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Atypical hemolytic uremic syndrome (aHUS) is a rare but severe type of thrombotic microangiopathy that is triggered by the abnormal activation of the alternative complement pathway. Previous studies have reported that three completely linked coding variants of CFHR1 form two haplotypes, namely, CFHR1 *A (c.469C, c.475C, c.523G) and CFHR1 *B (c.469T, c.475G, c.523C). CFHR1 *B is associated with susceptibility to aHUS. To explore the genetic mechanism by which CFHR1 isoforms contribute to aHUS, we compared the structures of FHR1*A and FHR1*B by homology modeling and found differences in the angles between SCR3 and SCR4-SCR5, as FHR1*B had a larger angle than FHR1*A. Then, we expressed FHR1*A and FHR1*B recombinant proteins and compared their functions in complement system regulation and inflammation. We found that FHR1*B presented a significantly higher capacity for binding C3b and necrotic cells than FHR1*A. In a cofactor assay, the FHR-1*B showed stronger influence on FH mediated cofactor function than the FHR-1*A, resulted in fewer C3b cleavage products. In the C3 convertase assays, FHR1*B showed more powerful effect compared with FHR1*A regarding to de-regulate FH function of inhibition the assembling of C3bBb. Additionally, we also found that FHR1*B triggered monocytes to secrete higher levels of IL-1β and IL-6 than FHR1*A. In the present study, we showed that variants of CFHR1 might differently affect complement activation and sterile inflammation. Our findings provide a possible mechanism underlying the predisposition to aHUS caused by CFHR1 isoform CFHR1 *B., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Kang, Du, Guo, Zhu and Zhang.)

The aims of this study was to investigate the influences of hsa_circ_0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has_circ_0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa_circ_0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ_0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa_circ_0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa_circ_0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa_circ_0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.

Aim: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Results: Alemtuzumab-Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Conclusion: In this proof-of-concept study, a CHO-CD52 cell line has been developed and used to detect the presence of anti-alemtuzumab neutralizing antibodies. This platform provides the basis of an assay for routine screening of serum for neutralizing antibodies from patients treated with alemtuzumab.

In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences is required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmarks, e.g., binding energy of -11 kcal/mol, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated in the analyses of two small molecule targets, biotin and digoxin. Low nanomolar limits of detection were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.

Programmed death ligand 1 (PD-L1)-mediated immunosuppression regulates peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but also binds to B7-1 (CD80) to regulate T-cell function. The binding interaction of PD-L1 with B7-1 and its functional role need further investigation to understand differences between PD-1 and PD-L1 tumor immunotherapy. We examined the molecular orientation of PD-L1 binding to B7-1 using cell-to-cell binding assays, ELISA, and flow cytometry. As expected, PD-L1-transfected cells bound to PD-1-transfected cells, and B7-1 cells bound to CD28 or CTLA-4-transfected cells; however, PD-L1 cells did not bind to B7-1 cells. By ELISA and flow cytometry with purified proteins, we found PD-L1 and B7-1 had a strong binding interaction only when PD-L1 was flexible. Soluble PD-1 and B7-1 competed for binding to PD-L1. Binding of native PD-L1 and B7-1 in cis on the same cell surface was demonstrated with NanoBiT proximity assays. Thus, PD-L1-B7-1 interaction can occur in cis on the same cell but not in trans between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7-1 in cis , in a manner that can competitively block the binding of PD-L1 to PD-1 or of B7-1 to CD28. This binding orientation emphasizes the functional importance of coexpression of PD-L1 and B7-1 on the same cell. We found such coexpression on tumor-infiltrating myeloid cells. Our findings may help better utilize these pathways in cancer immunotherapy. Cancer Immunol Res; 6(8); 921-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.

Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2β). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of protein modifications might occur in diabetic patients in hyperglycaemic conditions that may be recognised as foreign molecules and can induce autoantibodies. Increased level of anti-Amadori-albumin autoantibodies may be used as a biomarker in disease diagnosis and its progression., Competing Interests: The authors have declared that no competing interests exist.

Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were determined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.

The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies.

Affinity proteins have advanced the field of targeted therapeutics due to their generally higher specificity compared to small molecular compounds. However, side effects caused by on-target binding in healthy tissues are still an issue. Here, we design and investigate a prodrug strategy for improving tissue specificity of Affibody molecules in future in vivo studies. The prodrug Affibody (pro-Affibody) against the HER2 receptor was constructed by fusing a HER2-specific Affibody (ZHER2) to an anti-idiotypic Affibody (anti-ZHER2). The linker was engineered to comprise a substrate peptide for the cancer-associated matrix metalloprotease 1 (MMP-1). The hypothesis was that the binding surface of ZHER2 would thereby be blocked from interacting with HER2 until the substrate peptide was specifically hydrolyzed by MMP-1. Binding should thereby only occur where MMP-1 is overexpressed, potentially decreasing on-target toxicities in normal tissues. The pro-Affibody was engineered to find a suitable linker and substrate peptide, and the different constructs were evaluated with a new bacterial display assay. HER2-binding of the pro-Affibody was efficiently masked and proteolytic activation of the best variant yielded over 1,000-fold increase in apparent binding affinity. Biosensor analysis revealed that blocking of the pro-Affibody primarily affected the association phase. In a cell-binding assay, the activated pro-Affibody targeted native HER2 on cancer cells as opposed to the non-activated pro-Affibody. We believe this prodrug approach with proteolytic activation is promising for improving tissue specificity in future in vivo targeting applications and can hopefully be extended to other Affibody molecules and similar affinity proteins as well.

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Interleukin-1β (IL-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1β from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1β was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1β is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1β and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1β, canakinumab binds IL-1β on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1β:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1β binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII.

T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions., (© 2014 by The American Society of Hematology.)

Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Context: As a new antibody concept, natural bispecific antibodies (nBsAbs) have been detected in long-term passive immunization and some diseases, but their potential immunomodulatory role remains unclear. Hashimoto thyroiditis (HT) appears to fulfill the condition for nBsAb production but has not yet been characterized., Objective: The objective of the study was to identify a new nBsAb against thyroid peroxidase (TPO) and thyroglobulin (Tg) in HT patients and to preliminarily explore its immunomodulatory role., Design, Setting, and Patients: Serum samples were obtained from 136 HT patients, 92 diseased controls, and 99 healthy controls for anti-TPO/Tg nBsAb detection. The relationship between anti-TPO/Tg nBsAb and other clinical parameters was also analyzed., Main Outcome Measures: The anti-TPO/Tg nBsAb was detected using a double-antigen sandwich ELISA. Higher nBsAb levels were found to be associated with decreased inflammation in HT patients., Results: The prevalence of anti-TPO/Tg nBsAb in HT was 44.9% (61 of 136), significantly higher than that of diseased controls (2.2%, 2 of 92) (P < .0001) and healthy controls (0%, 0 of 99) (P < .0001). HT patients who were nBsAb positive were prone to have significantly lower levels of serum C-reactive protein and TNF-α compared with the nBsAb-negative individuals (P < .05). The serum amyloid A and interferon-γ levels also showed a similar trend in the two groups. The IgG subclass of anti-TPO/Tg nBsAb was IgG4. Further analysis showed a negative correlation between anti-TPO/Tg nBsAb and serum total IgG4 (r = -0.697, P = .025) in IgG4 thyroiditis patients., Conclusions: A new type of nBsAb against TPO and Tg in HT patients is identified. Our data also indicate a protective effect of anti-TPO/Tg nBsAb in the pathogenesis of HT and extend prior knowledge about nBsAb in diseases.

Autoreactive T cells infiltrating the target organ can possess a broad TCR affinity range. However, the extent to which such biophysical parameters contribute to T cell pathogenic potential remains unclear. In this study, we selected eight InsB9-23-specific TCRs cloned from CD4(+) islet-infiltrating T cells that possessed a relatively broad range of TCR affinity to generate NOD TCR retrogenic mice. These TCRs exhibited a range of two-dimensional affinities (∼ 10(-4)-10(-3) μm(4)) that correlated with functional readouts and responsiveness to activation in vivo. Surprisingly, both higher and lower affinity TCRs could mediate potent insulitis and autoimmune diabetes, suggesting that TCR affinity does not exclusively dictate or correlate with diabetogenic potential. Both central and peripheral tolerance mechanisms selectively impinge on the diabetogenic potential of high-affinity TCRs, mitigating their pathogenicity. Thus, TCR affinity and multiple tolerance mechanisms converge to shape and broaden the diabetogenic T cell repertoire, potentially complicating efforts to induce broad, long-term tolerance., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Human ficolin-2 (L-ficolin/p35) is a lectin-complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during chronic hepatitis C virus (HCV) infection. In this study, we found that ficolin-2 inhibits the entry of HCV at an early stage of viral infection, regardless of the viral genotype. Ficolin-2 neutralized and inhibited the initial attachment and infection of HCV by binding to the HCV envelope surface glycoproteins E1 and E2, blocking HCV attachment to low-density lipoprotein receptor (LDLR) and scavenger receptor B1, and weakly interfering with CD81 receptor attachment. However, no interference with claudin-1 and occludin receptor attachment was observed. The C-terminal fibrinogen domain (201-313 aa) of ficolin-2 was identified as the critical binding region for the HCV-E1-E2 N-glycans, playing a critical role in the anti-HCV activity. More importantly, we found that apolipoprotein E (ApoE)3, which is enriched in the low-density fractions of HCV RNA-containing particles, promotes HCV infection and inhibits ficolin-2-mediated antiviral activity. ApoE3, but not ApoE2 and ApoE4, blocked the interaction between ficolin-2 and HCV-E2. Our data suggest that the HCV entry inhibitor ficolin-2 is a novel and promising antiviral innate immune molecule, whereas ApoE3 blocks the effect of ficolin-2 and mediates an immune escape mechanism during chronic HCV infection. HCV may be neutralized using compounds directed against the lipoprotein moiety of the viral particle, and ApoE3 may be a new target to combat HCV infection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Allopurinol (ALP) hypersensitivity is a major cause of severe cutaneous adverse reactions and is strongly associated with the HLA-B*58:01 allele. However, it can occur in the absence of this allele with identical clinical manifestations. The immune mechanism of ALP-induced severe cutaneous adverse reactions is poorly understood, and the T cell-reactivity pattern in patients with or without the HLA-B*58:01 allele is not known. To understand the interactions among the drug, HLA, and TCR, we generated T cell lines that react to ALP or its metabolite oxypurinol (OXP) from HLA-B*58:01(+) and HLA-B*58:01(-) donors and assessed their reactivity. ALP/OXP-specific T cells reacted immediately to the addition of the drugs and bypassed intracellular Ag processing, which is consistent with the "pharmacological interaction with immune receptors" (p-i) concept. This direct activation occurred regardless of HLA-B*58:01 status. Although most OXP-specific T cells from HLA-B*58:01(+) donors were restricted by the HLA-B*58:01 molecule for drug recognition, ALP-specific T cells also were restricted to other MHC class I molecules. This can be explained by in silico docking data that suggest that OXP binds to the peptide-binding groove of HLA-B*58:01 with higher affinity. The ensuing T cell responses elicited by ALP or OXP were not limited to particular TCR Vβ repertoires. We conclude that the drug-specific T cells are activated by OXP bound to HLA-B*58:01 through the p-i mechanism.

A key component of immunity against viruses, CD4(+) T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg-cell responses are diminished upon secondary infection, and Treg-cell response dynamics are associated more with T-cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA(b) M209 epitope of respiratory syncytial virus is recognized by both CD4(+) Treg and Teff cells, while the IA(b) M226 epitope is recognized almost exclusively by CD4(+) Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4(+) T-cell response during primary and secondary infections. IA(b) M209 -Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7-expressing IA(b) M209 -Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA(b) M209 -Teff cells outcompete IA(b) M209 -Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC-peptide-TCR interactions can produce different kinetic and functional profiles in CD4(+) T-cell populations even when responding to the same epitope., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.

It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the nonobese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant "don't eat me" signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. The increased affinity of BALB/c SIRPA for human CD47 was mounted at least through the BALB/c-specific L29V SNP within the IgV domain. Thus, the mouse strain effect on xenogeneic engraftment might be ascribed mainly to the binding affinity of strain-specific polymorphic SIRPA with human CD47. This information should be useful for developing a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

F.W.R. Brambell deduced the existence of a protective receptor for IgG, the neonatal Fc receptor (FcRn), long before its discovery in the early to mid-1990s. With the coincident, explosive development of IgG-based drugs, FcRn became a popular target for tuning the pharmacokinetics of monoclonal antibodies (mAbs). One aspect of Brambell's initial observation, however, that is seldom discussed since the discovery of the receptor, is the compliance in the mechanism that Brambell observed (saturating at 10s-100s of μM concentration), vs. the comparative stiffness of the receptor kinetics (saturating in the nM range for most species). Although some studies reported that increasing the already very high Fc-FcRn affinity at pH 6.0 further improved mAb half-life, in fact the results were mixed, with later studies increasingly implicating non-FcRn-dependent mechanisms as determinants of mAb pharmacokinetics. Mathematical modelling of the FcRn system has also indicated that the processes determining the pharmacokinetics of mAbs have more nuances than had at first been hypothesised. We propose, in keeping with the latest modelling and experimental evidence reviewed here, that the dynamics of endosomal sorting and trafficking have important roles in the compliant salvage mechanism that Brambell first observed nearly 50 years ago, and therefore also in the pharmacokinetics of mAbs. These ideas lead to many open questions regarding the endosomal trafficking of both FcRn and mAbs and also to what properties of a mAb can be altered to achieve an improvement in pharmacokinetics., (Published by Elsevier Ltd.)

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Aims: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes., Methods and Results: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells., Conclusions: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Recently, we identified the mimotope UH-CIS6 as a novel candidate antibody target for clinically isolated syndrome (CIS) and relapsing-remitting (RR) multiple sclerosis (MS). The purpose of this study was to further validate UH-CIS6 as an antibody target for CIS and MS and to identify the in vivo antibody target of UH-CIS6. First, a UH-CIS6 peptide ELISA was optimized. Next, we investigated the antibody response toward UH-CIS6 in cerebrospinal fluid (CSF) from patients with CIS (n = 20), MS (n = 43) and other neurological diseases (n = 42). Immunoprecipitation of anti-UH-CIS6 antibodies on a normal human brain lysate was performed to identify the in vivo antibody target of UH-CIS6. The cellular expression of an in vivo candidate target was investigated by immunohistochemistry using MS brain tissue sections. Antibody reactivity toward UH-CIS6 was detected in a significantly increased proportion of CSF samples from CIS and RR-MS patients as compared with neurological controls (p = 0.046). We identified and confirmed coronin-1a as the in vivo antibody target for UH-CIS6. Furthermore, coronin-1a was expressed by T cells and macrophages in an active MS lesion. Together, these results demonstrate that coronin-1a is a novel antibody target for CIS and MS., (© 2013 International Society for Neurochemistry.)

Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.

Purpose: Immunotherapy targeting disialoganglioside GD(2) emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD(2)-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD(2) antibodies of the 14.18 family., Experimental Design and Results: Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG(1)). This antibody bound to anti-GD(2) antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD(2) antibodies to the nominal antigen GD(2) as well as GD(2)-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD(2) surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD(2) antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD(2)-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD(2)-specific killing of neuroblastoma cells., Conclusion: We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.

Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance., Objective: We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα., Methods: Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated., Results: Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen., Conclusion: A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Interleukin-7 (IL-7) is essential to T-cell survival as well as homeostatic proliferation, and clinical trials that exploit the mitogenic effects of IL-7 have achieved success in treating human diseases. In mice, the in vivo potency of IL-7 improves dramatically when it is administered as a complex with the anti-IL-7 neutralizing monoclonal antibody clone M25. However, the mechanism whereby M25 augments IL-7 potency is unknown. We have analyzed the discrete contributions of the antibody constant (Fc) and IL-7-binding (Fab) domains to the mechanism. By engaging the neonatal Fc receptor the Fc domain extends the in vivo lifespan of IL-7/M25 complexes and accounts for the majority of their activity. Unexpectedly, the IL-7-neutralizing Fab domain provides an additional, albeit smaller, contribution, possibly by serving as a cytokine depot. This study is the first to demonstrate that the neutralizing aspect of the monoclonal antibody is directly involved in enhancing the potency of a cytokine with a single form of receptor. Lessons from the mechanism of IL-7/M25 complexes inform the design of next-generation cytokine therapeutics.

The Fc region of IgG-based molecules plays an important role in determining their in vivo pharmacokinetic profile by its pH-dependent binding to the neonatal Fc receptor (FcRn) which is expressed on the endothelial cells lining blood vessels. By virtue of this pH-specific interaction with IgG-Fc, FcRn mediates IgG homeostasis in human adults by maintaining serum IgG levels, and also transfers maternal IgGs from mother to fetus via the placenta. The Fc-FcRn interaction is also critical for keeping IgG-based therapeutic molecules in circulation thereby enhancing their serum half life. A homogeneous cell-based flow cytometric FcRn binding assay was established to characterize the Fc-FcRn interaction of therapeutic IgG-based molecules. It is a competition-based assay, wherein the IgG-Fc containing test molecule competes with a fixed concentration of fluorescently-labeled IgG-Fc moiety in solution for binding to the cell-expressed FcRn. The cell-bound fluorescence is read on a flow cytometer. Response of the test sample is analyzed relative to the standard sample and the results are reported as % relative binding. The assay is robust and meets the qualification criteria for specificity, method linearity, accuracy and precision over the relative binding range of 60%-160%. This assay was shown to effectively characterize altered Fc-FcRn interactions for photo-stressed, heat-stressed, oxidized, and Fc mutant samples. It was observed that the relative binding of the IgG-Fc to the cell-surface-expressed FcRn in the assay varies across different molecules, even within the same IgG subclass. This indicates that the Fc-FcRn binding can be influenced by the antigen-binding region of the molecules in addition to the IgG subclass. Overall, this assay is reflective of the in vivo mechanism of immunoglobulin binding to membrane-bound FcRn, and can be used as an analytical tool for assessing lot-to-lot consistency and stability testing across different batches of the same molecule. Additionally, the assay can be used as an effective tool for elucidating the amino acids in the IgG-Fc domain that are critical for FcRn binding and also for comparing the binding of different IgG-Fc containing molecules to FcRn., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Biotherapeutic proteins induce undesired immune responses that can affect drug efficacy and safety. For this reason, immunogenicity assessment is an integral part of drug development and is mandated by the regulatory authorities. Immunogenicity is typically evaluated by a tiered approach consisting of a screening assay followed by a competitive inhibition with unlabeled drug serving as confirmatory assay and additional characterization of the immune response. The confirmatory assay is intended to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. The positive-negative sample decisions are based on screening and confirmatory assay cut points that are statistically derived through evaluation of drug-naive samples. In this paper, we describe the analysis of cut point data for the presence of statistical correlation between the screening and confirmatory results. Data were obtained from validations of solution-phase bridging assays for detection of anti-drug antibodies against monoclonal antibody therapeutics. All data sets showed moderate to strong positive correlation, indicating that the screening and confirmatory assays were not independent and were likely to generate similar information. We present theoretical evidence that correlated results may be a general feature of the tiered approach when the same test platform is used for both screening and confirmatory assays. The competitive inhibition test, therefore, may be of limited value beyond reduction of the overall false positive rate. Our results indicate that similar sample results could be obtained by using just the screening assay with the false positive rate set to 1%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAcβ3Galβ4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.

Unlabelled: Humans express four MHC-like CD1 molecules CD1a, b, c and d that are capable of presenting a wide variety of self or foreign lipid antigens to T cells. Much progress has been made in elucidating the function of CD1d-restricted NKT cells in both innate and adaptive immune responses. However, knowledge of the other CD1 molecules is less well defined in terms of lipid presentation and immune regulation. We have previously shown that immunoglobulin-like transcript 4 (ILT4) binds to CD1d and inhibits its recognition by NKT cells. In this study, we show that CD1c can also interact specifically with ILT4 with a higher affinity than that of CD1d. Furthermore, changes in CD1c expression seem to modulate CD1d function; up-regulation of CD1c enhances NKT recognition of CD1d and down-regulation reduces CD1d recognition. We propose that CD1c can act as a sink for the inhibitory receptor ILT4: when CD1c is up-regulated, ILT4 is recruited to CD1c, thus reducing the inhibitory effect of ILT4 on CD1d recognition. Consequently, CD1c could be a potential target for modulating NKT activity., Keywords: NKT, CD1d, CD1c, ILT4, antigen presentation.

The enzyme-linked immunosorbent assay (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found non-specific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent., (Published by Elsevier B.V.)

DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of the PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind to mDEC205-expressing cells. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC(50), intrinsic dissociation t(1/2), and DM-mediated dissociation t(1/2) for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1 and compared these properties to the presence and magnitude of peptide-specific CD4(+) T cell responses. We found that MHC II-peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from nonrecognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation t(1/2) is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

Aims: To study the immunogenicity of advanced glycation end product (AGE) modified IgG (AGE-IgG) in experimental animals., Main Methods: Human IgG was subjected to in vitro glycation with glucose and the formation of N(ε)-(carboxymethyl)lysine (CML) was evaluated by high performance liquid chromatography (HPLC). The immunogenicity of native and AGE-IgG was investigated by raising polyclonal antibodies against them in rabbits. The induced antibodies were purified on a Protein-A agarose affinity column. Specific binding of antibodies was screened by competitive inhibition assay and band shift assay. Cross reactions of induced antibodies with various proteins or amino acids and their glycated conformers were ascertained by competitive inhibition ELISA., Key Findings: We detected the CML formation in AGE-IgG. The AGE-IgG was found to be highly immunogenic due to the generation of neo-epitopes on it. Affinity purified antibodies exhibited high degree of specific binding with AGE-IgG in comparison to the native IgG. Antibodies against AGE-IgG exhibited diverse antigen binding characteristics and the glycated conformers of various proteins and amino acids were found to be effective inhibitors of antibody-immunogen interaction in cross reaction studies. Band shift assay reiterated the results obtained by direct binding and competitive inhibition assay., Significance: The induced antibodies against AGE-IgG resembled the diverse antigen binding characteristics of autoantibodies found in rheumatoid arthritis (RA). IgG modified by AGEs under oxidative stress presents unique neo-epitopes which may be one of the factors for the induction of autoantibodies in RA patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.

Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.

Objective: Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system. Circulating autoantibodies (NMO-immunoglobulin [Ig]G) against astrocyte water channel aquaporin-4 (AQP4) cause complement- and cell-mediated astrocyte damage with consequent neuroinflammation and demyelination. Current NMO therapies, which have limited efficacy, include immunosuppression and plasma exchange. The objective of this study was to develop a potential new NMO therapy based on blocking of pathogenic NMO-IgG binding to its target, AQP4., Methods: We generated nonpathogenic recombinant monoclonal anti-AQP4 antibodies that selectively block NMO-IgG binding to AQP4. These antibodies comprise a tight-binding anti-AQP4 Fab and a mutated Fc that lacks functionality for complement- and cell-mediated cytotoxicity. The efficacy of the blocking antibodies was studied using cell culture, spinal cord slice, and in vivo mouse models of NMO., Results: In AQP4-expressing cell cultures, the nonpathogenic competing antibodies blocked binding of NMO-IgG in human sera, reducing to near zero complement- and cell-mediated cytotoxicity. The antibodies prevented the development of NMO lesions in an ex vivo spinal cord slice model of NMO and in an in vivo mouse model, without causing cytotoxicity., Interpretation: Our results provide proof of concept for a therapy of NMO with blocking antibodies. The broad efficacy of antibody inhibition is likely due to steric competition because of its large physical size compared to AQP4. Blocker therapy to prevent binding of pathogenic autoantibodies to their targets may be useful for treatment of other autoimmune diseases as well., (Copyright © 2011 American Neurological Association.)

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.