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.

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.

Characterization of a receptor for interleukin 5 on human eosinophils: variable expression and induction by granulocyte/macrophage colony-stimulating factor.

Interleukin 5 (IL-5) acts on eosinophil differentiation and activation, suggesting the existence of a membrane receptor for IL-5 on eosinophils. Here, we report that 125I-labeled recombinant human IL-5 bound, at 4 degrees C, to high affinity receptors on human eosinophils. The association constant was higher for hypodense eosinophils (1.93 x 10(9) M-1) than for normodense cells (0.39 x 10(9) M-1), with a closely related number of receptor sites per cell. No specific binding occurred on neutrophils. The specific binding of IL-5 was induced by overnight incubation at 37 degrees C of human eosinophils with granulocyte/macrophage (GM)-CSF. The levels of increase were significantly higher for normodense than for hypodense eosinophils, suggesting a previous in vivo activation of the later subpopulation by GM-CSF. IL-3 was ineffective by itself but synergistically enhanced the effect of GM-CSF. Specificity studies showed that the binding of 125I-labeled IL-5 was inhibited by IL-5, but not by other cytokines, on human eosinophils. These results show the existence of a specific binding site for IL-5 on human eosinophils with a variable affinity on eosinophil hypodense or normodense subpopulations, as previously reported for other membrane receptors.

Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/epsilon BP) of the S-type lectin family: role in IgE-dependent activation.

It has been suggested that neutrophils may be involved in the late-phase reaction of immunoglobulin E (IgE)-dependent hypersensitivity states. However, the identity of neutrophil-associated molecules inducing the release of mediators remains unclear. In this report, we demonstrate that human neutrophils from normal donors or from patients with inflammatory disorders could bind myeloma IgE proteins, especially after desialylation. Northern blot, immunoprecipitation, and flow cytometry analyses revealed that neutrophils did not express Fc epsilon RII/CD23, but rather Mac-2/epsilon binding protein (BP), belonging to the S-type lectin family. Similarly to IgA used as positive control, myeloma IgE proteins, as well as polyclonal IgE antibodies with or without antibody specificity, were both capable of inducing a neutrophil respiratory burst. Anti-Mac-2 but not anti-CD23 mAb strongly decreased the IgE-dependent activation of neutrophils, induced either by the specific antigen or by anti-IgE antibodies. These findings open new perspectives on the functional role of neutrophils in IgE-associated diseases including allergic states or parasitic infections.

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.

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.

Eosinophil IgE receptor and CD23.

In the present review, eosinophil Fc epsilon RII was compared to CD23, a differentiation marker of B cells. Biochemical analysis revealed that molecules of similar molecular weight were immunoprecipitated from eosinophils and B cells by an anti-CD23 monoclonal antibody (mAb) or by BB10, and anti-eosinophil Fc epsilon RII. By flow cytometry, a correlation was found between the binding of anti-CD23 mAb and myeloma IgE. However, a low expression of different epitopes of CD23 was observed in various hypereosinophilic patients. Northern blot analysis of eosinophil RNA with the cDNA probe of CD23 revealed a weak message in only 3 of the 6 patients expressing membrane CD23. The inhibition by anti-CD23 mAbs of IgE-mediated cytotoxicity and IgE binding to eosinophils clearly indicated the participation of CD23 or a related molecule in IgE-dependent eosinophil functions. However, the differential effects of anti-CD23 mAbs on eosinophils and B cells suggest major differences in the characteristics of the molecule expressed by eosinophils and by B cells.

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.

The interleukin 2 receptor in the hypereosinophilic syndrome.

The hypereosinophilic syndrome (HES) has been previously described as a clinicobiological entity characterized by a blood eosinophil count of over 1.5 x 10(9)/L of unknown cause associated with several clinical complications. In reality, HES is a heterogeneous group of diseases with variable and unpredictable progress in visceral lesions, thought to be related to the deleterious effects of tissue eosinophil infiltration. Various criteria for discrimination between benign and severe forms of HES have been described. These previous retrospective clinical investigations, using biological and clinical markers, have defined different stages of HES. It appears more relevant, however, to consider elements of disease activity by studying mechanisms of induction of persistent hypereosinophilia. The T-cell dependence of blood eosinophilia has led us to evaluate various markers of T-cell activation in particular. In the present review, we report previous results and perspectives suggested by the study of the interleukin 2 receptor in HES.

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.

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.

[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.

[Visceral lesions in hypereosinophilia]. / Lésions viscérales des hyperéosinophilies.

The hypereosinophilic syndrome is an ill-defined nosological entity with predominant risks of cardiac and/or neurological lesions. In the light of new data on the effector cytotoxic effects of eosinophils, we have tried to establish new criteria of severity by purifying the circulating eosinophils of 14 patients with hypereosinophilic syndrome and testing their toxicity. This study has revealed the existence of low density ("hypodense") eosinophils with potential cytotoxicity in vitro. The worst clinical forms of the syndrome were observed in the group of patients with positive eosinophil toxicity tests. The significance of these cellular changes (hypodensity, eosinotoxicity) and their relationship with the clinical manifestations are discussed.

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.

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.