Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Comparative Infra-Red, Raman, and Natural Bond Orbital Analyses of King's Sultam

By means of [1]H-NOESY- and Raman-spectroscopic analyses, we experimentally demonstrated the presence of the equatorial N—Me conformer of King's sultam 4b in solution, resulting from a rapid equilibrium. As a consequence, the value of the N lone-pair anomeric stabilization should be revised to 1.5-1.6 kcal/mol. Independently from the N tilting, natural bond orbital (NBO)-comparative analyses suggest that the S d* orbitals do not appear as primordial and stereospecific acceptors for the N lone pair. Second, the five-membered-ring sultams do not seem to be particularly well-stabilized by the S—C[sigma]* orbital in the N-substituted pseudo-axial conformation, as opposed to an idealized anti-periplanar situation for the six-membered-ring analogues. In this latter case, the other anti-periplanar C—C[sigma]* and C(1')—H/C(2')[sigma]* orbitals are as important, if not more, when compared to the S—C[sigma]* participation. In the pseudo-equatorial conformation,[upsilon]-sultams particularly benefit from the N lone-pair hyperconjugation with the anti-periplanar S—O[1][sigma]* and C(2)—H/C or C(1')—H/C[sigma]* orbitals. This is also the case for -sultams when the steric requirement of the N-substituent exceeds 1.6 kcal/mol. When both axial and equatorial conformations are sterically too exacting, the N-atom is prone to sp[2] hybridization or/and conformational changes (i.e., 12c). In that case also, the mode of stereoelectronic stabilization differs from [upsilon]- to [delta]-sultams

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic Nbs against several cell surface ecto-enzymes and ligand-gated ion channels