Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies

Abstract

Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data.

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

“…For instance, its molecular weight is only ~ 15 kDa and it can penetrate the blood brain barrier. And it can be produced with low cost and high yield and stability in not only yeast, but bacteria as well [2,19]. Recently, the first nanobody drug, Caplacizumab, was approved by the European Commission.…”

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

“…8 Most camelid V H H and V H genes 18 are homologous to human IGHV3-family genes (~75–90% identity) and encode distinctive solubilizing residues in FR2 (Phe/Tyr42, Glu49, Arg50 and Gly 52 using IMGT numbering; these positions map to the V H :V L interface in conventional antibodies), although functional V H Hs lacking this consensus have been isolated. 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.…”

“…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 ). V H Hs may also acquire somatic insertions and deletions at higher rates than conventional antibodies, 24 and may under some circumstances undergo secondary rearrangement events using a cryptic recombination signal sequence in FR3.…”

Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naïve nurse sharks

“…The lengths of V H H CDR3 are much longer than conventional VH from camel IgG. The median CDR3 length of V H H is 20 amino acids (similar with shark V NAR ), whereas median length for camel VH CDR3 is only 15 amino acids [32]. However, there is no comprehensive study on the cysteine numbers and locations in camel V H H sequences for comparison with shark V NAR .…”