Abstract
C-terminal mutations of CD20 constitute part of the mechanisms that resist rituximab therapy. Most CD20 having a C-terminal mutation was not recognized by L26 antibody. As the exact epitope of L26 has not been determined, expression and localization of mutated CD20 have not been completely elucidated. In this study, we revealed that the binding site of L26 monoclonal antibody is located in the C-terminal cytoplasmic region of CD20 molecule, which was often lost in mutated CD20 molecules. This indicates that it is difficult to distinguish the mutation of CD20 from under expression of the CD20 protein. To detect comprehensive CD20 molecules including the resistant mutants, we developed a novel monoclonal antibody that recognizes the N-terminal cytoplasm region of CD20 molecule. We screened L26-negative cases with our antibody and found several mutations. A rituximab-binding analysis using the cryopreserved specimen that mutation was identified in CD20 molecules indicated that the C-terminal region of CD20 undertakes a critical role in presentation of the large loop in which the rituximab-binding site locates. Thus, combination of antibodies of two kinds of epitope permits the identification of C-terminal CD20 mutations associated with irreversible resistance to rituximab and may help the decision of the treatment strategy.
Highlights
We reported that gene mutations of CD20 were somehow involved in resistance to rituximab therapy, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy.[1]
These results indicate that L26 antibody recognizes the C-terminal cytoplasm region of CD20 molecules and that its epitope is present in the amino-acid sequence of 264–281
We reported that mutations in the CD20 gene were found with some frequency in patients treated with rituximab
Summary
We reported that gene mutations of CD20 were somehow involved in resistance to rituximab therapy, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy.[1] Many of these C-terminal truncated CD20 molecules were not recognized by the L26 monoclonal antibody used routinely in most clinical laboratories. An immunohistochemical study using a polyclonal antibody showed that some kind of C-terminal truncated CD20 was present in cytoplasm, so it was possible that the epitope of L26 was lost by gene mutations.[1] L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported.[2,3] In this study, we determine a recognition site of L26 by using a series of deletion mutants of CD20 molecules. To detect every one of the mutated CD20 molecules, we developed new antibodies
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