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Activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Agonistic anti-CD40 antibodies currently in development require Fcγ-receptor-mediated crosslinking of CD40 molecules for meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely Fc-independent manner. These novel Fc-silenced anti-CD40 antibodies induce upregulation of costimulatory receptors CD80 and CD86 and cytokine release by dendritic cells with an efficacy exceeding that of existing antibodies. Binding to the CD40L interaction region on CD40 appears to be a prerequisite to achieving such strong activities. Finally, the most active identified anti-CD40 antibody shows evidence of activity in terms of the expected markers of canonical CD40 signaling when injected in humanized mice. There are no signs of obvious toxicities whereas the clinical-stage anti-CD40 antibody CP-870,893 induced severe signs of toxicity in these animals despite a lower dose compared with the novel Fc-silenced canonical agonist. These studies thus demonstrate potent activation of antigen-presenting cells with anti-CD40 antibodies lacking Fcγ-receptor-binding activity and open the possibility of an efficacious and safe combination therapy for cancer patients.One Sentence SummaryTreatment of antigen-presenting cells and humanized mice with novel Fc-silenced CD40 antibodies demonstrates an Fcγ-receptor-independent canonical agonistic mode of action for therapeutic use.

Background Antibody based cancer therapies have achieved convincing success rates combining enhanced tumor specificity and reduced side effects in patients. Trastuzumab that targets the human epidermal growth factor related receptor 2 (HER2) is one of the greatest success stories in this field. For decades, trastuzumab based treatment regimens are significantly improving the prognosis of HER2-positive breast cancer patients both in the metastatic and the (neo-) adjuvant setting. Nevertheless, ≥ 50% of trastuzumab treated patients experience de-novo or acquired resistance. Therefore, an enhanced anti-HER2 targeting with improved treatment efficiency is still aspired. Methods Here, we determined cellular and molecular mechanisms involved in the treatment of HER2-positive BC cells with a new rabbit derived HER2 specific chimeric monoclonal antibody called “B100″. We evaluated the B100 treatment efficiency of HER2-positive BC cells with different sensitivity to trastuzumab both in vitro and in the presence of a human immune system in humanized tumor mice. Results B100 not only efficiently blocks cell proliferation but more importantly induces apoptotic tumor cell death. Detailed in vitro analyses of B100 in comparison to trastuzumab (and pertuzumab) revealed equivalent HER2 internalization and recycling capacity, similar Fc receptor signaling, but different HER2 epitope recognition with high binding and treatment efficiency. In trastuzumab resistant SK-BR-3 based humanized tumor mice the B100 treatment eliminated the primary tumor but even more importantly eradicated metastasized tumor cells in lung, liver, brain, and bone marrow. Conclusion Overall, B100 demonstrated an enhanced anti-tumor activity both in vitro and in an enhanced preclinical HTM in vivo model compared to trastuzumab or pertuzumab. Thus, the use of B100 is a promising option to complement and to enhance established treatment regimens for HER2-positive (breast) cancer and to overcome trastuzumab resistance. Extended preclinical analyses using appropriate models and clinical investigations are warranted.

Bacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori , the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cag pathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.

The human gastric pathogen Helicobacter pylori is able to establish an infection in a hostile environment with virtually no competitors. For this purpose, it has elaborated a set of colonization factors which facilitate both survival under acid exposure, motility and orientation in a highly viscous mucus layer, and adherence to epithelial surfaces. A more intimate interaction with gastric epithelia provides the basis to influence gene expression profiles as well as morphological transitions via signaling cascades or via direct activities of virulence factors. H. pylori is also one of the most genetically diverse of organisms, and variations are not only found in outer membrane adhesins, but also in two major virulence factors, the VacA cytotoxin and the cag pathogenicity island. Both factors are able to target different cell types and different interaction partners to induce a wide range of possible cellular effects. Despite the fact that H. pylori elicits a strong inflammatory response, the immune system fails to clear the infection, suggesting that immune evasion strategies are used. The mechanisms for immune evasion include the induction of a strongly polarized immune response, a modulation of phagocytosis and neutrophil function, and an inhibition of lymphocyte proliferation. Prolonged inflammation and direct action of bacterial factors may lead to impairment of gland function and eventually to carcinogenesis.

The human gastric pathogen Helicobacter pylori is able to establish an infection in a hostile environment with virtually no competitors. For this purpose, it has elaborated a set of colonization factors which facilitate both survival under acid exposure, motility and orientation in a highly viscous mucus layer, and adherence to epithelial surfaces. A more intimate interaction with gastric epithelia provides the basis to influence gene expression profiles as well as morphological transitions via signaling cascades or via direct activities of virulence factors. H. pylori is also one of the most genetically diverse of organisms, and variations are not only found in outer membrane adhesins, but also in two major virulence factors, the VacA cytotoxin and the cag pathogenicity island. Both factors are able to target different cell types and different interaction partners to induce a wide range of possible cellular effects. Despite the fact that H. pylori elicits a strong inflammatory response, the immune system fails to clear the infection, suggesting that immune evasion strategies are used. The mechanisms for immune evasion include the induction of a strongly polarized immune response, a modulation of phagocytosis and neutrophil function, and an inhibition of lymphocyte proliferation. Prolonged inflammation and direct action of bacterial factors may lead to impairment of gland function and eventually to carcinogenesis.

SummaryHelicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the β2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.