Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens

Self-reactivity in the dimeric intravenous immunoglobulin fraction

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg

Clinical properties of a novel liquid intravenous immunoglobulin: Studies in patients with immune thrombocytopenic purpura and primary immunodeficiencies

Background: We have developed a novel liquid intravenous immunoglobulin (IVIG-F10). It consists of a nanofiltered 12% immunoglobulin G (IgG) solution, stabilized with nicotinamide, L-proline and L-isoleucine. The efficacy, tolerability, safety, and pharmacokinetics of this product were assessed in patients with chronic immune thrombocytopenic purpura (ITP) and primary humoral immunodeficiencies (PID). Patients and Methods: 33 chronic ITP patients with platelet counts of < 20 × 109/l were treated with IVIG-F10 or Sandoglobulin at doses of 0.4 g/kg body weight on 5 consecutive days. The primary efficacy endpoint was an increase in platelet counts to ≥50 × 109/l. Secondary endpoints were time to and duration of platelet response and regression of bleeding. 34 PID patients with X-linked agammaglobulinemia, common variable immunodeficiency or IgG subclass deficiency were treated for 6 months with IVIG-F10 or Sandoglobulin at doses of 0.3-0.8 g/kg, infused at 3- or 4-week intervals. The primary efficacy endpoint was the number of days absent from school/work. Secondary endpoints were feeling of well-being, days of hospitalization, and use of antibiotics. Results: In ITP patients, the primary endpoint was met by 12/16 patients on IVIG-F10 and by 12/17 patients on Sandoglobulin (p = 1.000). Results of the secondary endpoints were comparable in the two study groups. In the PID study, 10/17 patients on IVIG-F10 and 9/17 patients on Sandoglobulin missed days at school/work, with monthly mean absences of 0.7 and 0.6 days (p = 0.746), respectively. There were no significant differences in the outcome of the secondary endpoints. Pharmacokinetics showed constant peak and trough serum IgG levels in PID patients. The median half-life (t1/2) of IgG was 33 days in the IVIG-F10 group and 41 days in the Sandoglobulin group. For anti-HBsAg, median t1/2 values were shorter, i.e. 17 and 19 days for IVIG-F10 and Sandoglobulin, respectively. In the ITP study, adverse events related to study drug were suspected in 9 and 14 patients treated with IVIG-F10 and Sandoglobulin, respectively; in the PID study adverse events occurred in 8 and 9 patients, respectively. Viral safety was ascertained in both studies by serology supplemented with nucleic acid amplification testing. Serum levels of the stabilizers transiently increased after infusion of IVIG-F10, but were back to baseline at the following day. Conclusions: The clinical studies in patients with chronic ITP and PID showed that the efficacy, tolerability, safety, and pharmacokinetics of IVIG-F10 were comparable to the properties of Sandoglobulin.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

<i>In vivo</i> pharmacokinetics of different IgG preparations, proline and glycine in rats.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, repectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and analyzed for total IgG by Nephelometry and Proline and Glycine concentration by HPLC. The elimination profile for IgG is shown in (A) and for proline and glycine in (B).</p

<i>In vivo</i> comparison of the binding to Aβ, Actin, tetanus toxoid and Varicella Zoster Virus by ELISA.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, respectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and the binding capacity to oligomeric Aβ (A and B), actin (C and D), Tetanus Toxoid (E and F), and VZV (G and H) was analyzed by ELISA. (*p<0.05, **p<0.01). Each ELISA was performed 3 times n = 3 (with all animals n = 30) except VZV t = 2 min n = 2 (with all animals n = 30) due to limited sample volume.</p

<i>In vitro</i> Comparison of reformulated IgG preparations activity to Aβ, actin and tetanus toxoid by ELISA and Octet.

<p>Unformulated Privigen (in H₂O) was formulated in Proline pH 4.8, Glycine pH 4.7 or Glycine pH 4.2. (A) ELISA measurements on plate-bound Aβ oligomers. The panel shows combined results from repeated measurements (n = 3). (B) Octet binding measurements to immobilized Aβ and <b>C</b> to bound tetanus toxoid. Privigen Fc fragments were used as negative controls.</p

Intravenous Immunglobulin Binds Beta Amyloid and Modifies Its Aggregation, Neurotoxicity and Microglial Phagocytosis <i>In Vitro</i>

<div><p>Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aβ) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aβ -specific antibodies (pAbs-Aβ) on aggregation, toxicity and phagocytosis of Aβ <i>in vitro</i>, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aβ specifically bound to Aβ and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aβ inhibited Aβ-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aβ binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aβ also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aβ by BV-2 microglia. Phagocytosis of Aβ depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aβ-mediated LTP inhibition in mouse hippocampal slices. Therefore, our <i>in vitro</i> results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.</p></div