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Review
, 198 (3), 157-74

Single Domain Antibodies: Promising Experimental and Therapeutic Tools in Infection and Immunity

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Review

Single Domain Antibodies: Promising Experimental and Therapeutic Tools in Infection and Immunity

Janusz Wesolowski et al. Med Microbiol Immunol.

Abstract

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.

Figures

Fig. 1
Fig. 1
Distinguishing structural features of camelid and shark hcAbs. Conventional Abs composed of heavy (H) and light (L) chains are found in all vertebrates. Heavy chain antibodies are found in camelids and sharks. Immunoglobulin domains are depicted as ellipsoid domains. Interchain disulfide bonds are indicated by black lines, glycosylation sites as hexagons. The antigen-binding paratope (P) of conventional Abs is formed by the variable domains of heavy and light chains (VH and VL), while the paratope of heavy chain Abs is formed only by the heavy chain variable domain, which is designated VHH in camelid hcAbs and VNAR in shark IgNARs. VH and VL domains in conventional Abs display hydrophobic binding interactions, while the corresponding region in hcAbs is hydrophilic (depicted in pink). Note that in camelid hcAbs the V-domains are directly connected to the hinge region, owing to the lack of the CH1 domain. Camelid hcAbs come in two variants, distinguished on the basis of the lengths of their hinge region and designated as long- and short-hinge isotypes (as depicted schematically in the diagrams)
Fig. 2
Fig. 2
Schematic diagram of the VHH domain of a camelid heavy chain antibody. a The three complementarity determining regions (CDRs) of the antigen-binding paratope are depicted as colored loops: CDR1 red, CDR2 green, and CDR3 blue. b The canonical disulfide bond connecting framework regions 1 and 3 (FR1 and FR3) in the two β-sheets of the immunoglobulin domain is indicated in yellow. Many camelid antibodies contain an additional disulfide bond (S–S) connecting the CDR3 with the end of the CDR1 (camels) or the beginning of the CDR2 (llamas). h Hinge, M transmembrane domain of membrane isoform, G glycosylation site, S stop codon of secretory isoform
Fig. 3
Fig. 3
3D-structures of enzyme-inhibiting sdAbs derived from camel and shark hcAbs. Images were generated with the PyMOL program [123]. The three CDR loops are color-coded as in Fig. 2: CDR1 red, CDR2 green, CDR3 blue, and disulfide bonds are depicted in yellow. a Chicken lysozyme in complex with an inhibitory shark sdAb VNAR (pdb code 1t6v). The VNAR contacts the enzyme only with its CDR1 and CDR3 regions, the latter extends into and blocks the active site crevice [124]. b Chicken lysozyme in complex with an inhibitory camel sdAb (pdb code 1mel). The CDR3 extends into and fills out the active site crevice of the enzyme [5]. c Chicken lysozyme in complex with its substrate (pdb code 1lsz) [125]. d Chicken lysozyme in complex with the VL and VH domains of a conventional mouse mAb (pdb code 1mlc). The flat interaction surface with the conventional antibody lies outside of the active site crevice [126]
Fig. 4
Fig. 4
Amino-acid sequence alignment of heavy chain variable domains of conventional (VH), camelid (VHH), and shark (VNAR) antibodies. Amino acids are color-coded as in previous figures: CDR1 red, CDR2 green, CDR3 blue, hydrophilic motif in FR2 pink, and cysteine residues yellow. The sequences are derived from the anti-lysozyme VH (mAbVH), VHH (cAbLys3), and VNAR domains shown in Fig. 3a, b, and d. VHH s+16a is an enzyme-blocking sdAb derived from a llama immunized with the murine T-cell ecto-enzyme ART2.2 [29]
Fig. 5
Fig. 5
Schematic diagram of the M13 phage display vector used for cloning libraries of the VHH and VNAR repertoires of immunized camelids and sharks. The open reading frame of the V-domains from hcAbs are PCR amplified from cDNA generated from lymphocytes purified from peripheral blood, lymph node, or spleen cDNA of an immunized animal, typically obtained 4–14 days after the final boost. The purified PCR amplification products are cloned into the phagemid vector downstream of an inducible bacterial promoter (arrow), in-frame behind a periplasm signal sequence (BL = bacterial leader peptide) and upstream of one or two epitope tags (T), an amber stop codon followed by the coding sequence of the M13 phage head capsid protein gIIIp. The phagemids are transfected into an E. coli strain that can read through the amber stop codon. Following infection with a helper phage, libraries of recombinant phage particles are harvested from bacterial culture supernatants and phages displaying sdAbs of interest are selected by panning on immobilized antigen. Bound phages are subjected to one or more additional rounds of selection. Phagemids are recuperated from single colonies of infected E. coli and the cDNA insert subjected to sequencing
Fig. 6
Fig. 6
Expression and purification of sdAbs. Single domain antibodies carrying C-terminal epitope tags are readily generated following transfection of the phagemids into an E. coli strain that recognizes the amber stop codon. sdAbs can be purified from periplasmic lysates by affinity chromatography, e.g., on immobilized nickel ions, as illustrated in this Coomassie stain of a llama VHH (lanes 3 and 4) and of a shark VNAR (lanes 7 and 8) directed against the lymphocyte ecto-enzyme CD38. Periplasmic lysates obtained from the cell pellets of 100 ml E. coli cultures were subjected to affinity chromatography on 1 ml Ni-NTA columns. Aliquots of protein suspensions obtained during purification were size fractionated by SDS-PAGE and stained with Coomassie. Lanes 1 and 5: total proteins in periplasmic lysates as loaded onto the column, lanes 2 and 6: column flow through, lanes 34 and 78 proteins eluted from the Ni-NTA column by imidazole. The protein yield from the 100 ml E. coli culture was 0.5–1 mg of sdAb. M = molecular weight markers, size indicated in kilodalton (kD)
Fig. 7
Fig. 7
Schematic diagram of various formats of recombinant sdAbs. a Monovalent sdAbs can be obtained directly from E. coli transfected with phagemids selected as illustrated in Fig. 6. These sdAbs can be used to block enzymes, such as T-cell ecto-ADP-ribosyltransferase ART2.2, here symbolized by the Pacman [29]. b Monovalent sdAbs can be conjugated chemically to radioisotopes, fluorochromes, biotin, magnetic beads, affinity matrices, etc. Radiolabeled sdAbs can be used for in vivo imaging of tumors [43]. c Fusion of sdAbs to other proteins, such as green fluorescent protein (GFP) [127, 128] or the trypanolytic serum protein apolipoprotein A [101] is accomplished by recloning of the PCR-amplified insert into appropriate expression vectors. d Bivalent sdAbs are generated by tandem cloning of two V-domains separated by a flexible linker peptide of 12–20 amino acids [13]. e The same strategy can be applied to fuse two distinct sdAbs, e.g., one encoding an albumin-specific sdAb, the other encoding a cytokine-specific sdAb [16]. Inclusion of an albumin-specific sdAb results in a dramatically increased in vivo half-life upon binding to albumin. f A bivalent hcAb format can be regenerated by fusion of the V-domain to an IgG-Fc-domain [77]. The increased avidity is useful for diagnostic purposes. Moreover, the hcAb format has a dramatically increased serum half-life, which can be of great value for certain therapeutic applications. g Fusion of an sdAb to a bacterial outer membrane protein yields recombinant bacteria exposing hundreds of copies of the sdAb on the cell surface, as in the illustrated example of a rotavirus-specific VHH fused to an outer membrane protease of a commensal gut bacterium [17]. h Intracellular sdAbs are generated by recloning the VHH (without the leader peptide) into a eukaryotic expression vector. Transfection of cells with an SpvB-specific sdAb protects cells from the cytotoxic action of the Salmonella SpvB ADP-ribosyltransferase (here indicated by the Pacman). Targeting to organelles can be achieved by fusion of appropriate targeting sequences (not shown) [19, 20]. i sdAbs directed against multimeric lumazine synthase (LS) can be used for the construction of highly immunogenic molecular assembly vaccines. This could be used to enhance the antibody response against an ADP-ribosyltransferase (here illustrated as a Pacman) fused to an LS-specific sdAb. j sdAbs directed against the idiotype of a conventional antibody can be used for molecular mimicry of the target antigen [64]
Fig. 8
Fig. 8
3D-structures of leukocyte ecto-enzymes and toxin enzymes with active site crevices targeted by sdAbs. a Murine T-cell ecto-ADP-ribosyltransferase ART2.2 with substrate NAD (pdb code 1og3). b Human lymphocyte ecto-NAD-glycohydrolase CD38 with substrate NAD (pdb code 2i65). cSalmonella enterica virulence protein SpvB, an actin ADP-ribosyltransferase, with NAD (pdb code 2gwl). dClostridium difficile toxin B, a rho glucosyltransferase (pdb code 2bvl). Images were generated with the PyMOL program [123]. We have already generated enzyme-blocking sdAbs against ART2.2 and SpvB from immunized llamas. Current work aims at generating similar antibodies against CD38 and Toxin B from immunized llamas and sharks

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References

    1. {'text': '', 'index': 1, 'ids': [{'type': 'PubMed', 'value': '12509759', 'is_inner': True, 'url': 'http://pubmed.ncbi.nlm.nih.gov/12509759/'}]}
    2. Brekke OH, Sandlie I (2003) Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat Rev Drug Discov 2(1):52–62. doi:10.1038/nrd984 - PubMed
    1. {'text': '', 'index': 1, 'ids': [{'type': 'PubMed', 'value': '8502296', 'is_inner': True, 'url': 'http://pubmed.ncbi.nlm.nih.gov/8502296/'}]}
    2. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448. doi:10.1038/363446a0 - PubMed
    1. {'text': '', 'index': 1, 'ids': [{'type': 'PubMed', 'value': '11526908', 'is_inner': True, 'url': 'http://pubmed.ncbi.nlm.nih.gov/11526908/'}]}
    2. Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74(4):277–302 - PubMed
    1. {'text': '', 'index': 1, 'ids': [{'type': 'PubMed', 'value': '16151406', 'is_inner': True, 'url': 'http://pubmed.ncbi.nlm.nih.gov/16151406/'}]}
    2. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23(9):1126–1136. doi:10.1038/nbt1142 - PubMed
    1. {'text': '', 'index': 1, 'ids': [{'type': 'PubMed', 'value': '8784355', 'is_inner': True, 'url': 'http://pubmed.ncbi.nlm.nih.gov/8784355/'}]}
    2. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F, Hamers R, Muyldermans S, Wyns L (1996) Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol 3(9):803–811. doi:10.1038/nsb0996-803 - PubMed

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