Journal of Molecular Biology
Substantial Energetic Improvement with Minimal Structural Perturbation in a High Affinity Mutant Antibody
Introduction
The mechanisms differentiating very high affinity protein interactions (sub-picomolar dissociation constants) from high affinity (nanomolar dissociation constants) have seldom been possible to study,1, 2 because few protein interaction families that span this range of affinity have been identified from either nature or engineering efforts. Antibodies provide a natural affinity series of related proteins but, in vivo, rarely surpass a 0.1 nanomolar dissociation constant (Kd) due to physiological limits on the selection.3 Further affinity improvements are often desired for pharmaceutical and biotechnology uses, and can be attained through directed evolution protein engineering techniques.4, 5, 6, 7 Computational methods have also provided some limited predictions of specific changes needed to improve high affinity interactions,8 although they have yet to realize substantial improvements experimentally. To date, the transitions from micromolar to nanomolar9, 10, 11, 12, 13, 14 and from femtomolar back to picomolar2, 15 affinity have been studied, providing little guidance into the mechanistic means to engineer an enhancement from nanomolar to femtomolar affinity.
Previous studies of antibody maturation from weak binding (micromolar) to high affinity binding (nanomolar) have shown that a number of factors are involved in improving affinity across this range. Several studies have noted that decreased loss of entropy on binding, with creation of a lock and key fit mechanism, contribute to affinity improvements.11, 14, 16, 17 In a structural, thermodynamic, and computational energetic analysis of an esterolytic antibody bound to a p-nitrophenyl phosphonate hapten, affinity maturation occurred through a reorganization of the binding site geometry so as to optimize gaining favorable electrostatic interactions with the hapten and losing those with solvent during the binding process.9, 18 In a study of an anti-testosterone antibody affinity maturation, improvement was derived from small structural changes providing more comprehensive packing around the antigen.12 A study of an anti-(4-hydroxy-3-nitrophenyl) acetate affinity matured antibody suggests that “relief of cramped contacts” in the wild-type provided the increased affinity in the mutant.19 In the first structural mechanistic study of affinity matured antibodies to a protein antigen, the higher affinity was attributed to an increased burial of hydrophobic surface area on binding and improved shape complementarity between antigen and antibody.13 Across these affinity maturation studies, mutations were found in antigen contact and non-contact regions; however, it was not always clear whether all the cited mutations were contributing to the improvements. It is perhaps to be expected that insights derived from studies of micromolar to nanomolar affinity improvements may not translate directly to mechanisms involved in fine-tuning an interaction from nanomolar to femtomolar affinity levels.
The monoclonal antibody 4-4-20 directed against the hapten fluorescein was originally isolated to facilitate studies of immunological recognition.20 The 4-4-20 binding has been extensively studied as the whole IgG, the Fab, the Fv, and the scFv by thermodynamic, kinetic, structural, computational, spectroscopic, and mutational methods.21, 22, 23, 24, 25, 26, 27, 28 A recent comparative structural study compared the 4-4-20 Fab structure with an idiotypically related lower affinity Fab, 9-40.29 This study revealed a reorganization of the binding site and a more closed structure with fewer water molecules at the binding pocket for 4-4-20, compared with 9-40, to account for the change in binding affinity.
Ten femtomolar affinity mutants of 4-4-20 were previously identified through directed evolution with random mutagenesis over the whole Fv followed by screening with yeast surface display.7 Clone 4M5.3 from that study was chosen for further study here. This clone contains 14 mutations, including all ten of the consensus mutations observed in the final directed evolution round clones.
Here, we compare 4M5.3 to 4-4-20 by kinetic, thermodynamic, structural, and theoretical analyses. No individual structural change was unambiguously associated with the −4.5 kcal/mol improvement in binding free energy. However, these results implicate a sum of many small structural changes in combination with specific mutations that improve electrostatics, which together result in a large binding free energy change.
Section snippets
Kinetics and thermodynamics
The fluorescein-biotin equilibrium binding constant for 4-4-20 in both the soluble form and displayed on the surface of yeast has been determined previously, with a Kd of 0.7 nM at 25 °C for both.7 The 4M5.3 binding constant with fluorescein-biotin was previously determined to be 270 fM through the ratio of dissociation and association kinetic rates.7 To test for the presence of kinetic intermediates that would cause the ratio of the measured association and dissociation rates to differ from the
Discussion
The entropic and enthalpic components of protein–ligand binding free energy include: (1) covalent, van der Waals, hydrogen-bonding, and electrostatic interactions within and between protein and ligand; (2) solvation effects, including desolvation costs and the hydrophobic effect; and (3) overall translational and rotational, as well as internal (often represented as vibrational) degrees of freedom within the protein and ligand. It remains unclear how best to create substantial enhancements in
Over-expression and purification of scFv proteins
The 4-4-20 and 4M5.3 scFvs7 were subcloned into the pRS316 backbone with the Gal1-10 promoter.40 A Flag tag (amino acid sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was inserted between the EagI and NheI sites N-terminal to the scFv sequence and the first expressed amino acid was changed to alanine by site-directed mutagenesis (Stratagene, La Jolla, CA). The sequence expressed was Ala-Ala-Arg-Pro-(Flag-tag)-(scFv). The scFvs were expressed solubly in Saccharomyces cerevisiae strain YVH10, which
Acknowledgements
K.M. thanks Balaji Rao for extremely helpful discussions about the competition assays for Kd determination on the surface of yeast. Funding was provided from NCI CA96504 (to K.D.W.). S.M.L. was funded by an NSF Graduate Fellowship. Funding was provided from the Alfred P. Sloan Foundation (to C.L.D.). H.H.H. was funded by an NIH biotechnology training grant (T32-GM08334). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility
References (56)
- et al.
Structural studies of binding site tryptophan mutants in the high-affinity streptavidin–biotin complex
J. Mol. Biol.
(1998) - et al.
X-ray crystallographic studies of streptavidin mutants binding to biotin
Biomol. Eng.
(1999) - et al.
CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range
J. Mol. Biol.
(1995) - et al.
Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site
J. Mol. Biol.
(1996) - et al.
Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen
J. Mol. Biol.
(1999) - et al.
Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains
J. Biol. Chem.
(2002) - et al.
Thermodynamic and kinetic aspects of antibody evolution during the immune response to hapten
Mol. Immunol.
(2003) - et al.
Streptavidin-biotin binding energetics
Biomol. Eng.
(1999) - et al.
Maturation of an antibody response is governed by modulations in flexibility of the antigen-combining site
Immunity
(2000) - et al.
Structural plasticity and the evolution of antibody affinity and specificity
J. Mol. Biol.
(2003)
Partial elucidation of an anti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal anti-fluorescyl antibodies
Mol. Immunol.
Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies
J. Biol. Chem.
High resolution structures of the 4-4-20 Fab-fluorescein complex in two solvent systems: effects of solvent on structure and antigen-binding affinity
Biophys. J.
Mutational analysis of active site contact residues in anti-fluorescein monoclonal antibody 4-4-20
Mol. Immunol.
Three-dimensional structures of idiotypically related Fabs with intermediate and high affinity for fluorescein
J. Mol. Biol.
Shape complementarity at protein/protein interfaces
J. Mol. Biol.
Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition
J. Mol. Biol.
Amino–aromatic interactions in proteins
FEBS Letters
Dissecting the energetics of a protein–protein interaction: the binding of ovomucoid third domain to elastase
J. Mol. Biol.
Biochemistry by numbers: simulation of biochemical pathways with Gepasi 3
Trends Biochem. Sci.
Processing of X-ray difraction data collected in oscillation mode
Methods Enzymol.
Kinetic and affinity limits on antibodies produced during immune responses
Proc. Natl Acad. Sci. USA
Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity
Proc. Natl Acad. Sci. USA
Theoretical and experimental studies of biotin analogues that bind almost as tightly to streptavidin as biotin
J. Org. Chem.
Structural insights into the evolution of an antibody combining site
Science
Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb
Proc. Natl Acad. Sci. USA
A comparative analysis of the immunological evolution of antibody 28B4
Biochemistry
X-ray snapshots of the maturation of an antibody response to a protein antigen
Nature Struct. Biol.
Cited by (108)
Yeast display platform with expression of linear peptide epitopes for high-throughput assessment of peptide-MHC-II binding
2023, Journal of Biological ChemistryDevelopment of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence–based affinity maturation
2022, Journal of Biological ChemistryPhotoswitchable CAR-T Cell Function In Vitro and In Vivo via a Cleavable Mediator
2021, Cell Chemical BiologyCitation Excerpt :MALDI-TOF-MS: m/z C59H57N15NaO16S [M+Na]+1286.37, Found 1286.85; C59H57KN15O16S [M+K]+ 1302.35, Found 1302.83. A gene cassette containing the 4M5.3 anti-FITC scFv (Ma, et al., 2016; Midelfort, et al., 2004; Vaughan, et al., 1996), the CD8α hinge and transmembrane region, and the cytoplasmic domains of 4-1BB and CD3ζ was synthesized by Icartab Co., Ltd. (SuZhou, China) and cloned into a lentivirus vector. Lentivirus production and transduction of human T cells were performed as previously described (Kim, et al., 2015; Ma, et al., 2016).