INDI-integrated nanobody database for immunoinformatics.

Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

Exploiting antibody biology for the treatment of cancer.

Over the last decade, antibodies have become an important component in the arsenal of cancer therapeutics. High-specificity, low off-target effects, desirable pharmacokinetics and high success rate are a few of the many attributes that make antibodies amenable for development as drugs. To design antibodies for successful clinical applications, however, it is critical to have an understanding of their structure, functions, mechanisms of action and pharmacokinetic/pharmacodynamic properties. This review highlights some of these key aspects, as well as certain limitations encountered, with monoclonal antibody therapy. Further, we discuss rational combination therapies for clinical applications, some of which could help overcome the limitations.

Discovery of receptor-ligand interfaces in the immunoglobulin superfamily.

Cell-surface-anchored immunoglobulin superfamily (IgSF) proteins are widespread throughout the human proteome, forming crucial components of diverse biological processes including immunity, cell-cell adhesion, and carcinogenesis. IgSF proteins generally function through protein-protein interactions carried out between extracellular, membrane-bound proteins on adjacent cells, known as trans-binding interfaces. These protein-protein interactions constitute a class of pharmaceutical targets important in the treatment of autoimmune diseases, chronic infections, and cancer. A molecular-level understanding of IgSF protein-protein interactions would greatly benefit further drug development. A critical step toward this goal is the reliable identification of IgSF trans-binding interfaces. We propose a novel combination of structure and sequence information to identify trans-binding interfaces in IgSF proteins. We developed a structure-based binding interface prediction approach that can identify broad regions of the protein surface that encompass the binding interfaces and suggests that IgSF proteins possess binding supersites. These interfaces could theoretically be pinpointed using sequence-based conservation analysis, with performance approaching the theoretical upper limit of binding interface prediction accuracy, but achieving this in practice is limited by the current ability to identify an appropriate multiple sequence alignment for conservation analysis. However, an important contribution of combining the two orthogonal methods is that agreement between these approaches can estimate the reliability of the predictions. This approach was benchmarked on the set of 22 IgSF proteins with experimentally solved structures in complex with their ligands. Additionally, we provide structure-based predictions and reliability scores for the 62 IgSF proteins with known structure but yet uncharacterized binding interfaces.

Antibodies against recombinant alpha-galactosidase A in Fabry disease: Subclass analysis and impact on response to treatment.

BACKGROUND: Treatment of Fabry disease (FD) with recombinant alpha-galactosidase A (r-αGAL A) is complicated by the formation of anti-drug antibodies in the majority of male patients with the classical disease phenotype. Detailed information regarding antibody subtypes, onset and persistence of antibody development and their effect on treatment efficacy is sparse. METHODS: A retrospective study was carried out in 39 male patients with classical FD, treated with either agalsidase-alfa or agalsidase-beta (mean follow up of 10 years). With six to twelve months intervals plasma-induced in vitro inhibition of enzyme activity, lysoglobotriaosylsphingosine (lysoGb3) levels and renal function were assessed. In a subset of 12 patients, additionally anti- r-αGAL A IgM, IgA and IgG1, 2, 3 and 4 levels were analyzed. RESULTS: In 23 out of 39 patients, plasma-induced in vitro inhibition of r-αGAL A activity was observed (inhibition-positive). The inhibition titer was strongly negatively correlated to the decrease in lysoGb3: agalsidase-alfa (FElog10(inhibition) = -10.3, P ≤.001), agalsidase-beta (FElog10(inhibition) = -4.7, P ≤.001). Inhibition-positive patients had an accelerated decline in renal function (FE = 1.21, p = .042). During treatment IgG1 anti-r-αGAL A levels increased only in inhibition-positive patients (p = .0045). IgG4 anti-r-αGAL A antibodies developed in 7 out of 9 inhibition-positive patients. Other antibody subclasses were either not present or too low to quantify. CONCLUSION: Development of inhibiting antibodies against r-αGAL A negatively affects the biochemical response to ERT and resulted in an accelerated decline in renal function. The presence of IgG1 and IgG4 anti-r-αGAL A antibodies is associated with in vitro αGAL A activity inhibition.

Novel Approaches to Analyze Immunoglobulin Repertoires.

Analysis of immunoglobulin (Ig) repertoires aims to comprehend Ig diversity with the goal of predicting humoral immune responses in the context of infection, vaccination, autoimmunity, and malignancies. The first next-generation sequencing (NGS) analyses of bulk B cell populations dramatically advanced sampling depth over previous low-throughput single-cell-based protocols, albeit at the expense of accuracy and loss of chain-pairing information. In recent years the field has substantially differentiated, with bulk analyses becoming more accurate while single-cell approaches have gained in throughput. Additionally, new platforms striving to combine high throughput and chain pairing have been developed as well as various computational tools for analysis. Here we review the developments of the past 4-5 years and discuss the open challenges.

Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma.

In recent years, several studies on large series of multiple myeloma (MM) patients have demonstrated the clinical utility of flow cytometry monitoring of minimal residual disease (flow-MRD) in bone marrow (BM), for improved assessment of response to therapy and prognostication. However, disturbing levels of variability exist regarding the specific protocols and antibody panels used in individual laboratories. Overall, consensus exists about the utility of combined assessment of CD38 and CD138 for the identification of BM plasma cells (PC); in contrast, more heterogeneous lists of markers are used to further distinguish between normal/reactive PCs and myeloma PCs in the MRD settings. Among the later markers, CD19, CD45, CD27, and CD81, together with CD56, CD117, CD200, and CD307, have emerged as particularly informative; however, no single marker provides enough specificity for clear discrimination between clonal PCs and normal PCs. Accordingly, multivariate analyses of single PCs from large series of normal/reactive vs. myeloma BM samples have shown that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient. However, the specific antibody clones, fluorochrome conjugates and sources of the individual markers determines its optimal (vs. suboptimal or poor) performance in an eight-color staining. Assessment of clonality, via additional cytoplasmic immunoglobulin (CyIg) κ vs. CyIgλ evaluation, may contribute to further establish the normal/reactive vs. clonal nature of small suspicious PC populations at high sensitivity levels, provided that enough cells are evaluated.

Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging.

Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the "professional phagocyte" of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity in vivo. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.

Significant Impact of Immunogen Design on the Diversity of Antibodies Generated by Carbohydrate-Based Anticancer Vaccine.

Development of an effective vaccine targeting tumor associated carbohydrate antigens (TACAs) is an appealing approach toward tumor immunotherapy. While much emphasis has been typically placed on generating high antibody titers against the immunizing antigen, the impact of immunogen design on the diversity of TACA-specific antibodies elicited has been overlooked. Herein, we report that the immunogen structure can significantly impact the breadth and the magnitude of humoral responses. Vaccine constructs that induced diverse TACA-binding antibodies provided much stronger recognition of a variety of Tn positive tumor cells. Optimization of the breadth of the antibody response led to a vaccine construct that demonstrated long lasting efficacy in a mouse tumor model. After challenged with the highly aggressive TA3Ha cells, mice immunized with the new construct exhibited a statistically significant improvement in survival relative to controls (0% vs 50% survival; p < 0.0001). Furthermore, the surviving mice developed long-term immunity against TA3Ha. Thus, both the magnitude and the breadth of antibody reactivity should be considered when designing TACA-based antitumor vaccines.

Sequencing the functional antibody repertoire--diagnostic and therapeutic discovery.

The development of high-throughput DNA sequencing technologies has enabled large-scale characterization of functional antibody repertoires, a new method of understanding protective and pathogenic immune responses. Important parameters to consider when sequencing antibody repertoires include the methodology, the B-cell population and clinical characteristics of the individuals analysed, and the bioinformatic analysis. Although focused sequencing of immunoglobulin heavy chains or complement determining regions can be utilized to monitor particular immune responses and B-cell malignancies, high-fidelity analysis of the full-length paired heavy and light chains expressed by individual B cells is critical for characterizing functional antibody repertoires. Bioinformatic identification of clonal antibody families and recombinant expression of representative members produces recombinant antibodies that can be used to identify the antigen targets of functional immune responses and to investigate the mechanisms of their protective or pathogenic functions. Integrated analysis of coexpressed functional genes provides the potential to further pinpoint the most important antibodies and clonal families generated during an immune response. Sequencing antibody repertoires is transforming our understanding of immune responses to autoimmunity, vaccination, infection and cancer. We anticipate that antibody repertoire sequencing will provide next-generation biomarkers, diagnostic tools and therapeutic antibodies for a spectrum of diseases, including rheumatic diseases.

Construção e expressão de anticorpo humanizado a partirdo anticorpo monoclonal contra proteína de 70 kDa de Sporotrix schenckii (P6E7) / Construction and expression of humanized antibody from the monoclonal antibody against the protein of 70 kDa Sporotrix schenckii (P6E7)

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana

Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis

Re-evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis.

Excessive amounts of melanin pigments may hamper histopathological assessments of melanocytic lesions by obscuring cellular morphology and hindering antibody-antigen interactions. To determine the optimal melanin-bleaching conditions for histopathological examination, heavily pigmented melanomas were treated with warm hydrogen peroxide (H2O2) diluted with various diluents (1% disodium hydrogen phosphate 12H2O (Na2 HPO4); phosphate buffer 0.05 M, pH 7.4 (PB); and PBS 0.05 M, pH 7.4) at varying temperatures (50°C, 55°C, and 60°C) and for varying incubation times (0.5, 1, 2, and 3 h). The effect of the sequential order of antigen retrieval and bleaching on preserving tissue morphology was then evaluated. Additionally, the effect of melanin bleaching using warm diluted H2O2 on the antigenicity of melanoma-related markers (HMB-45, MART-1, and S-100) and other markers used for histopathology was examined in amelanotic melanomas and tonsil tissue. Optimal and complete bleaching was achieved using warm 3% H2O2 in PB treatment at 55°C for 2 h following antigen retrieval with microwaving or digestion with trypsin. Under these conditions, the tissue morphology and antigenicity of various immunohistochemical markers were also well preserved. Bleaching with warm 3% H2O2 PB is a fast and efficient method of bleaching melanin pigments and performing immunohistochemical examination in heavily melanin-pigmented lesions.

Anti-Heat Shock Protein 70 antibody levels are increased in myasthenia gravis and Guillain-Barré syndrome.

Myasthenia gravis (MG) is an autoimmune disorder where patients develop autoantibodies towards skeletal muscle proteins (e.g. acetylcholine receptor and muscle specific kinase), causing weakness in striated muscles. Ocular MG (OMG) represents a subtype of (MG) affecting only the periocular muscles. The pathogenesis of this phenotype remains unclear. Heat Shock Protein 70 (Hsp70) plays a role in immune regulation. Antibodies against this protein are associated with several autoimmune diseases, and its biological significance has been shown in vivo. We have therefore examined the concentration of anti-Hsp70 antibodies in sera from 35 OMG patients and 94 patients with generalized MG (GMG) using ELISA assays. The antibody concentrations were compared to those in patients with multiple sclerosis (MS), Guillain-Barré syndrome (GBS) and to healthy controls. MG patients had significantly higher anti-Hsp70 antibody concentrations than both MS patients and healthy controls. GBS patients had higher antibody levels than all other groups. No difference in antibody levels was found when comparing OMG and GMG. Our results suggest that patients with MG and GBS have a previous or current increased exposure to Hsp70 antigens. The similarity between GMG and OMG strengthens the hypothesis that OMG represents a systemic disease, similar to GMG.

Glatiramer acetate immunization induces specific antibody and cytokine responses in ALS patients.

We assessed humoral and cytokine responses in monthly plasma samples from ALS patients who received glatiramer acetate (GA) immunization every day or every other week, or remained untreated (control) from a six-month phase II trial. Samples were evaluated by GA-specific ELISA assays for detection of combined immunoglobulin (Ig) classes (IgM,A,G), IgG alone, and IgG subclasses (IgG1, IgG2, IgG3, and IgG4). T-helper (Th) type 1 and 2 (Th1 and Th2) cytokine levels were determined by flow cytometric cytokine bead arrays. Fourteen of 21 GA-immunized patients produced anti-GA Ig responses. Those treated every day produced anti-GA responses within one month, while those treated every other week exhibited responses by month two. All anti-GA IgG subclass concentrations were increased in excess of 4.2-fold in plasma from treated patients, and anti-GA IgG1 comprised the majority of the humoral response. Mean plasma cytokine levels were statistically indistinguishable between treatment regimens; however, stratification by patient and time on study showed more prevalent trends in changes of Th1 or Th2 cytokine levels following GA treatment every other week or every day, respectively. These data show significant humoral responses and cytokine trends following GA immunization in ALS patients.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs.

BACKGROUND: Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. METHODS: Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. RESULTS: Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. CONCLUSIONS: The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

Optimization of antibodies for detection of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using a biotin-free visualization system.

Testing for microsatellite instability (MSI) has become an important step in the planning of therapeutic and follow-up procedures for patients with colorectal cancer, both as a prognostic marker and as a screening tool for hereditary non-polyposis colorectal cancer. Today the gold standard for MSI testing is based on the polymerase chain reaction. Immunohistochemistry may represent an alternative or complement to molecular MSI testing. Antibodies against the protein products of the most commonly affected mismatch repair genes (hMLH1, hMSH2, hMSH6, and hPMS2) have been available for some time now. However, the quality of the primary antibody and optimization of the antigen retrieval methods are essential to get reproducible results. The aim of the present study was to test and optimize a panel of antibodies against the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using biotin-free, polymer-based visualization systems. The antibodies were tested on multitissue blocks containing normal tissue and tumor tissue from patients with known microsatellite-stable and microsatellite-instable tumors. For all four antibody groups, the chosen clones gave specific and reproducible staining. Furthermore, with the PowerVision+ detection system, the influence of endogenous biotin was eliminated, the incubation time with the primary antibody was significantly reduced, and the primary antibody could be further diluted. The authors found that immunohistochemistry may provide a cost-effective and time-saving complement to the molecular MSI analysis, and using the PowerVision+ detection system has greatly decreased the turnaround time as well as reduced the cost of immunohistochemistry in the authors' laboratory.

VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula).

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.

Production and purification of monoclonal and polyclonal antibodies against cholera toxin.

The aim of this study was to produce monoclonal and polyclonal antibodies against cholera toxin (CT). Hyperimmune ICR mice produced polyclonal antibodies (PAbs) after injection with 0.5 mL of pristane and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). After these mice were immunized four times and given a final boost, their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the hypoxanthine, aminopterin, and thymidine (HAT)-RPMIX medium. Anti-CT antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Eleven murine hybridoma producing anti-CT MAbs were obtained and designated CT-A2, CT-B4, CT-B11, CT-C7, CT-D7, CT-E8, CT-F4, CT-F2, CT-F8, CT-E3, CT-E6. Isotypes of MAbs were identified as IgM heavy chain and all were lambda light chain. Hitrap rProtein A and Hitrap IgM purification columns were used for the purification of PAbs and MAbs, respectively.

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.

Antibodies in myasthenia gravis and related disorders.

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.