by Yoko Akazawa-Ogawa and Yoshihisa Hagihara
National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan
VHH antibody is a variable domain of camelid heavy-chain antibody and the smallest antigen binding formats that originates from a natural mammalian antibody [1, 2]. The VHH antibodies has a number of distinctive features as compared to conventional immunoglobulin G (IgG), and their excellent stability is especially emphasized in many literatures. The term “protein stability” includes many meanings, such as biostability, protease resistance, shelf life and physical stability against harsh conditions. In this article, we focused on the topic related to the thermal stability of VHH antibody.
One important indicator of protein thermal stability is equilibrium thermodynamic stability, which is described by the difference of Gibbs free energy (ΔGU) between the native and the various denatured states. The latter of the small globular structural unit including Ig fold domain, which is denatured with two state manner, is the unfolded state. Estimation of equilibrium thermodynamic stability requires reversible conformational transition of protein. In the case of large and multi-domain proteins, such as IgG, it is often difficult to evaluate precise equilibrium thermodynamic stability. To estimate the equilibrium thermodynamic stability, structural changes by heat or denaturant were measured and transition curves were analyzed. Mid-point temperature of thermal unfolding (Tm) is an intuitive index to approximate the equilibrium thermodynamic stability. Although Tm is not directly correlated to ΔGU, the comparison of Tm is often enough to discuss the difference of equilibrium thermodynamic stability of similar proteins or mutants. In addition to equilibrium thermodynamic stability, heat resistance is another popular indicator of the stability of protein against heat, and is often estimated by measuring the residual activity and/or native structure after heat treatment. Heat resistance is mechanistically more ambiguous than equilibrium thermodynamic stability and affected by several factors: aggregation of folding intermediates, aggregation of the unfolded state, chemical modifications, and generation of misfolded monomers.
Increasing the equilibrium thermodynamic stability is major target of protein engineering. Introduction of artificial disulfide bond into protein has been eagerly tested to improve the industrial enzymes. Trials to introduce the artificial disulfide bonds have given mixed result; it dramatically stabilized a protein but there was no effect in another, depending on the proteins and the position of artificial disulfide bond. Two groups (including authors’ group) attempted to stabilize VHH antibody by the same artificial disulfide bond, where the amino acid positions of 49 and 69 of Kabat sequence scheme were mutated to cysteines, and the result was fairly good. The introduced disulfide bond connected two β-sheets of frameworks 2 and 3 increasing the Tm about 10 °C, and only slightly affected antigen binding affinity [3-5]. This stabilizing method is now widely used in many VHH antibodies that recognize a variety of antigens . Another group showed that this method is even applicable to other Ig fold domains than VHH antibody .
Tm of VHH antibody at neutral pH usually ranged from 60 to 80 °C [8-12]. Tm of other Ig fold domain from conventional IgG including VL, VH and CL domains exhibits Tm similar to VHH antibody . Thus equilibrium thermodynamic stability of VHH antibody is similar to that of other Ig fold domain. Heat resistance of VHH antibody, however, stands in sharp contrast to that of conventional IgG. In many cases of VHH antibodies, their antigen binding affinities are preserved after incubation at high temperature [10, 14-19]. At the same experimental condition, IgG almost completely loses its antigen binding ability. Considering the equilibrium thermodynamic stability, VHH antibody unfolds at 90 °C similar to Ig fold domains of IgG. Thus, the exceptional heat resistance of VHH antibody is considered to come from the efficient recovery of the unfolded structure at 90 °C to the native structure at room temperature .
Even the highly heat resistant VHH antibody is at last inactivated by prolonged incubation time at high temperature. Recently, we carried out quantitative analysis of heat induced inactivation of anti-hCG and anti β-lactamase VHH antibodies and found that heat-induced inactivation of VHH antibody was dominantly caused by chemical modifications of amino acids . To identify the origin of inactivation, we first carried out “repetitive” and “continuous” heating experiments; anti-hCG and anti β-lactamase VHH antibodies were subjected to a given number of heating (90 °C for 5min) and cooling (20 °C for 5 min) cycles as former experiments, and continuous heating (90 °C) for a given period of time as latter experiments, then their residual activities were estimated. The difference between repetitive and continuous experiments was the number of refolding and unfolding processes. Thus, the aggregation of folding intermediates can be estimated by the comparison of the residual activities of the two experiments. The residual activities curves of repetitive and continuous experiments, which were plotted against total incubation time at 90 °C, were well matched to each other in both anti-hCG and anti β-lactamase VHH antibodies, suggesting that the refolding from heat-induced unfolding state is extremely efficient. Then, we found that heat-induced inactivation of anti-hCG VHH antibody was only slightly dependent on protein concentration at the range from 1 µg/mL to 1 mg/mL, and monomeric species of VHH antibody still remained even after long-term treatment at 90 °C. Incubation of inactivated VHH antibody with high concentration of denaturant and subsequent dilution of sample solution did not affect the residual antigen binding activity. These indicated that the heat-induced inactivation of VHH antibody predominantly occurred by chemical modifications of amino acids. Indeed, the replacement of asparagine, which is susceptible to chemical modifications, increased the heat resistance of VHH antibody.
In contrast to VHH antibody, the heat-induced inactivation of anti-hCG single-chain antibody (scFv) was protein concentration dependent, and thus its heat-induced inactivation would be driven by aggregation at high temperature. IgG and Fab fragment were inactivated very fast by heat and include domains of scFv. We consider that IgG and Fab are inactivated by aggregation of the unfolded state and possibly by the aggregation of folding intermediates. Thus, the mechanisms of heat-induced inactivation of antibody and its fragments are considered different, depending on the molecular structure (Figure 1).
The equilibrium thermodynamic stability of VHH antibody is ordinary as an Ig fold domain, however, protein engineering that inducing disulfide bond can dramatically increase its thermodynamic stability. Also, improvement of heat resistance of VHH antibody seems to be easier than other antibody formats as chemical modification can be controlled easier than aggregation, which is related to the structure of the folding intermediates and/or the unfolded state. For applications requiring resistance against harsh condition, VHH antibody is better than other antibody formats because of its potential for engineering of both equilibrium thermodynamic stability and heat resistance improvements.
About the Authors
Yoko Akazawa-Ogawa is the Researcher of National Institute of Advanced Industrial Science and Technology (AIST). She received her Ph.D. from Kobe University.
Yoshihisa Hagihara obtained his Ph.D. from Osaka University. After four years of post-doctoral training at Whitehead Institute for Biomedical Research and Protein Research Institute in Osaka University, he was appointed as Researcher at National Institute of Advanced Industrial Science and Technology (AIST). He is now the Group Leader at AIST.
- Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains. Nature 1993; 363:446-8.
- Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem 2013; 82:775-97.
- Hagihara Y, Mine S, Uegaki K. Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region. J Biol Chem 2007; 282:36489-95.
- Saerens D, Conrath K, Govaert J, et al. Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains. J Mol Biol 2008; 377:478-88.
- Hagihara Y, Saerens D. Improvement of single domain antibody stability by disulfide bond introduction. Methods Mol Biol 2012; 911:399-416.
- Hagihara Y, Saerens D. Engineering disulfide bonds within an antibody. Biochim Biophys Acta 2014; 1844:2016-23.
- Kim DY, Kandalaft H, Ding W, et al. Disulfide linkage engineering for improving biophysical properties of human VH domains. Protein Eng Des Sel 2012; 25:581-9.
- Hussack G, Riazi A, Ryan S, et al. Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility. Protein Eng Des Sel 2014; 27:191-8.
- Hussack G, Hirama T, Ding W, et al. Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS One 2011; 6:e28218.
- Turner KB, Liu JL, Zabetakis D, et al. Improving the biophysical properties of anti-ricin single-domain antibodies. Biotechnology Reports 2015; 6:27-35.
- Turner KB, Zabetakis D, Goldman ER, et al. Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection. Protein Eng Des Sel 2014; 27:89-95.
- Hagihara Y, Matsuda T, Yumoto N. Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains. J Biol Chem 2005; 280:24752-8.
- Kim DY, Hussack G, Kandalaft H, et al. Mutational approaches to improve the biophysical properties of human single-domain antibodies. Biochim Biophys Acta 2014; 1844:1983-2001.
- van der Linden RH, Frenken LG, de Geus B, et al. Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta 1999; 1431:37-46.
- Perez JM, Renisio JG, Prompers JJ, et al. Thermal unfolding of a llama antibody fragment: a two-state reversible process. Biochemistry 2001; 40:74-83.
- Dumoulin M, Conrath K, Van Meirhaeghe A, et al. Single-domain antibody fragments with high conformational stability. Protein Sci 2002; 11:500-15.
- Ladenson RC, Crimmins DL, Landt Y, et al. Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 2006; 78:4501-8.
- Omidfar K, Rasaee MJ, Kashanian S, et al. Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia. Biotechnol Appl Biochem 2007; 46:41-9.
- Anderson GP, Liu JL, Hale ML, et al. Development of antiricin single domain antibodies toward detection and therapeutic reagents. Anal Chem 2008; 80: 9604-11.
- Ewert S, Cambillau C, Conrath K, et al. Biophysical properties of camelid V(HH) domains compared to those of human V(H)3 domains. Biochemistry 2002; 41:3628-36.
- Akazawa-Ogawa Y, Takashima M, Lee YH, et al. Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications. J Biol Chem 2014; 289:15666-79.