Cédric Bès

Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contribute to antigen binding.

Overlapping peptide scans prepared by Spot synthesis have been used to map interaction sites in several systems. Here we report our experience with this approach to identify peptides from the variable parts of anti-hapten, anti-peptide and anti-protein antibodies that retain their specific antigen-binding capacity in the Spot format. In general, the identification by the Spot method of antigen-reactive peptides was confirmed by using soluble peptides which demonstrated antigen-binding capacity in ELISA or Biacore and, biological activity for some peptides derived from anti-CD4 antibodies. The Spot method was also used to map precisely key residues from the antibody paratope. The identification of critical residues from an anti-troponin I antibody of diagnostic interest is reported as well as the compiled results from the analysis of five other antibodies of various specificities. A critical assessment of our results is provided by comparing results obtained by our approach in the mapping of antibody residues critical for antigen binding with data from the literature concerning the structural analysis of antigen-antibody complexes.

Synthetic Peptides Derived from the Variable Regions of an Anti-CD4 Monoclonal Antibody Bind to CD4 and Inhibit HIV-1 Promoter Activation in Virus-infected Cells

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1–CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nm. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81–92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven β-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat β-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

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

A systematic exploration of the VH2/Vκ12–13 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.

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.

Corrigendum to: Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1 (FEBS 25428): [FEBS Letters 508 (2001) 67-74]

Corrigendum to: E¤cient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1 (FEBS 25428) [FEBS Letters 508 (2001) 67^74]C Cedric Be©sa, Laurence Briant-Longuetb, Martine Cerrutic, Piergiuseppe De Berardinisd, Gerard Devauchellec, Christian Devauxb, Claude Graniera, Thierry Charde©sc; aCNRS-UMR 5094, Faculte de Pharmacie, Montpellier, France bCNRS-EP2104, Institut de Biologie, Montpellier, France cINRA-CNRS-UMR 5087, Saint-Christol-Lez-Ale©s, France dInstitute of Protein Biochemistry and Enzymology, C.N.R., Naples, Italy