Pathway Signal Of Type Ⅱ Anti-CD20 Antibody Induced Cell Death In B-cell Lyphoma

BACKGROUD AND OBJECTIVENon-Hodgkin’s lymphoma (NHL) is among the hematological malignancies with high prevalence worldwide, causing estimated 385 741 new cases and 199 630 deaths in 2012. The disease, ranks 8th and 11th among the most common cancers in men and women respectively, contributing 5.1% of all cancer cases and 2.7% of all cancer deaths.According to the GLOBOCAN 2012 data published by the IARC (International Agency for Research on Cancer), is the most important cause of death in developed countries; while second most important cause of death in developing countries and is responsible for about 8.3 million deaths in 2012, world-wide. NHL is more frequent in developed areas, and are also increasing in certain developing countries such as Thailand and China. About 80% to 90% NHLs are of B-cell origin, while rest are of T-cell origin.CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target formonoclonal antibodies (mAb), such as rituximab and ofatumumab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows mAb to persist on the cell surface for extended periods and deliver sustained immunological attack from complement and FcR-expressing innate effectors, particularly macrophages. CD20 can also generate transmembrane signals when engaged by certain mAb which, although unproven, might provide an important element of the therapeutic success of anti-CD20 mAb. These favorable characteristics have led to anti-CD20 mAb being developed and exploited for use in immunotherapy, where they have proven remarkably efficacious in both the treatment of malignant disease and autoimmune disorders by deleting malignant or normal B cells, respectively.Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of apoptosis. The relative contributions of these different mechanisms of action are still a matter of debate. Based on the ability to redistribute CD20 into lipid raft the anti-CD20 mAbs can be simply divided into two groups:type Ⅰ mAbs (Rituximab and most anti-CD20 antibodies) are able to efficiently shift CD20 complex into raft and mediate CDC but is relatively ineffectual at inducing cell apoptosis, type Ⅱ(B1) canNot promote CD20 segregation nor cause CDC effect but is more effective to trigger cell apoptosis. Both types of mAb are equally potent in ADCC with FcR-bearing myeloid effectors.Monoclonal antibody (mAb) therapy is of growing interest due to their importance in treating malignancies and certain autoimmune diseases. To date, more than ten anticancer mAb drugs have been approved by the Food and Drug Administration (FDA), USA. Rituximab (IDEC C2B8) is the first approved mAb drug for use in relapsed or refractory low-grade or follicular B-cell non-Hodgkin’s lymphoma (NHL) in 1997 which targets CD20, a pan-B cell marker. The great success of Rituximab has encouraged extensive efforts to search for new anti-CD20 mAbs or to improve the existing ones. Despite rituximab has been widely used in the treatment of lymphoma, only 48% of patients respond to the treatment and complete responses are less than 10%. There is an urgent need to develop more effective CD20-targeting antibody agents for the treatment of B-cell lymphomas.In this article, we explore and discuss the mechanisms of killing by anti-CD20 monoclonal antibodies. These antibodies may be a proming agent for the treatment of B-cell lymphoma. Moreover, our study may provide a new idea for the design of better antibody drug.METHODS1) Flow cytometry was applied to identify cell death in vitro by using dye of cell death stained with the different concentrations treatment of antibodies. And observe the cell dead form with different antibodies treatment by transmission electron microscopy, identify the morphology of cell death.2) Westen Bolt was applied to test the crucail protein in cell death pathways to indentify or exclude the signaling pathway induced cell death. Confocal was applied to observe the protein released and determine the role of each protein wether assosciated to cell death by acting inhbitors.3) Co-IP, Western Blotting and immunofluorescence confocal were applied to test the protein of CD20 complex in cells with different anti-CD20 antibodies treatment in Vitro. Block the signal of cell membrane receptor was applied to find whether CD20 and other cell membrane receptor are synergies to activte downstram pathway. SiRNA was applied to down-regulate expression of crucial protein, and then flow cytometry was applied to test the cell death of low expression to find how the protein modulates cell death.4) Immunofluorescence confocal and flow cytometry were applied to test lysosome associate protein release to find how the signal passed to regulate the cell death. Different inhibitors was applied to inhibit the series of protein expression, to find what lead to lysosome release and induce cell death.RESULTS1) Type Ⅱ anti-CD20 antibody-induced cell death was necrosis:type Ⅰ anti-CD20 antibody Rituximab was applied to be as a control, type Ⅱ anti-CD20 antibody 11B8 and mutant antibodies HY102K was applied to be as object, to found that type Ⅱ anti-CD20 antibody induced notapoptosis. And even in the case of cross-linking, type Ⅱ anti-CD20 antibody was was applied to induce cell death was significantly stronger than type Ⅰ anti-CD20 antibody. The cell death was concentration-related.2) Type Ⅱ anti-CD20 antibody-induced cell death was Caspase-indepandent cell death and lysosome dependent. Westen-blot was appliy to test the crucial protein of caspase singnal way. We found that type Ⅱ anti-CD20 antibody induced the cell death is a Caspase-independent pathway. Then Confocal was appliy to lysosome release during the cell death. And the downsteam protein kinea CathepsinB was detective by confocal. This cell death can be inhibited by CathepsinB inhibitors.3) CD20 interact with TNFR and activate downstream pathways:Co-IP was appliy to anlysis variety of proteins contained by CD20-associated complex. The silver staining and westen-blot were used to identified whether TNFR natural binding with CD20, and activate downstream signaling, causing the cell death. This study sugguset this cell death is a programed necrosis called necroptosis. Use the inhibitor of necroptosis necosistain can significantly inhibit cell death. TNFR blocking agent also can inhibite antibody-induced cell death. Co-immunoprecipitation (Co-IP) was applied to found a variety of necroptosis associated protein, and indentified by westen-blot. RIP1 combined with RIP3 were crucial for necroptosis. We found only type Ⅱ anti-CD20 antibody could activate programmed necrosis (necroptosis) pathway. After using lentiviral system down-regulation the expression of RIP1, necroptosis is inhibited.4) Ceramide changes downstream pathway:We have already known, the cell death caused by lysosome broken and release CathepsinB. Applied to series inhibitor tested, it suggest the activatation of sphingolipid-delta-4-desaturase by FADS7 led D-erythro-sphinganie turn to Derythro-sphgosine, trigger Ceramide activation, then made the lysosome broken release CathepsinB, inducing cell death. This result has verified by Confocal.CONCLUSIONWe found that the cell death caused by type Ⅱ anti-CD20 antibody is a program cell death apoptosis rather than necrosis. Such Caspase-independent pathway, lysosome-dependent cell death. After the type Ⅱ anti-CD20 antibody binding with CD20, a synergy singnal was triggered by with TNFR, activate downstream signal pathways, inducing cell death. On the other hind, the downstream signal point out, ubiquitination of FADS7 activate sphingolipid-delta-4-desaturase, led D-erythro-sphinganie turn to Derythro-sphgosine, trigger Ceramide activation, then made the lysosome broken release CathepsinB, inducing cell death. This data suggest the mechanism of type Ⅱ anti-CD20 antibody inducing cell death was a programed necrosis, necroptosis. It is the first time CD20 and TNFR was set as a model to pass a synergy signal inducing cell death. And it is also the first time to reveal the mechism of type Ⅱ anti-CD20 antibody inducing cell death. This will appliy theoretical basis to clinical antibody treatment of lymphoma, and explain its signaling pathway. We hope this signal pathway could apply some new ideas for the future antibody design and a better treatment of antibody or drugs combination with antibody for B-cell lymphoma.