Histology of biopsies from saline- or zalutumumab-injected skin (part I of the study).

<p>Representative sections are shown of skin biopsies of one subject for part I of the study (following two injections of 10 μg of zalutumumab). Sections were stained with HE to show nucleated cells and (patho-)histological features. In the saline-injected skin (A, C), inflammatory cells were virtually absent and the hair follicle structure was intact. Note that after zalutumumab injection (B, D), the hair follicle structure was destroyed and extensive influx of inflammatory cells (including neutrophils) in the vicinity of the destroyed hair follicle/glandular structures can be observed (asterisks). Scale bars are 100 µm (A, B) and 20 µm (C, D).</p

Histology and immunohistochemistry of biopsies from zalutumumab-injected skin treated with HuMab-10F8 or untreated.

<p>Data shown are representative sections of skin biopsies of one subject for part III of the study. Biopsies were taken at day 14 after two injections with 100 µg of zalutumumab with [A, C, E, G, I, K] or without HuMab-10F8 [B, D, F, H, J, L]. A, B: HE staining; C, D: staining for EGFR; E–H: staining for macrophages and I–L: staining for neutrophils. Extensive influx of inflammatory cells in the vicinity of the hair follicle with histological signs of folliculitis was observed in zalutumumab-injected skin (right panels), whereas in the zalutumumab-injected skin treated with HuMab-10F8 (left panels) inflammatory cells were virtually absent and sebaceous glands and hair follicles showed normal histological features. Inflammatory infiltrate in the zalutumumab-injected skin was mainly composed of neutrophils (neutrophil elastase positive cells, J, L) and to a lesser extent of macrophages; (CD68 positive cells F, H). Scale bars are 100 µm (A–F, I, J) and 20 µm (G, H, K, L). G, H, K and L are magnifications of the squared areas indicated in E, F, I and J.</p

Papulopustular rash was induced by local injection of zalutumumab.

<p>The rash observed at the injection site of one subject in part II of the study, following two injections of 100 µg of zalutumumab each, is shown.</p

Trial design.

<p>Injection schemes for the three different parts of the study are shown. Subjects were injected with different doses of zalutumumab at different sites, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039706#pone-0039706-t001" target="_blank">Table 1</a>. Sc – screening of patient.</p

Dose escalation schedule for zalutumumab injections.

<p>Every injection of zalutumumab was accompanied by a control injection of an identical volume of isotonic saline at a different site. In the third part the dose escalation started at 100 μg.</p

Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange

Bispecific antibodies (bsAbs) are one of the most versatile and promising pharmaceutical innovations for countering heterogeneous and refractory disease by virtue of their ability to bind two distinct antigens. One critical quality attribute of bsAb formation requiring investigation is the potential randomization of cognate heavy (H) chain/light (L) chain pairing, which could occur to a varying extent dependent on bsAb format and the production platform. To assess the content of such HL-chain swapped reaction products with high sensitivity, we developed cysteine-stable isotope labeling using amino acids in cell culture (SILAC), a method that facilitates the detailed characterization of disulfide-bridged peptides by mass spectrometry. For this analysis, an antibody was metabolically labeled with ¹³C₃,¹⁵N-cysteine and incorporated into a comprehensive panel of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody technology). This technology is a postproduction method for the generation of bispecific therapeutic IgGs of which several have progressed into the clinic. Herein, two parental antibodies, each containing a single heavy chain domain mutation, are mixed and subjected to controlled reducing conditions during which they exchange heavy–light (HL) chain pairs to form bsAbs. Subsequently, reductant is removed and all disulfide bridges are reoxidized to reform covalent inter- and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up) approach focusing on the characterization of both “left-arm” and “right-arm” HL interchain disulfide peptides and observed that native HL pairing was preserved in the whole panel of bsAbs produced by controlled Fab-arm exchange

E345 and E430 limit Fc:Fc interactions.

<p>(A) Left: overview of the IgG1 antibody hexamer observed in the IgG1-b12 crystal structure (PDB access code 1HZH [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002344#pbio.1002344.ref020" target="_blank">20</a>]). Right: zoomed-in view of two neighboring Fc domains with enhancing amino acid positions highlighted. (B) Summary of CDC inhibition data. The surface view is rotated 90° relative to panel A to show the Fc:Fc interface. Daudi cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate the unmodified amino acids of the two heavy chain regions composing a single Fc domain. All mutants assessed were ranked by efficiency per amino acid position, and the resulting median lysis efficacy is indicated by coloring per position; orange and red colors indicate positions with reduced lysis. Blue: >60% lysis; orange: 30%–60% lysis; red: <30% lysis. (C) Summary of CDC enhancement data. Wien 133 cells were opsonized with mutants of anti-CD38 mAb IgG1-005 (1.0 μg/mL) and assessed for CDC. Light and dark grey colors indicate unmodified amino acids as described under (B). The maximal lysis of the most efficient mutant per amino acid position is displayed with green indicating positions for which CDC was enhanced. Blue: <20% lysis; light green: 20%–40% lysis; dark green: >40% lysis. (D) Dose response in CDC of Wien 133 cells by IgG1-005 and selected mutants. A representative of 3 replicate experiments is shown. (E) Dose response in CDC of Ramos cells induced by RTX and selected mutants. A representative experiment of 3 replicates is shown. (F) Structural view zoomed to residue E345. Amino acids facing E345 at the Fc:Fc interface are indicated in blue and named with apostrophe. (G) Structural view zoomed to residue E430, forming a salt bridge with K338 at the intramolecular C_H2–C_H3 domain interface.</p

Promoting Fc:Fc interactions alleviates CDC sensitivity to target and mCRP expression.

<p>(A) Cell lines were opsonized with saturating amounts of wild-type or mutated RTX and analyzed by CDC assay. Left panel: cell lines with increasing CD20:mCRP ratio (left to right); right panel: cell lines with decreasing CD20:mCRP ratio (left to right). (B) In vivo analysis of subcutaneous tumor growth in a severe combined immunodeficiency (SCID) xenograft model. SCID mice were injected with luciferase-expressing Raji cells. Eight days after tumor cell injection, mice were randomized at an average tumor volume of 85 mm³ per group and treated with 50 μg IgG1 antibody. Tumor volumes were monitored over time and depicted as average tumor volume ± standard error of the mean (SEM) (left panel), and as Kaplan-Meier curves with time to progression cutoff set at a tumor volume of >700 mm³ (right panel).</p

Biophysical characterization of antibody hexamerization in solution.

<p>(A) Native MS analysis of solutions of IgG1-005 and mutant antibodies at 2 μM. IgG1-005-RGY (RGY), a previously described triple mutant, is used as a positive control. The charge envelope of the IgG monomers appears around <i>m/z</i> 5,500 (~ 147 kD Mw) and that of the IgG hexamers around <i>m/z</i> 12,500 (~890 kD Mw). The signals of the hexameric species are displayed at a 100-fold or 10-fold (RGY only) amplification. (B–C) Dynamic light scattering analysis of 7D8 and mutant solutions formulated in PBS pH 7.4. Three independent experiments are shown. (B) Linear regression of diffusion coefficient versus Ab concentration. (C) Hydrodynamic radii (Rhs) of mutants measured by dynamic light scattering (DLS) were divided by Rh of wild-type 7D8 to correct for viscosity without masking the contribution of Fc:Fc self-association. (D) HP-SEC analysis of 100 mg/mL 7D8 and mutant antibody solutions formulated in PBS, incubated for three months at 5°C. Multimer detection limit of 1% is indicated by a dashed line.</p

CDC enhancement by promoting Fc:Fc interactions is broadly applicable.

<p>A representative example of three replicates is shown. Each graph compares CDC in dose response for the wild-type and E345R-mutated mAb. (A) CDC of Wien 133 (left) and Daudi cells (right) opsonized with anti-CD20 mAbs RTX and 11B8. (B) CDC of Wien 133 (left) and Raji cells (right) opsonized with anti-CD52 mAb ALM. (C) CDC of Wien 133 cells (left) and Daudi cells (right) opsonized with anti-CD38 mAbs IgG1-003 and IgG1-005. (D) CDC assay of A431 cells opsonized with anti-EGFR mAb 2F8.</p