Impaired phagocytosis and reactive oxygen species production in phagocytes is associated with systemic vasculitis

BACKGROUND: Anti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) is a group of autoimmune diseases, characterized by small vessel inflammation. Phagocytes such as neutrophils and monocytes are the main effector cells found around the inflamed vessel wall. Therefore, we wanted to investigate aspects of function and activation of these cells in patients with AAV.METHODS: Flow cytometry was used to evaluate: the expression of activation markers (CD11c, CD62L, CD177 and C5aR); the number of recently released neutrophils from bone marrow, defined as CD10(-)D16(low) cells in peripheral blood; and the capacity of peripheral blood monocytes and polymorphonuclear leukocytes (PMN) to produce reactive oxygen species and to phagocytose opsonized bacteria.RESULTS: AAV patients (n = 104) showed an increase of CD10(-)CD16(low) neutrophils and total PMN in peripheral blood, suggesting a combination of increased bone marrow release and prolonged survival. An increased percentage of AAV PMN expressed CD177 but no other signs of activation were seen. A decreased production of reactive oxygen species was observed in AAV phagocytes, which was associated with disease activity. Moreover, granulocytes from patients with microscopic polyangiitis showed lower oxidative burst capacity compared to patients with granulomatosis with polyangiitis or eosinophilic granulomatosis with polyangiitis. In addition, decreased phagocytosis capacity was seen in PMN and monocytes.CONCLUSION: Our results indicate that phagocytes from AAV patients have impaired function, are easily mobilized from bone marrow but are not particularly activated. The association between low reactive oxygen species formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor

Tumor-directed immunotherapy can generate tumor-specific T cell responses through localized co-stimulation

The most important goals for the field of immuno-oncology are to improve the response rate and increase the number of tumor indications that respond to immunotherapy, without increasing adverse side effects. One approach to achieve these goals is to use tumor-directed immunotherapy, i.e., to focus the immune activation to the most relevant part of the immune system. This may improve anti-tumor efficacy as well as reduce immune-related adverse events. Tumor-directed immune activation can be achieved by local injections of immune modulators in the tumor area or by directing the immune modulator to the tumor using bispecific antibodies. In this review, we focus on therapies targeting checkpoint inhibitors and co-stimulatory receptors that can generate tumor-specific T cell responses through localized immune activation

Kick-starting the cancer-immunity cycle by targeting CD40

Stimulation of CD40 on dendritic cells to expand and activate tumor-specific T cells and generate anticancer immunity is an attractive therapeutic approach. Since CD40 agonists exert their effects upstream of checkpoint inhibitors, including PD-1 or PD-L1 antagonists, they are ideal candidates for combination regimens

Human monoclonal antibodies with different fine specificity for digoxin derivatives: cloning of heavy and light chain variable region sequences.

Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH1114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had mu heavy chains, two of them (MO6 and LH114) had kappa light chains and one (LH92) lambda light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in M13 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies

The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T cell dependent tumor immunity.

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 has been investigated in human and murine in vitro models. The in vivo effect has been investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. Results: Activation of dendritic cells (DCs) by ADC-1013 results in up-regulation of the co-stimulatory molecules CD80 and CD86, and secretion of IL-12. ADC-1013 also activates dendritic cells from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated dendritic cells induce antigen-specific T cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant anti-tumor effects and long-term tumor-specific immunity. Further, ADC-1013 demonstrates significant anti-tumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. Conclusions: Our data demonstrate that ADC-1013 induces long-lasting anti-tumor responses and immunological memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer

Pre-assembly of the extracellular domains of CD40 is not necessary for rescue of mouse B cells from anti-immunoglobulin M-induced apoptosis

CD40 is a tumour necrosis factor receptor (TNFR) family member of central importance for the adaptive immune system. To elucidate the functional role of the different extracellular domains of CD40, we have created a set of truncated CD40 molecules where domains, or parts of domains, have been removed. These CD40 proteins, which contain a peptide tag in the N-terminal end, have been expressed in a murine B-cell line, WEHI 231. It was found that ligation of these engineered CD40 proteins via the peptide tag, was sufficient to rescue as well as to promote proliferation of apoptotic WEHI 231 cells, even when all the extracellular domains of CD40 were absent. Our results suggest that pre association of CD40 in the cell membrane plays no critical role for the CD40 signalling pathway. Furthermore, our data imply that conformational changes initiated in the extracellular domains of CD40 are not essential for signal transduction