METHODS AND RESULTS: Analysis of publicly available DLBCL microarray data sets showed that TRPM4 transcripts were up-regulated in DLBCL compared to normal germinal centre B (GCB) cells, were expressed more highly in the activated B cell-like DLBCL (ABC-DLBCL) subtype and higher TRPM4 transcripts conferred worse overall survival (OS) in R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL cases (P < 0.05). Our immunohistochemical analysis showed that TRPM4 was expressed in various human tissues but not in normal B cells within lymphoid tissues (reactive tonsil, lymph node and appendix). TRPM4 protein was present in 26% (n = 49 of 189) of our cohort of R-CHOP-treated DLBCL cases and this was associated significantly with more aggressive clinical parameters, including higher lactate dehydrogenase (LDH), Eastern Cooperative Oncology Group (ECOG) scores or stage (P < 0.01 for each of the parameters) and the ABC-DLBCL subtype (P = 0.016). TRPM4 positivity conferred significantly worse OS (P = 0.004) and progression-free survival (PFS) (P = 0.005). Worse OS remained associated significantly with TRPM4 positivity in multivariate analysis, including higher International Prognostic Index (IPI) or the non-GCB DLBCL phenotype (P < 0.05).
CONCLUSIONS: TRPM4 protein expression is up-regulated in DLBCL cases compared to non-malignant B cells with preferential expression in ABC-DLBCL cases, and it confers significantly poorer DLBCL patient outcomes.
Methods: The antibody binding pattern of the epitope was analyzed using bioinformatics tools. The IgG production in mice were examined by FACS Calibur™ Flow cytometer.
Results: The epitope bound the 72A1 monoclonal antibody at the same site as GP350/220 protein, indicating that the epitope should stimulate B cells to produce antibody. Moreover, in vivo administration of EBVepitope successfully induced IgG expression from B cells, compared with controls. Further investigation indicated that the relative number of B cells expressing IgE in EBVepitope-treated mice was lower than controls.
Conclusions: Our data suggest that this EBV GP350 epitope is able to induce IgG expression in vivo without causing allergic reactions, and represents a potential EBV vaccine candidate.
METHODS: From bioinformatics data, mismatched mouse amino acids in variable light and heavy chain amphipathic regions were identified and substituted with those common to human antibody framework. Appropriate synthetic DNA sequences inserted into vectors were transfected into HEK293 cells to produce the humanized antibody.
RESULTS: Humanized antibodies showed specific binding to CD20 and greater cytotoxicity to cancer WIL2-NS cell proliferation than rituximab in vitro.
CONCLUSION: A humanized version of rituximab with potential to be developed into a biobetter for treatment of B-cell disorders has been successfully generated using a logical and bioinformatics approach.