Kinetic Analysis of Recombinant Antibody–Antigen Interactions: Relation Between Structural Domains and Antigen Binding

Borrebaeck, Carl; Malmborg, Ann Christin; Furebring, Christina; Michaelsson, Anne; Ward, Sally; Danielsson, Lena; Ohlin, Mats

doi:10.1038/nbt0692-697

Cited by 83 publications

(31 citation statements)

References 8 publications

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“…39 The resulting K D of 65 nM compares with the value measured by ELISA. In addition, the measured values of k on (4.1 ϫ 10 4 M -1 s -1 ) and k off (2.7 ϫ 10 -3 s -1 ) compare with those measured previously for the lysozyme specific antibody D1.3: V H domain alone (10 ϫ 10 4 M -1 s -1 and 1.0 ϫ 10 -3 s -1 , respectively) 43 or scFv (90.0 ϫ 10 4 M -1 s -1 and 30.0 ϫ 10 -3 s -1 , respectively). 44 Despite the reduced size of the single-domain antibody, binding is comparable to that of two-domain fragments of classic immunoglobulins.…”

Section: Resultsmentioning

confidence: 87%

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate

et al. 1998

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Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds.

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Section: Resultsmentioning

confidence: 87%

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate

et al. 1998

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“…These two phenomena have been widely observed, both with the BIAcore and the Fisons IAsys sensor, when protein ligands are covalently immobilized on a carboxymethyl dextran matrix through amide bonds with their free side-chain amine groups. As a result of the immobilization method, the ligand molecules are randomly distributed over and within the matrix, and presented in several different configurations (Borrebaeck et al, 1992;O'Shannessy et al, 1993;Edwards et al, 1995).…”

Section: (A) Expression and Purification Of The Fv-male Hybridsmentioning

confidence: 99%

Energetic and Kinetic Contributions of Contact Residues of Antibody D1.3 in the Interaction with Lysozyme

1997

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Fully functional variable fragments (Fv) of D1.3, a mouse antibody directed against the hen egg lysozyme, were readily produced as hybrids (Fv-MalE) with the maltose-binding protein of Escherichia coli and purified independently of their antigen-binding properties. We used site-directed mutations of residues in the complementarity-determining regions (CDRs) of D1.3 as local conformational probes, and compared their effects on the binding of Fv and Fv-MalE to lysozyme. We found that the MalE moiety did not significantly interfere with the interaction between the antigen and the antibody Fv fragment. We then determined the contribution of several potential contact residues of D1.3 in the interaction with lysozyme, by assaying the effect of site-directed mutations on the kinetics of association and dissociation of the complex between Fv-MalE and immobilized lysozyme, using the BIAcore apparatus. While the k on values were virtually unaffected by the mutations, the k off values varied by more than three orders of magnitude. Both charged (aspartate and arginine) and aromatic (tyrosine and tryptophan) residues in the CDR3 regions of the heavy and light chains of D1.3, which form the center of its antigen-combining site, played a preponderant part in the binding of lysozyme. Our results also showed that indirect hydrogen bonds, bridged by water molecules, contributed significantly to the interaction between D1.3 and lysozyme, and that their energy could be estimated at 1 to 2 kcal‚mol -1 .A large number of high-resolution crystallographic studies have lead to a better understanding of the molecular details of protein-protein interfaces (Janin & Chothia, 1990). Potential contacts between the partners can be defined with great precision by such methods, but structural analysis cannot show by itself which interactions are energetically important for tight binding nor how protein-protein docking kinetically occurs. Comprehensive studies of the part played by each contact atom in the association and the dissociation processes are fundamental to understand the chemical basis of macromolecular recognition and should, in turn, open up the pathway for rational protein and drug design.Interaction between antibodies and protein antigens is an excellent model for studying protein-protein interaction (Webster et al., 1994;Davies & Cohen, 1996). One of the best-characterized complexes is that between the hen egg white lysozyme (HEL) 1 and the mouse monoclonal antibody D1.3. Three-dimensional structures of the heterodimeric variable fragment (Fv) in both its liganded and unliganded states have been determined by X-ray crystallography at 1.8 Å resolution . These data suggest that residues from all six complementarity-determining regions (CDR) of D1.3 contact HEL, through as many as 20 hydrogen bonds and 44 Van der Waals contacts. Several indirect hydrogen bonds, bridged by water molecules, could also help stabilize the complex between the antibody and the antigen.Three studies in which contact residues of D1.3 were mutated t...

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“…59 In this way, hypervariable regions are formed (called the complementarity determining regions [CDRs]), and there are three each on the VL and the VH chains that determine specificity, diversity, and affinity; the remainder of the VH and VL domains are called framework regions and support the loops. 8 In human and mouse VHs, the number of loop architecture combinations is limited.…”

Section: Notesmentioning

confidence: 99%

“…Following the research hypothesis of enhancing potency with reducing size, third-generation fragment technology delivers single-cell and miniaturized Abs (∼11-15 kDa). The size advantage is, however, counterbalanced by lower affinities, 8 difficulties in mass production, 9 a very short serum half-life that disfavors the slower uptake kinetics, 10 and a tendency to aggregate. 11 The discovery in the early 1990s that Camelidae (bactrian camels, dromedaries, vicugnas, and llamas) produce fully functional Ab structures that retain only the most essential antigen-binding regions 12 demonstrated the superiority of nature in downsizing, but it also verified the research hypothesis.…”

Section: Introductionmentioning

confidence: 99%

Nanobodies as novel agents for disease diagnosis and therapy

Siontorou¹

2013

IJN

123

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Kinetic Analysis of Recombinant Antibody–Antigen Interactions: Relation Between Structural Domains and Antigen Binding

Cited by 83 publications

References 8 publications

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate

Energetic and Kinetic Contributions of Contact Residues of Antibody D1.3 in the Interaction with Lysozyme

Nanobodies as novel agents for disease diagnosis and therapy

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