The anti-CD20 mAb 2H7 was purchased from Alexis (Lausen, Switzerland).

The anti-CD20 mAbs B1 (Coulter, Hialeah, FL), 1F5 (ECACC, Salisbury, United Kingdom), LT20, 17 CAT13.6, 4 and AT80 16 were a kind gift from Dr M.S. The anti-CD20 rituximab (chimeric IgG1) and its isotype-matched TNFα-specific mAb infliximab were purchased from IDEC Pharmaceutical Corporation (San Diego, CA) and Centocor (Malvern, PA), respectively. Unless otherwise specified, all chemicals were purchased from BDH Chemicals (Poole, United Kingdom). 4Įlectrophoresis reagents were purchased from Bio-Rad Laboratories (Hercules, CA). 15 Support for this view is provided by the heterogeneity of the effector functions 16, 17 displayed by rituximab and other anti-CD20 mAbs (ie, mAb B1), which apparently recognize the same CD20 epitope. Even so, there is convincing evidence that these mechanisms do not completely explain the cytotoxic effect of rituximab and hence that other unidentified mechanisms, which probably depend on its fine specificity, may operate following mAb infusion. 7-9 Antibody-dependent cell-mediated cytotoxicity (ADCC), 10 complement-dependent cytotoxicity (CDC), 11 and induction of apoptosis 12, 13 have all been postulated as explanations of B-cell ablation in vivo 14 and the therapeutic effect of rituximab. A possible rituximab interaction with ASMLPD is suggested.ĬD20 sequence analysis predicts a 4-transmembrane domain with intracellular termini and only 1 extracellular 44-amino acid loop (from amino acid 142 to 182), which is the contact site of most anti-CD20 mAbs available, 4 including rituximab, which has proven effective for the treatment of lymphoma 5, 6 or even certain autoimmune diseases. 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. Peptides induced antibodies with specificity and effector functions similar to those of rituximab. Anti-CD20 mAb 1F5 only displayed a reactivity profile similar to that of rituximab, which also reacted with ASMLPD-derived peptides. The WPXWLE motif expressed by the linear PDPL-derived clone insert sequences could only be aligned to the reverse-oriented 161WPXWLE 156 of acid sphingomyelinase-like phosphodiesterase 3b precursor (ASMLPD), though linear peptides bearing WPXWLE competed with cyclic ones for rituximab-paratope binding. P 172 was the most critical for rituximab binding, since its replacement with S 172 (of mouse CD20) abolished the reactivity. Rituximab-specific c7c PDPL-derived clone insert sequences expressed the motif A(S)NPS overlapping the human CD20 170ANPS 173. 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. 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.

Serial cloner linear to circular