We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Clefts on protein surfaces are avoided by antigen-combining sites of conventional antibodies, in contrast to heavy-chain antibodies (HCAbs) of camelids that seem to be attracted by enzymes' substrate pockets. The explanation for this pronounced preference of HCAbs was investigated. Eight single domain antigen-binding fragments of HCAbs (VHH) with nanomolar affinities for lysozyme were isolated from three immunized dromedaries. Six of eight VHHs compete with small lysozyme inhibitors. This ratio of active site binders is also found within the VHH pool derived from polyclonal HCAbs purified from the serum of the immunized dromedary. The crystal structures of six VHHs in complex with lysozyme and their interaction surfaces were compared to those of conventional antibodies with the same antigen. The interface sizes of VHH and conventional antibodies to lysozyme are very similar as well as the number and chemical nature of the contacts. The main difference comes from the compact prolate shape of VHH that presents a large convex paratope, predominantly formed by the H3 loop and interacting, although with different structures, into the concave lysozyme substrate-binding pocket. Therefore, a single domain antigen-combining site has a clear structural advantage over a conventional dimeric format for targeting clefts on antigenic surfaces.antibody-lysozyme structures ͉ camel single domain antibody ͉ enzyme inhibitor ͉ epitope-paratope interactions S ix hypervariable antigen-binding loops constitute the antigen-combining sites of conventional antibodies. These loops, three (H1-H3) from the variable domain of the heavy chain (VH), three (L1-L3) from the variable domain of the light chain (VL) are juxtaposed forming a continuous surface (paratope) that is complementary to a surface on the antigen (epitope) (1). The paratope is essentially planar for protein antigens and forms a groove or cavity to interact with peptides and haptens (2, 3). The loops L1-L3 and H1-H2 fold into a limited number of canonical structure classes, determined by the loop length and the presence of conserved residues at key positions within the hypervariable and framework regions (4, 5). The extreme length and sequence variability of H3 makes the structure prediction of this loop extremely difficult (6).The structures of antigen-binding sites and loops, as well as the canonical loop determining residues, are well established (1,4,5). In contrast, the elucidation of the molecular basis for the recognition of particular epitopes by antibodies remains a major challenge. Our knowledge and paradigms of protein-epitope recognition by antibodies is largely based on the analysis of the immune response toward hen egg white lysozyme (HEWL). This is due to the high antigenicity, the large number of natural variants of HEWL (7), and the availability of eleven different crystal structures of Fab or Fv antibody fragments (8-10) in complex with lysozyme, collected over the last two decades. Six structures represent Fabs or Fvs that are clearly clonally unrelated (8)...

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...

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, ␤-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T m ‫ס‬ 60-80°C), and display high conformational stabilities (⌬G(H 2 O) ‫ס‬ 30-60 kJ mole −1 ). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.Keywords: Camel heavy-chain antibodies; protein stability; protein folding; circular dichroism; fluorescence; Fourier transform infrared spectroscopy; surface plasmon resonance; high pressure Article and publication are at

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII ␤-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those ␤-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 ␤-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of ␤-lactamases.

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Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::␤-lactamase fusion activating a prodrug.