The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20

Abstract

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

“…Generally speaking, antibodies that bind to different epitopes on the same protein may have different cytotoxic capabilities (e.g., anti-CD20 mAbs). 40 However, our results revealed that cetuximab and Pan exhibited identical ADCC activity in the luciferase reporter assay despite binding to different epitopes on EGFR.…”

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

“…Interestingly, when the sCAR hinge was shortened to the 12-aa IgG4m hinge, the trend for OFA switches was reversed and C-terminal PNE switches afforded better activity than N-terminal switches. Although the structure of CD20 antibody is also unknown, the OFA antibody is reported to bind a small, membrane proximal loop of the multipass transmembrane CD20 antigen (31). In this case, it is likely that the distance created by the N-terminal OFA switches with the IgG4m sCAR is too short to enable efficient synapse formation between the sCAR and CD20.…”

“…Along with CD19, CD20 is an important pan-B-cell marker and the most widely targeted B-cell antigen in the clinic. To develop the anti-CD20 switch, the OFA antibody was chosen because it binds a membrane proximal epitope (31), which might lead to enhanced activity because of the close association of the T cell and cancer cell. Switches were generated in a fashion similar to the CD19 switches.…”

Preclinical Characterization of AMG 330, a CD3/CD33-Bispecific T-Cell–Engaging Antibody with Potential for Treatment of Acute Myelogenous Leukemia

“…The scFv was incubated with the peptides and specific binding detected using an anti-hexahistidine antibody. Binding signals were measured in a 384-well ELISA reader (19,20).…”