Structural And Functional Study Of Chimeric Anti-CD20Monoclonal Antibody

Non-Hodgkin’s Lymphoma (NHL) is one of the most common malignant tumororiginating from blood system, often occurs in middle-aged people. CD20antigen, aprotein that is expressed on more than90%of B-cell NHL cells, is considered to be anideal therapeutic target for monoclonal antibody medicines. The chimeric monoclonalantibody rituximab which targets against CD20, is the first monoclonal antibody approvedby FDA for the treatment of NHL. Numerous clinical practices have shown that rituximabhas significant therapeutic acticity on NHL. However, there are still some challenges needto be overcome: some patients were unresponsive to rituximab, some responsive patientsdeveloped resistance to further treatment. In this study, the National Engineering ResearchCenter of Antibody Medicine of China (NERCAM) designed and produced a variant ofrituximab (CMAB304) by optimizing the cell culture and manufacturing conditions.SDS-PAGE analysis indicated CMAB304has a similar molecule weight to rituximab, andfurthermore, these two proteins were demonstrated to share the same amino acid sequencesby LC/MS peptide mapping.In this study, we took rituximab as control, conducted a series of research onCMAB304. The detailed structures information between CMAB304and rituximab,including amino acid sequences and glycosylation profiles, were successfully characterizedat levels of intact protein subunits and free oligosaccharides using liquid chromatographyelectrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS).The combined data revealed that CMAB304has distinct glycosylation patterns fromrituximab. Furthermore, it is confirmed that CMAB304possesses much higher G0and G1glycan levels。This result suggested that CMAB304maybe has more powerful positiveeffect in stimulating the ADCC activity.In vitro biological analysis, CMAB304exhibited a higher FcγRIIIa（Fc gammareceptor IIIa, CD16a）binding affinity and ADCC activity than rituximab, but the sameCDC acticity as innovator, which further confirmed that CMAB304has a differentproportion of glycoslation.The sponsor-initiated phase I and IIa clinical trials of CMAB304were conducted toevaluate the clinical tolerability and preliminary response in accordance with the CommonToxicity Criteria version3(CTCAE v3.0) and WHO response evaluation criteria,respactively. Patient serum levels of CMAB304were tested using competition assays by flow cytometric analysis. The pharmacokinetic profile analysis, which was fitted bydifferent modeling methods using WinNonLin software, suggested that CMAB304andrituximab shared similar PK profiles. In clinical trials, intravenously infusion ofCMAB304was well-tolerated in NHL patients, indicating the glycan proportiondifferences in the Fc fragment did not lead to the PK behavior and tolerability changes.Clinical study showed that a rapid and significant suppression of CD20antigens wasobserved during treatment, as a median percentage change from81.4%to88.1%in thethree single-dose groups. No anti-CMAB304antibodies were detected during the wholestudy. The decreases in CD19+, CD20+were dose-dependent. Data of historical controlshowed that CMAB304exhibited a better therapeutic effect than rituximab.In conclusion, the lower fucosylated saccharide proportion of CMAB304canincreased the ADCC effect and, in addition, lead to a better treatment effect clinically,suggesting that CMAB304has the potential to become as a new drug candidate for NHLtreatment.