Construction, Expression And Characterization Of Anti-CD20 Chimeric Monoclonal Antibody And Discovery Of Interaction Protein Of CD20

Non-hodgkin's lymphoma is the most common hematologic cancer in adults, and one of top ten life-threatening malignancies. Some indolent or slow growing B-cell non-Hodgkin's lymphomas are incurable. In contrast, others which are aggressive or very aggressive and may be rapidly fatal are often curable. Until presently, no chemotherapeutic regimen provided a survival benefit. The anti-CD20 monoclonal antibody rituximab with better curative effect and less side-effect, first approved for clinical use in 1997, has changed the standard of care for many patients with NHL. The overall response rate was 48%, with 6% of the patients having complete remissions; better response rates correlated with fewer previous treatments. The median duration of the response was about one year.There are two reasons for the good therapeutic effect of rituximab listed as follow:On one hand, Rituximab is a chimeric monoclonal antibody composed of murine variable regions from the anti-CD20 antibody 2B8 that are linked to a human Fc component directed against CD20 on B cells. Therefore, Rituximab has lower immunogenicity than murine monoclonal antibody. Furthermore, this results in lower incidence rate of HAMA than murine monoclonal antibody. And chimeric monoclonal antibodies have longer half life than other micromolecule recombination antibodies in human body. Moreover, it is obvious that human Fc fragment has diverse immunological functions which involve in kill lymphoma.On the other hand, CD20 plays an important role in the mechanism of action of Rituximab. Approximately 85% of non-Hodgkin's lymphomas in adults are of B cell origin. CD20 is a 33-37 kD, non-glycosylated phosphoprotein expressed on the surface of normal B lymphocytes and about 95% of malignant B cells. This fact has lead that CD20-based immunotherapy have been successfully employed in the treatment, and that over one million patients accept Rituximab-based treatment world-wide with diseases such as Non-Hodgkins Lymphoma and various autoimmune disorders. Despite such clinical success and recent progress, researchers still remain largely ignorant of how anti-CD20 mAb operate in vivo. Expression of CD20 is restricted to the B-cell lineage from the pre-B-cell stage until terminal differentiation into plasma cells. Treatment with rituximab induces a pronounced, rapid and prolonged near-depletion of circulating B-cells. Circulating B-cells are replenished from bone marrow pro-B-cells within 4 to 12 months, sometimes longer. Interestingly, naive B-cells appear to recover faster than memory B-cells. Antibodies kill tumor cells through complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and induction of apoptosis. Despite the expression of antigens such as CD20, patients may not have a response to antibody therapy or resistance to the therapy may develop. Therefore, to clarity the foundation of CD20 is imperative to justify the mechanisms of resistance to anti-CD20 monoclonal antibodies potentially.Some researchers claimed that CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. They provide direct evidence of CD20 homo-oligomerization into tetramers. After activation, BCR-CD20 complexes dissociated and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. So they deem that CD20 is a"store-operated"calcium entry (SOCE). Controversy, other researches data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and"hijack"the signaling potential of the BCR. But these researches did not provide not only specify of CD20 function but also determine of interaction protein of CD20.This study means to construction, expression and characterization of two anti-CD20 chimeric monoclonal antibodies. The new genes of anti-CD20 chimeric monoclonal antibodies come from modification of another anti-CD20 chimeric antibody which has been preserved in our laboratory. At the moment, we try to find the native interaction protein of CD20 in order to explain some mechanisms of resistance to anti-CD20 monoclonal antibodies potentially, learn the foundation of CD20 and look for the new development way of anti-CD20 monoclonal antibodies. Methods and Results1. Construction of 2 anti-CD20 chimeric monoclonal antibody eukaryotic expression vector.According to the patents of anti-CD20 antibodies, the synthesis of new anti-CD20 chimeric monoclonal antibodies light chain and VH gene is amplified by overlap extension PCR. The templates are the plasmids which were sroryed by our laboratory. Then, light chain, VH and Fc fragment were cloned into pBudCE4.1 different multiple clone sites. Each eukaryotic expression vector has two open reading frames, and regular two gene expressions.2. Construction of various BCR various and EGFP fusion expression vectors.BCR is composed of Igα, Igβand sIgM (or sIgD). The sIgM is the surface IgM monomer. The extracellular regions of Igαand Igβare synthesized by PCR and the templates are human cDNA. IgM heavy chain is spliced into 3 kinds of regions which are CH1CH2, CH2CH3 and CH3CH4. These regions gene is amplified by polymerase chain reaction (PCR) from the human cDNA. And then these gene and EGFP gene was linked and cloned into a mammalian expression vector pcDNA3.1. These vectors called after Igα, Igβ, IgM1, IgM2 and IgM3.3. Purification of anti-CD20 antibody protein.After anti-CD20 antibody eukaryotic expression vectors transfection into CHO-K1 cell, the positive clones were obtained by the screening of zeocin antibiotics. Then, the positive clones which are increased expression clones detected by ELISA inoculate into roller bottles. Supernatant of roller bottles culture with serum free medium load in Protein A affinity chromatography column. And then the proteins can be eluted with a high concentration of the carbohydrate. The collected protein can be detection by SDS-PAGE by compared molecular weights of antibody proteins. After BCA assay, the purity of proteins can be calculated. SDS-PAGE assay show that 20R and 20S protein molecular weight are about 150 kD. And their concentrations are 120 mg/L and 77 mg/L.4. Anti-CD20-mediated apoptosis.Anti-CD20-mediated apoptosis assays fall into two different methods. (1) Apoptosis assay kits analysis of apoptosis.Briefly, 5 x103 to 5 x104 Raji, Ramos, and HL60 cells were resuspended in 100μL culture medium and plated into 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 24 hours with 20R 20S (density is 2μg/ml,5μg/ml,20μg/ml), the cells are added Annexin V-FITC and PI. The dyeing result can be observed with fluorescence microscope (wave length in 488nm). The result shows that 20R and 20S can both induct apoptosis of CD20+ cell and neither apoptosis of CD20- cells. And the effects of apoptosis present dose-respones relationship.(2) Flow cytometric analysis of apoptosis.Flow cytometric analysis of cellular DNA was performed following propidium iodide staining according to the method of Fried et al [5]. Briefly, after 3 x 106 Raji cells were incubated with 20R, 20S (density of 1μg/ml and 5μg/ml) 24 hours, the cells are washed in phosphate-buffered saline (PBS), and then are gently resuspended in 1mL PBS. Then PI is added into the cells. Samples were stored in the dark at 4°C until flow cytometric analysis of individual nuclei using a FACScan flow cytometer could be performed. The result show that 20R have better perform at induction of apoptosis of CD20+ cells.5. Anti-CD20-mediated CDC.First, 1 x105 Raji resuspended in 50μl culture medium and plated in 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 15 minutes with 20R 20S (density is 2μg/ml,5μg/ml,10μg/ml), the cells are added 10μl human blood serum. Then, about 30 minutes later, the cells are added 10μl CCK8. When the colors of negative control become orange red (means to OD450nm exceed 1.0), the wells should be reading at OD450nm. The result shows that the ability of 20R-mediated CDC as 66% as that of Rituximab. And ability of 20S-mediated CDC is much worse than 20R.6. Five kinds of Cells express the fusion protein.After fusion expression vectors transfecting 293 cells, the positive clones were obtained by the screening of G418. The protein expressions of these cells can be analyzed by Western Blotting assay.The results show that the lysis solutions of cells contain interest proteins. 7. GST-Pull DownAfter CD20-GST co-incubated with Igα, Igβ, IgM1, IgM2, IgM3, negative control and positive control two hours at 4℃, the mixed liquor can be added GST Bind Resin. Two hours later, wash resins five times with GST Bing Buffer. Then, samples are heated toghter loading buffer to 100℃for 5 minutes. At least, analyze sample by Western Blotting. The result shows that the interaction protein of CD20 is Igβand IgM. And the region of interaction of IgM is CH2 region.In sum, this study involves in construction two kind of anti-CD20 chimeric monoclonal antibodies. The two antibodies both can induct the apoptosis of CD20+ cells. And the effects of the assay and antibodies have dose-effect relationship. However, the effects of two antibodies mediated CDC are worse than Rituximab. And effect of 20R is about as 66% as Rituximab. This result illustrate that the region of mutation may be key to antibody affinity to CD20 or to maintain the constitution of anti-CD20 Fab'.To surprised, this study finds the native interaction protein of CD20, as demonstrates that the foundation of CD20 is relevant to BCR. And the new discovery may result in constructions of new anti-CD20 antibody. The method of design of anti-CD20 would be changed.