©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Affinity Maturation of Anti-4-hydroxy-3-nitrophenylacetyl Mouse Monoclonal Antibody
A CALORIMETRIC STUDY OF THE ANTIGEN-ANTIBODY INTERACTION (*)

(Received for publication, May 16, 1995; and in revised form, July 12, 1995)

Hidetaka Torigoe (1)(§) Tomonori Nakayama (2) Mami Imazato (2) Ichio Shimada (2) Yoji Arata (2)(¶) Akinori Sarai (1)

From the  (1)Gene Bank, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan and (2)Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 °C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.


INTRODUCTION

In affinity maturation of the immune response, the average affinity of the immunized serum generally increases with time after immunization(1) . To investigate the relationship between the primary sequence diversity of antibodies and the progressive change in binding affinity, extensive analyses of antibodies have been carried out by using several haptens, 4-hydroxy-3-nitrophenylacetyl (NP)(^1)(2, 3, 4, 5) , p-azophenylarsonate(6) , phosphorylcholine(7) , and 2-phenyl-5-oxazolone(8) .

A series of anti-NP mouse monoclonal IgG antibodies used in the present study were produced by the immune response of C57BL/6 mice against NP coupled to T cell-dependent carrier, chicken -globulin (9, 10) . The variable regions of the primary response anti-NP antibodies show low affinity for NP and carry few, if any, somatic mutations(2, 4) , whereas those of the secondary response anti-NP antibodies usually exhibit increased affinity for NP and are somatically mutated(5) . The secondary response antibodies are divided into two groups by carrying or lacking a somatic Trp Leu exchange at position 33 in the variable region of the heavy chain(11, 12) . In the present study we compare N1G9, a primary response anti-NP antibody, with 3B44 and 3B62, which are secondary response anti-NP antibodies with and without Trp Leu exchange, respectively.

Thermodynamic aspect of antigen-antibody association is essential in order to understand the mechanism of the high affinity and specificity of antigen-antibody interaction. Thermodynamic parameters such as Gibbs free energy change, DeltaG, enthalpy change, DeltaH, entropy change, DeltaS, and heat capacity change, DeltaCp, can provide useful information to identify fundamental forces involved in the antigen-antibody interaction. For instance, the magnitude of DeltaCp is usually related to the contribution of the hydrophobic effect to molecular association (13, 14, 15, 16) .

With the recent improvement in the sensitivity and reliability of the calorimeter(17, 18) , isothermal titration calorimetry (ITC) has become a powerful tool for the direct measurement of thermodynamic parameters in various biological interactions, such as protein-protein interactions(19, 20, 21, 22) , oligosaccharide-lectin associations(23, 24, 25) , and ligand binding to proteins(26, 27) . Recently this method has been applied to the quantitative thermodynamic analyses of antibody binding to various types of antigens, e.g. haptens(28, 29, 30, 31) , oligosaccharides(32, 33, 34) , and proteins(35, 36, 37, 38, 39, 40, 41, 42, 43, 44) . However, no calorimetric studies have been reported on the antigen-antibody interaction in affinity maturation.

In the present study we show the ITC analyses of the antigen-antibody associations in affinity maturation. We examined the interactions between 4-hydroxy-3-nitrophenylacetyl caproic acid (NP-Cap) antigen and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62. On the basis of the obtained thermodynamic data, we have found that the binding constants, K, for NP-Cap correlate with each of the DeltaH, DeltaCp, and DeltaSu values. As the logarithm of the K values increases in the course of affinity maturation, the magnitudes of the corresponding DeltaH, DeltaCp, and DeltaSu values increase almost linearly. Although the interactions between a series of monoclonal antibodies and their same antigen have been investigated in several cases(35, 36, 37, 39, 43, 44, 45) , the linear relationship between log K and each of DeltaH, DeltaCp, and DeltaSu shown in the present study has not yet been observed. On the basis of this correlation of the thermodynamic data along with the previously reported nuclear magnetic resonance (NMR) data(46) , we will discuss the mechanism of the affinity maturation.


MATERIALS AND METHODS

Chemicals

4-Hydroxy-3-nitrophenylacetic acid was purchased from Sigma. NP-Cap was synthesized from 4-hydroxy-3-nitrophenylacetic acid and -amino-n-caproic acid. All other chemicals were of reagent grade and used without further purification.

Preparation of Fab Fragments

Anti-NP mouse monoclonal IgG antibodies, N1G9, 3B44, and 3B62, were purified from C57BL/6 mice hybridoma cell lines kindly provided by Professor K. Rajewsky, as described previously(46) . The Fab fragments of these antibodies were prepared by papain digestion according to the procedure described previously(47) . For brevity, the Fab fragments derived from N1G9, 3B44, and 3B62 will be designated as Fab(N1G9), Fab(3B44), and Fab(3B62), respectively.

Concentration Determination

The concentration of NP-Cap was determined at 430 nm with use of the molar absorption coefficient, 4230 M cm. The concentration 1 mg/ml of the Fab solution is equivalent to the absorbance at 280 nm, 1.73 (N1G9 and 3B62), and 1.57 (3B44).

ITC

Isothermal titration experiments were carried out on a Microcal OMEGA or MCS calorimeter interfaced with a microcomputer(18) . The Fab solution was prepared by extensive dialysis against the experimental buffer, and the antigen was dissolved in the same dialysis buffer. The antigen solution was injected 20 times in 5-µl increments and 3-min intervals into the Fab solution. The heat for each injection was subtracted by the heat of dilution of the injectant, which was measured by injecting the antigen solution into the dialysis buffer. Each corrected heat was divided by the moles of NP-Cap injected and analyzed with Microcal Origin software supplied by the manufacturer.


RESULTS

Determination of Thermodynamic Parameters

Fig. 1a shows a typical ITC profile for the interaction between NP-Cap and Fab(3B62) at 30.0 °C. An exothermic heat pulse was observed after each injection of NP-Cap into Fab(3B62). The magnitude of each peak decreased gradually with each new injection, and a small exothermic peak was still observed at a molar ratio (NP-Cap)/(Fab(3B62)) = 2. The area of this exothermic peak was equivalent to the heat of dilution measured in a separate experiment by injection of NP-Cap into the buffer solution. The area under each peak was integrated, and the heat of dilution of NP-Cap was subtracted from the integrated values. The corrected heat was divided by the moles of NP-Cap injected, and the resulting values were plotted as a function of the molar ratio (NP-Cap)/(Fab(3B62)), as shown in Fig. 1b. The resultant titration plot was well fitted to a sigmoidal curve by using a nonlinear least-squares method. The binding constant, K(a), and the enthalpy change, DeltaH, were obtained from the fitted curve. Further, the Gibbs free energy change, DeltaG, and the entropy change, DeltaS, were calculated from the equation, DeltaG = -RT lnK(a) = DeltaH - TDeltaS. The thermodynamic parameters obtained for Fab(3B62) are the following: K(a) = 8.0 ± 0.7 times 10^6M, DeltaH = -20.3 ± 0.1 kcal mol, DeltaS = -35.5 ± 0.5 cal mol K, and DeltaG = -9.6 ± 0.1 kcal mol.


Figure 1: Typical isothermal titration calorimetric profiles of the interaction between NP-Cap and Fab(3B62) at 30.0 °C. a, the NP-Cap solution (1.0 mM in 5 mM sodium phosphate buffer, 200 mM sodium chloride, pH 8.0) was injected 20 times in 5-µl increments into 38 µM Fab(3B62) solution, which was dialyzed against the same buffer. Injections were occurred over 10 s at 3-min intervals. b, integrated areas for the above peaks were plotted against the molar ratio (NP-Cap)/(Fab(3B62)). The data were fitted using a nonlinear least-squares method.



The magnitudes of the DeltaS and DeltaG values are dependent on the concentration units for the standard state. In order to obtain unitary entropy change, DeltaSu, and unitary Gibbs free energy change, DeltaGu, which are independent of the concentration units chosen for the standard state since, in essence, solute concentrations are measured in mole fraction units, we used the following equations(48) :

The cratic contribution to the entropy change, R lnX(M), is R ln(1/55.6) = -7.98 cal mol K, where 55.6 M is the concentration of water in dilute aqueous solution(13) . In the case of Fig. 1DeltaSu and DeltaGu are -27.5 ± 0.5 cal mol K and -12.0 ± 0.1 kcal mol, respectively. The thermodynamic parameters for the interaction between NP-Cap and each of Fab(N1G9) and Fab(3B44) were obtained in the same way.

Temperature Dependence of the Antigen-Antibody Interaction

As a function of temperature between 25 and 45 °C, we analyzed the interaction between NP-Cap and each of Fab(N1G9), Fab(3B44), and Fab(3B62) by ITC. The thermodynamic parameters for these interactions are summarized in Table 1. The thermodynamic parameters, DeltaGu, DeltaH, and -TDeltaSu are plotted as a function of temperature in Fig. 2. For all of these interactions, both the DeltaH and DeltaSu values are negative and exhibit strong temperature dependencies. By contrast, the negative DeltaGu values show only a weak dependence on temperature. Since the temperature dependencies of DeltaH and -TDeltaSu have the opposite signs, their contributions to the temperature dependence of DeltaGu are almost canceled out. The enthalpy-entropy compensation has been observed previously for other antigen-antibody interactions(41, 44, 45) .




Figure 2: Thermodynamic parameters (DeltaH (circle), DeltaGu (), and -TDeltaSu (up triangle)) for the associations between NP-Cap and each of Fab(N1G9), Fab(3B44), and Fab(3B62) as a function of temperature. All experiments were performed in 5 mM sodium phosphate buffer, 200 mM sodium chloride, pH 8.0, at the experimental temperature. The solid lines were obtained from a linear least-squares regression. DeltaCp was calculated from the slope of the regression line of DeltaH (circle).



The associations between NP-Cap and the three anti-NP Fab are mainly driven by favorable negative changes in DeltaH. The negative DeltaH values decrease with increasing temperature and show linear dependence on temperature in the range of 25-45 °C (Fig. 2). The DeltaCp value for each interaction can be determined from the slope of the temperature dependence of DeltaH. The negative DeltaCp values of -309 ± 27, -363 ± 12, and -415 ± 12 cal mol K are observed for Fab(N1G9), Fab(3B44), and Fab(3B62), respectively (Table 1).

Fig. 3shows that the K(a) values for NP-Cap correlate with each of the DeltaH, DeltaCp, and DeltaSu values. As the logarithm of the K(a) values increases in the order of Fab(N1G9), Fab(3B44), and Fab(3B62), the magnitudes of the corresponding DeltaH, DeltaCp, and DeltaSu values increase almost linearly.


Figure 3: Correlations between the log K values at 25 °C and each absolute value of DeltaH at 25 °C (a), DeltaCp (b), and DeltaSu at 25 °C (c).



Ionic Strength Dependence of the Antigen-Antibody Interaction

Existence of positively charged amino acid residues was suggested in the combining site of anti-NP Fab fragments(49) . In order to analyze the electrostatic effect, the interaction between NP-Cap and each of Fab(N1G9), Fab(3B44), and Fab(3B62) was investigated by ITC as a function of the sodium chloride concentration between 20 mM and 400 mM. As the logarithm of the sodium chloride concentration increases, the logarithm of the K(a) values of Fab(N1G9), Fab(3B44), and Fab(3B62) for NP-Cap decreases linearly as shown in Fig. 4. The slopes of the regression lines in Fig. 4are -0.57 ± 0.09, -0.59 ± 0.10, and -0.48 ± 0.07 for Fab(N1G9), Fab(3B44), and Fab(3B62), respectively. Thus, the dependence of the K(a) values of Fab(N1G9), Fab(3B44), and Fab(3B62) on the sodium chloride concentration is similar within experimental errors.


Figure 4: Dependence of the log K values of Fab(N1G9) (circle), Fab(3B44) (), and Fab(3B62) (up triangle) for NP-Cap upon the logarithm of the sodium chloride concentration at 30 °C. All experiments were performed in 5 mM sodium phosphate buffer, pH 8.0, containing the experimental concentration of sodium chloride.




DISCUSSION

In the present study we have carried out the ITC analyses of the antigen-antibody associations in affinity maturation, in order to understand the mechanism of affinity maturation. The present results reveal that all of the associations between NP-Cap and the three Fab are mainly driven by favorable negative changes in DeltaH. Van der Waals interactions and hydrogen bondings are usually considered to be the major potential sources of the negative DeltaH values (40, 43, 50) . Thus, we suggest that van der Waals interactions and hydrogen bondings play a fundamental role in the interactions between NP-Cap and the three Fab. Also, the increase in the magnitude of the negative DeltaH with the increase in log K(a) (Fig. 3a) suggests that, in the course of the affinity maturation, the increase in the van der Waals interactions and hydrogen bondings promotes the increase in the binding affinity of the anti-NP antibody.

The negative DeltaCp values in Table 1are within the range of -100 to -650 cal mol K, which were previously reported for various antigen-antibody associations (28, 29, 35, 37, 38, 40, 41, 43-45). In general, the negative DeltaCp values for protein folding and protein-ligand association are proportional to the reduction in water-accessible nonpolar surface areas of the molecules, and related to the contribution of hydrophobic effect to molecular association(13, 14, 15, 16, 51) . In order to interpret our data quantitatively, the empirical method of Sturtevant (52) was used to estimate the hydrophobic and intramolecular vibrational contribution to DeltaCp (Table 2). For all the three Fab, the calculated hydrophobic contribution to DeltaCp is larger than the calculated vibrational contribution. Therefore, we suggest that the observed negative change in DeltaCp may primarily result from the hydrophobic effect, that is, the decrease in solvent exposure of both the aromatic antigen and the nonpolar groups in the binding site of the three Fab caused by the antigen-antibody association. Furthermore, the increase in the magnitude of the negative DeltaCp with the increase in log K(a) (Fig. 3b) suggests that, in the course of the affinity maturation, the increase in the hydrophobic effect contributes to the increase in the binding affinity of the anti-NP antibody. This is consistent with the previous NMR result that the binding site of NP-Cap is located in a similar position, but the combining site of Fab(3B62) with higher affinity for NP-Cap is composed of more Tyr residues than that of Fab(N1G9) with lower affinity for NP-Cap(46) .



The hydrophobic effect, which drives the association of nonpolar surfaces of molecules by excluding water from the interface, would contribute to the positive change in DeltaSu. However, we observed the unfavorable negative DeltaSu values in the range of 25-45 °C, as shown in Table 1. Negative DeltaSu has been observed previously for other antigen-antibody interactions (29, 30, 31, 32, 35, 36, 39, 40, 41, 42, 43, 44) . Consequently, we conclude that some other factors should counteract the hydrophobic effect and make larger contributions to the negative DeltaSu. Such effect to the negative DeltaSu can be produced by the following factors: 1) the constraint of intramolecular vibrational flexibility of Fab due to the antigen binding(52) ; 2) the reduction in the translational and overall rotational degrees of freedom upon the complex formation(53, 54) ; and 3) the conformational freezing of the amino acid residues of Fab caused by the antigen binding(55) . The previously reported estimate of the factor 2) was almost constant for different antigen-antibody complexes (TDeltaS = 7-11 kcal mol, where DeltaS is an amount of translational and overall rotational entropy change) (53, 54, 55) . The estimation of the factor 3 was previously reported in the interactions between lysozyme and a few anti-lysozyme monoclonal antibodies(55) . We applied the empirical method of Sturtevant (52) in order to estimate the hydrophobic and intramolecular vibrational (described above as the factor 1) contribution to DeltaSu (Table 2). For all three Fab, the sign of the calculated hydrophobic contribution is positive, but that of the calculated vibrational contribution is indeed negative.

The obtained K(a) values shown in Table 1increase in the order of Fab(N1G9), Fab(3B44), and Fab(3B62), which is consistent with the previously reported results(2, 4, 5) . As the logarithm of the sodium chloride concentration increases, the logarithm of the K(a) values of the three Fab for NP-Cap decrease linearly. The dependence of the K(a) values on the sodium chloride concentration is similar for the three Fab (Fig. 4). These results suggest that the electrostatic effect is involved in the antigen-antibody associations, but the proportion of the electrostatic effect to the NP-Cap binding is similar for the three Fab. We conclude that, in the course of the affinity maturation, the electrostatic effect does not significantly contribute to the increase in the binding affinity of the anti-NP antibody.

We have found a linear correlation between log K(a) and each of DeltaH, DeltaCp, and DeltaSu, as shown in Fig. 3. As the logarithm of the K(a) values increases in the course of affinity maturation, the magnitudes of the corresponding DeltaH, DeltaCp, and DeltaSu values increase almost linearly. Although the interactions between a series of monoclonal antibodies and their same antigen have been investigated in several cases(35, 36, 37, 39, 43, 44, 45) , the linear relationship between log K(a) and each of DeltaH, DeltaCp, and DeltaSu shown in the present study has not been observed yet.

This linear relation of log K(a), DeltaH, and DeltaCp (Fig. 3, a and b) implies that the surface complementarity of the anti-NP antibody with the antigen increases in the course of the affinity maturation, which may reflect increased van der Waals interactions and hydrogen bondings between specific functional groups, and increased hydrophobic interactions with more exclusion of water molecules from the interface. However, the constancy of the electrostatic effect in the course of the affinity maturation (Fig. 4) suggests that the number of electrostatic interactions involved in the interface remains unchanged with the increase in the surface complementarity. On the other hand, we observed more pronounced effect of unfavorable negative DeltaSu in the course of the affinity maturation (Fig. 3c). This apparently contradicting effect seems feasible, because more enhanced surface complementarity would make more restraint in the intramolecular vibrations of the complex.

We conclude that, in the course of the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity. The attainment of a better surface complementarity may be produced by an increase in the number of Tyr side chains in the antibody combining site.


FOOTNOTES

*
This research was supported in part by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency and by grants from the Ministry of Education, Science, and Culture of Japan (04403035, 03671024, and 04557101). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Gene Bank, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan. Tel: 81-298-36-9082; Fax: 81-298-36-9080; torigoe{at}rtcs1.riken.go.jp.

Present address: Water Research Institute, Tsukuba, Ibaraki 305, Japan.

(^1)
The abbreviations used are: NP, 4-hydroxy-3-nitrophenylacetyl; NP-Cap, 4-hydroxy-3-nitrophenylacetyl caproic acid; DeltaCp, heat capacity change; Fab, antigen binding fragment composed of the light chain and the N-terminal half of the heavy chain; DeltaGu, unitary Gibbs free energy change; ITC, isothermal titration calorimetry; K, binding constant; NMR, nuclear magnetic resonance; DeltaSu, unitary entropy change.


ACKNOWLEDGEMENTS

We thank Professor K. Rajewsky, Institut für Genetik der Universität zu Köln, for generously providing us with the anti-NP mouse monoclonal antibody N1G9, 3B44, and 3B62 cell lines.


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