Dataset Information

Affinity transfer to a human protein by CDR3 grafting of camelid VHH.

ABSTRACT: VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 loop and the Cys residue of CDR1. Human origin protein structures with the same orientation were searched in PDB and ubiquitin was selected. Ubi-(1RI8), the CDR3-grafted ubiquitin, had antigen-binding ability, though the affinity was relatively low compared to cAb-CA05-(1RI8). The thermodynamic parameters of Ubi-(1RI8) binding to HEWL were different from cAb-CA05-(1RI8). Hydrogen-deuterium exchange experiments showed decreased stability around the CDR3 grafting region of Ubi-(1RI8), which might explain the decreased antigen-binding ability and the differences in thermodynamic properties. We concluded that the orientation of the CDR3 sequence of Ubi-(1RI8) could not be reconstructed correctly.

SUBMITTER: Inoue H

PROVIDER: S-EPMC3302641 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Affinity transfer to a human protein by CDR3 grafting of camelid VHH.

Inoue Hidetoshi H Iihara Akiko A Takahashi Hideo H Shimada Ichio I Ishida Isao I Maeda Yoshitake Y

Protein science : a publication of the Protein Society 20111005 12

VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 l ...[more]

PMID: 21910150

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Project description:Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Dataset Information

Affinity transfer to a human protein by CDR3 grafting of camelid VHH.

Publications

Affinity transfer to a human protein by CDR3 grafting of camelid VHH.

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