Context-dependent mutations predominate in an engineered high-affinity single chain antibody fragment

A mutational analysis of the femtomolar-affinity anti-fluorescein antibody 4M5.3, compared to its wild-type progenitor, 4-4-20, indicates both context-dependent and -independent mutations are responsible for the 1800-fold affinity improvement. 4M5.3 was engineered from 4-4-20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were studied here. Affinities of the 4-4-20 single mutant addition and 4M5.3 single site reversion mutants were compared. These experiments identified four mutations, of these seven, that were context-dependent in their contribution to higher affinity. A simplified mutant containing only these seven mutations was created to analyze complete double mutant cycles of selected sets of mutations. Specific mutational sets studied included the ligand contact mutations, the heavy chain CDR3 mutations, the heavy chain CDR3 mutations plus the neighboring residue at site H108, and the early and late acquired mutations on the directed evolution pathway. The heavy chain CDR3 mutational set and the ligand-contacting mutations were shown to provide -1.4 and -2.0 kcal/mol, respectively, of the total -3.5 kcal/mol change in free energy of binding of the seven-site consensus mutant. The mutations acquired late in the directed evolution rounds provided much of the change in free energy without the earlier acquired mutations (-3.1 kcal/mol of the total -3.5 kcal/mol). Prior structural data and electrostatic calculations presented several hypotheses for the higher affinity contributions, some of which are supported by these mutational data.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display

The recognition molecule L1 plays important functional roles in the nervous system and in non-neural tissues. Since antibodies to L1 are of prime importance to study its functional properties, we have generated affinity matured human single chain variable fragment (scFv) antibodies against mouse L1 by introducing random mutations in the complementarity determining regions (CDRs) of a previously isolated scFv antibody heavy chain (CDR1 and CDR2) and light chain (CDR3). After biopanning the mutant library, a clone (5F7) that gave the strongest ELISA signal was expressed, purified, and characterized. The dissociation constant of 5F7 (2.86 x 10(-8)M) was decreased 60-fold compared to the wild type clone G6 (1.72 x 10(-6)M). 5F7 detected L1 by Western blot analysis in mouse brain homogenates and recognized L1 in L1 transfected cells and cryosections from mouse retina and optic nerve by immunofluorescence. Bivalent 5F7 scFv antibody (5F7-Cys) was also generated and showed a dissociation constant of 5.22 x 10(-9)M that is 5.5-fold lower than that of monomeric 5F7 antibody. The bivalent affinity matured L1 scFv antibody thus showed stronger binding by a factor of 310 compared to the wild type clone. This antibody should be useful in various biological assays.     

Cooperative hydrogen bond interactions in the streptavidin-biotin system

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.     

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ～50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.     

Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.     

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

Thermodynamic basis of resistance to HIV-1 protease inhibition: calorimetric analysis of the V82F/I84V active site resistant mutant

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The active site mutant V82F/I84V has been shown to lower the binding affinity of protease inhibitors in clinical use. To identify the origin of this effect, we have investigated the binding thermodynamics of the protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir to the wild-type HIV-1 protease and to the V82F/I84V resistant mutant. The main driving force for the binding of all four inhibitors is a large positive entropy change originating from the burial of a significant hydrophobic surface upon binding. At 25 degrees C, the binding enthalpy is unfavorable for all inhibitors except ritonavir, for which it is slightly favorable (-2.3 kcal/mol). Since the inhibitors are preshaped to the geometry of the binding site, their conformational entropy loss upon binding is small, a property that contributes to their high binding affinity. The V82F/I84V active site mutation lowers the affinity of the inhibitors by making the binding enthalpy more positive and making the entropy change slightly less favorable. The effect on the enthalpy change is, however, the major one. The predominantly enthalpic effect of the V82F/I84V mutation is consistent with the idea that the introduction of the bulkier Phe side chain at position 82 and the Val side chain at position 84 distort the binding site and weaken van der Waals and other favorable interactions with inhibitors preshaped to the wild-type binding site. Another contribution of the V82F/I84V to binding affinity originates from an increase in the energy penalty associated with the conformational change of the protease upon binding. The V82F/I84V mutant is structurally more stable than the wild-type protease by about 1.4 kcal/mol. This effect, however, affects equally the binding affinity of substrate and inhibitors.     

Mean-field analysis of protein-protein interactions

Calculations were performed on the D1.3-E5.2 antibody-antibody complex estimating the binding affinities of the wild-type and 16 alanine substitutions. Analyzed were structural models of the interfacial region containing a zinc ion and crystallographic waters. A continuum approach was used to evaluate the electrostatic free energies and the hydrophobic effect was calculated by employing a buried molecular surface area relationship. Estimates of the absolute binding affinity reproduced the experimental value within the uncertainty of assessing entropic and strain energy contributions. The best correlation for mutants with experimental data was achieved when the hydrophilicity of created cavities were considered, and yielded a correlation coefficient of 0.7 and an average error of +/-1.4 kcal/mol. Empirically fitting the free energy function produced a smaller error of +/-1.0 kcal/mol. Depending on the electrical potential and electrostatic reorganization, scaling the 'protein dielectric constant' to approximately 10 may improve the accuracy of continuum models for evaluating amino acid substitutions.     

Thermodynamic characterization of affinity maturation: the D1.3 antibody and a higher-affinity mutant

Understanding the structural and dynamic determinants of binding free energy in the antigen-antibody bond is of great interest. Much work has focused on selective mutations in order to locate key interaction residues, but this generally results in reduced affinity. The present work instead examines a higher-affinity mutant to characterize the thermodynamic pathway of the affinity maturation process. We have compared the antigen binding energetics of scFv D1.3, an anti-hen egg lysozyme single chain antibody, with a higher-affinity mutant (Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. (1993). J. Mol. Biol. 234, 958-964). The mutant has five-fold higher affinity for lysozyme but nearly the same enthalpy and heat capacity change upon binding, as measured by isothermal titration calorimetry. Thus, much of the binding free energy difference can be attributed to entropic effects. Fluorescence quenching with acrylamide indicates that this more favorable entropy change may result from a more flexible mutant-lysozyme complex and thus be a configurational entropy effect.     

Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.     

Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity, coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K(Me)) by > or =10(3) M(-1). Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (DeltaG(coupling) approximately 0 kcal mol(-1)). In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (DeltaG(coupling) > or = +3.0 kcal mol(-1)). 1H-(15)N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K(Me).     

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Antigen-antibody interactions and structural flexibility of a femtomolar-affinity antibody

The femtomolar-affinity mutant antibody (4M5.3) generated by directed evolution is interesting because of the potential of antibody engineering. In this study, the mutant and its wild type (4-4-20) were compared in terms of antigen-antibody interactions and structural flexibility to elucidate the effects of directed evolution. For this purpose, multiple steered molecular dynamics (SMD) simulations were performed. The pulling forces of SMD simulations elucidated the regions that form strong attractive interactions in the binding pocket. Structural analysis in these regions showed two important mutations for improving attractive interactions. First, mutation of Tyr102(H) to Ser (sequence numbering of Protein Data Bank entry 1FLR ) played a role in resolving the steric hindrance on the pathway of the antigen in the binding pocket. Second, mutation of Asp31(H) to His played a role in resolving electrostatic repulsion. Potentials of mean force (PMFs) of both the wild type and the mutant showed landscapes that do not include obvious intermediate states and go directly to the bound state. These landscapes were regarded as funnel-like binding free energy landscapes. Furthermore, the structural flexibility based on the fluctuations of the positions of atoms was analyzed. It was shown that the fluctuations in the positions of the antigen and residues in contact with antigen tend to be smaller in the mutant than in the wild type. This result suggested that structural flexibility decreases as affinity is improved by directed evolution. This suggestion is similar to the relationship between affinity and flexibility for in vivo affinity maturation, which was suggested by Romesberg and co-workers [Jimenez, R., et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100, 92-97]. Consequently, the relationship was found to be applicable up to femotomolar affinity levels.     

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.     

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).