Adcitmer® , a new CD56-targeting monomethyl auristatin E-conjugated antibody, is a potential therapeutic approach in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. OBJECTIVES: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer® ) targeting CD56 in preclinical models of MCC. METHODS: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. RESULTS: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. CONCLUSIONS: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors.

Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system.

Monoclonal antibodies (mAbs) are usually delivered systemically, but only a small proportion of the drug reaches the lung after intravenous injection. The inhalation route is an attractive alternative for the local delivery of mAbs to treat lung diseases, potentially improving tissue concentration and exposure to the drug while limiting passage into the bloodstream and adverse effects. Several studies have shown that the delivery of mAbs or mAb-derived biopharmaceuticals via the airways is feasible and efficient, but little is known about the fate of inhaled mAbs after the deposition of aerosolized particles in the respiratory system. We used cetuximab, an anti-EGFR antibody, as our study model and showed that, after its delivery via the airways, this mAb accumulated rapidly in normal and cancerous tissues in the lung, at concentrations twice those achieved after intravenous delivery, for early time points. The spatial distribution of cetuximab within the tumor was heterogeneous, as reported after i.v. injection. Pharmacokinetic (PK) analyses were carried out in both mice and macaques and showed aerosolized cetuximab bioavailability to be lower and elimination times shorter in macaques than in mice. Using transgenic mice, we showed that FcRn, a key receptor involved in mAb distribution and PK, was likely to make a greater contribution to cetuximab recycling than to the transcytosis of this mAb in the airways. Our results indicate that the inhalation route is potentially useful for the treatment of both acute and chronic lung diseases, to boost and ensure the sustained accumulation of mAbs within the lungs, while limiting their passage into the bloodstream.

Efficiency of immunoglobulin G replacement therapy in common variable immunodeficiency: correlations with clinical phenotype and polymorphism of the neonatal Fc receptor.

Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An 'efficiency' index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P < 0·001) and with the presence of at least one CVID disease-related phenotype (lymphoproliferation, autoimmune cytopenia or enteropathy) (P < 0·001). High IgG efficiency was noted in patients homozygotes for the variable number tandem repeat (VNTR) 3/3 polymorphism of the neonatal Fc receptor gene [IgG Fc fragment receptor transporter alpha chain (FCGRT)] promoter, and this was particularly significant in patients treated with IVIG (P < 0.01). In a multivariate analysis, FCGRT VNTR 3/3 genotype (P = 0·008) and high serum albumin (P < 0·001) were associated independently with increased efficiency of i.v. Ig.

Evidence for a protective role of the STAT5 transcription factor against oxidative stress in human leukemic pre-B cells.

STAT5 transcription factors are involved in normal B lymphocyte development and in leukemogenesis. We show that the inhibition of STAT5A expression or activity in the NALM6, 697 and Reh leukemic pre-B cell lines, results in a higher spontaneous apoptosis and an increased FAS-induced cell death. However, the molecular mechanisms underlying the altered pre-B cell survival are unclear. We used a proteomic approach to identify proteins that are differentially regulated in cells expressing (NALM6Δ5A) or not a dominant negative form of STAT5A. Among the 14 proteins identified, six were involved in the control of the oxidative stress like glutathione (GSH) synthetase and DJ-1. Accordingly, we showed increased levels of reactive oxygen species (ROS) in NALM6Δ5A cells and suppression of the increased sensitivity to Fas-mediated apoptosis by the GSH tripeptide. Similar results were observed when NALM6 cells were treated with TAT-STAT5Δ5A fusion proteins or STAT5A shRNA. In addition, the 697 and Reh pre-B cells were found to share number of molecular changes observed in NALM6Δ5A cells including ROS generation, following inhibition of STAT5 expression or function. Our results point out to a hitherto undescribed link between STAT5 and oxidative stress and provide new insights into STAT5 functions and their roles in leukemogenesis.

STAT3 transcription factor is constitutively activated and is oncogenic in nasal-type NK/T-cell lymphoma.

Nasal-type natural killer (NK) cell lymphoma is an infrequent aggressive malignant disease with very poor prognosis. We aimed to explore the possible role of the transcription factor STAT3 in the pathophysiology of this malignancy, as it was involved in oncogenesis and chemoresistance. For this, we established and characterized a continuous interleukin 2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK-cell lymphoma. Cells harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-gamma, interleukin-10 and vascular-endothelium growth factor in vitro. STAT3 was phosphorylated in Y705 dimerization residue in MEC04 cells and restricted to the nucleus. Y705 STAT3 phosphorylation involved JAK2, as exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation. By using recombinant transducible TAT-STAT3-beta (beta isoform), TAT-STAT3Y705F (a STAT3 protein mutated on Y705 residue, which prevents STAT3 dimerization) and peptides inhibiting specifically STAT3 dimerization, we inhibited STAT3 phosphorylation and cell growth, with cell death induction. Finally, STAT3 was phosphorylated in Y705 residue in the nuclei of lymphoma cells in eight/nine patients with nasal-type NK/T-cell lymphoma and in YT, another NK cell line. Our results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK-cell lymphomas, and may represent a promising therapeutical target.

[Prevalence of celiac disease markers in a French cohort of children and adolescents with type 1 diabete mellitus]. / Prévalence des marqueurs biologiques de la maladie coeliaque dans une cohorte d'enfants et d'adolescents diabétiques de type I.

The aim of this study was to determine the prevalence of celiac disease (CD) markers in a French cohort of 84 children type 1 diabetics. Detection of antitransglutaminase (AtTG), antiendomysium (AEA) and antigliadin (AGA) antibodies was performed. Group 1 included 81 (96.4%) diabetic patients with negative antibodies. Group 2 included 3 patients (3.6%) with positive serological markers: 1 AGA-AEA-AtTG and 1 AEA-AtTG with proved histological diagnosis and 1 AGA positive with negative histology. No statistically significant difference was observed between the groups with regard to age, duration of diabetes, familial target stature, and ratios Height/Age and Weight/Height. Presence of CD serological markers was related to a lower level of HbA1c. Prevalence of CD serological markers is important in this French cohort but lower than other countries.

Primary Sjögren's syndrome associated agranulocytosis: a benign disorder?

OBJECTIVE: To report on an uncommon association of agranulocytosis in primary Sjögren's syndrome (SS). METHODS: The clinical, haematological, and immunological features of seven patients with primary SS associated with a chronic (>6 months) agranulocytosis, and the outcome of the patients, were analysed. RESULTS: Patients were white women with an unexplained agranulocytosis. They all had non-erosive arthritis and three had a thrombocytopenia or Evan's syndrome. In three patients, transient or durable expansion of T lymphocytes was present in the peripheral blood or in the bone marrow, but evolved independently from neutrophil counts. There was no paroxysmal nocturnal haemoglobinuria clone or antibodies to neutrophil surface antigens. In vitro bone marrow culture was normal (four patients) or showed a decrease in colony forming unit-granulocyte monocyte (CFU-GM) and colony forming unit-erythroblast (CFU-E) (one patient). Serum levels of soluble Fas ligand (sFasL) were normal, and granulocyte-colony stimulating factor (G-CSF) concentrations were either normal or raised. One patient was treated with steroids associated with intravenous immunoglobulins and achieved a lasting response. Two other patients were treated with steroids and methotrexate, with poor efficacy. Short courses of subcutaneous G-CSF produced a transient and mild response in all three patients. Complete recovery of the neutrophils occurred temporarily during pregnancy in two patients. After a mean follow up of 34.8 months (range 6-139) all patients were alive and none developed serious infections. CONCLUSION: A subset of patients with primary SS and non-destructive arthritis may develop a chronic but well tolerated agranulocytosis that is usually poorly responsive to steroids and oral methotrexate.

Dexamethasone inhibits apoptosis of eosinophils isolated from hypereosinophilic patients.

Glucocorticoids reduce in vivo the number of eosinophils and are effective in treatment of blood and tissue hypereosinophilia. Dexamethasone is a synthetic glucocorticoid known to induce in vitro apoptosis of eosinophils of healthy donors, and apoptosis may be a mechanism induced by glucocorticoids to reduce eosinophilia. Here we confirm that dexamethasone exerts a pro-apoptotic effect on eosinophils isolated from healthy subjects but that dexamethasone is not always a pro-apoptotic agent towards eosinophils from hypereosinophilic patients. Implications of these results in the resistance of some patients to a treatment by glucocorticoids are discussed.

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line.

Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.

Activated Stat related transcription factors in acute leukemia.

Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and Stat5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and Stat5 proteins are described. The mechanisms of such activation remain to be clarified.

Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.

Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines.

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation.

IL-5, which is produced mainly by activated T cells and allergically triggered mast cells, is a major survival and differentiation factor for eosinophils, and therefore, is of relevance to diseases associated with this type of cell infiltration, most importantly asthma. In this study, we have examined the transcriptional regulation of human IL-5 in a mouse mast cell line, CPII, stimulated with IgE and Ag. We report that an inducible activity in the region between -177 and -80, and a constitutive activity between -80 and -70, in the promoter of the human gene, are both necessary for the allergically triggered activation. A computer-assisted search for transcription factor binding motifs revealed a nuclear factor of activated T cell (NF-AT) and a GATA consensus site in the two regions. Corresponding binding activities were detected to be present in nuclear extracts from the mouse mast cell line by defined NF-AT and GATA binding sites as probes for a gel shift analysis. Competition analysis, in combination with probes from the human IL-5 promoter, confirmed that these factors indeed bind to the consensus sequences identified by computer analysis. An oligonucleotide spanning the IL-5 NF-AT consensus site is shown to confer allergic stimulation to a basal IL-5 promoter only in conjunction with the GATA site downstream, indicating that an inducible NF-AT-like factor cooperates with a constitutive member of the GATA transcription factor family in mediating the allergic stimulation of the human IL-5 gene.