Interaction Of CD20 With BCR And Study Of Anti-B Lymphoma Antibody

CD20 molecule is a four transmembrane, non-glycosylated B-cell surface antigen, composed of 297 amino acid, molecular weight of 33 ~ 37kD. Except pro-B cells and plasma cells, CD20 molecules expressed on B-cells of all periods and are specific marker of the B cell surface. CD20 molecule is also expressed in the majority of non-Hodgkin's lymphoma cells, but stem cells, pro-B cells, normal plasma cells or other normal tissue cells do not express CD20 molecules. In addition, CD20 is not shed from the cell surface or internalized after antibody binding, so CD20 molecule is a very important target for the monoclonal antibodies treatment of B-cell lymphoma. Although the anti-CD20 monoclonal antibody Rituximab in the treatment of a variety of malignant B lymphoma has achieved great success, and is studied to be applied to other diseases, such as some autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, the biological function of CD20 antigen remains unclear. CD20's natural ligands are still uncertain.BCR(B cell receptor)is composed of cell surface immunoglobulin M (sIgM), Ig-αand Ig-β. BCR is a very important cell surface receptor complex, and in a small number of B cells, cell surface immunoglobulin D (sIgD) instead of sIgM. sIgM is noncovalently associated with heterodimer of Ig-αand Ig-β. sIgM is involved in the combination of BCR and antigens, while the heterodimers of Ig-αand Ig-βmainly as BCR signal transduction molecules. The intracellular regions of both Ig-αand Ig-βhave immune receptor tyrosine-based activation motif (ITAM), which can be combined with a variety of protein kinases. And phosphorylations of ITAM can propagate cell signals.When infection occurs, sIgM binds with the antigen, and Ig-αand Ig-βtranslate the antigen binding into intracellular signals to promote B-cell activation. In addition to activating B cells for invasion by pathogens, BCR play a very important role in the regulation of B-cell development, B-cell survival and elimination of B cells.Recent literatures show that CD20 molecule probably interacts with BCR. In this paper, using GST pull down assay and fluorescence resonance energy transfer(FRET), we have proved that which regions of CD20 and BCR probably interacts. At the same time, we find the relationships between the BCR and the CD20 are very complicated from some current literatures and not only the interaction between CD20 and sIgM can happen, and interactions between CD20 and Ig-β, sIgM and Ig-β, sIgM and Ig-αcan also occur. And even CD20 itself can form oligomers from self-interaction. For this reason, in this paper we construct three heavy-chain antibody-like eukaryotic expression vectors to transfect HEK293 cell line, expecting to obtain heavy-chain antibody-like molecular proteins, and then evaluate their activity of anti-B lymphoma.To achieve the above aims, we made the following work and get some conclusions:1. Construct the eukaryotic expression vectors of fusion protein of different parts of BCR and the EGFP. Then, we transfectted them into the HEK293 cells and successfully expressed five kinds of EGFP fusion protein:Ig-α(33-118AA)-EGFP, Ig-β(26-135AA)-EGFP, Cμ1Cμ2(169-366AA)-EGFP, Cμ2Cμ3(266-472AA)-EGFP and Cμ3Cμ4(383-586AA)-EGFP. We use GST-CD20(1-297AA), obtained from purchase, and five cell lysates of above transfectted cells for GST pull down assay. We found that full-length of CD20 molecule interact with Ig-β(26-135AA)-EGFP, Cμ1Cμ2(169-366AA)-EGFP and Cμ2Cμ3(266-472AA)-EGFP.2. Construct the donor and acceptor plasmids of FRET experiments, through the FRET experiments to further verify that there is interaction between CD20(140-186AA) and Cμ2Cμ3(266-472AA). And this is the interaction between both of their extracellular regions.3. Construct the prokaryotic expression vector of GST-Cμ2(266-382AA), and transform the vector into the BL21 E.coli cells to obtain the protein of GST-Cμ2(266-382AA). We use GST-Cμ2(266-382AA) and the cell lysate of CD20(140-186AA)-EYFP for GST pull down assay. We confirmed that CD20 molecule probably interacts with the single domain Cμ2.4. Construct the eukaryotic expression vectors of three heavy-chain antibody-like molecules: CD20(140-186AA)-CH1Fc, Ig-α(33-118AA)-CH1Fc, and Ig-β(26-135AA)-CH1Fc. By transfecting HEK293 cells,expressing and purifying, we have already obtained the protein of Ig-α(33-118AA)-CH1Fc. The expression and purification of other two kinds of heavy-chain antibody-like molecules is still in progress.The interaction of CD20 with BCR probably provides new ideas for the treatment of B lymphoma. We hope that three heavy-chain antibody-like molecules could locate on the surface of B lymphoma, and then use the Fc-induced effects of CDC and ADCC, as well as antibodies induced apoptosis to kill the B lymphoma cells, providing new methods for the treatment of B lymphoma.