A new approach to comparing anti-CD20 antibodies: importance of the lipid rafts in their lytic efficiency

The view that B lymphocytes are pathogenic in diverse pathological settings is supported by the efficacy of B-cell-ablative therapy in lymphoproliferative disorders, autoimmune diseases and graft rejection. Anti-B-cell antibodies (Abs) directed against CD20 have therefore been generated, and of these, rituximab was the first anti-CD20 monoclonal Ab (mAb) to be applied. Rituximab-mediated apoptosis, complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity differ from one disease to another, and, for the same disease, from one patient to another. This knowledge has prompted the development of new anti-CD20 mAbs in the hope of improving B-cell depletion. The inclusion of CD20/anti-CD20 complexes in large lipid rafts (LRs) enhances the results of some, but not all, anti-CD20 mAbs, and it may be possible to include smaller LRs. Lipid contents of membrane may be abnormal in malignant B-cells, and could explain resistance to treatment. The function of these mAbs and the importance of LRs warrant further investigation. A detailed understanding of them will increase results for B-cell depletion in lymphoproliferative diseases

Therapeutic activity of two xanthones in a xenograft murine model of human chronic lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>We previously reported that allanxanthone C and macluraxanthone, two xanthones purified from <it>Guttiferae </it>trees, display <it>in vitro </it>antiproliferative and proapoptotic activities in leukemic cells from chronic lymphocytic leukemia (CLL) and leukemia B cell lines.</p> <p>Results</p> <p>Here, we investigated the <it>in vivo </it>therapeutic effects of the two xanthones in a xenograft murine model of human CLL, developed by engrafting CD5-transfected chronic leukemia B cells into SCID mice. Treatment of the animals with five daily injections of either allanxanthone C or macluraxanthone resulted in a significant prolongation of their survival as compared to control animals injected with the solvent alone (<it>p </it>= 0.0006 and <it>p </it>= 0.0141, respectively). The same treatment of mice which were not xenografted induced no mortality.</p> <p>Conclusion</p> <p>These data show for the first time the <it>in vivo </it>antileukemic activities of two plant-derived xanthones, and confirm their potential interest for CLL therapy.</p

Floridoside Extracted from the Red Alga Mastocarpus stellatus Is a Potent Activator of the Classical Complement Pathway

Many biological properties of algae have been found to have useful applications in human health, particularly in the fields of oncology and immunology. Floridoside, extracted from the red alga Mastocarpus stellatus, has a structure similar to the xenoantigen Gal alpha 1–3 Gal. This xenoantigen has been described to induce a high immune response in human xenografts and is mediated by natural anti-gal antibodies that activate the classical complement pathway. Based on this property, we analyzed the potential activities of floridoside on the immune system. We demonstrated that floridoside activates a complement cascade via the classical complement pathway, through the recruitment and activation of natural IgM. This algal molecule could represent an important step in the development of a potent new anticomplementary agent for use in therapeutic complement depletion

Inhibition of NF-κB-mediated signaling by the cyclin-dependent kinase inhibitor CR8 overcomes pro-survival stimuli to induce apoptosis in chronic lymphocytic leukemia cells

Purpose: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. Experimental Design: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry–based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. Results: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. Conclusions: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL

HLA-DR expression in melanoma: from misleading therapeutic target to potential immunotherapy biomarker

Since the advent of anti-PD1 immune checkpoint inhibitor (ICI) immunotherapy, cutaneous melanoma has undergone a true revolution with prolonged survival, as available 5-year updates for progression-free survival and overall survival demonstrate a durable clinical benefit for melanoma patients receiving ICI. However, almost half of patients fail to respond to treatment, or relapse sooner or later after the initial response to therapy. Little is known about the reasons for these failures. The identification of biomarkers seems necessary to better understand this resistance. Among these biomarkers, HLA-DR, a component of MHC II and abnormally expressed in certain tumor types including melanoma for unknown reasons, seems to be an interesting marker. The aim of this review, prepared by an interdisciplinary group of experts, is to take stock of the current literature on the potential interest of HLA-DR expression in melanoma as a predictive biomarker of ICI outcome

Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Abstract Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody\u2019s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt\u2019s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal\u3b1(1\u21923)Gal\u3b2(1\u21924)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the \u3b1-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab- Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt\u2019s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia

<p>Abstract</p> <p>Background</p> <p>The t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of <it>ETV6 </it>(<it>TEL</it>) and <it>RUNX1 </it>(<it>AML1</it>) genes and defines a relatively uniform category, although only some patients suffer very late relapse. <it>TEL/AML1</it>-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.</p> <p>Results</p> <p>We compared the leukemia cell gene expression profiles of 16 <it>TEL/AML1</it>-positive ALL patients to those of 44 <it>TEL/AML1</it>-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -<it>RUNX1, TCFL5, TNFRSF7, CBFA2T3</it>, <it>CD9</it>, <it>SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7</it>, <it>SEMA6A, CTGF, LSP1, TFPI </it>– highlighting the biology of the <it>TEL/AML1 </it>sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of <it>RUNX1 (AML1) </it>was further investigated and in one third of the patients correlated with cytogenetic findings.</p> <p>Conclusion</p> <p>Gene expression analyses of leukemia cells from 60 children with <it>TEL/AML1</it>-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the <it>TEL/AML1</it>-positive ALL sub-group.</p