Michael P. Rudolf

Oral anti‐IgE immunization with epitope‐displaying phage

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope‐displaying M13 bacteriophages as carriers for an experimental oral anti‐IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti‐idiotypic Fab of the non‐anaphylactogenic monoclonal anti‐IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII‐expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti‐IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti‐phage but no anti‐IgE antibodies. After feeding a single dose of mimotope‐displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope‐displaying bacteriophages can be used to induce an epitope‐specific antibody response via the oral route.

Preclinical In Vitro and In Vivo Characterization of the Fully Human Monoclonal IgM Antibody KBPA101 Specific for Pseudomonas aeruginosa Serotype 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/κ antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 × 107 M−1 ± 2.8 × 107 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 μg/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.

Molecular Basis for Nonanaphylactogenicity of a Monoclonal Anti-IgE Antibody

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcεRI) on mast cells and basophils. Therefore, the IgE/FcεRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcεRI, and interferes with binding of IgE to FcεRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cε3 and one within the Cε4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cε3 region predominantly blocks binding of IgE to FcεRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cε4 region may explain how BSW17 recognizes FcεRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti–allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

Cloning of Human Anti-lgE Autoantibodies and Their Role in the Regulation of IgE Synthesis

In vivo experiments using anti-IgE antibodies have clearly documented that they inhibit IgE production. In vitro experiments showed that not only IgE synthesis but also the effector phase of the allergic response may be influenced, because anti-IgE antibodies can prevent basophil sensitization with IgE or even remove IgE-receptor-bound IgE molecules. However, the question remains whether naturally occurring anti-IgE autoantibodies possess similar biological activity. To generate such antibodies for the necessary in vitro studies, we have cloned human Ig variable genes and selected anti-IgE antibodies using phage display libraries. Most of the human anti-IgE antibodies were anti-idiotypes, but anti-isotypes were also isolated.

Anti-IgE in allergic sensitization

Anti‐IgE autoantibodies exist predominantly in the sera of patients with atopic disease. For some time such anti‐IgE autoantibodies have been considered a phenomenon that may not be of clinical importance. The cloning of such anti‐IgE autoantibodies has eliminated doubts of whether these antibodies exist, but it is still unclear whether such autoantibodies play a pathophysiological role. However, there are ongoing clinical trials that use humanized anti‐IgE antibodies for passive immunization of atopic individuals. While this approach may not definitely clarify the role of anti‐IgE autoantibodies, it will nevertheless clarify the role of IgE.

Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation

Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.

MOLECULAR MIMICRY OF THE UNIDENTIFIED ANTIGEN OF MYELOMA ANTIBODY IGE-ND

Antigen‐specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266‐derived IgE‐ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE‐ND were isolated from phage‐display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE‐ND‐binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE‐ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE‐ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen‐specific IgE responses.

Can Active Immunization Redirect an Anti-IgE Immune Response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.

Development of a 4-valent genotyping assay for direct identification of the most frequent Pseudomonas aeruginosa serotypes from respiratory specimens of pneumonia patients

Pseudomonas aeruginosa is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of P. aeruginosa, we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of P. aeruginosa. The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of P. aeruginosa or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).

Natural And Recombinant ANTI-IgE Autoantibodies

Since the first description of anti-IgE autoantibodies by Williams et al. in 19721, these antibodies have remained a much disputed subject. Most immunologists considered such natural autoantibodies as low affinity antibodies of no importance. Despite the fact that Manning et al. described only 4 years later that repeated injections of a rabbit anti-Ig antiserum to neonatal mice suppressed not only IgM but also IgG, IgA and IgE synthesis2, it was still not generally accepted that anti-IgE autoantibodies may have the same profound effect as heterologous anti-IgE antisera. Subsequent animal experiments, showing that anti-IgE antibodies are strongly immunoregulatory and lead to the suppression of IgE synthesis in vivo have not been associated with the possibility to use such antibodies for therapy3,4, or to postulate that such antibodies may represent a normal physiological control. Only recently, both possibilities have become feasible. Here we demonstrate that anti-IgE antibodies, isolated from the human genome, as recombinant antibodies, may possess the necessary fine specificity for future therapeutic application.