Dual beneficial effect of interloop disulfide bond for single domain antibody fragments.

The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.

Nanobodies as tools for in vivo imaging of specific immune cell types.

UNLABELLED: Nanobodies are single-domain antigen-binding fragments derived from heavy-chain antibodies that are devoid of light chains and occur naturally in Camelidae. We have shown before that their small size and high affinity and specificity for their target antigen make Nanobodies ideal probes for in vivo tumor imaging. In the present study, we have evaluated the use of Nanobodies as a generic method for imaging the in vivo biodistribution of specific immune cell types, using myeloid cells as an example. METHODS: The cellular specificity of Nanobodies raised against murine bone marrow-derived dendritic cells was verified using flow cytometry on a range of myeloid and nonmyeloid cell types. The Nanobodies were then labeled with (99m)Tc and their biodistribution was analyzed using SPECT. The biodistribution was also assessed by measuring radioactivity in various organs and tissues. To verify whether the observed biodistribution was due to specific targeting through the antigen-binding loops, rather than retention in organs because of effects of the framework regions, we genetically grafted the antigen-binding loops of the Nanobodies onto the framework region of a Nanobody scaffold that by itself showed low background retention in the periphery. The cellular specificity and biodistribution of these grafted Nanobodies were determined as before. RESULTS: Nb-DC2.1, which recognizes a wide range of myeloid cells, targets most strongly to the liver, spleen, and lungs. Nb-DC1.8, which recognizes immature bone marrow-derived dendritic cells in vitro, gives a much smaller signal in the liver and spleen than does Nb-DC2.1 but mainly targets to the lungs and gives a pronounced signal in the skin. Grafting of the antigen-binding loops of Nb-DC1.8 or Nb-DC2.1 to the scaffold of Nb-BCII10 alters the observed biodistribution of the Nanobodies to resemble that of the Nanobody from which the antigen-binding loops have been derived. CONCLUSION: The observed in vivo biodistribution of the Nanobodies reflects the main in vivo locations of the cells recognized by the Nanobodies and is determined by the antigen-binding loops of the Nanobodies. Thus, Nanobodies represent elegant targeting probes for imaging the in vivo biodistribution of specific immune cell types.

A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire.

It is well established that, in addition to conventional Abs, camelids (such as Camelus dromedarius and Lama glama) possess unique homodimeric H chain Abs (HCAbs) devoid of L chains. The Ag-binding site of these HCAbs consists of a single variable domain, referred to as VHH. It is widely accepted that these VHHs, with distinct framework-2 imprints evolved within the V(H) clan III-family 3, are exclusively present on HCAbs. In this study, we report the finding of a distinct leader signal sequence linked to variable genes displaying a high degree of homology to the clan II, human VH(4) family that contributes to the HCAb Ag-binding diversity. Although the VHH framework-2 imprints are clearly absent, their VH(4)-D-JH recombination products can be rearranged to the H chains of both classical and HCAbs. This suggests that for these V domains the presence of a L chain to constitute the Ag-binding site is entirely optional. As such, the capacity of this promiscuous VH(4) family to participate in two distinct Ab formats significantly contributes to the breadth of the camelid Ag-binding repertoire. This was illustrated by the isolation of stable, dendritic cell-specific VH(4) single domains from a VH(4)-HCAb phage display library. The high degree of homology with human VH(4) sequences is promising in that it may circumvent the need for "humanization" of such single-domain Abs in therapeutic applications.