Soluble carcinoembryonic antigen activates endothelial cells and tumor angiogenesis.

Carcinoembryonic antigen (CEA, CD66e, CEACAM-5) is a cell-surface-bound glycoprotein overexpressed and released by many solid tumors that has an autocrine function in cancer cell survival and differentiation. Soluble CEA released by tumors is present in the circulation of patients with cancer, where it is used as a marker for cancer progression, but whether this form of CEA exerts any effects in the tumor microenvironment is unknown. Here, we present evidence that soluble CEA is sufficient to induce proangiogenic endothelial cell behaviors, including adhesion, spreading, proliferation, and migration in vitro and tumor microvascularization in vivo. CEA-induced activation of endothelial cells was dependent on integrin ß-3 signals that activate the focal-adhesion kinase and c-Src kinase and their downstream MAP-ERK kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt effector pathways. Notably, while interference with VEGF signaling had no effect on CEA-induced endothelial cell activation, downregulation with the CEA receptor in endothelial cells attenuated CEA-induced signaling and tumor angiogenesis. Corroborating these results clinically, we found that tumor microvascularization was higher in patients with colorectal cancer exhibiting higher serum levels of soluble CEA. Together, our results elucidate a novel function for soluble CEA in tumor angiogenesis.

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.

Mimotopes identify conformational B-cell epitopes on the two major house dust mite allergens Der p 1 and Der p 2.

House dust mite allergy occurs in 10-20% of the population. Improvement of the present immunotherapy requires detailed knowledge about the structure of the allergens. Mimotopes selected from phage peptide libraries imitate the conformational epitopes of a natural allergen. The aim of our study was to generate epitope mimics for the two major allergens of house dust mite. When the monoclonal anti-Der p 1 and anti-Der p 2 antibodies were used for biopannings, mimotopes were selected which bound also specific IgE from human allergic patients' sera. The conformational matching of these mimotopes on the 3D structure of the natural allergens determined discontinuous epitopes in both cases, representing conformational B-cell epitopes relevant for binding of human IgE. Therefore, these mimotopes are potential candidates for the directed induction of blocking antibodies and epitope-specific immunotherapy of mite allergy.

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.

Induction of IgG antibodies against the GD2 carbohydrate tumor antigen by vaccination with peptide mimotopes.

The disialoganglioside GD2, a carbohydrate antigen, is expressed on all tumors of neuroectodermal origin, including melanoma, neuroblastoma, sarcoma and small cell lung cancer. Due to its specific expression on tumor surfaces, GD2 is an attractive target for immunotherapies. The mouse/human chimeric anti-GD2 mAb ch14.18 is already applied in melanoma and neuroblastoma trials as a passive immunotherapy. To establish an active immunotherapy alternative, we aimed to replace the poorly immunogenic ganglioside with immunogenic peptides. Previously, we used the ch14.18 antibody to select GD2 peptide mimics from a phage display library. In the present study, two mimics of the ch14.18 epitope were coupled to keyhole limpet hemocyanin and used for immunizing BALB/c mice. Induction of a specific humoral immune response towards the original antigen GD2, both purified and expressed on neuroblastoma and melanoma cells, could be demonstrated in ELISA, Western blot, and immunofluorohistochemistry. As the elicited antibodies were of the IgG isotype, the mimotope conjugates were capable of recruiting T cell help and inducing memory phenomena. In conclusion, we show that an epitope of the carbohydrate antigen GD2 can successfully be translated into immunogenic peptide mimotopes. Our immunization experiments indicate that GD2 mimotopes are suitable for active immunotherapy of GD2-expressing tumors.