The crystal structure of a llama heavy chain variable domain

Spinelli, S.; Frenken, Leon; Bourgeois, Dominique; Ron, LU; Bos, Wil; Verrips, Theo; Anguille, Christelle; Cambillau, Christian; Tegoni, Mariella

doi:10.1038/nsb0996-752

Cited by 137 publications

(146 citation statements)

References 27 publications

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“…Furthermore, the monomeric state of the Nanobodies is not compromised by the Glu-49 3 Gly and Arg-50 3 Leu mutations as observed from the single symmetrical elution peak on size-exclusion chromatography, despite a more sticky behavior on the gel matrix. Several studies already demonstrated that other inherent features in the sequences of single-domain antibodies of Camelidae also contribute to this soluble and monomeric behavior (54,55). Moreover, the mutations at positions 49 and 50 seem to be completely neutral for the antigen-binding capacity of the Nanobodies we studied.…”

Section: Discussionmentioning

confidence: 92%

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Vincke

Loris

Saerens

et al. 2009

Journal of Biological Chemistry

482

437

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Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 3 Gly and Arg-50 3 Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 3 Val and Gly/Ala-52 3 Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.Minimizing the size of antigen-binding entities from a multidomain protein such as a monoclonal antibody to a singlechain variable fragment or even a single domain has been one of the primary goals of antibody engineering. For drug therapy, these smaller formats can be beneficial in various aspects such as immunogenicity, biodistribution, renal clearance, serum half-life, tissue penetration, and target retention. However, the minimal sized antibody fragments need to retain sufficiently high antigen specificity and affinity, be expressed in high yields, and should have a low tendency to aggregate so as to maintain maximal potency and reduce possible risks of immunogenicity. Moreover functionality in adverse environments such as high concentrations of denaturant or elevated temperatures, and a concomitant increased shelf-life are additional assets.A significant proportion of the functional antibodies within species of the Camelidae are devoid of light chains. These immunoglobulins are referred to as heavy-chain antibodies (1), and their antigen-binding fragment is reduced to a single domain (referred to as VHH or Nanobody), with a molecular size of only ϳ15 kDa, which is smaller in comparison to singlechain variable fragment fragments (30 kDa), Fab fragments (60 kDa), and whole antibodies (150 kDa). All Nanobodies belong to the same sequence family, closely related to that of the human VH 3...

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Section: Discussionmentioning

confidence: 92%

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Vincke

Loris

Saerens

et al. 2009

Journal of Biological Chemistry

482

437

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“…1, A and B). Amino acid pairs matching the following criteria were searched for in the three-dimensional structure of VHH (Protein Data Bank code 1HCV) (17), for which the antigen is hCG. First, we selected pairs containing combinations of Ala, Val, Ala and Val, and Ala and Ile, which were reported previously to be stable at 30°C in more than two different immunoglobulin fold domains (12).…”

Section: Introduction Of An Artificial Disulfide Bond Into Vhh-wementioning

confidence: 99%

“…In the three-dimensional structure of llama VHH raised against the ␣-subunit of human chorionic gonadotropin (hCG) (17), Ala 49 and Ile 70 are buried in the domain, and the distance between ␤-carbons of these amino acids is within a normal distance of naturally occurring ␤-carbons of cystines. We replaced these two amino acids with Cys in the wild-type and mutant domains, substituting the native disulfide bond with the Trp/Ala amino acid pair.…”

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confidence: 99%

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Hagihara

Mine

Uegaki

2007

Journal of Biological Chemistry

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We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala 49 and Ile 70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10°C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala 49 and Ile 70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.One of the major objectives of antibody engineering is to stabilize the three-dimensional structure of the antibody. Disulfide bonds often significantly stabilize the structure of native proteins. Thus, the introduction of artificial disulfide bonds is recognized as a useful protein engineering technique to increase conformational stability. Although this technique has been applied to many proteins, there are no reports of engineered disulfide bonds in an immunoglobulin fold framework.Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1-4). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein. There are several examples of artificial disulfide bonds that can replace a pair of buried hydrophobic residues, the accessible surface areas of which were Ͻ20% (5-11). The introduction of a disulfide bond into the buried hydrophobic core of human carbonic anhydrase (A23C/L203C) markedly stabilizes this enzyme; the midpoint temperature of thermal unfolding (T m ) of the mutant is 10°C higher than that of the wild-type protein (6). The engineered disulfide bonds in alkaline protease AprP (G199C/F236C) (11), xylanase (V98C/ A152C) (7), and manganese peroxidase (A48C/A63C) (10) mutants increase their tolerance against heat inactivation. On the other hand, subtilisin BPNЈ mutants (V26C/A232C and A29C/M119C) exhibit similar or slightly lower stability to irreversible thermal inactivation (8). Tolerance against heat denaturation is not directly correlated with conformational stability. Only the mutational effect on human carbonic anhydrase II (6) was examined in a reversible system. Little information is available about the thermodynamic effe...

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“…A V HH antibody from llama that binds the α chain of hCG, "V HH -H14", was obtained. The x-ray (Spinelli, et al, 1996) and NMR solution structures of free V HH -H14 have been reported. Because V HH -H14 is only 117 amino acids, the interactions between V HH -H14 and PepH14 could be studied using heteronuclear ( 1 H -15 N) single-quantum correlation (HSQC) spectrometry.…”

Section: Mapping Epitopes In Lipopolysaccharide From Legionellamentioning

confidence: 99%

Mapping the Epitopes of Antibodies

Ladner¹

2007

Biotechnology and Genetic Engineering Reviews

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The crystal structure of a llama heavy chain variable domain

Cited by 137 publications

References 27 publications

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Mapping the Epitopes of Antibodies

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