Surface display of a single-domain antibody library on Gram-positive bacteria.

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

Bacterial two hybrid: a versatile one-step intracellular selection method.

Many antibody fragments, selected ex vivo by phage display, fail to form functional antigen-binding entities when expressed and used intracellularly (i.e., as intrabodies) because the interior of the cell poses significant challenges on the folding of antibodies. Such dropout can be avoided by employing intracellular selection methods like yeast or bacterial two hybrid systems. These involve four facile steps: construction of plasmids, transformation of microbial cells, intracellular expression of fusion proteins, and selection for reporter activity. Using E. coli as host instead of yeast offers the advantages of a faster growth and a higher transformation efficiency allowing to screen larger repertoires. This chapter describes the protocol, optimized to identify antigen-specific single domain antibodies (sdAbs), by bacterial two hybrid selection.

A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.

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.

Modulation of protein properties in living cells using nanobodies.

Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venom.

Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.

Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies.

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDR-H loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJmol(-1)), good expression level (5 mgl(-1) in E.coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity.