The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance.

The occurrence of an immune response against therapeutic proteins poses a major risk for the development of biologics and for successful treatment of patients. Generation of anti-drug antibodies (ADAs) can lead to formation of immune complexes (ICs), consisting of drug and ADAs, with potential impact on safety, efficacy and exposure. Here, we focus on the effects of IC formation, i.e., specific IC sizes, ADA and drug properties, on drug pharmacokinetics. Pre-formed IC preparations of an IgG1 drug (with wild type or with an ablated effector function at the Fc domain) and different ADA surrogates (directed against the complementarity-determining regions or Fc domain of the drug) were administered to rats and collected serum was analyzed to determine the total drug concentration. A combination of size-exclusion chromatography and ELISA enabled a size-specific evaluation of IC profiles in serum and their changes over time. Within five minutes, total drug concentration decreased by ~20-60% when the drug was complexed. Independent of the ADA surrogate and drug variant used, increasing IC size led to increased clearance. Comparing ICs formed with the same ADA surrogate but different IgG1 variants, we observed that complexed drug with a wildtype Fc domain showed faster clearance compared to immune effector function modified drug. Data generated in this study indicated that clearance of drug due to ADA generation is driven by size and structure of the formed ICs, but also by the immune effector functions of the Fc domains of IgGs.Abbreviations Ab: antibody, ADA: anti-drug antibody, AUC: area under the curve, Bi: biotin, CDR: complementary-determining region, cmax: maximal concentration, Dig: digoxigenin, ELISA: enzyme-linked immunosorbent assay, Fc: fragment crystallizable, FcRn: neonatal Fc receptor, HMW: high molecular weight, IC: immune complex, IC-QC: immune complex quality control, IgG: immunoglobulin G, mAb: monoclonal antibody, mADA: monoclonal ADA, pAb: polyclonal antibody, pADA: polyclonal ADA, PD: pharmacodynamics; PK: pharmacokinetic, QC: quality control, SEC: size-exclusion chromatography, WT: wildtype.

Green tea extract inhibits the onset of periodontal destruction in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Green tea extract exerts a variety of biological effects, including anti-inflammatory activities. However, there has been no report on the effect of green tea extract on loss of attachment, which is an important characteristic of periodontitis. Here, we examined the inhibitory effects of green tea extract on the onset of periodontitis in a rat model. MATERIAL AND METHODS: Rats were immunized intraperitoneally with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 12) received a topical application of LPS onto the palatal gingival sulcus every 24 h. The green tea extract group (n = 12) received a topical application of LPS mixed with green tea extract, sunphenon BG, every 24 h. The phosphate-buffered saline (PBS) group (n = 6) received a topical application of PBS every 24 h. The levels of anti-LPS immunoglobulin G (IgG) in serum were determined using ELISA. Rats in the LPS and green tea extract groups were killed after the 10th and 20th applications. Rats in the PBS group were killed after the 20th application. Loss of attachment, level of alveolar bone and inflammatory cell infiltration were investigated histopathologically and histometrically. RANKL-positive cells and the formation of immune complexes were evaluated immunohistologically. RESULTS: There was no significant difference in the serum levels of anti-LPS IgG between the LPS group and the green tea extract group. In contrast, loss of attachment, level of alveolar bone, inflammatory cell infiltration and RANKL expression in the green tea extract group were significantly decreased compared with those in the LPS group. CONCLUSION: These findings demonstrate that green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat model of experimental periodontitis.

Optimization of ELISA conditions to quantify colorectal cancer antigen-antibody complex protein (GA733-FcK) expressed in transgenic plant.

The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 µg/well). Among the amounts of the capture antibody, 1 and 0.5 µg/well (capture antibody) showed similar absorbance, whereas 0.25 µg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 µg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R(2) between the ELISA quantified value and protein density was 0.85 (p<0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant.

The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation.

This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 µg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 µg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 µg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.

Direct electrical detection of antigen-antibody binding on diamond and silicon substrates using electrical impedance spectroscopy.

The integration of biological molecules with semiconducting materials such as silicon and diamond has great potential for the development of new types of bioelectronic devices, such as biosensors and bioactuators. We have investigated the electrical properties of the antibody-antigen modified diamond and silicon surfaces using electrical impedance spectroscopy (EIS). Frequency dependent measurements at the open-circuit potential show: (a) significant changes in impedance at frequency >10(4) Hz when the surface immobilized IgG was exposed to anti-IgG, and (b) only little or no change when exposed to anti-IgM. Mott-Schottky measurements at high frequency (200 kHz) show that the impedance is dominated by the space charge layer of the semiconducting substrates. Silicon surfaces modified in a similar manner to the diamond surface are compared; n-type and p-type samples show complementary behavior, as expected for a field effect. We also show it is possible to directly observe antigen-antibody interaction at a fixed frequency in real time, and with no additional labeling.

Determination of the minimum temperature required for selective photothermal destruction of cancer cells with the use of immunotargeted gold nanoparticles.

Laser photothermal therapy of cancer with the use of gold nanoparticles immunotargeted to molecular markers on the cell surface has been shown to be an effective modality to selectively kill cancer cells at much lower laser powers than those needed for healthy cells. To elucidate the minimum light dosimetry required to induce cell death, photothermal destruction of two cancerous cell lines and a noncancerous cell line treated with antiepidermal growth factor receptor (anti-EGFR) antibody-conjugated gold nanoparticles is studied, and a numerical heat transport model is used to estimate the local temperature rise within the cells as a result of the laser heating of the gold nanoparticles. It is found that cell samples with higher nanoparticle loading require a lower incident laser power to achieve a certain temperature rise. Numerically estimated temperatures of 70-80 degrees C achieved by heating the gold particles agree well with the measured threshold temperature for destruction of the cell lines by oven heating and those measured in an earlier nanoshell method. Specific binding of anti-EGFR antibody to cancerous cells overexpressing EGFR selectively increases the gold nanoparticle loading within cancerous cells, thus allowing the cancerous cells to be destroyed at lower laser power thresholds than needed for the noncancerous cells. In addition, photothermal therapy using gold nanoparticles requires lower laser power thresholds than therapies using conventional dyes due to the much higher absorption coefficient of the gold nanoparticles.

[Immunospecific detection of the potato virus X by the surface plasmon resonance].

Highly specific and proximal method of laboratory diagnostics of the viral disease, has been developed using the structural protein of the potato virus X, as a model, and monospecific antibodies to it. The immunospecific determination of the potato virus X was carried out by the surface plasmon resonance method using specific IgG-antigen complexes, immobilized on the sensor surface modified by rodanide and protein A of Staphylococcus aureus.

[Detection of immune complexes using atomic force microscopy]. / Vyiavlenie immunnykh kompleksov s pomoshchiu atomno-silovoi mikroskopii.

Complex formation between immunoglobulins and ligands immobilized on mica was studied by atomic force microscopy in two different systems. In the first system, 60-kDa ligands possessing only one site for antibody recognition were used. In the other system, a more complex interaction of human immunoglobulin with immobilized polyclonal antibodies was studied. In both systems, specific complexes with proper ligand appeared, and unspecific interaction was not detected. The method of revealing immunocomplexes by image atomic force microscopy can be used in the development of modern diagnostic systems.

Multi-analyte analysis system using an antibody-based biochip.

A multi-analyte detection system using a unique antibody (Ab) biochip is described. The Ab-based biochip, also referred to as the protein biochip, uses a sensor array based on a complementary metal oxide silicon (CMOS) integrated circuit. The Ab-biochip has a sampling platform of four-by-four microarrays of antibodies deposited onto a Nylon membrane substrate. The micro-arrayed antibodies can be interrogated simultaneously or sequentially using the biochip sensing array detector with the use of a diffractive optical element illuminating each antibody spot individually. The usefulness of the Ab biochip is illustrated by the measurements of immunoglobulin G (IgG) used as the model analyte system. The detection limit for Cy5-labeled IgG molecules was 13 pg.

Blocking of human anti-pig xenoantibodies by soluble GAL alpha 1-3Gal and Gal alpha 1-2Gal disaccharides; studies in a pig kidney in vitro perfusion model.

Depletion of anti-pig xenoantibodies reduces cell cytotoxicity of human serum to pig endothelial cells and lymphocytes. The aim of this study was to test, in a pig kidney xenoperfusion model, the ability of soluble alpha Gal terminated disaccharides to prevent the hyperacute rejection process in an organ. Porcine kidneys were perfused with whole human blood lacking saccharide and blood supplemented with Gal alpha 1-3GAL, Gal alpha 1-2Gal and lactose. Parameters evaluated were, urine production, renal blood flow, vascular resistance, renal clearance, blood cell counts, xenoantibody titers, complement activation and histopathology. The blood flow was higher in the Gal alpha 1-3Gal (155 +/- 31 ml/min x 100 g-1 kidney tissue) group compared to Gal alpha 1-2Gal (138 +/- 16), lactose (92 +/- 78) and controls (69 +/- 16), When calculated as percent of the blood flow value at 1 min, the blood flow at 30 min was 157% for the Gal alpha 1-3Gal and for 187% the Gal alpha 1-2Gal. The corresponding values for the lactose and control groups were 102% and 74%, respectively. Urine production in the lactose/control groups was lower (0.7 ml/min x 100 g-1 kidney tissue) compared to Gal alpha 1-3Gal (3.0) and Gal alpha 1-2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Gal alpha alpha 1-groups during the perfusions. An increase in urine potassium excretion was found in the Gal alpha alpha 1-groups while a reduction occurred in the lactose/control experiments. An initial 40-50% reduction in platelet count was observed in all groups while the leukocyte count showed a continuous decrease. Immunohistochemistry revealed less deposition of IgM, IgG, C3 and C1 q in the Gal alpha alpha 1-saccharide groups compared to the lactose/control groups. Soluble Gal alpha a1-disaccharides improved both functional and histological parameters. However, significant pathological changes were still present indicating that this approach to inhibit HAR must be used in combination with additional therapeutic approaches such as solid phase xenoantibody immunoadsorption and blocking of complement activation.

Presence of high levels of non-degraded gliadin in breast milk from healthy mothers.

BACKGROUND: Secretion of dietary antigens into breast milk has been extensively documented. The presence of these antigens is of relevance because they could be involved in the modulation of the immune response in neonates. The objective of this study is to determine the gliadin concentration in milk, colostrum, and serum samples from healthy lactating mothers on a normal diet. Gliadin levels in milk samples from a group of six mothers after a brief period of gluten restriction were also determined. The molecular weight of secreted gliadins was also analysed. METHODS: Gliadin concentration was determined with a highly sensitive competitive enzyme-linked immunosorbent assay, modified so as to eliminate anti-gliadin antibody interference. The level of gliadin/IgA anti-gliadin immune complexes in milk, colostrum, and serum samples was determined. RESULTS: Gliadin was detected in all 49 milk samples. Its concentration varied between 5 and 1200 ng/ml (mean, 178 ng/ml). In colostrum (n = 14) gliadin levels were higher (range, 28-9000 ng/ml; mean, 883 ng/ml), not being detectable in one case. Gliadin was detectable in 14 of 31 serum samples, in which levels were lower than in milk and colostrum samples (mean, 41 ng/ml). Neither a correlation between gliadin levels in milk, colostrum, and serum samples from the same subject nor a relation between gluten intake and gliadin concentration in milk samples from six subjects under a 3-day gluten-free diet could be found. Higher levels of immune complexes were observed in colostrum samples than in milk and serum samples. No correlation was detected between gliadin concentration and the level of immune complexes. The analysis of milk and colostrum samples by immunoblotting showed bands of immunoreactive gliadin presenting Mr similar to those of native proteins from wheat extracts. CONCLUSIONS: Very high levels of gliadin were detected in milk samples from healthy mothers on an unrestricted diet. Gliadin levels were higher than those reported for dietary antigens in other studies. Breast milk contained non-degraded gliadins and gliadin/anti-gliadin IgA immune complexes.

Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein.

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence (LIF). Exploiting recombinant technology and the natural fluorescence of GFP presents the capability of preempting the need for fluorescent derivatization. Such an approach would circumvent the obstacles typically associated with covalently labeling and purifying analytes that undergo fluorescent labeling. The unique property of GFP to fluoresce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly or in vitro with wild-type GFP isoforms isolated from native jellyfish Aequorea victoria. Second, GFP antisera were reacted with wild-type GFP and the formation of the GFP antigen-antibody complex was monitored. A simple borate buffer, pH 8.5, was ample for resolving the two isoforms of the naturally fluorescent GFP in less than 5 min. The separation of GFP from GFP-Ab was complete in less than 7 min with the individual components detectable at the picogram level. A number of factors influence CE separation and/or LIF detection including sample buffer pH and incorporation of the additive 1,4-diamino butane. Remarkably, conditions that severely impair fluorescence detection of free GFP do not diminish fluorescence detection of the GFP antigen-antibody complex in a similar manner. Thus, the antibody appears to preserve the natural fluorophore of GFP. These data lend credence to the utility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient separation and detection of an immunoreaction. In principle, a fusion protein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually required in LIF detection.

Evaluation of immune complexes after immunotherapy with wheat flour in bakers' asthma.

Inhalant food allergy has been described many times in literature, but double-blind clinical trials to support successful hyposensitization to these allergens has seldom been reported. Some authors have suspected that certain adverse reactions after immunotherapy may be mediated by immune complexes. Furthermore, the FDA does not recommend injection therapy with food extracts. We present a study on the detection of adverse effects after immunotherapy with an inhalant food (wheat flour) in a double-blind clinical trial in 26 patients with bakers' asthma. We investigated the presence of circulating immune complexes (CICs) after 2 years of treatment with hyposensitization to wheat flour.

Anti-IgE autoantibodies mistaken for specific IgG.

During isolation of specific IgE by affinity chromatography, we obtained IgG acting as "specific IgG" in dot immunoassay and RAST. Further analysis of this fraction revealed that this specific IgG was indeed an immune complex consisting of IgG anti-IgE autoantibodies and specific IgE that falsely appeared as specific IgG in the immunoassays. After disruption of complexes, more IgE became accessible, resulting in increased counts per minute in the RAST. Thus, anti-IgE autoantibodies may hinder determination of specific IgE in in vitro immunoassays, since they hide specific IgE immunologically. To demonstrate the isotope specificity of such immune complex-derived IgG, the purified anti-IgE autoantibodies were radiolabeled and used as detection antibodies in immunoblotting. These autoantibodies recognized different IgE preparations, including a recombinant IgE peptide.

A five day course of high dose corticosteroids has no effect on the clearance of soluble aggregates of human immunoglobulin G in healthy volunteers.

It is thought that glucocorticosteroids impair the clearance of immune complexes by the mononuclear phagocyte system (MPS). We studied the effect of a five day course of prednisone (1 mg/kg body weight per day) on MPS function in 10 healthy volunteers, using soluble radiolabeled aggregates of human immunoglobulin G as a probe. MPS function was assessed before steroid treatment, and again 24 hours after the last dose of prednisone. Elimination kinetics and the uptake of the immunoglobulin aggregates by liver and spleen did not change after prednisone treatment. This suggests that, in contrast to general belief, high doses of glucocorticosteroids have no direct, acute effect on MPS function.

Disappearance of immune deposits with EDTA chelation therapy in a case of IgA nephropathy.

In this report, we describe the development of renal function impairment in a 33-year-old patient with mesangial IgA nephropathy and a history of recent gout. Increased body lead burden was identified with a positive EDTA mobilization test. The patient was treated with 1 g of edetate disodium calcium weekly for 2 months until normalization of urinary lead excretion. Improvement of renal function and proteinuria were noted. It was even more interesting to find that both immunofluorescence and electron microscopy studies of the second biopsy specimen revealed the loss of previous mesangial immune deposits. Our case demonstrated that lead may be a nonspecifically damaging factor related to the deterioration of renal function in patients with preexisting renal disease. Moreover, the disappearance of mesangial immune deposits after chelation therapy has not been previously documented. The pathogenetic basis of this observation is unknown, and its causal relationship with lead requires further elucidation.