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PURPOSE: This study assessed clinical activity, safety and immunogenicity of MAGE-A3 immunotherapeutic in patients with MAGE-A3-positive metastatic melanoma. PATIENTS AND METHODS: In this open-label, multicentre, uncontrolled, Phase II study (ClinicalTrials.gov NCT00896480), patients received ≤24 doses of MAGE-A3 immunotherapeutic (4-cycle schedule). At screening, two skin lesions were biopsied for MAGE-A3 expression analysis and presence/absence of a previously identified gene signature (GS) associated with favourable clinical outcome. Clinical activity was assessed in terms of clinical response, time-to-treatment failure (TTF) and progression-free survival (PFS). Adverse events (AEs) and serious AEs (SAEs) were recorded. MAGE-A3-specific immune responses were assessed. Clinical activity and immunogenicity were analysed overall and separately in patients with 2/2 (GS+/+), 1/2 (GS+/-) or 0/2 (GS-/-) biopsies presenting GS. RESULTS: Of 49 screened patients, 32 had MAGE-A3-positive tumours; 24 (8 GS+/+, 8 GS+/-, 8 GS-/-) were treated. Two complete (GS+/+ patients) and two partial responses (one GS+/+, one GS+/-) were reported; of note, one of the two complete responses was unlikely to be related to the study treatment. Median TTF and PFS were 14.8 and 7.2 months for GS+/+, 2.3 and 2.8 months for GS+/- and 2.4 and 2.9 months for GS-/- patients. Three grade 3 AEs and two SAEs unrelated to treatment were reported. All patients were seropositive for MAGE-A3 antibodies on vaccination with no differences between the different GS profiles. MAGE-A3-specific CD4+ and CD8+ T cell immunogenicity was detected; 12/16 (75.0%) of patients presented CD4+ T cell responses. CONCLUSION: Treatment with MAGE-A3 immunotherapeutic showed signs of clinical activity in GS+/+ patients. Treatment was well tolerated and immunogenic. No differences in immune responses according to GS status were observed. TRIAL REGISTRATION NUMBER: NCT00896480 (Results).

We assessed safety, immunogenicity and clinical activity of recombinant MAGE-A3 antigen combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) in association with dacarbazine in patients with metastatic melanoma. In this open-label, phase I/II, uncontrolled multicentre trial conducted in Belgium and France, patients with MAGE-A3-positive melanoma received up to 24 doses of MAGE-A3 immunotherapeutic (four cycles) coadministered with eight doses of dacarbazine. Adverse events (AE) were recorded until 31 days postvaccination, and serious AEs (SAE), until 30 days following the last dose. MAGE-A3-specific antibodies were measured by ELISA. Clinical activity of MAGE-A3 immunotherapeutic was assessed in patients positive/negative for previously identified gene signature (GS) associated with clinical outcome. Forty-eight patients were enrolled and treated (32 GS+, 15 GS-, 1 unknown GS status); two patients completed the study. All patients reported AEs, the most common were 'general disorders and administration site conditions' (94%). Treatment-related AEs were reported by 85% of patients; the most common was pain at injection site (38%). Sixteen SAEs were reported by 21% of patients; two were considered as treatment related (neutropenia and thrombocytopenia; grade 4). Postdose 4, all patients were seropositive for MAGE-A3-specific antibodies, with a geometric mean titre of 2778.7 ELISA units (EU)/mL (95% CI 1638.3 to 4712.8). One complete and three partial responses were reported (only in GS+ patients). Median overall survival was 11.4 months for GS+ and 5.3 months for GS- patients. Although this trial shows poor results compared with the new results with checkpoint inhibitors, it gives an interesting insight in rapidly developing fields like combinations of immunotherapy and chemotherapy, new generation vaccines and the use of gene profile as a predictive marker. NCT00849875.

This Phase I dose-escalation study (NCT000 58526) assessed the safety and immunogenicity of an anticancer immunotherapeutic (recombinant HER2 protein (dHER2) combined with the immunostimulant AS15) in patients with early-stage HER2-overexpressing breast cancer (BC). Sixty-one trastuzumab-naive patients with stage II–III HER2-positive BC received the dHER2 immunotherapeutic after surgical resection and adjuvant therapy. They were allocated into four cohorts receiving different doses of dHER2 (20, 100, 500 μg) combined with a fixed AS15 dose. Safety and immunogenicity (dHER2-specific antibody responses) were assessed. After completing the immunization schedule (three or six doses over 14 weeks) and a six-month follow-up, the patients were followed for 5 years for late toxicity, long-term immunogenicity, and clinical status. The immunizations were well tolerated, and increasing doses of dHER2 had no impact on the frequency or severity of adverse events. Few late toxicities were reported, and after 5 years 45/54 patients(83.3 %) were still alive, while 28/45 (62 %) with known disease status were disease free. Regarding the immunogenicity of the compound, a positive association was found between the dHER2 dose, the immunization schedule, and the prevalence of dHER2-specific humoral responses. Among the patients receiving the most intense immunization schedule with the highest dHER2 dose, 6/8 maintained their dHER2-specific antibody response 5 years after immunization. The dHER2 immunotherapeutic had an acceptable safety profile in early HER2-positive BC patients. dHER2-specific antibody responses were induced, with the rate of responders increasing with the dHER2 dose and the number and frequency of immunizations., SCOPUS: ar.j, SCOPUS: ar.j, info:eu-repo/semantics/published

This Phase I dose-escalation study (NCT00058526) assessed the safety and immunogenicity of an anti-cancer immunotherapeutic (recombinant HER2 protein (dHER2) combined with the immunostimulant AS15) in patients with early-stage HER2-overexpressing breast cancer (BC). Sixty-one trastuzumab-naive patients with stage II-III HER2-positive BC received the dHER2 immunotherapeutic after surgical resection and adjuvant therapy. They were allocated into four cohorts receiving different doses of dHER2 (20, 100, 500 µg) combined with a fixed AS15 dose. Safety and immunogenicity (dHER2-specific antibody responses) were assessed. After completing the immunization schedule (three or six doses over 14 weeks) and a six-month follow-up, the patients were followed for 5 years for late toxicity, long-term immunogenicity, and clinical status. The immunizations were well tolerated, and increasing doses of dHER2 had no impact on the frequency or severity of adverse events. Few late toxicities were reported, and after 5 years 45/54 patients (83.3 %) were still alive, while 28/45 (62 %) with known disease status were disease free. Regarding the immunogenicity of the compound, a positive association was found between the dHER2 dose, the immunization schedule, and the prevalence of dHER2-specific humoral responses. Among the patients receiving the most intense immunization schedule with the highest dHER2 dose, 6/8 maintained their dHER2-specific antibody response 5 years after immunization. The dHER2 immunotherapeutic had an acceptable safety profile in early HER2-positive BC patients. dHER2-specific antibody responses were induced, with the rate of responders increasing with the dHER2 dose and the number and frequency of immunizations.

Modulation of the immune system for therapeutic ends is now increasingly being employed in patients with advanced cutaneous melanoma, a deadly cancer arising from melanocytes. Cancer immunotherapy, which harnesses the protective capacity and specificity of the immune system toward eliminating cancer cells, generally capitalizes on the specific recognition of tumor antigens by cognate CTLs (cytolytic T lymphocytes). This recognition requires efficient presentation of antigenic peptides on the surface of tumor cells. The aim of this thesis was to explore the mechanisms of transcriptional regulation of genes encoding melanoma antigens in order to identify potential strategies to increase their repertoire and the expression levels. A group of CG (cancer-germline) genes uses primarily DNA methylation for transcriptional repression in somatic cells, whereas genome hypomethylation in tumors results in their activation and presentation of relevant antigenic peptides recognized by cognate CTLs. In the first part of this thesis, we explored the potential of protein BORIS (Brother of the Regulator of Imprinted sites) to induce and/or maintain the activation of MAGE-A1 (Melanoma AntiGEn A1) and other CG genes in melanoma cells. BORIS expression on its own was insufficient to produce these effects and hence may not serve as a general means to increase the repertoire of melanoma antigens. The quantitative analysis of BORIS transcripts revealed the expression pattern characteristic to CG genes. BORIS activation in a substantial proportion of melanoma samples indicates the potential for immunotherapeutic targeting of BORIS-derived antigenic peptides. The identity of such peptides remains to be investigated. The second part of this thesis focused on the mechanisms of the transcriptional regulation of MITF-M, the melanocyte-specific isoform of MITF (Microphthalmia-associated transcription factor). MITF-M is a critical transcriptional activator of melanocyte differentiation genes such, Une modulation du système immunitaire dans un but thérapeutique est de plus en plus utilisée chez les patients atteints de mélanome avancé, un cancer mortel issu des mélanocytes. L’immunothérapie du cancer tire parti de la capacité protectrice du système immunitaire et de sa spécificité pour éliminer la tumeur en se basant de façon générale sur la reconnaissance spécifique des antigènes tumoraux par les CTL (lymphocytes T cytolytiques). Cette reconnaissance requiert une présentation efficace de peptides antigéniques à la surface des cellules tumorales. Le but de ce travail de thèse était d’étudier les mécanismes de régulation transcriptionnelle des gènes qui donnent naissance aux antigènes de mélanome pour identifier les stratégies potentielles d’augmentation de leur répertoire et de leur niveau d’expression. Un groupe de gènes cancer-lignée germinale (CG) utilise la méthylation de l’ADN comme mécanisme de répression transcriptionnelle dans les cellules somatiques, tandis que l’hypométhylation du génome caractéristique des tumeurs aboutit à l’activation de ces gènes et à la présentation de peptides antigéniques reconnus par les CTL spécifiques. Nous avons exploré le potentiel de la protéine BORIS (en anglais, Brother of the Regulator of Imprinted sites) d’induire et/ou maintenir l’activation de MAGE-A1 (en anglais, Melanoma AntiGEn A1) et d’autres gènes du groupe CG dans les cellules de mélanome. L’expression de BORIS n’était pas une condition suffisante pour produire ces effets et, ainsi, ne peut pas servir d’outil pour augmenter le répertoire des antigènes de mélanome. L’analyse quantitative des transcrits de BORIS a révélé le profil d’expression caractéristique pour des gènes du groupe CG. L’activation de BORIS dans une proportion substantielle des échantillons de mélanome indique le potentiel de ciblage thérapeutique des antigènes dérivés de la protéine BORIS. L’identité de ces antigènes reste à explorer. La deuxième partie de cette thèse s’est focalisée sur des, (SBIM 3) -- UCL, 2011

IL-9 contributes to lung inflammatory processes such as asthma, by promoting mast cell differentiation, B cell activation, eosinophilia, and mucus production by lung epithelial cells. The observation that IL-9 overexpressing mice show increased mast cell numbers in the intestinal mucosa suggests that this cytokine might also play a role in intestinal inflammation. In colons from IL-9 transgenic mice, the expression of Muc2, a major intestinal mucin gene, was up-regulated, together with that of CLCA3 chloride channel and resistin like alpha, which are goblet cell-associated genes. Additional IL-9 up-regulated genes were identified and included innate immunity genes such as angiogenin 4 and the PLA2g2a phospholipase A(2), which are typical Paneth cell markers. Histochemical staining of Paneth cells by phloxine/tartrazine showed that IL-9 induces Paneth cell hyperplasia in Lieberkuhn glands of the small intestine, and in the colonic mucosa, where this cell type is normally absent. Expression of Paneth cell markers, including angiogenin 4, PLA2g2a, and cryptdins, was induced in the colon of wild-type mice after two to four daily administrations of IL-9. By crossing IL-9 transgenic mice with IL-13(-/-) mice, or by injecting IL-9 into IL-4R(-/-) mice, we showed that IL-13 was required for the up-regulation of these Paneth cell-specific genes by IL-9. Taken together, our data indicate that Paneth cell hyperplasia and expression of their various antimicrobial products contribute to the immune response driven by TH2 cytokines, such as IL-9 and IL-13 in the intestinal mucosa. The Journal of Immunology, 2009, 182: 4737-4743.

Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.

In tick salivary glands, genes induced during blood feeding result in the expression of new proteins secreted into tick saliva. These proteins are potentially involved in modulation of vertebrate host immune and hemostatic responses. In this study, subtractive and full-length cDNA libraries were constructed by use of mRNA extracted from salivary glands of unfed and 5-day engorged Ixodes ricinus. Sequences from these 2 libraries were compared with European Molecular Biology Laboratory (EMBL)/GenBank databases, which led to their classification into 2 major groups. The first group comprises cDNAs that failed to match or showed low homology to genes of known function. The second group includes sequences that showed high homology to genes of known function--for example, anticoagulants, inhibitors of platelet aggregation, and immunomodulatory proteins. Analyses of corresponding proteins suggest that they may be secreted by salivary gland cells. To study the properties of the recombinant proteins, selected cDNAs were expressed in mammalian or bacterial systems.