Heterogeneity of the effector functions displayed by rituximab and other anti-CD20 monoclonal antibodies (mAbs) apparently recognizing the same CD20 epitope suggests that additional mechanisms, probably related to mAb fine specificity, are responsible for B-cell depletion. To improve our understanding of rituximab's function, its fine specificity was investigated by means of phage display peptide library (PDPL)-expressing 7-mer cyclic (c7c) or 7-/12-mer linear peptides. Rituximab-specific c7c PDPL-derived clone insert sequences expressed the motif A(S)NPS overlapping the human CD20 170 ANPS 173 . P 172 was the most critical for rituximab binding, since its replacement with S 172 (of mouse CD20) abolished the reactivity. The WPXWLE motif expressed by the linear PDPL-derived clone insert sequences could only be aligned to the reverse-oriented 161 WPXWLE 156 of acid sphingomyelinase-like phosphodiesterase 3b precursor (ASMLPD), though linear peptides bearing WPXWLE competed with cyclic ones for rituximab-paratope binding. Anti-CD20 mAb 1F5 only displayed a reactivity profile similar to that of rituximab, which also reacted with ASMLPD-derived peptides. Peptides induced antibodies with specificity and effector functions similar to those of rituximab. Our results show a unique fine specificity of rituximab, define the molecular basis for the lack of rituximab reactivity with mouse CD20 (mCD20), and the potential of targeting CD20 in an active immunotherapy setting. A possible rituximab interaction with ASMLPD is sug-

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An attractive, whether alternative or complementary, approach to passive immunotherapy (IT) with the anti-CD20 mAb rituximab for the treatment of autoimmune diseases is to stimulate the host to produce an anti-CD20 immune response by using peptides that mimic CD20 (mimotopes). The only mimotope reported to target CD20 antigen is a 43-mer polypeptide corresponding to the exposed domain of the molecule (from amino acid 142 to 184). Owing to its length, however, it failed to efficiently induce a CD20-specific response. A search has now been made for a smaller mimotope by biopanning a phage-display peptide library with rituximab. A total of 10 positive phage clones expressing six distinct sequences were isolated. Their alignment produced a motif that did not match any portion of the CD20 extracellular loop, whereas the motif bearing the 12-mer linear peptide Rp10-L specifically reacted with rituximab and inhibited its binding to CD20. Furthermore, in BALB/c mice Rp10-L-induced antibodies that reacted with the CD20(+) B lymphoid cell line Raji but not with the C20(-) T lymphoid cell line CEM. This peptide is currently being investigated to determine the effectiveness of CD20-based active IT for the treatment of autoimmune diseases.

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