Antibody variable domains vary widely in their intrinsic thermodynamic stability. Despite the mutual stabilization of the domains in the scFv fragment, most scFv derived from monoclonal antibodies without further engineering show poor to moderate stability. The situation gets more complex for Fab fragments and fullsized antibodies: while the disulfide-linked CL/CH heterodimer shows very limited thermodynamic stability, its unfolding kinetics are very slow. The same is true for Fab fragments, which, due to this kinetic stabilization, appear to be more stable than their thermodynamic stability suggests. However, suboptimal variable domains can be engineered for improved stability and folding efficiency while preserving their antigen-binding specificity and affinity, either by a limited number of point mutations or by grafting their antigen specificity to superior variable domain frameworks.
Keywords
- Variable Domain
- Epithelial Cell Adhesion Molecule
- Denaturant Concentration
- Immune Effector Function
- Engineer Antibody
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References
Auf der Maur A, Escher D, Barberis A (2001) Antigen-independent selection of stable intracellular single-chain antibodies. FEBS Lett 508:407-412
Auf der Maur A, Zahnd C, Fischer F et al. (2002) Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J Biol Chem 277:45075-45085
Berman HM, Westbrook J, Feng Z et al. (2000) The Protein Data Bank. Nucl Acids Res 28:235-242
Bird RE, Hardman KD, Jacobson JW et al. (1988) Single-chain antigen-binding proteins. Science 242:423-426
Chothia C, Lesk AM (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196:901-917
Davies J, Riechmann L (1994) ‘Camelising’ human antibody fragments: NMR studies on VH domains. FEBS Lett 339:285-290
Davies J, Riechmann L (1995) Antibody VH domains as small recognition units. Bio-Technology 13:475-479
Di Paolo C, Willuda J, Kubetzko S et al. (2003) A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity. Clin Cancer Res 9:2837-2848
Eigenbrot C, Randal M, Presta L et al. (1993) X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J Mol Biol 229: 969-995
Ewert S, Honegger A, Pl ückthun A (2003) Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42:1517-1528
Ewert S, Cambillau C, Conrath K et al. (2002) Biophysical properties of camelid VHH domains compared to those of human VH 3 domains. Biochemistry 41:3628-3636
Ewert S, Huber T, Honegger A et al. (2003) Biophysical properties of human antibody variable domains. J Mol Biol 325:531-553
Hamers-Casterman C, Atarhouch T, Muyldermans S et al. (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446-448
Harris LJ, Larson SB, Hasel KW et al. (1997) Refined structure of an intact IgG2a monoclonal antibody. Biochemistry 36:1581-1597
Helms LR, Wetzel R (1996) Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis. J Mol Biol 257:77-86
Honegger A, Pl ückthun A (2001) The Influence of the buried glutamine or glutamate residue in position 6 on the structure of immunoglobulin variable domains. J Mol Biol 309:687-699
Honegger A, Pl ückthun A (2001) Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol 309:657-670
Hubbard SJ, Thornton JM (1993) NACCESS. computer program
Huston JS, Levinson D, Mudgett-Hunter M et al. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 85:5879-5883
Jones PT, Dear PH, Foote J et al. (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522-525
Jung S, Pl ückthun A (1997) Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Prot Eng 10:959-966
Jung S, Honegger A, Pl ückthun A (1999) Selection for improved protein stability by phage display. J Mol Biol 294:163-180
Kabat EA, Wu TT, Terry H et al. (1991) Sequences of proteins of immunological interest, NIH Publication No. 91-3242
Kaufmann M, Lindner P, Honegger A et al. (2002) Crystal Structure of the Anti-His Tag Antibody 3D5 Single-chain Fragment Complexed to its Antigen. J Mol Biol 318:135-147
Knappik A, Ge L, Honegger A et al. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57-86
Kortt AA, Lah M, Oddie GW et al. (1997) Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer. Prot Eng 10:423-433
Lefranc MP, Giudicelli V, Ginestoux C et al. (1999) IMGT, the international ImMunoGeneTics database. Nucl Acids Res 27:209-212
Muyldermans S, Atarhouch T, Saldanha J et al. (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Prot Eng 7:1129-1135
Myklebust AT, Beiske K, Pharo A et al. (1991) Selection of anti-SCLC antibodies for diagnosis of bone marrow metastasis. Brit J Cancer Suppl 14:49-53
Proba K, Honegger A, Pl ückthun A (1997) A natural antibody missing a cysteine in VH : consequences for thermodynamic stability and folding. J Mol Biol 265:161-172
Proba K, W örn A, Honegger A et al. (1998) Antibody scFv fragments without disulfide bonds made by molecular evolution. J Mol Biol 275:245-253
R öthlisberger D, Honegger A, Pl ückthun A (2005) Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J Mol Biol 347:773-789
Tanha J, Xu P, Chen Z et al. (2001) Optimal design features of camelized human single-domain antibody libraries. J Biol Chem 276:24774-24780
Tomlinson IM, Walter G, Marks JD et al. (1992) The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. J Mol Biol 227:776-798
Tomlinson IM, Cox JP, Gherardi E et al. (1995) The structural repertoire of the human V kappa domain. EMBO J 14:4628-4638
Vermeer AWP, Norde W (2000) The thermal stability of immunoglobulin: Unfolding and aggregation of a multi-domain protein. Biophys J 78: 394-404
Vermeer AWP, Norde W, van Amerongen A (2000) The unfolding/ denaturation of immunogammaglobulin of isotype 2b and its F-ab and F-c fragments. Biophys J 79:2150-2154
Waibel R, Alberto R, Willuda J et al. (1999) Stable one-step technetium-99m labeling of Histagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat Biotech 17:897-901
Weber-Bornhauser S, Eggenberger J, Jelesarov I et al. (1998) Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4. Biochemistry 37:13011-13020
Willuda J, Kubetzko S, Waibel R et al. (2001) Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides. J Biol Chem 276:14385-14392
Willuda J, Honegger A, Waibel R et al. (1999) High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res 59:5758-5767
W örn A, Pl ückthun A (1998a) Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochemistry 37:13120-13127
W örn A, Pl ückthun A (1998b) An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly. FEBS Lett 427:357-361
W örn A, Auf der Maur A, Escher D et al. (2000) Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors. J Biol Chem 275:2795-2803
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Honegger, A. (2008). Engineering Antibodies for Stability and Efficient Folding. In: Chernajovsky, Y., Nissim, A. (eds) Therapeutic Antibodies. Handbook of Experimental Pharmacology, vol 181. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-73259-4_3
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