Comparison of llama VH sequences from conventional and heavy chain antibodies

Abstract

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.