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

Abstract

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. 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. Antibody (Ab) 4The abbreviations used are: Ab, antibody; H, heavy chain; L, light chain; GXM, glucuronoxylomannan; mAb, monoclonal antibody; Ag, antigen; C, constant region; V, variable region; SPR, surface plasmon resonance. binding to its antigen (Ag) is a fundamental step for the development of protective adaptive immune responses. Understanding the biophysical properties of antigen-antibody interactions is essential to comprehend the evolution of the adaptive immune response. Like other protein-protein associations, antigen-antibody complexes arise from noncovalent interactions, including electrostatic and van der Waals forces, hydrogen bonds, and hydrophobic effects (1Janin J. Biochimie (Paris). 1995; 77: 497-505Crossref PubMed Scopus (8) Google Scholar, 2Mariuzza R.A. Poljak R.J. Curr. Opin. Immunol. 1993; 5: 50-55Crossref PubMed Scopus (78) Google Scholar). A critical condition for Ab-Ag binding is the formation of a specific complex between the Ab and the Ag. Understanding the interaction of these two biological macromolecules requires detailed knowledge of the structure and functional characteristics of the complex. The structure of the Ab-Ag complex can be described using x-ray crystallography and computer-generated structural models. The functional activity can be described by the kinetic rate constants, equilibrium constants, and thermodynamic binding parameters of the complex. Historically, it was widely assumed that the antibody heavy constant (CH) domain determines Ab isotype without directly affecting Ag binding affinity and/or specificity. This concept dates to the discovery that when B cells switch from one CH region to another they maintain the same variable (V) regions, leading to the inference that the avidity and effector functions of an Ab change without altering the specificity for the antigen (3Tonegawa S. Nature. 1983; 302: 575-581Crossref PubMed Scopus (3177) Google Scholar). Hence, the classical view of Ab function was that of a bifunctional molecule with the V domains being solely responsible for Ab affinity and specificity, whereas the C region was responsible for the biological properties such as complement activation, Fc receptor binding, avidity, and serum half-life (4Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1286) Google Scholar). However, in recent years this dogma has unraveled with the accumulation of new data, which suggest that the CH region can affect V region structure, thereby affecting Ab affinity and specificity (5Greenspan N.S. Dacek D.A. Cooper L.J. FASEB J. 1989; 3: 2203-2207Crossref PubMed Scopus (33) Google Scholar, 6Schreiber J.R. Cooper J. Diehn S. Dahlhauser P.A. Tosi M.F. Glass D.D. Patawaran M. Greenspan N.S. J. Infect. Dis. 1993; 167: 221-226Crossref PubMed Scopus (46) Google Scholar, 7Morelock M.M. Rothlein R. Bright S.M. Robinson M.K. Graham E.T. Sabo J.P. Owens R. King D.J. Norris S.H. Scher D.S. J. Biol. Chem. 1994; 269: 13048-13055Abstract Full Text PDF PubMed Google Scholar, 8McLean G.R. Torres M. Elguezabal N. Nakouzi A. Casadevall A. J. Immunol. 2002; 169: 1379-1386Crossref PubMed Scopus (78) Google Scholar, 9McCloskey N. Turner M.W. Steffner P. Owens R. Goldblatt D. Immunology. 1996; 88: 169-173Crossref PubMed Scopus (34) Google Scholar, 10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 11Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. PLoS ONE. 2007; 2: e1310Crossref PubMed Scopus (34) Google Scholar, 12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar, 13Torres M. Casadevall A. Trends Immunol. 2008; 29: 91-97Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Perhaps the strongest evidence for this effect comes from surface plasmon resonance (SPR) studies showing that V region-identical antibodies differing in C region manifest large differences in binding to univalent antigens (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 11Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. PLoS ONE. 2007; 2: e1310Crossref PubMed Scopus (34) Google Scholar). Those results indicated kinetic and thermodynamic variations that implied diverse Ab-Ag interactions within IgG molecules expressing different CH regions but identical V regions. Although SPR is a very powerful and useful technique for studying protein-ligand interactions, this method is vulnerable to possible artifacts. For example, SPR measurements can be affected by mass transport effects, excluded volume effects, surface concentration, and the possibility that protein immobilization affects its affinity for antigen. Furthermore, interpretation of the SPR data required data analysis by fitting to binding models, which introduces additional uncertainty. Consequently, it is important to validate conclusions derived from SPR data by other techniques. In this work, we used isothermal titration calorimetry (ITC) and a univalent peptide (P1) (Table 1) to investigate the thermodynamic binding properties of the GXM-binding mAb 3E5 (IgG3) and its IgG switch variants. These Abs have identical V regions but differ in their CH domains (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). ITC simultaneously and directly determines the enthalpic and entropic contributions, as well as the binding constant and stoichiometry in solution. ITC revealed differences in the binding energetics of V region-identical mAbs differing in isotype for a peptide mimetic thus establishing the influence of the CH region in Ag-Ab binding interactions. The results have important implications for Ab engineering and for the use of therapeutic Abs of different isotype.TABLE 1Amino acid sequence of P1 and PA1PeptideSequenceP1SPNQHTPPWMLKPA1GLQYTPSWMLVG Open table in a new tab Monoclonal Antibodies and Peptides—GXM-binding mAb 3E5 (IgG3) was produced by a hybridoma isolated from a mouse immunized with a GXM-Tetanus Toxoid conjugate vaccine (14Casadevall A. Mukherjee J. Devi S.J. Schneerson R. Robbins J.B. Scharff M.D. J. Infect. Dis. 1992; 165: 1086-1093Crossref PubMed Scopus (154) Google Scholar). The IgG1, IgG2a, and IgG2b switch variants were generated in vitro by sib selection (15Spira G. Scharff M.D. J. Immunol. Methods. 1992; 148: 121-129Crossref PubMed Scopus (33) Google Scholar). The mAb 3E5 family shares identical VH and VL sequences (12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar). All mAbs were purified by protein A or G affinity chromatography (Pierce) from hybridoma culture supernatants and were dialyzed against phosphate-buffered saline. mAb concentration was determined by enzyme-linked immunosorbent assay and Bradford protein measurements. All Abs were tested by PAGE to verify their integrity and correct molecular weight. The mAbs of the 3E5 family were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry at the Laboratory for Macromolecular Analysis at the Albert Einstein College of Medicine to confirm the absence of mAb aggregates (12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar). Peptide mimetic of GXM P1 (SPNQHTPPWMLK) (Table 1) (12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar) was synthesized and biotinylated by the Rockefeller University Proteomics Resource Center. Isothermal Titration Microcalorimetry—ITC experiments were performed using a VP-ITC instrument from Microcal (Northampton, MA). Injections of 6 μl of peptide solution were added from a computer-controlled microsyringe at an interval of 4 min into the sample solution of antibody (cell volume = 1.43 ml) with stirring at 350 rpm. An example of an ITC experiment is shown in Fig. 1 for IgG2a binding to peptide P1 at 27 °C. The concentration range of the antibodies was 7–25 μm, and the peptide was 1–2 mm. Titrations were done at pH 7.2 using 20 mm phosphate-buffered saline. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with ΔH (binding enthalpy kcal mol–1), Ka (association constant), and n (number of binding sites per monomer), as adjustable parameters. The quantity c = Ka Mt(0), where Mt(0) is the initial macromolecule concentration, is of importance in titration microcalorimetry (16Wiseman T. Williston S. Brandt J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar). All experiments were performed with c values 1 < c < 200. The instrument was calibrated using the calibration kit containing RNase A and 2′-CMP supplied by the manufacturer. Thermodynamic parameters were calculated from the Gibbs free energy equation, ΔG =ΔH – TΔS =–RT lnKa, where ΔG, ΔH, and ΔS are the changes in free energy, enthalpy, and entropy of binding, respectively. T is the absolute temperature, and R = 1.98 cal mol–1 K–1. Our previous serological studies (8McLean G.R. Torres M. Elguezabal N. Nakouzi A. Casadevall A. J. Immunol. 2002; 169: 1379-1386Crossref PubMed Scopus (78) Google Scholar, 12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar) revealed that 3E5 IgG3 and its switch variants IgG1, IgG2a, and IgG2b possessed different fine specificities for GXM. Specificity differences among these variants with identical V domains reinforce the notion that the C region of an antibody plays important roles in determining its specificity. We studied the kinetics and thermodynamics of binding of a 12-mer peptide mimetic (P1) to these variants by employing SPR to examine whether quantitative data support this view (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This study shows that Ab switch variants with identical V domains bound to P1 with different kinetics and thermodynamics. Molecular modeling suggests that amino acid sequence polymorphisms in the CH region influence the secondary structure of the antigen-binding site leading to specificity differences (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, the thermodynamic data determined by SPR are indirect and may be affected by certain experimental conditions and intrinsic limitations of the technique. Therefore, in this study, we used ITC to determine the thermodynamic binding parameters of 3E3 IgG3 and its switch variants IgG1, IgG2a, and IgG2bfor P1. ITC can directly determine the association constant, stoichiometry, enthalpy, and entropy of binding in a single experiment. This method measures binding parameters in solution and is free of the limitations associated with solid phase binding techniques such as SPR. Peptide mimetics of GXM bind to the antigen-binding site of an antibody and are useful tools for determining the fine specificities of mAbs with identical V regions (Valadon et al. (24Valadon P. Nussbaum G. Boyd L.F. Margulies D.H. Scharff M.D. J. Mol. Biol. 1996; 261: 11-22Crossref PubMed Scopus (127) Google Scholar). The present study was done with P1 (Table 1), a peptide mimetic of GXM representing a sequence isolated from the screening of peptide-expressing phage library with 3E5 IgG3. The phage expressing P1 showed differences in its binding reactivity with the 3E5 switch variants (12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar). Binding Stoichiometry of the Antibodies for the Peptide P1—ITC can directly and unambiguously determine the number of binding site (n) of a macromolecule. The n values of mAb 3E5 and its switch variants range from 1.9 to 2.2 (Table 2). These values indicate that the antibodies possess two binding sites for the peptides, and each intact antibody molecule can bind to two molecules of P1, as would be expected for an IgG. The excellent correlation between the predicted and experimentally determined binding stoichiometries for the mAb 3E5 variants provides an important internal control for the high quality of the immunoglobulin preparations. Furthermore, the near-2.0 ratio of peptide to immunoglobulin molecule indicates that each V region is interacting with a single ligand, ruling out avidity effects. Anti-p24 (HIV-1) monoclonal antibody CB4-1 shows similar stoichiometry for four epitope-related and three library-derived peptides (17Welfle K. R. J. Mol. PubMed Scopus Google binding parameters of mAb 3E5 and its switch variants with P1 at pH and 27 with biotin 3E5 3E5 3E5 peptide 3E5 3E5 3E5 3E5 Open table in a new tab Peptide Binding of 3E5 and data that the association Ka of 3E5 IgG3 and its variants for P1 peptide are different (Table 2). The Ka values of mAb IgG3 and its switch variants IgG1, IgG2a, and IgG2b for P1 peptide were and at 27 °C. The Ka values with biotinylated P1 in similar conditions were and The of the mAbs determined by ITC are greater than determined by SPR, whereas the differences in the of the are in intrinsic limitations associated with SPR have to the of the of the antibodies a range of Ka values for different For example, the of the (HIV-1) mAb CB4-1 for a of peptides from to (17Welfle K. R. J. Mol. PubMed Scopus Google Scholar). antibody to an a functional of the with a Ka of A of the with affinity to the same binding site of the antibody S. A. PubMed Scopus Google Scholar). The functional of P1 is we are to the affinity of the to that of its mimetic peptide P1. Furthermore, in the and differences between SPR and it is that the method antibody binding to whereas the measures the interaction of in solution. differences in the of effects by SPR and techniques that V region-identical antibodies manifest differences in the energetics of binding to a 12-mer peptide that changes in Ag-Ab interactions by differences in domain of and to the Binding of P1 to antibodies is associated with favorable entropy = and favorable enthalpy to (Table 2). The absolute values of ΔH and TΔS among the the are The favorable entropic values indicate that the binding may be dominated by hydrophobic interactions and are with the of molecules from the site and/or peptide The ΔH values in suggest the formation of a number of hydrogen in the from the structure of the mAb complex P. Casadevall A. Scharff M.D. J. Mol. Biol. PubMed Scopus Google Scholar) support these The favorable entropic in P1 binding is with other interactions. For example, in another binding of peptides to mAbs CB4-1 and is and is by entropic effects (17Welfle K. R. J. Mol. PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar). However, it is to that binding of a and other to to is associated with favorable entropy = and hydrophobic interactions significant to binding 2002; PubMed Scopus Google Scholar). were in the binding of a of a to mAb J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). from the of mAb have the of mAb mAb to Cryptococcus neoformans with a acid peptide (Table 1) P. Casadevall A. Scharff M.D. J. Mol. Biol. PubMed Scopus Google Scholar). mAb is sequence with mAb and the binding site of these two antibodies are (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). the other and P1 (SPNQHTPPWMLK) (Table 1) to a of peptides that possess a The of a indicates that these are essential for binding the peptides to the x-ray studies have shown that with mAb and P. Casadevall A. Scharff M.D. J. Mol. Biol. PubMed Scopus Google Scholar). These are present in the region of P1. mAbs and 3E5 are antibodies and and P1 possess a structural from complex may of the thermodynamic binding data with mAb 3E5 and P1. in other peptides, secondary of the of a between and of the the of between and is into a in the into the site of mAb of its structural with P1 may similar that can its binding to mAb The site of mAb is and is of from the light and and heavy and In a the surface of a peptide bound to an antibody and results in a fitting in the site 1992; PubMed Scopus Google Scholar). The site of mAb is hydrophobic in peptides with hydrophobic such as and would with the binding The containing and is a structure for the peptide to bind to the the of the peptide within the that into the binding site are In two hydrophobic are between of the and and of P1 may be in hydrophobic interactions with mAb 3E5 the and of the The favorable TΔS values associated with P1 binding to mAb and its switch variants strongly support this interaction and of the peptide has for an binding to mAb S. A. PubMed Scopus Google Scholar). of hydrogen with mAb of which four are by two and interactions of are with in complexes M. S. A. Biochem. 1993; PubMed Scopus Google Scholar). All the affinity peptides possess at (24Valadon P. Nussbaum G. Boyd L.F. Margulies D.H. Scharff M.D. J. Mol. Biol. 1996; 261: 11-22Crossref PubMed Scopus (127) Google Scholar). The and of P1 can similar with mAb The ΔH values with P1 to (Table are with the formation of a number of between P1 and mAb In the is 20 and to in a binding enthalpy = S. A. PubMed Scopus Google Scholar). of Peptide Binding techniques such as enzyme-linked immunosorbent assay that assay properties use biotinylated to antigens solid For example, we have used biotinylated peptides to assay their antibody binding (12Torres M. May R. Scharff M.D. Casadevall A. J. Immunol. 2005; 174: 2132-2142Crossref PubMed Scopus (73) Google Scholar). We to examine the biotin molecules to peptides this the affinity and other binding biotinylated P1 peptides tested in this study showed ΔH values with P1 to (Table 2). The ΔH values from additional hydrogen the biotin However, this enthalpic was by entropic a the biotinylated P1 significant changes in affinity with P1. have binding to A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, 1991; Google Scholar) and antibodies J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, PubMed Scopus (78) Google Scholar, D.A. D. G. J. J. 1993; PubMed Scopus Google Scholar) and are to the properties of 1991; Google Scholar). A change in binding enthalpy is by the change of binding entropy. an enthalpic interaction such as a hydrogen or a is a a certain of entropy is the other when new are is of binding interactions in plays a in molecules bind to the the hydrophobic surface of the as the binding sites of a protein and its hydrogen in enthalpic to the site of a these molecules are and a certain of enthalpy is This is by favorable entropy. a of with their the same the enthalpy and entropy a D.A. D. G. J. J. 1993; PubMed Scopus Google Scholar). A of the values at for P1 binding to the mAbs in shows that it is 2). The shows a with a of and the correlation of the in Fig. is similar to for other interactions in that their are than J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, D.A. D. G. J. J. 1993; PubMed Scopus Google in to interactions where the is greater than G.R. Biochem. PubMed Scopus Google Scholar, Chem. Rev. 1989; Google Scholar). A greater than that the free energy of binding is by enthalpy, whereas a than indicates entropy The of the indicates that the binding of P1 to mAb 3E5 and its switch variants is dominated by entropy. the data with biotinylated P1 were the from to of the values of the biotinylated peptide P1. However, the is than mAb 3E5 and Binding for P1—ITC data in that the C region of the antibodies to their binding thermodynamics. identical V the enthalpy and entropy of binding of 3E3 and its variants IgG1, IgG2a, and IgG2b are very different for P1. The range of ΔH was to whereas TΔS from to (Table 2). of interactions have to be by the of amino that are from the binding site D. M. 1996; PubMed Scopus Google Scholar). Molecular modeling revealed that are three regions of structural differences within the domain of 3E5 IgG (10Torres M. Fernandez-Fuentes N. Fiser A. Casadevall A. J. Biol. Chem. 2007; 282: 13917-13927Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). acid in these regions of different may be in electrostatic and hydrophobic interactions with other and molecules in influence the structure and the of the site of the The structural differences shown by modeling may be by x-ray However, that these differences can the binding thermodynamics of the which are in by Hence, ITC provides unambiguous thermodynamic evidence for differences in Ag-Ab interactions by constant region