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. 2022 Oct 20;14(6):1521–1526. doi: 10.1007/s12551-022-01008-7

Structural, functional, and physiological properties of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies during the course of affinity maturation

Masayuki Oda 1,✉
PMCID: PMC9842813  PMID: 36659986

Abstract

Structural and functional analyses of antibodies in the affinity maturation pathway can help us understand the molecular mechanisms of protein recognition. Using one of the haptens, (4-hydroxy-3-nitrophenyl)acetyl (NP), various monoclonal antibodies have been obtained, either at the early or late stage of immunization. The variable regions of monoclonal antibodies and their site-directed mutants can also be obtained as single-chain Fv (scFv) antibodies. The change in antigen-binding affinity and avidity of matured-type antibodies from germline-type antibodies could be evaluated based on binding kinetics and thermodynamics, proposing the antigen recognition mode. Crystal structures of a germline-type antibody, N1G9, and a matured-type antibody, C6, in complex with NP were determined, revealing different antigen-binding mode at atomic resolution. Notably, the Tyr to Gly mutation at the 95th residue of the heavy chain is critical for changing the configuration of complementarity determining region 3, which is involved in antigen binding. Furthermore, thermal stability analyses of scFv antibodies have revealed trade-off between antigen-binding affinity and thermal stability in the antigen-unbound state. To increase affinity, the stability of the variable region may be decreased, possibly due to protein architecture. The high stability of germline-type antibodies and the low stability of matured-type antibodies, which increase upon antigen binding, can be explained by the stability of antibodies required at the respective stages of immunization.

Keywords: Affinity and avidity, Antibody, Binding kinetics and thermodynamics, Crystal structure, Thermal stability

Introduction

Antibodies possessing a basic four-chain structure with two combining sites interact with various types of antigens and also act as antigen receptors on B cells. Antibodies specifically bind to antigens via complementarity determining region (CDR) loops in variable heavy and light chains (VH and VL). Affinity maturation is known to increase the binding affinity and specificity of antibodies towards their immunogen by somatic hypermutation (Eisen and Siskind 1964; Tonegawa 1983; Milstein and Rada 1995; Azuma 1998). Haptens such as (4-hydroxy-3-nitrophenyl)acetyl (NP) and phosphorylcholine and 2-phenyl oxazolone have been used to analyze the structural basis of affinity maturation (Bothwell et al. 1981; Gearhart et al. 1981; Kaartinnen et al. 1983; French et al. 1989; Alzari et al. 1990). The equilibrium association constant (Ka) of germline-type antibodies towards the hapten is as low as 105–106 M–1, and that of affinity-matured antibodies reaches 109 M–1 at maximum (Foote and Eisen 1995; Furukawa et al. 1999; Oda and Azuma 2000). In the analysis of anti-NP antibodies during affinity maturation, Rajewsky et al. found that the Trp to Leu mutation at VH33 by somatic hypermutation is critical for maturation (Allen et al. 1988). Azuma et al. found that the Tyr to Gly mutation at VH95 could be classified as a matured-type antibody at the late stage of immunization (Furukawa et al. 1999). The DNA bases translating to the residue at VH95 are located at the boundary of the V and D gene junctions (Kabat et al. 1991; Hofle et al. 2000), and the residue at VH95 is located at the bottom of H-CDR3, enabling a variety of H-CDR3 conformations (Shirai et al. 1998; Furukawa et al. 2001). In this review article, some anti-NP monoclonal antibodies are referred to as typical antibodies for affinity maturation; N1G9 and F8 are germline-type possessing Trp33H and Tyr95H, B2 is affinity matured-type possessing Leu33H and Tyr95H, and 9T7, C6, and E11 are affinity matured-type possessing Trp33H and Gly95H, respectively. In addition to monoclonal antibodies, we recently constructed expression plasmids of anti-NP single-chain Fv (scFv) antibody in Escherichia coli and analyzed their structural, functional, and physiological properties. The VL and VH regions of anti-NP antibodies were connected by a linker region, comprising three repeats of the peptide GGGGS. The scFv protein was expressed in Escherichia coli as the insoluble fraction, and solubilized using urea, followed by refolding by dilution. One of the advantages of generating scFv antibodies is that the specific site of an scFv can be changed by site-directed mutagenesis which could provide antibodies that are not obtained as monoclonal antibodies, and the effects on antigen binding, tertiary structure, and stability can be analyzed. In addition to the scFvs of monoclonal antibodies such as N1G9, 9T7, C6, and E11, scFvs of N1G9_W33L, 9TG, and C6_W33L were generated and are referred to in this article. N1G9_W33L and C6_W33L are site-directed mutants of W33L at the VH of N1G9 and C6, respectively. 9TG is a putative germline-type of 9T7.

Antigen binding

During affinity maturation, antigen-binding affinity and specificity generally increase. Many antibodies with somatic hypermutations have been generated and selected, and some are expressed from plasma cells. The Trp to Leu mutation at VH33 of the anti-NP antibody results in an increase in affinity towards NP by approximately tenfold (Allen et al. 1988). Antibodies with mutations including that from Tyr to Gly at VH95 have 10–103-fold affinity (Furukawa et al. 1999). It should also be noted that most antibodies with the Leu33H/Tyr95H-type appeared within 2–3 weeks of immunization, while those with the Trp33H/Gly95H type appeared later. Binding kinetics and thermodynamics provide additional information on the increased affinity. During affinity maturation, both association and dissociation rate constants (kon and koff) contribute to increased antigen-binding affinity (Oda and Azuma 2000, 2016; Sato et al. 2016, 2017). Using isothermal titration calorimetry, binding thermodynamic parameters such as enthalpy and entropy changes (ΔH and ΔS) were obtained for NP binding to anti-NP monoclonal and scFv antibodies (Table 1). Using a surface plasmon resonance biosensor, binding kinetic parameters such as kon and koff could be obtained (Table 2). The antibodies bind to NP with favorable ΔH, which is partially compensated by unfavorable ΔS (Torigoe et al. 1995; Furukawa et al. 1999; Nishiguchi et al. 2022). For most antibodies with increasing affinity, ΔH becomes more favorable. In contrast, we also found that, among the antibodies originating from a common ancestor clone, an increase in affinity was obtained by an increase in ΔS (Sagawa et al. 2003). Antibodies with more favorable ΔS bind to NP with decreased kon and koff values. Taken together, the antigen–antibody interaction would be shifted from a “induced-fit” type to a “lock-and-key” type during antibody evolution. Using scFv, we analyzed the effects of Trp to Leu mutation at VH33. Upon mutation of N1G9, antigen-binding affinity increased (Table 1), similar to that of B2, Leu33H/Tyr95H-type, on affinity maturation. In contrast, Trp to Leu mutation on C6, C6-W33L, which is classified as Leu33H/Gly95H-type and is not obtained in anti-NP monoclonal antibodies, results in decreased antigen-binding affinity (Table 1).

Table 1.

Thermodynamic parameters for NP binding to anti-NP antibody at 25 °C

Ka ∆G ∆H T∆S
(M–1) (kJ mol–1) (kJ mol–1) (kJ mol–1)
(Trp33H/Tyr95H)
  F8 IgGa 2.7 × 105  − 31.0  − 56.0  − 25.0
  N1G9 scFvb 1.85 × 106  − 35.4  − 46.0  − 10.6
(Leu33H/Tyr95H)
  B2 IgGa 3.4 × 106  − 37.2  − 75.7  − 38.5
  N1G9_W33L scFvb 8.31 × 106  − 39.4  − 69.2  − 29.8
(Leu33H/Gly95H)
  C6_W33L scFvb 1.40 × 107  − 40.7  − 46.0  − 5.2
(Trp33H/Gly95H)
  9TG scFvc 3.72 × 105  − 31.8  − 33.5  − 1.7
  9T7 scFvc 7.16 × 106  − 39.1  − 51.6  − 12.5
  C6 scFvb 5.68 × 107  − 44.2  − 52.1  − 7.9
  E11 scFvc 1.47 × 108  − 46.6  − 72.3  − 25.7
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aData were taken from Furukawa et al. (1999)

bData were taken from Sato et al. (2017)

cData were taken from Nishiguchi et al. (2022)

Table 2.

Kinetic parameters for NP binding to anti-NP antibody at 25 °C

kon (M−1 s−1) koff (s−1) Ka (M–1)
(Trp33H/Tyr95H)
  N1G9 scFva 3.73 × 105 1.16 × 10–1 3.22 × 106
(Leu33H/Tyr95H)
  B2 IgGb 8.7 × 104 8.1 × 10–2 1.1 × 106
  N1G9_W33L scFva 6.87 × 105 3.05 × 10–2 2.25 × 107
(Leu33H/Gly95H)
  C6_W33L scFva 2.22 × 106 3.14 × 10–2 7.07 × 107
(Trp33H/Gly95H)
  C6 scFva 1.28 × 106 7.81 × 10–3 1.64 × 108
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aData were taken from Sato et al. (2017)

bData were taken from Oda and Azuma (2000)

Avidity effects should be considered for antibody binding, particularly for antibody function in vivo. IgG has two combining sites, whereas secreted IgM has ten combining sites because it forms a pentamer. In comparison with monovalent binding to antigens, divalent binding strength would be higher. We showed that the divalent binding affinity of anti-NP antibodies, either germline- or matured-type, was around the nanomolar level of equilibrium dissociation constant (Kd) (Oda and Azuma 2000). Notably, the increased ratio of affinity from monovalent to divalent binding is much larger for the germline-type relative to the matured-type. This may be due to the ceiling avidity (affinity) for naturally evolving antibodies (Foote and Eisen 1995; Murakami et al. 2010). In contrast, the engineered antibody could increase the affinity to a higher level (Boder et al. 2000). Ceiling affinity also applies to secreted IgM (Tobita et al. 2004). The nanomolar level of Kd may be sufficient for immunity against antigens to be defended by antibodies. At the molecular level, the binding entropy loss for divalent binding energy may be larger in IgG with high monovalent antigen-binding affinity. It seems reasonable that the IgM pentamer and IgG with low affinity first appear at the early stage of immunization, followed by IgG with increased affinity at the late stage of immunization.

Crystal structures and structural dynamics

Difference in H-CDR3 was significant in both the lengths and sequences of amino acid sequences of N1G9 and C6. Antigen-binding affinity of C6 scFv was 30–50 times higher than that of N1G9 scFv (Tables 1 and 2). Satow et al. reported the crystal structure of N1G9 Fab in complex with NP at 2.4 Å resolution (PDB ID; 1NGP) (Mizutani et al. 1995). We reported the crystal structure of C6 scFv in complex with NP at 1.65 Å resolution (PDB ID; 6K4Z) (Nishiguchi et al. 2019). The NP phenyl group was recognized by π-π stacking with Trp91L in both C6 and N1G9. The contact geometry of π-π stacking in C6 appears to be preferable to that of N1G9, owing to the almost complete parallel geometry of the two aromatic rings of NP and C6 Trp91L. In comparison with the N1G9 crystal structure, most of the C6 residues interacting with NP were the same, except for Arg58H and His102H (Fig. 1). Among the 17 residues of somatic hypermutation in C6, Lys58HArg in H-CDR2 was the only mutation directly involved in the interaction with NP. Although Lys58H seemed to be more favorable for NP binding than Arg58H, mutational analysis demonstrated that the NP binding affinity of the C6_R58K scFv mutant was slightly lower than that of C6 scFv (unpublished results). The residue His102H is located at H-CDR3, of which two residues are shorter than N1G9. Crystal structure analysis showed a large difference in H-CDR3 between N1G9 and C6. Bulky side-chains of Tyr95H and Tyr97H occupied N1G9, but Gly95H occurred in C6, resulting in a large space enabling His102H to form a hydrogen bond to NP. In addition, the main-chain nitrogen of Gly99H forms a hydrogen bond with NP. Conversely, in N1G9, the side chains of Tyr95H and Tyr97H were involved in NP binding. Despite the availability of high-resolution crystal structures, it seems to be difficult to determine the contribution of respective sites to increased affinity. Both the change in residues and the overall conformation, especially that of H-CDR3, would contribute to increased NP binding. One of the key residues is His35H. While His35H of N1G9 forms a hydrogen bond with NP, that of C6 is flipped away from NP, resulting in the formation of hydrogen bond with His102H that interacts with NP. Together with the contribution of antibody structural dynamics (Sato et al. 2016), the residue change from N1G9 to C6 would make it possible to increase NP-binding affinity by a sophisticated recognition mechanism.

Fig. 1.

Fig. 1

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Close-up view of crystal structure of C6 scFv in complex with NP. The antigen, NP (magenta), and the side-chains of residues interacting with NP are indicated by stick-model. The figure was drawn by PyMOL (Schrodinger, LLC)

Using the recombinant scFv antibody, a His-tag can be added at the N- or C-terminus of scFv and uniformly immobilized on the Ni-nitrilotriacetic acid substrate, making it possible to bind antigens in a fashion similar to that in solution. Diffracted X-ray tracking was used to analyze the structural dynamics of C6 scFv (Sato et al. 2016). In this method, gold-nanocrystals were labeled on the surface of the scFv for motion tracing. The results revealed that that the fluctuations of C6 scFv were suppressed upon antigen binding, and the antigen-binding energy determined based on the angular diffusion coefficients was in agreement with that calculated from the kinetics analysis.

Antibody stability

Using scFv, we analyzed the thermal stability of antibodies during the affinity maturation process in the presence or absence of antigens (Sato et al. 2017; Nishiguchi et al. 2022). One of the advantages of using scFv is that it consists of a minimum unit of antibody for antigen binding and provides information on its structure and function without perturbation of other domains. The thermodynamic parameters, denaturation temperature (Td), and calorimetric enthalpy change (ΔHcal), analyzed using differential scanning calorimetry are summarized in Table 1. The thermal stability of scFvs in the antigen-bound state was higher than that in the antigen-unbound state. Notably, the stability of germline-type antibodies in the antigen-unbound state was higher than that of affinity matured-type antibodies. Upon antigen binding, the stability of matured-type antibodies increases more than that of germline-type antibodies, and the two become similar. The increased stability correlates well with antigen-binding affinity (Fig. 2). This was the first report of the trade-off phenomena of binding and stability in correlation with affinity maturation of antibodies. Antibody structure and flexibility are closely correlated to antigen binding and stability. Structure instability might result from increasing antigen-binding affinity by somatic hypermutation from germline-type to matured-type antibodies. However, it is difficult to determine the instability of C6 scFv based on its crystal structure (Nishiguchi et al. 2019). In comparison with the crystal structure of N1G9 Fab (Mizutani et al., 1995), we cannot conclude the stability difference between them. In terms of immunity, at a late stage of immunization, under conditions of antigen excess, antibody instability might not be critical because the antibody could bind to its antigen to be stabilized. In contrast, at an early stage of immunization, high stability of germline-type antibodies might be required to maintain antigen-binding ability at low antigen concentrations. Furthermore, when comparing Gly95H-type with Tyr95H-type, ΔHcal of 9TG scFv was larger than that of N1G9 scFv (Table 3). This suggests that the Gly95H-type antibody architecture affords to decrease stability to maintain the native structure at physiological temperature when somatic hypermutation results in increased affinity. One of the most critical features of the antibody structure of Gly95H-type is the hydrogen bond between His35H and His102H (Nishiguchi et al. 2019). Although ΔHcal of C6 scFv is similar to that of N1G9 scFv, Td of C6 scFv is much lower than that of N1G9 scFv (Table 3). These results indicate that the decreased stability of C6 scFv in the antigen-unbound state was mainly due to unfavorable ΔS. Although it is difficult to evaluate the contribution of antibody flexibility based only on crystal structure information, the results of folding thermodynamics suggest that C6 matures to complement the antigen better than N1G9, and antigen binding occurs via a “lock-and-key” mode with decreasing entropy in the antigen-unbound state. This is also supported by the binding thermodynamics, which reveal that ΔS of C6 is more favorable than that of N1G9 (Table 1). The thermodynamic parameters could be calculated using the heat capacity change, showing that the decreased stability of C6 scFv was due to unfavorable ΔS, partially compensated by favorable ΔH (Nishiguchi et al., 2022).

Fig. 2.

Fig. 2

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Plots of –ΔG values for NP binding vs ΔTd. The ΔG and ΔTd values are taken from Tables 1 and 3, respectively

Table 3.

Folding thermodynamics of scFvs in the absence or presence of antigen

Td (°C) ∆Td (°C) ∆Hcal (kJ mol−1)
(Trp33H/Tyr95H)
  N1G9 scFva 66.2 395
  N1G9 scFv + NPa 68.8 2.6 452
(Leu33H/Tyr95H)
  N1G9_W33L scFva 64.0 418
  N1G9_W33L scFv + NPa 69.8 5.8 493
(Leu33H/Gly95H)
  C6_W33L scFva 47.4 405
  C6_W33L scFv + NPa 60.2 12.8 459
(Trp33H/Gly95H)
  9TG scFvb 68.4 567
  9TG scFv + NPb 70.7 2.3 666
  9T7 scFvb 52.7 402
  9T7 scFv + NPb 62.5 9.8 644
  C6 scFva 48.5 384
  C6 scFv + NPa 67.0 18.5 585
  E11 scFvb 50.1 570
  E11 scFv + NPb 70.1 20.0 795
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+ NP; molar ratio of scFv and NP is 1:10

∆Td = Td in the presence of NP − Td in the absence of NP

aData were taken from Sato et al. (2017)

bData were taken from Nishiguchi et al. (2022)

Concluding remarks

Affinity maturation of antibodies, including somatic hypermutation, is a form of protein evolution in nature (Wedemayer et al. 1997; Vajda et al. 2021). The mutation is introduced in either the direct antigen-contact region or the indirect region, including the framework regions, enabling changes in antibody architecture and structural dynamics. The combination of biophysical methods, such as crystal structure, binding kinetics and thermodynamics, and folding thermodynamics, can provide information on the protein structure–activity relationship. Our recent analysis clearly showed that antibodies increase antigen-binding affinity but destabilize in a trade-off manner. Destabilization of the matured-type antibody is recovered upon antigen binding to a level similar to that of the germline-type antibody. These stability characteristics appear to be reasonable for the respective antibodies required at different stages of immunization. We can learn a lot from affinity maturation, not only antigen binding, but also stability, both of which result from the antibody architecture. Further analysis, especially of structural dynamics, could aid our understanding of the structural basis in correlation with the function applicable to protein engineering and drug design. We are in the progress of generalizing the trade-off phenomenon for other antibodies during the affinity maturation process.

Acknowledgements

The study of anti-NP monoclonal antibodies was started from 1999 under the guidance of Prof. Takachika Azuma of Tokyo University of Science. I greatly appreciate Prof. Haruki Nakamura for giving me a special chance, a turning point of my research life, when I was a researcher in his group of Protein Engineering Research Institute (PERI) and Biomolecular Engineering Research Institute (BERI) from 1993 to 1997.

Funding

This study was partly supported by JSPS KAKENHI Grant Number 18K06161.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher’s note

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