Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: Role of the heavy-chain CDR3 residues

Da, Brummell; Vp, Sharma; Nn, Anand; Bilous, D.; Dubuc, G.; Michniewicz, J.; Cr, MacKenzie; Sadowska, Joanna; Bw, Sigurskjold; Sinnott, B.

doi:10.1021/bi00055a024

Cited by 72 publications

(69 citation statements)

References 27 publications

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“…Enthalpy-Entropy Compensation-Enthalpy-entropy compensation plots have been observed previously during carbohydrate binding to lectins (25,26) and antibodies (21,27,28) and are attributed to the unique properties of water (26). A change in binding enthalpy is always compensated by the change of binding entropy.…”

Section: Relative Contributions Of Enthalpy and Entropy To The Bindinmentioning

confidence: 59%

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

Dam

Torres

Brewer

et al. 2008

Journal of Biological Chemistry

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The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9 -2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.

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Section: Relative Contributions Of Enthalpy and Entropy To The Bindinmentioning

confidence: 59%

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

Dam

Torres

Brewer

et al. 2008

Journal of Biological Chemistry

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“…c Data also presented in Table II. residues with all 19 other amino acids, and none of the mutants showed any improvement in oligosaccharide binding (1). Phage-display techniques were then used to modify additional CDR residues, and modest improvements were found for some CDR H2 mutants (30,33).…”

Section: Discussionmentioning

confidence: 99%

“…One means of exploring this question is to attempt to generate mutant anti-carbohydrate antibodies with higher affinities, either by design of site-directed mutants or by randomizing selected codons in a phage library. However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1)(2)(3)(4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen.…”

mentioning

confidence: 99%

“…However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1)(2)(3)(4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen. Mutants of the anti-Lewis Y antibody BR96 were obtained as phage-displayed scFv; although the mutant scFv binding affinity had increased by up to 6-fold versus the native antibody, its specificity was altered (2,3).…”

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confidence: 99%

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Structure of an Anti-blood Group A Fv and Improvement of Its Binding Affinity without Loss of Specificity

Thomas¹,

Patenaude²,

MacKenzie³

et al. 2002

Journal of Biological Chemistry

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The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the antiblood group A antibody AC1001 has been determined to 2.2-Å resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the Apentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity.The affinity of anti-carbohydrate antibodies for their antigens is commonly observed to be 3-5 orders of magnitude lower than affinities of anti-protein or anti-peptide antibodies for their antigens, yet there is no clear mechanism to explain this phenomenon. One means of exploring this question is to attempt to generate mutant anti-carbohydrate antibodies with higher affinities, either by design of site-directed mutants or by randomizing selected codons in a phage library. However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1-4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al. (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen. Mutants of the anti-Lewis Y antibody BR96 were obtained as phage-displayed scFv; although the mutant scFv binding affinity had increased by up to 6-fold versus the native antibody, its specificity was altered (2, 3). More recently, higher affinity antibodies against levan, a model for the polysaccharide capsules of bacteria, were obtained by mutational analysis though their fine specificity varied from that of the parent antibody (5). In contrast, significant enhancements have been achieved with antibodies to protein antigens. For example, a mutant scFv of an anti-c-erbB-2 tumor antigen obtained by chain shuffling and phage display had a 5-6-fold increase in affinity (4). In a later study by the same group (6) sequential mutation of light and heavy chains of the scFv yielded 16-and 9-fold increases in affinity for the protein antigen. Yang et al. (7) obtained mutant anti-HIV-1 Fab by phage-display techniques with a 96-fold increase in binding, even though the native antibody fragments already possessed high affinity for the protein antigen. Though phage-display techniques have been reported to yield higher binding mutant scFvs, the resulting antibodies often contain mult...

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“…It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4)(5)(6)(7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8)(9)(10)(11).…”

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confidence: 99%

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

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Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: Role of the heavy-chain CDR3 residues

Cited by 72 publications

References 27 publications

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

Structure of an Anti-blood Group A Fv and Improvement of Its Binding Affinity without Loss of Specificity

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

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