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

Abstract

Several novel anti-CD20 monoclonal antibodies are currently in development with the aim of improving the treatment of B cell malignancies. Mutagenesis and epitope mapping studies have revealed differences between the CD20 epitopes recognized by these antibodies. Recently, X-ray crystallography studies confirmed that the Type I CD20 antibody rituximab and the Type II CD20 antibody obinutuzumab (GA101) differ fundamentally in their interaction with CD20 despite recognizing a partially overlapping epitope on CD20. The Type I CD20 antibodies rituximab and ofatumumab are known to bind to different epitopes. The differences suggest that the biological properties of these antibodies are not solely determined by their core epitope sequences, but also depend on other factors, such as the elbow hinge angle, the orientation of the bound antibody and differential effects mediated by the Fc region of the antibody. Taken together, these factors may explain differences in the preclinical properties and clinical efficacy of anti-CD20 antibodies.

Figure 1. (A) The structure and topology of CD20 and the epitopes recognized by rituximab, ofatumumab and GA101. (B) Sequence alignment of CD20 epitopes recognized by CD20 antibodies based on published information. Core epitope residues are boxed in light blue. For 2F2 (ofatumumab), core epitope assignment is based on published work from Teeling et al. 46. For residues labeled in blue experimental evidence suggests a role in 2F2 binding. For the other antibodies, the following coloring scheme has been applied based on Pepscan results and FACS binding data of amino acid exchange mutants: green = almost any exchange tolerated at this position; brown = non-conservative exchange tested and not tolerated at this position; orange = conservative exchange tested and tolerated at this position; red = also conservative exchanges not tolerated at this position; black = position has not yet been evaluated. Italic font indicates that Pepscan and FACS binding results are discordant. Since the FACS binding results better reflect the native protein context, the coloring in such instants was based on the FACS binding data.

Figure 2. Hypothetical model for the 2:1 binding ratio of Type I and Type II CD20 antibodies binding to CD20 (tetramers, depicted in red). An explanation to explain the 2:1 binding stoichiometry between Type I and Type II CD20 antibodies is to assume that A) Type I antibodies bind between CD20 tetramer (inter-tetramer, depicted in red) resulting in accumulation in lipid rafts together with FcγRIIb (gray oval). In contrast, as Type II antibodies may bind within one tetramer (intra-tetramer).

Figure 3. Hypothetical model for CD20 binding of Type I and Type II CD20 antibodies explaining the impact of FcγRIIb on internalization. A) Type I antibodies such as rituximab may bind to CD20 in a conformation that allows simultaneous binding to FcγRIIb and subsequent signaling followed by internalization in lipid rafts. B) Type II antibodies such as GA101 may bind in a conformation that does not allow simultaneous binding to FcγRIIb, thus resulting in reduced internalization.

Figure 4. Published crystal structures of CD20 antibodies. A) rituximab-CD20 complex,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.

Figure 5. Comparison of A) rituximab (Type I) and B) GA101 (Type II) crystal structures in complex with CD20 peptide.29 While for rituximab N171 is deeply immersed and N176 has no contacts with the rituximab CDRs, N171 is not deeply immersed in the the GA101 CDRs and vice versa N176 makes contacts to residues F52/D57/D59 of GA101 supporting the C-terminal shift of the GA101 epitope.

Figure 6. Three-dimensional models of A) rituximab and B) GA101. GA101 binds to the same binding epitope region of CD20 as rituximab, but in a different binding orientation. The molecular models were created by combining known structural data with the current knowledge and general understanding of antibody structure and membrane protein topology. The CD20 membrane protein model was created by combining the structural fragments of the crystallized CD20 antibody binding epitope and the transmembrane part of the HER2 receptor as a typical example of a membrane spanning molecule with known 3D information, and CD20 topology information.