High-affinity lamprey VLRA and VLRB monoclonal antibodies

Abstract

Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and beta-galactosidase (beta-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, beta-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications.

Fig. 1.

Antigen binding by VLRB antibodies. (A) Plasma from HEL-immunized adult lamprey sampled and boosted as indicated. Direct ELISA with 1 μg/well of HEL, anti-VLRB mAb for detection, and streptavidin as a control. (B) The pYSD2 vector. VLR were directionally cloned in 2 different SfiI sites, between the Flo1p leader and C terminus, under control of the GAL1 promoter. An intraplasmid homologous recombination cassette consisted of two 49-nucleotide direct repeats separated by a PmeI restriction site, for plasmid linearization. Primers HR.F and HR.R served for rolling-circle amplification across the plasmid. (C) Yeast surface display of VLR fused to the Flo1p anchor. The HA-tag served for VLR detection via Alexa 488-conjugated antibodies. Biotinylated ligands were detected via SAPE. (D) Enrichment of HEL-binding VLRB clones from HEL-immunized larval library. From left to right: the unsorted library, enrichment by antibiotin magnetic microbeads (with MACS), sorting of the double-positive cells in the gate, output of the first sort, output of the second sort, and the resulting clones. (E). Comparison of naïve and immune YSD libraries enriched for binders of β-gal, CTB, and B-trisaccharide. A representative clone for each antigen is shown.

Fig. 2.

Affinity maturation in vitro of VLRB antibodies. (A) The sequence of VLRB.HEL.2D aligned with 5 mutant clones selected after in vitro random mutagenesis. Dots indicate identity to the top sequence. The LRRCT hypervariable loop region is shaded yellow. (B) Sorting LRRCT loop-swapped mutants. The second MACS output was labeled with 1 nM HEL, and a representative mutant clone VLRB.CTMut.5 was stained with HEL as indicated. (C) The sequence of VLRB.HEL.1 aligned with mutant clone VLRB.CTMut.5. The swapped LRRCT region is delineated by PCR primers (forward, reverse). (D) SPR sensogram of the interaction between immobilized NeutrAvidin-biotin-VLRB.CTMut.5 with 2-fold HEL serial dilutions (7.66–0.00299 nM). K_D = 119 pM. RU, resonance units.

Fig. 3.

Monoclonal VLRA antibodies cloned from a HEL-immunized adult lamprey. (A) Sorting anti-HEL VLRA. The second MACS output was labeled with 20 nM HEL. Clones were sorted using gates R2–R4. (B) Protein sequence alignment of 13 anti-HEL VLRA; only residues 3–246 are shown. Tiled below: germline VLRA gene portions (boxes 1 and 12) and the corresponding genomic LRR cassettes (boxes 2–11; 3a and 3b alternative cassettes). Dots indicate identity to the top sequence. (C) VLRA YSD antigen titrations. Normalized mean fluorescence intensity of biotin-HEL-SAPE plotted against 2-fold serial HEL dilutions (for R2.1, 10–0.025 nM; for R2.6, 100–0.25 nM; for R4.9, 200–0.5 nM). Bars indicate SEs of triplicate samples. (D) Neighbor-joining tree of the VLRA nucleotide sequences. The corresponding K_D values calculated by YSD antigen titration are given in parentheses.