Generation of IgE-based immunotherapies against HER-2 overexpressing tumours

Ergänzend zu Chemotherapie und Strahlentherapie ist die passive Immuntherapie mit monoklonalen Antikörpern eine moderne Behandlungsmethode in der Krebstherapie. Zwei Eigenschaften von Antikörpern werden dabei genutzt: i) über das Fab Fragment binden sie spezifisch Tumorantigene und ii) über den Fc Teil rekrutieren sie Effektorzellen und aktivieren das Komplementsystem. Einer dieser Antikörper ist Trastuzumab (Herceptin®), ein wachstumshemmender, humanisierter, monoklonaler IgG1 Antikörper. Trastuzumab ist gegen das Tumorantigen HER-2 gerichtet, welches in 30% aller Brusttumore überexprimiert wird. Alle Antikörper, die bis jetzt in Form von passiver Immuntherapie in der Klinik eingesetzt werden, sind ausschließlich der IgG Subklasse zu zuordnen. Antikörper der IgE Subklasse sind hingegen bekannt für ihre schädliche Funktion in der Typ I Allergie. Es ist jedoch kaum bekannt, dass IgE Antikörper eine anti-Tumor Wirkung haben und diese Eigenschaft für Immuntherapien in der Onkologie ausgenutzt werden könnte. Das Ziel dieser Doktorarbeit war es daher alternative Strategien zur Behandlung von Krebserkrankungen basierend auf IgE Antikörper zu untersuchen, sowie deren Wirksamkeit mit der von IgG zu vergleichen. Die orale Immunisierungsroute eignet sich sehr gut zur Induktion einer Th2 Immunantwort mit hoch affinen IgE Antikörpern, die gegen das applizierte Antigen gerichtet sind. Daher wird die Etablierung eines IgE-abhängiges Nahrungsmittelallergiemodells in Mäusen beschrieben. Dieses Modell haben wir anschließend für unsere Krebsstudien adaptiert. Mäuse wurden mit unterschiedlichen Konzentrationen von Ovalbumin unter gleichzeitiger Säuresuppression gefüttert. Dies führte zu einer Induktion von antigen-spezifischen IgE in einem Th2 Milieu. Dieses orale Immunisierungsschema wurde auch mit HER-2 Mimotopen, d.h. Peptide, die das Trastuzumab-Epitop imitieren, angewendet. Die durch das Mimotop induzierten IgE Antikörper erkannten spezifisch das Tumorantigen auf HER-2 überexprimierende Brustkrebszellen und führten zur Lyse der Tumorzellen. Ergänzend zu diesem aktiven immuntherapeutischen Ansatz wurde ein Trastuzumab IgE Antikörper für passive Immuntherapie konstruiert. Wir konnten zeigen, dass Trastuzumab IgE die gleiche Spezifität wie Trastuzumab IgG besitzt. In einem Three-Colour Flow Cytometric Assay zeigte sich, dass beide Antikörper Tumorzellen erfolgreich zerstören, dies aber durch unterschiedliche Mechanismen tun: Trastuzumab IgG führte zu „antibody-dependent cellmediated phagocytosis“ (ADCP), während Trastuzumab IgE „antibody-dependent cellmediated cytotoxicity“ (ADCC) auslöste. Ausgehend von den hier präsentierten Daten schließen wir, dass tumor-spezifisches IgE, aktiv induziert oder passiv verabreicht, eine vielversprechende Alternative zu Immuntherapien mit IgG gegen Krebs darstellt.In combination with chemotherapy or radiation, passive immunotherapy with monoclonal antibodies is state of the art in cancer therapy. For this purpose, two properties of antibodies are harnessed: i) via the Fab fragment they bind a specific tumour antigen and ii) via the Fc portion they recruit effector cells and activate the complement system. One of these antibodies is trastuzumab (Herceptin®), a growth-inhibitory humanized monoclonal IgG1 antibody recognizing the tumour antigen HER-2, which is overexpressed in 30% of human breast cancers. Interestingly, all antibodies applied for passive immunotherapy are so far exclusively of the IgG subclass. In contrast, antibodies of the IgE subclass are best-known for their detrimental function in type I hypersensitivity. It is little-known that IgE has anti-tumour capacity which could be exploited for immunotherapy of cancer. Thus, the aim of this doctoral thesis was to examine alternative strategies for cancer treatment based on IgE antibodies, and to compare their efficacy with that of IgG. The oral immunization route is well suited for the induction of a Th2 immunity including high affine IgE responses to administered antigens. Therefore, the establishment of an IgEdependent food allergy model in mice is described, which we applied also for our cancer studies. When mice were fed with different concentrations of ovalbumin under concomitant anti-acid medication, an antigen-specific IgE induction in a Th2 environment could be achieved. This oral vaccination regimen was also used for feeding HER-2 mimotopes, i.e. epitope-mimics of the anti-HER-2 IgG antibody trastuzumab. Indeed, these mimotopes induced IgE antibodies recognizing the tumour antigen which were able to bind HER-2 overexpressing breast cancer cells and led to tumour cell lysis. Complementary to this active immunotherapeutic approach a trastuzumab-like IgE antibody for passive immunotherapy was constructed. We could show that this trastuzumab IgE exhibited the same specificity as the original trastuzumab IgG. Moreover, a three-colour flow cytometric assay indicated that even though both antibodies were able to induce tumour cell killing, they used different mechanisms: trastuzumab IgG acted via antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE elicited mostly antibody-dependent cell-mediated cytotoxicity (ADCC). Based on the presented data we conclude that tumour-specific IgE, induced actively or applied in a passive manner, represents a potent alternative to IgG-based immunotherapies against cancer

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. Copyright (C) 2012 S. Karger AG, Base

Phase II study on the efficacy and safety of Lapatinib administered beyond disease progression and combined with vinorelbine in HER-2/neu– positive advanced breast cancer: results of the CECOG LaVie trial

Background Vinorelbine constitutes effective chemotherapy for metastatic breast cancer (MBC) and acts synergistically with trastuzumab in HER-2/neu positive disease. The present study was set out to evaluate the efficacy and safety of vinorelbine when combined with lapatinib, an anti-HER2 tyrosine-kinase inhibitor, as late-line regimen administered beyond previous disease progression on prior lapatinib in patients with HER-2/neu- positive MBC. Methods The CECOG LaVie study was designed as open-labeled, single-arm, multicenter phase II trial. Patients had to be pretreated with lapatinib plus chemotherapy, and received lapatinib at a daily dose of 1250 mg in combination with vinorelbine 20 mg/m i.v. on days 1 and 8 of a three-week cycle until disease progression, intolerable toxicity or withdrawal of consent. Progression-free survival (PFS) was defined as primary study endpoint; secondary endpoints included overall survival (OS), response rate according to RECIST 1.1, and safety. The study was terminated early due to poor accrual. Results A total number of nine patients were included; lapatinib administered beyond disease progression combined with vinorelbine resulted in a median PFS of 7.7 months (95 % CI 0.56-14.91) and a median OS of 23.4 months (95 % CI 16.6130.13), respectively. Partial remission was seen in one of nine patients, three patients had stable disease of > six months, whereas the remaining five patients had primary disease progression. In two patients, modification of vinorelbine dose due to toxicity became necessary; no dose modification was needed for lapatinib. The majority of reported adverse events (AE) were grade 1 and 2 in severity with diarrhea being the most commonly observed AE Conclusion In this heavily pretreated patient population, combination of vinorelbine plus lapatinib showed encouraging activity and was characterized by an acceptable safety profile. Despite the low patient number, lapatinib plus vinorelbine may constitute a potential treatment option in heavily pretreated patients with HER-2/neu-positive MBC previously exposed to lapatinib. Trial registration EudraCT number 2009-016826-15, (15. 10.2009)(VLID)510941

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area

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 sensitization impacts a subsequent sensitization in the offspring. Indeed, in stimulated splenocytes of these offspring we found a relative Th2-dominance, because the Th1- and T-regulatory cytokines were significantly suppressed. 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