Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features

Harmsen, Michiel M.; Ruuls, Robin; Nijman, Isaäc J; Niewold, Theodoor; Frenken, Leon; Geus, B. de

doi:10.1016/s0161-5890(00)00081-x

Cited by 197 publications

(166 citation statements)

References 50 publications

Supporting

Mentioning

157

Contrasting

Unclassified

Order By: Relevance

“…That more stringent selection for proteolytic stability results in the selection of clones from a single CDR group suggests that CDRs are the primary site of proteolysis. This is in accordance with the general view that CDRs form flexible surface-exposed loops (Padlan 1994;Harmsen et al 2000), which is a structure that is more susceptible to proteolysis (North 1989). However, the newly selected clones still varied considerably in proteolytic stability.…”

Section: Discussionsupporting

confidence: 90%

“…pUR4584 is a 2-μm-based LEU2-marked plasmid suitable for GAL7 promoter-controlled expression of VHHs fused to the yeast invertase signal peptide that does not encode a C-terminal c-myc tag but is further identical to pUR4585 (Harmsen et al 2000). VHH clones were transferred between pUR4676 and pUR4584 using the PstI and BstEII restriction sites.…”

Section: Phage Display Selection Of F4 Fimbriae Binding Vhhsmentioning

confidence: 99%

“…Single-domain antibody fragment encoding regions of pUR4676-derived plasmids were subjected to automated DNA sequencing as described previously (Harmsen et al 2000). The deduced VHH amino acid sequences were aligned according to the International ImMunoGeneTics Information System (IMGT) (Lefranc 2004) for alignment, numbering and complementarity-determining region (CDR) definition of immunoglobulins.…”

Section: Sequencing and Sequence Analysismentioning

confidence: 99%

“…Sequencing of their VHH encoding region revealed that clone K712 was isolated three times, and clone K910 was isolated four times, resulting in seven unique clones. The CDR3 of VHHs generally is most variable (Harmsen et al 2000) and is directly involved in antigen binding. The seven clones and the previously isolated clones K609 and K719 all had similar CDR3 sequences, and their overall sequence differed by only one to ten amino acid residues (Fig.…”

Section: Isolation Of F4 Binding Vhhs With Increased Proteolytic Stabmentioning

confidence: 99%

See 3 more Smart Citations

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

Harmsen

Solt

Bemmel³

et al. 2006

Appl Microbiol Biotechnol

107

View full text Add to dashboard Cite

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7-to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5-to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Phage Display Selection Of F4 Fimbriae Binding Vhhsmentioning

confidence: 99%

Section: Sequencing and Sequence Analysismentioning

confidence: 99%

Section: Isolation Of F4 Binding Vhhs With Increased Proteolytic Stabmentioning

confidence: 99%

See 2 more Smart Citations

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

Harmsen

Solt

Bemmel³

et al. 2006

Appl Microbiol Biotechnol

107

View full text Add to dashboard Cite

show abstract

“…19,20 Some camelid V genes may ‘promiscuously’ recombine with both heavy chain-only and conventional antibody constant region genes. 19 V H H domains bear unusually long CDR3 loops in comparison with human and murine conventional antibodies, 21,22 probably reflecting increased non-templated nucleotide addition, although this may be a feature of only a subset of V H Hs; 21 in some V H Hs, the long CDR3 loop serves a dual purpose, folding over the former V L interface as well as interacting with cognate antigen. The rearranged V H H exon is thought to undergo elevated rates of somatic hypermutation of both CDRs and FRs ( e.g ., FR1-encoding sequences immediately flanking CDR1; 23–25 FR2-encoding sequences which may play a role in structuring the CDR3 loop; 20,25 FR3-encoding sequences that form a β-turn which can make contact with antigen, sometimes called CDR4 24 ).…”

Section: Introductionmentioning

confidence: 99%

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Henry

MacKenzie

2018

mAbs

View full text Add to dashboard Cite

Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

show abstract

Single‐Domain Antibodies and Their Utility

2013

View full text Add to dashboard Cite

Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 ABSTRACTEngineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid heavy-chain antibodies. These nature-made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In this unit, we will present and discuss comprehensive and step-by-step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage-display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere.

show abstract

Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features

Cited by 197 publications

References 50 publications

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Single‐Domain Antibodies and Their Utility

Contact Info

Product

Resources

About