Structural Basis for Recognition of CD20 by Therapeutic Antibody Rituximab

Abstract

Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-Å resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro 172 ). The 170 ANPS 173 motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity.CD20 is a pan-B cell marker expressed from pre-B cells until B cells are differentiated into plasma cells (1). It is a tetraspan membrane protein that is predicted to contain a large extracellular loop (about residues 142 to 182) and to form oligomers on the cell surface (2)(3)(4). Although the precise function of CD20 remains unclear, biochemical and cell biological data have shown that it seems to form or regulate a voltage-independent calcium channel (3,5). Despite the limited knowledge about its function, several lines of evidence have clearly demonstrated that CD20 is an ideal target for passive immunotherapy of B-cell lymphoma: it is highly expressed in more than 80% of the B-cell lymphomas but not in stem cells, pro-B cells, normal plasma cells, or other normal tissues; it remains on the cell surface without substantial internalization after cross-linking with antibodies; and it is not shed to the circulation to inhibit the antibody therapy (6 -8).The CD20-targeted chimeric monoclonal antibody (mAb)3Rituximab (Rituxan, IDEC-C2B8) was the first Food and Drug Administration approved mAb drug for the treatment of malignancy. Although it was originally used for treating low-grade non-Hodgkin lymphoma, Rituximab has been proven to be also effective against other types of lymphomas (9, 10) and some autoimmune diseases (11-13). Multiple mechanisms have been proposed for the action of Rituximab in the depletion of B cells including its ability to mediate complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, and to induce cell apoptosis (reviewed in Refs. 14 and 15). With the expansion of the clinical application of Rituximab in the treatment of lymphoproliferative diseases, it has been noticed that intensity of CD20 expression on B cells varies in patients that may affect the binding and efficacy of Rituximab therapy (16,17). Therefore, it is...

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties

“…Rituximab is a Type I chimeric (human-mouse) immunoglobulin (Ig)G1 anti-CD20 antibody. The CD20 epitope recognized by rituximab and other mouse-derived antibodies spans amino acid residues 168-175 of the CD20 protein, with the ANPS motif at residues 170-173 on the large extracellular loop appearing to be of critical importance 29,33,48,50,53 (Fig. 1B).…”

“…the conformational stability of the epitope-antibody complex. 50 The 182 YCYSI 186 region at the C-terminus of the large extracellular loop of CD20 also appears to play a role in rituximab binding, 49 most likely through the formation of the disulfide bond that induces the cyclic conformation of the epitope 50 loop necessary for the binding of CD20 to rituximab. 55 Abrogating the internal disulfide bridge (C167-C183) of the large extracellular sequences 53 where P172 was found to have a particular importance, since rituximab binds the human ANPS sequence but not the corresponding murine SNSS sequence.…”

“…The structure of the rituximab:epitope complex has been determined by co-crystallizing a synthetic peptide mimic of the extracellular loop epitope of CD20 (residues 163-187) in complex with the antigen-binding fragment of rituximab. 50 The bound CD20 peptide forms a cyclic conformation owing to a disulfide bond between two cysteine residues, C167 and C183. This structure comprises a short N-terminal coil (residues 167-171), a 3 10 helix (residues 172-174), a small loop (residues 175-177) and a short C-terminal α-helix (residues 178 -184).…”

“…AME-133v (Ocaratuzumab, LY2469298, MENTRIK) is a humanized IgG1 Type I antibody in Phase 2 development. AME-133v is an optimized version of rituximab 48 (B) ofatumumab (no co-crystal structure is available), 50 (C) 2H7-CD20 complex, 49 and (D) GA101-CD20 complex. 29 The heavy chain is colored in darker shades, the peptides derived from CD20 are colored in red where appropriate.…”

Is it the time to offer rituximab as a cost-benefit treatment for immunoglobulin A nephropathy? A short-review to current concepts

“…B cell depletion potentially offers a new therapy (5). Rituximab (RTX) is a monoclonal antibody that specifically recognizes a large extracellular loop in CD20 molecule of B cells (6,7) and leads to significant depletion of these cells (8,9). This is FDA approved for non-Hodgkin's B cell lymphomas (10).…”