Generation of human IgG1 and IgG3 TA99 mAb with histidine-arginine rearrangements in Fc domains at amino acid position 435.

<p>Human IgG1 was mutated to contain an arginine at position 435 (IgG1 H435R), whereas the arginine at position 435 in human IgG3 was changed to histidine (IgG3 R435H). (<b>A</b>) Specific anti-human IgG1 or (<b>B</b>) anti-human IgG3 ELISA confirmed the correct isotype of mAbs. Staining of B16F10-gp75 with (<b>C</b>) different human TA99 mAb and (<b>D</b>) mouse TA99 IgG2a confirmed binding to surface gp75 and equal binding efficiency to gp75 of all human TA99 mAb. Concentration curves of human IgG1 and IgG3 mAb (<b>E</b>) and mouse IgG2a TA99 (<b>F</b>) on B16F10-gp75. Of note, scales of human (<b>E</b>) and mouse (<b>F</b>) antibodies are different.</p

Binding to FcγR by IVIg fractions.

<p>Sensograms of the binding of dimeric fraction (a,d) and monomeric fraction (b,e) to FcγRIIIb (a-b) or FcγRIIa (d-e) coupled to a biosensor chip through its C-terminal His-tag. Steady state apparent affinities calculated from the sensogram data were obtained from fitting a Langmuir binding model as shown in the inset panels, to calculate the apparent Kd values to FcγRIIIb (c) or FcγRIIa (f). One-way ANOVA; ** p < 0.01.</p

Neutrophil activation by IVIg and fractions thereof.

<p>Neutrophil activation, measured as elastase release of resting neutrophils, induced by filtered IVIg fractions at concentrations between 0.3 and 5 mg/mL in the presence of poloxamer (a), or using a previously established protocol without filtration and poloxamer (b). As a positive control, neutrophils were also stimulated by coated IgG, which leads to more than 80% degranulation at concentrations as low as 0.5 mg/mL (c). Activation of primed neutrophils using cytochalacin B (d) or TNF (e) by filtered IVIg fractions in the presence of poloxamer. Similarly, oxidation burst was evaluated in the presence of filtered IVIg fractions in the presence of poloxamer (f). One-way ANOVA (a, d, e, f) or Student’s t test (b, c); * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Cytotoxicity assays using murine macrophages and B16F10-gp75 tumour cells.

<p>(<b>A</b>) Remaining B16F10-gp75 cells after a 24 hour incubation with macrophages and 2 μg/ml TA99 mAbs (different isotypes). (<b>B</b>) FACS analysis of co-cultures of DiO labelled murine macrophages (FL1) and DiI labelled B16F10-gp75 (FL2) tumour cells after 24 hours of treatment with 1 μg/ml mouse IgG2a or human IgG1 or IgG3 TA99 mAb. Macrophages, which have phagocytosed B16F10-gp75 tumour cells are encircled in FACS plots. (<b>C</b>) Percentage of remaining viable tumour cells and (<b>D</b>) increase in number of macrophages, which have phagocytosed B16F10-gp75 tumour cells after treatment of co-cultures with different concentrations of mAb. Percentages of tumour cells after culture with isotype antibodies were set at 100%. Double-positive macrophages were depicted relative to the co-cultures with isotype antibodies (set to 1), as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177736#pone.0177736.ref004" target="_blank">4</a>]. Mouse MG4 or human HEPC mAb were used as isotypes controls, which were set to 100%. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001.</p

Complement activation by IVIg fractions.

<p>Detection of fluid-phase complement activation by measuring C4b/c in serum incubated at 37°C together with IVIg, dimeric, monomeric fraction and heated IgG (aggregates), or no stimulus, incubated at 37°C or on ice. One-way ANOVA; *** p < 0.001.</p

Data behind all figures.

This file contains all data points for all figures in the associated PLOS ONE manuscrip

Placental transport efficiency is relatively higher at lower maternal levels.

<p>In general, IgG1 is transported better than IgG4, both of which are transported better than IgG2 and IgG3, which have similar transport rates. Two-tailed Pearson correlation revealed a significant correlation for all subclasses, IgG1 R² = 0.379, P = 0.0018; IgG2 R² = 0.2910, P = 0.0096; IgG3 R² = 0.2415, P = 0.0202; IgG4 R² = 0.2881, P = 0.0121. Thus, for all subclasses, relatively more IgG was transported at lower maternal IgG.</p

No difference in half-life of IgG2 light chain isotypes κ and λ in mice.

<p>(A) Recombinant human IgG2κ and λ in Balb/C mice was injected and measured by total IgG ELISA for a two week period following injection of 200 µg IgG. Calculated half-lives were 7.2±1.48 and 6.4±0.84 days for IgG2κ and IgG2λ. (B) Enrichment of IgG2 κ isoforms was performed as described in Dillon et al 2008. HPLC elution profiles of IgG2 κA and κB structural isomeres on a Dionex ProPac WCX-10 (4.0_250 mm) column are depicted. IgG2κB isoform was generated by incubation of 3 mg/ml IgG2κ in 200 mM Tris buffer at pH 8 with 6 and 1 mM of cysteine and cystamine, respectively. For IgG2κA synthesis 0.9M guanidine hydrochloride (GuHCl) was also added. The samples were kept in the dark and placed at 4°C for 48–72 h. Following incubation the antibody was run on a Zeba spin desalting column (Pierce) for buffer exchange into PBS. (C) The clearance of IgG2λ, IgG2κA, and IgG2κB in Balb/C mice. Calculated half-lives were 4.0±0.58, 5.39±0.85, and 3.7±1.04 days for IgG2κA, IgG2κB and IgG2λ, respectively. (D) Clearance of IgG2κA and IgG2κB in WT and FcγR −/− C57Bl/6 mice. Calculated half-lives were 6.2±2.62, 6.43±1.69, and 7.5±1.89 days for IgG2κA, IgG2κB and IgG2λ, respectively, in WT mice but 1.08±0.28, 1.19±0.23, and 0.7±0.92 days for IgG2κA, IgG2κB and IgG2λ, respectively, in FcRn −/− mice. Graphs in (A, C–D) depict mean and standard deviations of results obtained for 4 mice per group. Half-lives were calculated assuming exponential decay and reported in days ± standard error of means. No significant difference in half-life was detected between the two isotypes.</p

Equal placental transport and serum clearance of IgG1 and 2 light chain isotypes κ and λ.

<p>(A) The average IgG1 and IgG2 placental transport (maternal/child) ratios were compared according to their light chain isotype. (B) Clearence of IgG1 and IgG2 κ and λ was investigated in humans by collecting blood from hypogammaglobulinemia patients four weeks after an IVIg transfusion. IgG1 and IgG2 light chain isotypes κ and λ were quantified in serum by subclass- and light chain specific ELISA and subclass composition was compared to that found in the IVIg used. No preferential clearance of one light chain isotype was detectable in either IgG subclass.</p

Determination of the FcRn binding properties of IgG1 and IgG2 light chain variants.

<p>Binding of titrated amounts of soluble human FcRn (62.5–8000 nM) at pH 6.0 to human IgG variants immobilized onto CM5 sensor flow cells. The relative affinity constants derived (KD) are indicated.</p