Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells.

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3-7% and ≤ 1%, respectively.

Immunoassay methods used in clinical studies for the detection of anti-drug antibodies to adalimumab and infliximab.

We examined the assay formats used to detect anti-drug antibodies (ADA) in clinical studies of the anti-tumour necrosis factor (TNF) monoclonal antibodies adalimumab and infliximab in chronic inflammatory disease and their potential impact on pharmacokinetic and clinical outcomes. Using findings of a recent systematic literature review of the immunogenicity of 11 biological/biosimilar agents, we conducted an ancillary qualitative review of a subset of randomized controlled trials and observational studies of the monoclonal antibodies against anti-TNF factor adalimumab and infliximab. Among studies of adalimumab and infliximab, the immunoassay method used to detect antibodies was reported in 91 of 111 (82%) and 154 of 206 (75%) adalimumab and infliximab studies, respectively. In most adalimumab and infliximab studies, an enzyme-linked immunosorbent assay or radioimmunoassay was used [85 of 91 (93%) and 134 of 154 (87%), respectively]. ADA incidence varied widely among assays and inflammatory diseases (adalimumab, 0-87%; infliximab, 0-79%). Pharmacokinetic and clinical outcomes were only reported for ADA-positive patients in 38 of 91 (42%) and 61 of 154 (40%) adalimumab and infliximab studies, respectively. Regardless of assay format or biological used, ADA formation was associated with lower serum concentrations, reduced efficacy and elevated rates of infusion-related reactions. Consistent with previous recommendations to improve interpretation of immunogenicity data for biologicals, greater consistency in reporting of assay methods and clinical consequences of ADA formation may prove useful. Additional standardization in immunogenicity testing and reporting, application of modern, robust assays that satisfy current regulatory expectations and implementation of international standards for marketed products may help to improve our understanding of the impact of immunogenicity to biologics.

Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules.

A mechanistic, multiscale mathematical model of immunogenicity for therapeutic proteins: part 1-theoretical model.

A mechanistic, multiscale mathematical model of immunogenicity for therapeutic proteins was formulated by recapitulating key biological mechanisms, including antigen presentation, activation, proliferation, and differentiation of immune cells, secretion of antidrug antibodies (ADA), as well as in vivo disposition of ADA and therapeutic proteins. This system-level model contains three scales: a subcellular level representing antigen presentation processes by dendritic cells; a cellular level accounting for cell kinetics during humoral immune response; and a whole-body level accounting for therapeutic protein in vivo disposition. The model simulations for in vivo responses against antigenic protein challenge are consistent with many known immunological observations. By simulating immune responses under various initial parameter conditions, the model suggests hypotheses for future experimental investigation and contributes to the mechanistic understanding of immunogenicity. With future experimental validation, this model may potentially provide a platform to generate and test hypotheses about immunogenicity risk assessment and ultimately aid in immunogenicity prediction.

A mechanistic, multiscale mathematical model of immunogenicity for therapeutic proteins: part 2-model applications.

A mechanistic, multiscale mathematical model of immunogenicity for therapeutic proteins was built by recapitulating key underlying known biological processes for immunogenicity. The model is able to simulate immune responses based on protein-specific antigenic properties (e.g., number of T-epitopes and their major histocompatibility complex (MHC)-II binding affinities) and host-specific immunological/physiological characteristics (e.g., MHC-II allele genotype, drug clearance rate). Preliminary validation was performed using mouse studies with antigens such as ovalbumin (OVA) or OVA-derived peptide. Further, using adalimumab as an example therapeutic protein, the model is able to simulate immune responses against adalimumab in individual subjects and in a population, and also provides estimations of immunogenicity incidence and drug exposure reduction that can be validated experimentally. This is a first attempt at modeling immunogenicity of biologics, so the model simulations should be used to help understand the immunogenicity mechanisms and impacting factors, rather than making direct predictions. This prototype model needs to be subjected to extensive experimental validation and refinement before fulfilling its ultimate mission of predicting immunogenicity. Nevertheless, the current model could potentially set up the starting framework to integrate various in silico, in vitro, in vivo, and clinical immunogenicity assessment results to help meet the challenge of immunogenicity prediction.

Association of antibodies to hepatitis C virus glycoproteins 1 and 2 (anti-E1E2) with HCV disease.

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, provide a target for host immune recognition. HCV genotypes are classified into six genetic groups. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the correlation between antibody level and viral load, genotype, disease severity and response to treatment was investigated. The levels of antibodies to HCV glycoproteins E1 and E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. Reactivity against UKN3A1.28 in sera from patients infected with genotype 3 was significantly higher than corresponding reactivity between patients infected with genotype 1 and H77c. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. The pre-treatment level of anti-E1E2 was higher in sustained responders to combination therapy. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.

Stability of a receptor-binding active human immunodeficiency virus type 1 recombinant gp140 trimer conferred by intermonomer disulfide bonding of the V3 loop: differential effects of protein disulfide isomerase on CD4 and coreceptor binding.

Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.

Severe fibrosis in hepatitis C virus-infected patients is associated with increased activity of the mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex.

Mannan-binding lectin (MBL) binds microorganisms via interactions with glycans on the target surface. Bound MBL subsequently activates MBL-associated serine protease proenzymes (MASPs). A role for MBL in hepatitis C virus (HCV) infection had been indicated by previous studies examining MBL levels and polymorphisms in relation to disease progression and response to treatment. We undertook this study to investigate a possible relationship between disease progression and functional MBL/MASP-1 complex activity. A functional assay for MBL/MASP-1 complex activity was employed to examine serum samples from patients with chronic HCV infection, non-HCV liver disease and healthy controls. Intrapatient consistency of MBL/MASP-1 complex activity levels was assessed in sequential samples from a subgroup of patients. Median values of MBL/MASP-1 complex activity were higher in sera from patients with liver disease compared with healthy controls. MBL/MASP-1 complex activity levels correlate with severity of fibrosis after adjusting for confounding factors (P = 0.003). MBL/MASP-1 complex activity was associated more significantly with fibrosis than was MBL concentration. The potential role of MBL/MASP-1 complex activity in disease progression is worthy of further study to investigate possible mechanistic links.

Collectins and their role in lung immunity.

The collectins are a small family of secreted glycoproteins that contain C-type lectin domains and collagenous regions. They have an important function in innate immunity, recognizing and binding to microorganisms via sugar arrays on the microbial surface. Their function is to enhance adhesion and phagocytosis of microorganisms by agglutination and opsonization. In the lung, two members of the collectin family, surfactant proteins A and D, are major protein constituents of surfactant. Another collectin, mannan-binding lectin, is also present in the upper airways and buccal cavity and may protect against respiratory infections. Recent work has shown that collectins have roles in resistance to allergy and in the control of apoptosis and clearance of apoptotic macrophage in the lung.

Humoral autoreactivity directed against surfactant protein-A (SP-A) in rheumatoid arthritis synovial fluids.

SP-A is found principally in the lung, and has been associated with lamellar bodies also found in the synovial joint. Both SP-A and C1q contain collagen-like regions, and SP-A and C1q have some structural similarities, both having a globular head region and a collagen-like tail. Here we are able to show that (i) autoreactivity to SP-A, as expressed by IgG and IgM autoantibodies, is present in synovial fluid (SF) isolated from patients with rheumatoid arthritis (RA); (ii) in absorption experiments only a limited degree of cross-reactivity between autoantibodies reactive with C1q and SP-A is observed; (iii) there is no cross-reactivity between autoantibodies reactive with type II collagen (CII) and those reactive with SP-A or C1q; (iv) autoantibodies react with polymeric (dimers and larger) SP-A, but not with monomeric SP-A subunits, indicating that a degree of quaternary structure is required for antibody binding. Unlike CII, which not accessible in the normal joint, both SP-A and C1q are available within the SF in patients with RA and may therefore provide antigens driving an autoimmune response directed against collagen-like structures.

Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells.

Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV.

A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo.

The pulmonary collectin, lung surfactant protein D (SP-D), plays a role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-D. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G protein from RSV (human, A2 strain) and both native and recombinant human SP-D. The effect of recombinant trimeric SP-D lectin domains (rSP-D) on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and in vivo infections in BALB/c mice. These studies show that full-length and recombinant SP-D bind to RSV G protein in a concentration-dependent manner. Both EDTA and mannan inhibited binding of full-length SP-D. These results indicate that binding occurs via the carbohydrate recognition domain of the SP-D. The recombinant SP-D inhibited RSV infectivity in cell culture in a dose-dependent manner, giving 100% inhibition of replication. Intranasal administration of recombinant SP-D to RSV-infected mice inhibited replication of the virus in the lungs, reducing levels of lung virus by 80%. These results suggest that SP-D plays a major role in clearing RSV from the lungs.

Induction of TNF-alpha release from human buffy coat cells by Pseudomonas aeruginosa is reduced by lung surfactant protein A.

Lipopolysaccharide (LPS) induction of TNF-alpha release is a central event in the pathophysiology of gram-negative bacterial septicaemia. Lung surfactant protein A (SP-A) mediates pathogen/host cell interactions. Binding of SP-A to Pseudomonas aeruginosa LPS and the effects of SP-A with LPS or whole bacteria on buffy coat cells were investigated. SP-A interacts with P. aeruginosa LPS in a concentration and calcium dependent manner, either through the lipid A portion of LPS or through another lectin/carbohydrate interaction. SP-A decreased TNF-alpha secretion induced by bacteria or LPS from buffy coat cells, in a concentration dependent manner.

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events.