Generation of biologically active linear and cyclic peptides has revealed a unique fine specificity of rituximab and its possible cross-reactivity with acid sphingomyelinase-like phosphodiesterase 3b precursor

Abstract

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-

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

“…49 Furthermore, when Perosa and colleagues screened phage-display peptide libraries containing a repertoire of sequences of random 7-or 12-amino acid peptides they found that, while cyclic peptides mimicking the CD20 epitope were dependent on the 170 ANPS 173 motif, linear mimics that also bound rituximab required a different motif-WPxWLE-that does not correspond to any sequence present in CD20 itself. 53,54 While the WPxWLE motif appears to share some rituximab contact points with 170 ANPS 173 , these regions are conformationally different and have been proposed as distinct epitopes. 54 However, the functional role and significance of the WPxWLE sequence is unclear.…”

“…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).…”

“…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. 53 Furthermore, mutation of the alanine and proline at positions 170 and 172 in human CD20 to serine was shown to abolish rituximab binding.…”

“…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. 53 Furthermore, mutation of the alanine and proline at positions 170 and 172 in human CD20 to serine was shown to abolish rituximab binding. 33,48 Asparagine 171 (N171) was also found to be a key residue for rituximab binding as any amino acid replacement at this position, except histidine, resulted in a substantial loss of binding affinity to peptides representing the extracellular CD20 loop.…”

Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

“…12 Rituximab, a chimeric antihuman CD20 monoclonal antibody, unexpectedly cross-reacted with this protein, and rituximab treatment prevented the degradation of SMPDL-3b and thereby averted the development of posttransplant proteinuria in patients with FSGS. 12,13 In this study, we examined whether rituximab inhibited the in vitro disruption of pig podocytes in an SMPDL-3b-dependent manner and evaluated its in vivo effect on proteinuria following preclinical pig-to-baboon xenogeneic GalTKO kindey transplant. We confirmed that (1) SMPDL-3b was expressed on pig kidneys; (2) rituximab bound to pig SMPDL-3b; (3) in vitro treatment of GalTKO pig podocytes with rituximab mitigated the damage caused by baboon anti-pig serum antibodies; and (4) rituximab administration in the peri-transplant period in vivo resulted in a marked delay in the development of proteinuria.…”