Anne-Laure Gagez

New anti-CD20 monoclonal antibodies: which is the best?

Rituximab (MabTh era â , Rituxan â ), a chimeric immunoglobulin G1 (IgG1) monoclonal antibody (Mab) targeting CD20, has dramatically improved the outcome of most B-cell lymphoproliferative disorders. Regarding the clinical effi cacy of this Mab, little is known however on how rituximab works in vivo , and it is speculated that rituximab induces both Fv-mediated (apoptosis) and Fc-mediated eff ects (antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and phagocytosis). A pioneering study [1] demonstrated that Fc γ RIIIa-158V/F polymorphism correlated with clinical response, patients with homozygous Fc γ RIIIa-158V showing a better response to rituximab in those with untreated follicular lymphoma. Similar results have been obtained with rituximab in other clinical indications [2,3], but also with other humanized IgG1 antibodies such as trastuzumab [4] and cetuximab [5]. Th ese studies underline the role of ADCC in the mechanism of action of these antibodies, but have also opened up a new area of development of therapeutic Mabs having increased affi nity for Fc γ RIIIa. Th is can be obtained either by mutation of amino acid residues of the Fc portion involved in Fc – Fc γ RIIIa interaction [6] (Fc-mutated Mab) or by modifi cation of the oligosaccharide located between the two Fc arms [7,8]. Th us, it has been demonstrated that a Mab with oligosaccharide containing a low fucose content (low-fucose Mab) or Fc-mutated Mab has an increased affi nity for Fc γ RIIIa, translating to higher ADCC in lymphoma cell lines. Very recently, a multicenter phase III trial demonstrated clearly that the fi rst low-fucose anti-CD20 Mab, obinutuzumab, signifi cantly increased the response rate (RR), molecular response and progressionfree survival compared to rituximab when associated with chlorambucil for unfi t patients with chronic lymphocytic leukemia [9]. Th ese results validate the clinical interest in Mabs with increased ADCC. However, obinutuzumab is also characterized by increased direct cytotoxic death and a lack of CDC compared to rituximab, in vitro . It is therefore diffi cult to discern the role of each mechanism (ADCC or direct cytotoxic death) in the clinical advantage observed in clinical trials. Ganjoo et al . report [10] a phase I/II study of ocaratuzumab (AME-133v) in patients with low affi nity Fc γ RIIIa with previously treated follicular lymphoma. Ocaratuzumab is the fi rst Fc-mutant Mab to be tested in a clinical trial. It is characterized by two amino acid changes introduced into its Fc portion [11]. Th ese amino acid changes led to increased ADCC in vitro (six-fold more potent that rituximab). Th ey

Obinutuzumab: a new class of anti-CD20 monoclonal antibody.

Purpose of review Obinutuzumab is a new anti-CD20 monoclonal antibody which demonstrated clinical superiority compared with rituximab in a recent phase III study. There is a need to better understand how this antibody differs from rituximab and why it could modify the landscape of the treatment of CD20+ malignancies in the near future. Recent findings Antibody-dependent cellular cytotoxicity plays a critical role in clinical activity of rituximab. To increase antibody-dependent cellular cytotoxicity, a strategy improving the affinity between the Fc portion of the antibody and Fc&ggr;RIIIa expressed by effector cells has been recently developed. This strategy modifies the carbohydrate located between the two Fc arms. Thus, the lack of fucose on IgG oligosaccharide improves binding to Fc&ggr;RIII and antibody-dependent cellular cytotoxicity. Obinutuzumab recognized a CD20 epitope different from that bound by rituximab. This property confers different features to obinutuzumab mechanisms of action with a noncaspase-dependent direct-cell death and the lack of complement-dependent cytotoxicity. Obinutuzumab demonstrated significant activity in animal models, and phase I or II studies showed clinical activity in different subtypes of CD20+ diseases. Summary Obinutuzumab, a type II glycoengineered monoclonal antibody, is characterized by an increased antibody-dependent cellular cytotoxicity and direct-cell death but no complement-dependent cytotoxicity. Recent clinical data demonstrated a superiority of obinutuzumab compared with rituximab, suggesting that this antibody should be, in the future, the backbone of the treatment of B-lymphoproliferative disorders.

Microwave-Assisted Extraction of Phycobiliproteins from Porphyridium purpureum.

In the present study, microwave-assisted extraction was first employed to extract the phycobiliproteins of Porphyridium purpureum (Pp). Freeze-dried Pp cells were subjected to microwave-assisted extraction (MAE) to extract phycoerythin (PE), phycocyanin (PC), and allophycocyanin (APC). MAE combined reproducibility and high extraction yields and allowed a 180- to 1,080-fold reduction of the extraction time compared to a conventional soaking process. The maximal PE extraction yield was obtained after 10-s MAE at 40xa0°C, and PE was thermally damaged at temperatures higher than 40xa0°C. In contrast, a flash irradiation for 10xa0s at 100xa0°C was the best process to efficiently extract PC and APC, as it combined a high temperature necessary to extract them from the thylakoid membrane to a short exposure to thermal denaturation. The extraction order of the three phycobiliproteins was coherent with the structure of Pp phycobilisomes. Moreover, the absorption and fluorescence properties of MAE extracted phycobiliproteins were stable for several months after the microwave treatment. Scanning electron microscopy indicated that MAE at 100xa0°C induced major changes in the Pp cell morphology, including fusion of the exopolysaccharidic cell walls and cytoplasmic membranes of adjacent cells. As a conclusion, MAE is a fast and high yield process efficient to extract and pre-purify phycobiliproteins, even from microalgae containing a thick exopolysaccharidic cell wall.

Antiproliferative Activity of Cyanophora paradoxa Pigments in Melanoma, Breast and Lung Cancer Cells

The glaucophyte Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg·mL−1. Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg·mL−1. Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, β-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and β-cryptoxanthin in melanoma cells.

Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster

AbstractnColistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC–MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4xa0kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41xa0kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Influence of FCGR3A-158V/F Genotype and Baseline CD20 Antigen Count on Target-Mediated Elimination of Rituximab in Patients with Chronic Lymphocytic Leukemia: A Study of FILO Group

Background and objectivesRituximab is an anti-CD20 monoclonal antibody approved in the first-line treatment of patients with chronic lymphocytic leukemia (CLL). Rituximab pharmacokinetics shows a time dependency possibly related to changes in the target antigen amount over time. The purpose of this study was to quantify the influence of both CD20 antigenic mass and the FcγRIIIA genetic polymorphism on rituximab pharmacokinetics in CLL.MethodsRituximab pharmacokinetics was described in 118 CLL patients using a semi-mechanistic model including a latent target antigen turnover, which allowed the estimation of rituximab target-mediated elimination in addition to the endogenous clearance.ResultsTarget-mediated elimination rate constant increased with the baseline CD20 count on circulating B cells (pxa0=xa00.00046) and in patients with the FCGR3A-158VV genotype (pxa0=xa00.0016). Physiologic elimination of antigen was lower in the Binet C disease stage (pxa0=xa00.00018). The effects of these covariates on rituximab concentrations were mainly visible at the beginning of treatment. Body surface area also increased central and peripheral volumes of distribution (pxa0=xa01.3xa0×xa010−5 and 0.0015, respectively).ConclusionsA pharmacokinetic model including target-mediated elimination accurately described rituximab concentrations in CLL and showed that rituximab ‘consumption’ (target-mediated elimination) increases with increasing baseline antigen count on circulating B cells and in FCGR3A-158VV patients.Clinical trial registration: NCT01370772.

miR-125b and miR-532-3p predict the efficiency of rituximab-mediated lymphodepletion in chronic lymphocytic leukemia patients. A French Innovative Leukemia Organization study

The underlying in vivo mechanisms of rituximab action remain incompletely understood in chronic lymphocytic leukemia. Recent data suggest that circulating micro-ribonucleic acids correlate with chronic lymphocytic leukemia progression and response to rituximab. Our study aimed at identifying circulating micro-ribonucleic acids that predict response to rituximab monotherapy in chronic lymphocytic leukemia patients. Using a hierarchical clustering of micro-ribonucleic acid expression profiles discriminating 10 untreated patients with low or high lymphocyte counts, we found 26 micro-ribonucleic acids significantly deregulated. Using individual real-time reverse transcription polymerase chain reaction, the expression levels of micro-ribonucleic acids representative of these two clusters were further validated in a larger cohort (n=61). MiR-125b and miR-532-3p were inversely correlated with rituximab-induced lymphodepletion (P=0.020 and P=0.001, respectively) and with the CD20 expression on CD19+ cells (P=0.0007 and P<0.0001, respectively). In silico analyses of genes putatively targeted by both micro-ribonucleic acids revealed a central role of the interleukin-10 pathway and CD20 (MS4A1) family members. Interestingly, both micro-ribonucleic acids were negatively correlated with MS4A1 expression, while they were positively correlated with MS4A3 and MSA47. Our results identify novel circulating predictive biomarkers for rituximab-mediated lymphodepletion efficacy in chronic lymphocytic leukemia, and suggest a novel molecular mechanism responsible for the rituximab mode of action that bridges miR-125b and miR-532-3p and CD20 family members. (clinicaltrials.gov Identifier: 01370772).

Identification and quantification of domoic acid by UHPLC/QTOF tandem mass spectrometry, with simultaneous identification of non-target photodegradation products

ABSTRACT Amnesic shellfish poisoning is a potentially lethal human toxic syndrome which is caused by domoic acid (DA), a neurotoxin produced by marine phytoplankton, principally from Pseudonitzschia genus. In this report, a method to identify and quantify the DA toxin, with simultaneous identification of its photodegradation products, has been developed. It uses an ultra high performance liquid chromatography coupled to a quadrupole-time-of-flight tandem mass spectrometer (UHPLC–QTOF) after solid-phase extraction (SPE). An unambiguous identification of DA was carried out by considering both the retention time of DA in UHPLC and the exact mass of protonated DA molecule ([M + H]+ = 312.1447 m/z) and of the most intense fragment ion (m/z 266.1391). The quantification was conducted using protonated DA molecule with protonated Glafenin as internal standard, obtaining a limits of detection of 0.75 µg L−1. Large screening with UHPLC–QTOF could also give structural information about degradation products of DA present in samples after UV-irradiation. This method was applied for the determination of DA in complex liquid samples after SPE and is applicable for environmental monitoring of this toxic substance in the aquatic environment.

Emergence and evolution of TP53 mutations are a key feature of disease progression in myelodysplastic patients with lower-risk del(5q) treated with lenalidomide.

The cytogenetic anomaly of isolated del(5q) characterizes a subgroup of patients with lower-risk myelodysplastic syndrome (MDS). In the 2016 World Health Organization classification, one additional karyotypic abnormality (except −7 or del7q) can be present in such patients.[1][1] Median survival

TMEM187-IRAK1 Polymorphisms Associated with Rheumatoid Arthritis Susceptibility in Tunisian and French Female Populations: Influence of Geographic Origin

Polymorphisms have been identified in the Xq28 locus as risk loci for rheumatoid arthritis (RA). Here, we investigated the association between three polymorphisms in the Xq28 region containing TMEM187 and IRAK1 (rs13397, rs1059703, and rs1059702) in two unstudied populations: Tunisian and French. The rs13397 G and rs1059703 T major alleles were significantly increased in RA patients (n = 408) compared with age-matched controls (n = 471) in both Tunisian and French women. These results were confirmed by a meta-analysis replication study including two independent Greek and Korean cohorts. The rs1059702 C major allele was significantly associated with RA, only with French women. In the French population, the GTC haplotype displayed a protective effect against RA, while the ATC, GCC, and GTT haplotypes conferred significant risk for RA. No association for these haplotypes was found in the Tunisian population. Our results replicated for the first time the association of the three Xq28 polymorphisms with RA risk in Tunisian and French populations and suggested that RA susceptibility is associated with TMEM187-IRAK1 polymorphisms in women. Our data further support the involvement of X chromosome in RA susceptibility and evidence ethnicities differences that might be explained by differences in the frequencies of SE HLA-DRB1 alleles between both populations.