PreclinicalIn VitroandIn VivoCharacterization of the Fully Human Monoclonal IgM Antibody KBPA101 Specific forPseudomonas aeruginosaSerotype IATS-O11

Abstract

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/ antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 ؋ 10 7 M ؊1 ؎ 2.8 ؋ 10 7 M ؊1 ) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 g/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

“…Intensive efforts over the last years have enabled the production of functionally active IgMs in mammalian cell culture at reasonably high levels (g/L) (5) for clinical applications (3,4). However, labor-intensive production and, as a consequence, high costs of recombinant IgMs prevented their widespread use.…”

“…Apart from their high avidity and agglutination efficiency, IgMs are exceptionally good in complement activation and germline-encoded native IgM plays an important role in defense against aberrant cells (1). Thus, monoclonal IgM antibodies have increasingly gained interest for the treatment of various diseases (2)(3)(4). However, despite the recognized relevance for pharmaceutical applications the (recombinant) production has long been considered a challenge (5,6).…”

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

“…Production of Microarray Chips-Microarrays for Mass Spectrometry (MAMS) were fabricated as described in Urban et al, and Kü ster et al (65,66) In brief, a coated ITO-glass slide (12)(13)(14)(15)(16)(17)(18) Ohm/m 2 , Sigma Aldrich) was structured using a laser ablation system to generate a checkerboard-like array of 2800 hydrophilic sample deposition areas of 300 m diameter and ϳ35 m depth each (720 m center-tocenter distance within one row).…”

“…Other antibody classes such as IgM or IgA show a higher complexity with respect to the number of glycosylation sites and variety of glycoforms (6,9). Only recently, also monoclonal IgM antibodies came into the focus of pharmaceutical industry, because they show great potential for the treatment of diseases (12)(13)(14).…”

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

“…In 2010, Horn et al evaluated the effects of a monoclonal IgM antibody employed against Pseudomonas aeruginosa infections (Horn et al 2010), serving as an example of how IgMs can be used for alternative therapies when facing increasing numbers of antibiotic-resistant microorganisms.…”