Regina Knittelfelder

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

Mimotope vaccination--from allergy to cancer.

Background: Mimotopes are peptides mimicking protein, carbohydrates or lipid epitopes and can be generated by phage display technology. When selected by antibodies, they represent exclusively B-cell epitopes and are devoid of antigen/allergen-specific T-cell epitopes. Coupled to carriers or presented in a multiple antigenic peptide form mimotopes achieve immunogenicity and induce epitope-specific antibody responses upon vaccination. Objective/methods: In allergy IgG antibodies may block IgE binding to allergens, whereas other IgG antibody specificities enhance this and support the anaphylactic reaction. In cancer, inhibitory antibody specificities prevent growth signals derived from overexpressed oncogenes, whereas growth-promoting specificities enhance signalling and proliferation. Therefore, the mimotope concept is applicable to both fields for epitope-specific vaccination and analysis of conformational B-cell epitopes for the allergen/antigen. Results/conclusions: Mimotope technology is a relatively young theme in allergology and oncology. Still, proof of concept studies testing allergen and tumour mimotope vaccines suggest that mimotopes are ready for clinical trials.

Dose-dependent food allergy induction against ovalbumin under acid-suppression: A murine food allergy model

BACKGROUND Animal models are essential for analyzing the allergenic potential of food proteins and for investigating mechanisms underlying food allergy. Based on previous studies revealing acid-suppression medication as risk factor for food allergy induction, we aimed to establish a mouse model mimicking the natural route of sensitization in patients. METHODS The effect of acid-suppressing medication on murine gastric pH was assessed by intragastric pH measurements after two injections of a proton pump inhibitor (PPI). To investigate dose-dependency, mice were fed different concentrations of ovalbumin (OVA; 0.2, 0.5, 1.0, 2.5 or 5.0mg) either with or without anti-ulcer medication. Additionally, different routes of exposure (i.p. vs. oral) were compared in a second immunization experiment. Sera were screened for OVA-specific antibody titers (IgG1, IgG2a and IgE) in ELISA and RBL assay. Clinical reactivity was evaluated by measuring rectal temperature after oral challenge and by type I skin tests. RESULTS Two intravenous injections of PPI significantly elevated the gastric pH from 2.97 to 5.3. Only oral immunization with 0.2mg OVA under anti-acid medication rendered elevated IgG1, IgG2a and IgE titers compared to all other concentrations. Protein feeding alone altered antibody titers only marginally. Even though also i.p. immunizations induced high levels of specific IgE, only oral immunizations under anti-acids induced anaphylactic reactions evidenced by a significant decrease of body temperature. CONCLUSION Only low-dosage ovalbumin feedings under anti-acid medication resulted in IgE mediated food allergy. Based on this knowledge we have established a suitable food allergy model for further investigations of food adverse reactions.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.

Anti-ulcer treatment during pregnancy induces food allergy in mouse mothers and a Th2-bias in their offspring

The treatment of dyspeptic disorders with anti‐acids leads to an increased risk of sensitization against food allergens. As these drugs are taken by 30–50% of pregnant women due to reflux and heartburn, we aimed here to investigate the impact of maternal therapy with anti‐acids on the immune response in the offspring in a murine model. Codfish extract as model allergen was fed with or without sucralfate, an anti‐acid drug, to pregnant BALB/c mice during pregnancy and lactation. These mothers developed a codfish‐specific allergic response shown as high IgG1 and IgE antibody levels and positive skin tests. In the next step we analyzed whether this maternal sensi‐tization impacts a subsequent sensitization in the offspring. Indeed, in stimulated splenocytes of these off‐spring we found a relative Th2‐dominance, because the Thl‐ and T‐regulatory cytokines were significantly sup‐pressed. Our data provide evidence that the anti‐acid drug sucralfate supports sensitization against food in pregnant mice and favors a Th2‐milieu in their offspring. From these results we propose that anti‐acid treatment during pregnancy could be responsible for the increasing number of sensitizations against food allergens in young infants.—Schöll, I., Ackermann, U., Özdemir, C., Blümer, N., Dicke, T., Sel, S., Sel, S., Wegmann, M., Szalai, K., Knittelfelder, R., Untersmayr, E., Scheiner, O., Garn, H., Jensen‐Jarolim, E., Renz, H. Anti‐ulcer treatment during pregnancy induces food allergy in mouse mothers and a Th2‐bias in their offspring. FASEB J. 21, 1264–1270 (2007)

Active induction of tumor-specific IgE antibodies by oral mimotope vaccination

A role of IgE antibodies in cancer surveillance has been implicated for a long time. Studies dealing with IgE antibodies directly targeted to tumor antigens have shown marked anticancer effects mediated by this antibody class. Thus, the basic function of IgE antibodies may be to control tumor growth. Thus far, cancer-specific IgE has only been applied passively. Consequently, the aim of this study was to establish an active vaccination protocol to induce tumor antigen-specific IgE antibodies, and to evaluate functional properties. We previously generated epitope mimics, so-called mimotopes, for the epitope recognized by the anti-HER-2 antibody trastuzumab. Upon i.p. immunizations, IgG antibodies with trastuzumab-like properties could be elicited. In the present study, we immunized BALB/c mice via the oral route with these trastuzumab mimotopes, under simultaneous neutralization and suppression of gastric acid. As shown in preceding experiments, this feeding regimen effectively induces Th2 immune responses. Oral immunizations with trastuzumab mimotopes under hypoacidic conditions indeed resulted in the formation of IgE antibodies towards the HER-2 antigen. Moreover, anti-HER-2 IgE-sensitized effector cells mediated SK-BR-3 target cell lysis in an antibody-dependent cytotoxicity assay. We conclude that directed and epitope-specific induction of IgE against tumor antigens is feasible with an oral mimotope vaccination regimen, and that these antibodies mediate anticancer effects.

Nitration of the Egg-Allergen Ovalbumin Enhances Protein Allergenicity but Reduces the Risk for Oral Sensitization in a Murine Model of Food Allergy

Background Nitration of proteins on tyrosine residues, which can occur due to polluted air under “summer smog” conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. Methodology/Principal Findings BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y107) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. Conclusions/Significance These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.

Immunization with Mimotopes Prevents Growth of Carcinoembryonic Antigen–Positive Tumors in BALB/c Mice

Purpose: The carcinoembryonic antigen (CEA) is a glycoprotein that is overexpressed in nearly 50% of all human and veterinarian tumors. At present, anti-CEA antibodies are being tested in clinical studies as passive immunotherapeutics. This study aims to establish an active immunotherapy for the poorly immunogenic CEA glycoprotein by generating antigen surrogates. Experimental Design: We used the monoclonal anti-CEA antibody Col-1 and the biopanning method to generate peptide mimics (mimotopes) of the Col-1 epitope. The peptide showing the highest specificity and mimicry was synthesized as an octameric multiple antigenic mimotope (MAM). Subsequently, immunogenicity of the selected mimotope was examined in BALB/c mice. We assessed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity mediated by the induced antibodies on CEA-expressing HT29 tumor cells. Furthermore, after immunization, the BALB/c mice were transplanted s.c. with Meth-A/CEA tumor cells. Results: When BALB/c mice were immunized with this MAM, they generated a specific humoral immune response against CEA. The mimotope-induced polyclonal and poly-isotypic antibodies induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro. Furthermore, when MAM-immunized mice were transplanted s.c. with Meth-A/CEA cells expressing human CEA, a suppressed tumor growth was observed. Conclusion: From our results, we can conclude that the Col-1 epitope of the glycoprotein CEA can be translated into an immunogenic peptide mimic. The mimotope-induced antibodies recognize CEA and do effectively inhibit growth of CEA-positive tumors. Based on these finding, we suggest that the generated mimotopes are candidates for active immunotherapy of CEA-expressing tumors.

Impact of the Specific Mutation in KRAS Codon 12 Mutated Tumors on Treatment Efficacy in Patients with Metastatic Colorectal Cancer Receiving Cetuximab-Based First-Line Therapy: A Pooled Analysis of Three Trials

Purpose: This study investigated the impact of specific mutations in codon 12 of the Kirsten-ras (KRAS) gene on treatment efficacy in patients with metastatic colorectal cancer (mCRC). Patients: Overall, 119 patients bearing a KRAS mutation in codon 12 were evaluated. All patients received cetuximab-based first-line chemotherapy within the Central European Cooperative Oncology Group (CECOG), AIO KRK-0104 or AIO KRK-0306 trials. Results: Patients with KRAS codon 12 mutant mCRC showed a broad range of outcome when treated with cetuximab-based first-line regimens. Patients with tumors bearing a KRAS p.G12D mutation showed a strong trend to a more favorable outcome compared to other mutations (overall survival 23.3 vs. 14–18 months; hazard ratio 0.66, range 0.43–1.03). An interaction model illustrated that KRAS p.G12C was associated with unfavorable outcome when treated with oxaliplatin plus cetuximab. Conclusion: The present analysis suggests that KRAS codon 12 mutation may not represent a homogeneous entity in mCRC when treated with cetuximab-based first-line therapy.

Characterization of intrinsic and extrinsic risk factors for celery allergy in immunosenescence

Recent studies indicated an underestimation of allergies in elderly. In our experimental food allergy model of protein feeding under acid-suppression we aimed to assess whether food allergy can be induced in immunosenescent mice. Furthermore, the impact of gastric digestion on celery allergenicity was evaluated in aged patients. Measurements of serum zinc and iron levels in senescent and adult BALB/c mice for definition of the nutritional status indicated a possible alteration of the immune response in the aged animals due to reduced zinc and iron levels. Feedings of mice with digestion-sensitive celery proteins under physiological gastric conditions induced IgG1 and IgG2a in the aged and preferentially IgG1 in the adult animals. In contrast, incomplete digestion due to acid-suppression rendered celery-specific IgE, positive skin tests and elevated IL-5 levels in both age groups. Also in aged celery allergic patients (mean age 72 years) properly digested celery showed decreased capacity to bind and crosslink IgE as evaluated by skin tests and IgE immunoblot. Thus, in the geriatric murine model, celery allergy was induced only if gastric digestion was hindered. Accordingly, gastric proteolysis decreased in vitro and in vivo IgE-reactivity against celery proteins in aged allergic patients.