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

Laune, Daniel; Molina, Franck; Ferrières, Gaëlle; Villard, Sylvie; Bès, Cédric; Rieunier, François; Chardès, Thierry; Granier, Claude

doi:10.1016/s0022-1759(02)00140-0

Cited by 79 publications

(46 citation statements)

References 32 publications

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“…The entire extracellular amino acid sequence of the ETB receptor was synthesized on a cellulose membrane using the previously described SPOT technique, 62 laying down overlapping 12-mer peptides, frameshifted by one residue. Rendomab-B4 epitope mapping was performed according to a protocol previously described, 61 incubating the membrane in 1 mg/mL of rendomab-B4 for 90 min at room temperature.…”

Section: Peptide Synthesis and Etb-binding Epitope Mappingmentioning

confidence: 99%

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Borrull

Allard

Wijkhuisen

et al. 2016

mAbs

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Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.

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Section: Peptide Synthesis and Etb-binding Epitope Mappingmentioning

confidence: 99%

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Borrull

Allard

Wijkhuisen

et al. 2016

mAbs

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“…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.…”

mentioning

confidence: 99%

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

Zhuang

Stahl

Watts

et al. 2014

Journal of Biological Chemistry

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Background: There is a need for alternative HIV-1 inhibitors. Results: An engineered antibody fragment (Fab) against the essential HIV-1 protein Rev significantly reduces reverse transcriptase activity in human cell culture, and synthetic antigen-binding peptides from the Fab block Rev polymerization. Conclusion: The Fab inhibits viral replication by blocking Rev function. Significance: The Fab and its antigen-binding peptides represent a new class of anti-HIV-1 agents.

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“…The data mostly suggest at least partial differences in binding mode between antibody and CDR-derived binders (Bè s et al, 2002;Heap et al, 2005;Williams et al, 1991 (Laune et al, 1997). From all residues identified as important for HEL-binding, only 14 involved antibody residues located at the HEL/HyHEL5 complex interface in the X-ray structure, while 16 corresponded to non-interface residues (Laune et al, 1997), and were thus identified for reasons other than their contribution to the paratope.…”

Section: Other Cdr Mimicry Studiesmentioning

confidence: 97%

“…From all residues identified as important for HEL-binding, only 14 involved antibody residues located at the HEL/HyHEL5 complex interface in the X-ray structure, while 16 corresponded to non-interface residues (Laune et al, 1997), and were thus identified for reasons other than their contribution to the paratope. Other common observations include an improvement of binding strength by cyclization of peptides (Figure 3), which has been attributed to an increase in the number of structural constraints (Berezov et al, 2001;Laune et al, 2002;Levi et al, 1993;Saragovi et al, 1991;Tsumoto et al, 2002). However, binding strength may be improved by decreasing the entropic cost related to the mobility of the free peptide, without necessarily achieving structural mimicry.…”

Section: Other Cdr Mimicry Studiesmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Timmerman

Shochat

Desmet

et al. 2010

J of Molecular Recognition

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We present data that reveal crucial differences between the binding mode of anti-gastrin17 (G17, pyroEGPW-LEEEEEAYGWMDF-NH 2 ) monoclonal antibodies (mAbs) and their CDR-derived synthetic binders (SBs) with G17. The mAbs recognize the N-terminal sequence of G17 ( pyroEGPWL) with nanomolar affinity and high sequence selectivity. Molecular simulations suggest that G17 recognition is based primarily on a multitude of weak antibody-ligand interactions (H-bonding, van der Waals, etc.) inside a structurally well-defined cleft-like binding pocket. Relatively small structural changes (e.g. G-2 to A for G17) have a drastic impact on affinity, which is characteristic for antibody-like binding. In contrast, SBs recognize various sequences, including G17-unrelated targets with affinities of 1:1 complexes estimated in the 0.1-1.0 mM range. In most cases however, the G17/SB complex stoichiometries are not well-defined, giving rise to multimer aggregate formation with high apparent complex stabilities. Mutational studies on both G17 and SBs reveal the importance of positively charged (K/R) and aromatic residues (W/Y/F) for G17/SB complex formation. We propose that the synthetic binders use combinations of electrostatic, hydrophobic, and/or cation-p interactions in a variety of ways due to their intrinsic flexibility. This may also be the reason for their relatively low target specificity. We speculate that our findings are of general relevance, in showing that high-affinity mAbs do not necessarily provide the optimal basis for functional mimics design.

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

Cited by 79 publications

References 32 publications

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

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

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

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