactive autoantibodies in the generation of P-C4d. To determine the association between P-C4d and aPL antibodies, the serum levels of aPL antibodies and P-C4d of 180 SLE patients were measured by enzyme-linked immunoassays and flow cytometry, respectively. To investigate the role of aPL antibodies, and possibly other autoantibodies as well, in mediating the generation of P-C4d, in vitro 2-step P-C4d induction experiments were performed. The results showed that the presence and levels of aPL antibodies in the serum were specifically elevated in SLE patients with positive P-C4d. The plasma and immunoglobulins purified from SLE patients who were positive for P-C4d and aPL were capable of inducing C4d deposition on normal platelets in vitro. The capacity of SLE plasma in inducing P-C4d appeared to correlate proportionately to the serum aPL levels. Collectively, the results demonstrate that both aPL and other platelet-reactive autoantibodies may participate in mediating the generation of P-C4d in SLE patients. Full article
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Open AccessArticle
Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops
Antibodies 2017, 6(2), 8; doi:10.3390/antib6020008 -
Abstract
Upregulation of the procoagulant activity of monocytes by antibodies to beta2-glycoprotein I (β2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by β2GPI/anti-β2GPI complexes and their binding sites on
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Upregulation of the procoagulant activity of monocytes by antibodies to beta2-glycoprotein I (β2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by β2GPI/anti-β2GPI complexes and their binding sites on β2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-β2GPI were compared to cells treated with dimerized domain V of β2GPI (β2GPI-DV) or point mutants of β2GPI-DV. We demonstrated that dimerized β2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on β2GPI is larger than previously thought and includes Lys308 in β2GPI-DV. Intact residues in two phospholipid-binding loops of β2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of β2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on β2GPI inducing procoagulant activity in monocytes can now be narrowed down to β2GPI-DV. The ability of β2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity. Full article
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Open AccessArticle
Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function
Antibodies 2017, 6(2), 7; doi:10.3390/antib6020007 -
Abstract
Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been
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Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function. Full article
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Open AccessArticle
Collagen Autoantibodies and Their Relationship to CCP Antibodies and Rheumatoid Factor in the Progression of Early Rheumatoid Arthritis
Antibodies 2017, 6(2), 6; doi:10.3390/antib6020006 -
Abstract
Serum autoantibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) are important markers for diagnosis and prognosis of rheumatoid arthritis (RA), but their autoantigens are not cartilage-specific. Autoantibodies to joint-specific type II collagen (CII) also occur in RA, and monoclonal antibodies of
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Serum autoantibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) are important markers for diagnosis and prognosis of rheumatoid arthritis (RA), but their autoantigens are not cartilage-specific. Autoantibodies to joint-specific type II collagen (CII) also occur in RA, and monoclonal antibodies of similar specificity induce collagen antibody-induced arthritis in animals, but their role in RA is uncertain. We utilized an enzyme-linked immunosorbent assay (ELISA) with the CB10 peptide of CII to compare the frequency of autoantibodies with those of anti-CCP and RF in stored sera from a prospective study of 82 patients with early RA to examine the outcome, defined as remission (n = 23), persisting non-erosive arthritis (n = 27), or erosions (n = 32). Initial frequencies of anti-CB10, anti-CCP and RF were 76%, 54%, and 57% in RA, and 4%, 0%, and 9% in 136 controls. The frequency of anti-CB10 was unrelated to outcome, but anti-CCP and RF increased with increasing severity, and the number of autoantibodies mirrored the severity. We suggest RA is an immune complex-mediated arthritis in which the three antibodies interact, with anti-CII inducing localized cartilage damage and inflammation resulting in citrullination of joint proteins, neoepitope formation, and a strong anti-CCP response in genetically-susceptible subjects, all amplified and modified by RF. Full article
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Open AccessReview
Epitope Specificity of Anti-Citrullinated Protein Antibodies
Antibodies 2017, 6(1), 5; doi:10.3390/antib6010005 -
Abstract
Anti-citrullinated protein antibodies are primarily associated with a progressive course in the autoimmune disease rheumatoid arthritis, a disease with a chronic and inflammatory nature. These antibodies do not appear to have any strict dependency for reactivity except from the presence of the non-genetically
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Anti-citrullinated protein antibodies are primarily associated with a progressive course in the autoimmune disease rheumatoid arthritis, a disease with a chronic and inflammatory nature. These antibodies do not appear to have any strict dependency for reactivity except from the presence of the non-genetically encoded amino acid citrulline, which is the result of a posttranslational modification, catalyzed by calcium-dependent peptidylarginine deiminase enzymes. Nevertheless, several amino acids surrounding the citrulline residue notably influence antibody reactivity, especially with a central-Cit-Gly-motif being essential for antibody reactivity. Most importantly, these antibodies have been proposed to be divided into two groups, based on their ability to recognize multiple citrullinated peptides. Thus, an “overlapping” antibody group, which appears to recognize several citrullinated peptides, and a “non-overlapping” antibody group, which only recognizes a limited number of citrullinated peptides, have been proposed. Based on these findings, we suggest that antibodies recognizing several citrullinated targets, also referred to as cross-reactive antibodies, primarily are backbone-dependent, whereas less cross-reactive antibodies primarily depend on the side chains of the amino acids comprising the epitopes for stable antibody-antigen interactions, which reduces the degree of cross-reactivity significantly. Clarifying the reactivity pattern of anti-citrullinated protein antibodies may contribute to determining their true nature of origin. Full article
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Open AccessReview
Neutrophil Extracellular Traps, Antiphospholipid Antibodies and Treatment
Antibodies 2017, 6(1), 4; doi:10.3390/antib6010004 -
Abstract
Neutrophil extracellular traps (NETs) are a network of extracellular fibers, compounds of chromatin, neutrophil DNA and histones, which are covered with antimicrobial enzymes with granular components. Autophagy and the production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are
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Neutrophil extracellular traps (NETs) are a network of extracellular fibers, compounds of chromatin, neutrophil DNA and histones, which are covered with antimicrobial enzymes with granular components. Autophagy and the production of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are essential in the formation of NETs. There is increasing evidence that suggests that autoantibodies against beta-2-glycoprotein-1 (B2GP1) induce NETs and enhance thrombosis. Past research on new mechanisms of thrombosis formation in antiphospholipid syndrome (APS) has elucidated the pharmacokinetics of the most common medication in the treatment of the disease. Full article
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Open AccessArticle
Pairing Alpaca and Llama-Derived Single Domain Antibodies to Enhance Immunoassays for Ricin
Antibodies 2017, 6(1), 3; doi:10.3390/antib6010003 -
Abstract
Previously, our group isolated and evaluated anti-ricin single domain antibodies (sdAbs) derived from llamas, engineered them to further increase their thermal stability, and utilized them for the development of sensitive immunoassays. In work focused on the development of therapeutics, Vance et al. 2013
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Previously, our group isolated and evaluated anti-ricin single domain antibodies (sdAbs) derived from llamas, engineered them to further increase their thermal stability, and utilized them for the development of sensitive immunoassays. In work focused on the development of therapeutics, Vance et al. 2013 described anti-ricin sdAbs derived from alpacas. Herein, we evaluated the utility of selected alpaca-derived anti-ricin sdAbs for detection applications, and engineered an alpaca-derived sdAb to increase its melting temperature, providing a highly thermal stable reagent for use in ricin detection. Four of the alpaca-derived anti-ricin A-chain sdAbs were produced and characterized. All four bound to epitopes that overlapped with our previously described llama sdAbs. One alpaca sdAb, F6, was found to possess both a high melting temperature (73 °C) and to work optimally with a thermally stable llama anti-ricin sdAb in sandwich assays for ricin detection. We employed a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond to further enhance the thermal stability of F6 to 85 °C. It is advantageous to have a choice of recognition reagents when developing assays. This work resulted in defining an additional pair of highly thermal stable sdAbs for the sensitive detection of ricin. Full article
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Open AccessEditorial
Acknowledgement to Reviewers of Antibodies in 2016
Antibodies 2017, 6(1), 2; doi:10.3390/antib6010002 -
Abstract
The editors of Antibodies would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2016.[...] Full article
Open AccessReview
Pathogenic and Protective Autoantibodies in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)
Antibodies 2017, 6(1), 1; doi:10.3390/antib6010001 -
Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare disorder caused by mutations in the autoimmune regulator (AIRE) gene, leading to defects in T cell selection. The disease manifestations include both autoimmune tissue destruction and immunodeficiency, with specific susceptibility to chronic mucocutaneous candidiasis.
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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare disorder caused by mutations in the autoimmune regulator (AIRE) gene, leading to defects in T cell selection. The disease manifestations include both autoimmune tissue destruction and immunodeficiency, with specific susceptibility to chronic mucocutaneous candidiasis. Studies have demonstrated a wide repertoire of high affinity tissue- and cytokine-specific antibodies in patients with APECED. Here, we review the antigenic targets and function of these disease-causing and disease-ameliorating antibodies. Full article
Open AccessReview
Current Controversies in Lupus Anticoagulant Detection
Antibodies 2016, 5(4), 22; doi:10.3390/antib5040022 -
Abstract
Antiphospholipid syndrome is an autoimmune, acquired thrombophilia diagnosed when vascular thrombosis or pregnancy morbidity are accompanied by persistent antiphospholipid antibodies. Lupus anticoagulants (LA) are one of the criteria antibodies but calibration plasmas are unavailable and they are detected by inference based on antibody
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Antiphospholipid syndrome is an autoimmune, acquired thrombophilia diagnosed when vascular thrombosis or pregnancy morbidity are accompanied by persistent antiphospholipid antibodies. Lupus anticoagulants (LA) are one of the criteria antibodies but calibration plasmas are unavailable and they are detected by inference based on antibody behaviour in a medley of coagulation-based assays. Elevated screening tests suggest the presence of a LA, which is confirmed with mixing tests to evidence inhibition and confirmatory tests to demonstrate phospholipid-dependence. At least two screening tests of different principle must be used to account for antibody heterogeneity and controversy exists on whether assays, in addition to dilute Russell’s viper venom time and activated partial thromboplastin time, should be employed. A variety of approaches to raw data manipulation and interpretation attract debate, as does inclusion or exclusion of mixing studies in circumstances where the presence of a LA is already evident from other results. Therapeutic anticoagulation compromises coagulation-based assays but careful data interpretation and use of alternative reagents can detect or exclude LA in specific circumstances, and this aspect of LA detection continues to evolve. This review focuses on the main areas of debate in LA detection. Full article
Open AccessReview
Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21
Antibodies 2016, 5(4), 21; doi:10.3390/antib5040021 -
Abstract
TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY
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TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21. Full article
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Open AccessArticle
Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally
Antibodies 2016, 5(4), 20; doi:10.3390/antib5040020 -
Abstract
Nanoparticles (NPs) are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n.) priming followed by i.n. or subcutaneous (s.c.) boosting immunization affects the humoral immune
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Nanoparticles (NPs) are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n.) priming followed by i.n. or subcutaneous (s.c.) boosting immunization affects the humoral immune response to chicken ovalbumin (Ova) and Ova conjugated to 20 nm NPs (NP-Ova). We show that i.n. priming with 20 mg of soluble Ova, a dose known to trigger oral tolerance when administered via gastric gavage, induced substantial systemic IgG1 and IgG2c, as well as mucosal antibodies. These responses were further boosted following a s.c. immunization with Ova and complete Freund’s adjuvant (Ova+CFA). In contrast, 100 µg of Ova delivered via NPs induced an IgG1-dominated systemic response, and primed the intestinal mucosa for secretion of IgA. Following a secondary s.c. or i.n. immunization with Ova+CFA or NP-Ova, systemic IgG1 titers significantly increased, and serum IgG2c and intestinal antibodies were induced in mice primed nasally with NP-Ova. Only Ova- and NP-Ova-primed mice that were s.c.-boosted exhibited substantial systemic and mucosal titers for up to 6 months after priming, whereas the antibodies of i.n.-boosted mice declined over time. Our results indicate that although the amount of Ova delivered by NPs was 1000-fold less than Ova delivered in soluble form, the antigen-specific antibody responses, both systemic and mucosal, are essentially identical by 6 months following the initial priming immunization. Additionally, both i.n.- and s.c.-boosting strategies for NP-Ova-primed mice were capable of inducing a polarized Th1/Th2 immune response, as well as intestinal antibodies; however, it is only by using a heterogeneous prime-boost strategy that long-lasting antibody responses were initiated. These results provide valuable insight for future mucosal vaccine development, as well as furthering our understanding of mucosal antibody responses. Full article
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Open AccessReview
Antibody Aggregation: Insights from Sequence and Structure
Antibodies 2016, 5(3), 19; doi:10.3390/antib5030019 -
Abstract
Monoclonal antibodies (mAbs) are the fastest-growing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregation of mAbs continues to be a major problem in their developability. Antibody aggregation could be triggered by partial unfolding
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Monoclonal antibodies (mAbs) are the fastest-growing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregation of mAbs continues to be a major problem in their developability. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and growth. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. In this review, we describe how the unfolding and aggregation susceptibilities of IgG could be related to their cognate sequences and structures. The impact of antibody domain structures on thermostability and aggregation propensities, and effective strategies to reduce aggregation are discussed. Finally, the aggregation of antibody-drug conjugates (ADCs) as related to their sequence/structure, linker payload, conjugation chemistry and drug-antibody ratio (DAR) is reviewed. Full article
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Open AccessReview
Antiphospholipid Antibodies: From General Concepts to Its Relation with Malignancies
Antibodies 2016, 5(3), 18; doi:10.3390/antib5030018 -
Abstract
Antiphospholipid syndrome (APS) is an adquired autoimmune pro-thrombotic disease characterized by arterial and/or venous thrombosis and/or fetal losses associated with the persistent presence of antiphospholipid antibodies (aPL) detectable by solid phase assays (anticardiolipin (aCL) and anti-β2 glycoprotein I, β2GPI) and/or functional coagulation test
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Antiphospholipid syndrome (APS) is an adquired autoimmune pro-thrombotic disease characterized by arterial and/or venous thrombosis and/or fetal losses associated with the persistent presence of antiphospholipid antibodies (aPL) detectable by solid phase assays (anticardiolipin (aCL) and anti-β2 glycoprotein I, β2GPI) and/or functional coagulation test (lupus anticoagulant (LA)). Most patients with typical APS manifestations have the presence of one or more of conventional aPL, but, some patients might exhibit clinical features related with APS but with persistent negative determinations of “classic” aPL (seronegative APS). Expanding the network of autoantibodies in patients highly suspected of having APS but who have normal results from a conventional test using new antibodies (i.e., phosphatidylserine/prothrombin and β2GPI domain 1) would increase the diagnosis. Thrombosis is one of the leading causes of death among patients with cancer, representing up to 15% of all deaths. Cancer increases the risk of thrombosis and chemotherapy is further associated with a higher risk of thrombosis. In addition, aPL may contribute to an increased risk of thrombosis in patients with malignancies, although the levels do not seem to reflect their pathogenicity. Several malignancies, particularly hematological and lymphoproliferative malignancies, may indeed be associated with the generation of aPL but do not necessarily enhance the thrombophilic risk in these patients. Full article