Loop Of CD20 Research Articles

Epitope Mapping of the Anti-CD20 Monoclonal Antibodies (C20Mab-11 and 2H7) Using HisMAP Method.

CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.

Target cell killing effects of CD20 targeting chimeric antigen receptor T cells derived from the type II anti-CD20 antibody.

e14548 Background: Chimeric Antigen Receptor T cells (CAR-Ts) targeting CD19 have shown very promising clinical outcomes in treatment of B-cell linage hematological malignancies. However, many patients with relapsed diseases were found to have down-regulated/loss of CD19 surface expression after CD19 CAR-T therapy. To solve this issue of CD19 single-targeting escape, we explored the application of another B-cell antigen, CD20, for targeted CAR-T therapy. Methods: We constructed four CD20 targeting CARs (all with 4-1BB co-stimulatory signaling) base on single-chain variable fragments (scFV) derived from four well-studied CD20 specific antibodies: Leu16, Rituximab, Obinutuzumab, and Ofatumumab. Leu16, Rituximab, and Obinutuzumab belong to the type I anti-CD20 antibody family and appear to bind to different epitopes located on the large loop of CD20, whereas Ofatumumab is the type II anti-CD20 antibody which has been shown to interact with the hydrophobic residues on the small loop surrounding a deep binding cleft. Results: All four CAR-T cells can specifically recognized CD20 positive target cells in our pre-clinical studies. They all showed up-regulated antigen-specific cell activation and high level of IFN-g release upon CD20 stimulation, and CAR-T20-Ofatumumab cells appeared to have significantly higher cell activation and more than 2-fold increase in IFN-g release compared to the other three CAR-T20 cells with their scFVs deriving from type I anti-CD20 antibodies. CAR-T20-Ofatumumab cells also showed higher degranulation upon stimulation, and it displayed ~50% of increase in ability to kill CD20 positive cells in cytotoxicity assays. Conclusions: Our data suggested that CAR-T20-Ofatumumab has better in vitro function and appears to be a CAR superior to those derived from other three antibodies. A possible explanation for this observation is that Ofatumumab interacts with the hydrophobic residues on the small loop, which is very close to cell membrane and confers more extensive binding to the small loop with striking slow off-rate. Our results suggest that CAR-Ts targeting CD20 with the scFVs from the type II anti-CD20 antibody may have superior cell killing effects.

Ofatumumab for newly diagnosed and relapsed/refractory chronic lymphocytic leukemia

Monoclonal antibodies have become an increasingly utilized treatment option for many hematological malignancies, including chronic lymphocytic leukemia (CLL). Ofatumumab is a second-generation fully human anti-CD20 monoclonal antibody that binds to the small extracellular loop of CD20, thereby producing complement-dependent cell lysis and antibody-mediated cell cytotoxicity in cells expressing CD20. Ofatumumab has shown efficacy in the treatment for relapsed or refractory CLL. This success has resulted in the recent US FDA approval of ofatumumab for the treatment of CLL in patients who are refractory to fludarabine and alemtuzumab-based regimens. Major side effects of ofatumumab include infusion reactions, neutropenia and increased risk for infection. This article provides an overview of the current data supporting the use of ofatumumab for CLL and projects the future role of ofatumumab as monotherapy and combination therapy.