Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR‐H1‐derived peptide CB1¹

Abstract

A systematic exploration of the V H 2/V U 12^13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest. ß

A Cell-penetrating Antibody Fragment against HIV-1 Rev Has High Antiviral Activity

“…Monoclonal antibodies and their derivatives, such as antigen binding fragments (Fab) 2 and the corresponding complementarity determining region (CDR) peptides, have been described as plausible agents for inhibiting HIV-1. These have been directed either against gp120, the envelope glycoprotein, or more commonly, the CD4 receptor (2)(3)(4)(5)(6)(7)(8). All have shown promise.…”

Mapping the Paratope of Anti-CD4 Recombinant Fab 13B8.2 by Combining Parallel Peptide Synthesis and Site-directed Mutagenesis

“…Reagents, Cell Lines, and Vectors-CD4-inserted recombinant baculovirus was constructed and further used for the production of recombinant human soluble CD4 as described (8,13). For Spot analysis, recombinant human CD4 (Repligen Inc., Needham, MA) was biotinylated using a commercial reagent (Amersham Biosciences) according to the manufacturer's instructions.…”

“…Seventeen hexapeptides covering those immunoreactive amino acid sequences and the six alanine analogs of each peptide were synthesized by the Spot method. Antigen reactivity of cellulose-bound peptides was assayed with biotinylated CD4 (1 g/ml) under conditions that yielded a blue precipitate on reactive spots as described (13). The reactivity of the spots was evaluated by scanning the membrane and measuring the intensities of the spots with NIH Image Version 1.61 software.…”

“…To this end, we previously characterized CD4-immunoreactive peptides in the 13B8.2 variable regions using the Spot method of parallel peptide synthesis (13). Whereas the Spot method has often been used to map interaction sites in epitopes (14), study of the antibody paratope (15) by probing overlapping peptides covering the variable regions of an antibody with labeled antigen was contrary to accepted dogma, probably due to the widely held view that the three-dimensional structure of the antibody is absolutely required for antigen binding.…”

Binding of CDR‐derived peptides is mechanistically different from that of high‐affinity parental antibodies

“…A wide variety of studies indicate that bioactive peptides can be generated using sequence information of antibody variable domains (Saragovi et al, 1991;Williams et al, 1991;Monnet et al, 1999;Levi et al, 1993Levi et al, , 2000Park et al, 2000;Laune et al, 1997Laune et al, , 2002Berezov et al, 2001;Tsumoto et al, 2002;Feng et al, 1998Feng et al, , 2005Bè s et al, 2001Bè s et al, , 2003Casset et al, 2003;Perosa et al, 2004; (wileyonlinelibrary.com) DOI:10.1002/jmr.1017 Heap et al, 2005). However, little experimental data are available to demonstrate that such peptides do indeed reproduce the structure and binding mode of the corresponding antibody paratope.…”