Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

Abstract

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule's most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding.

Keywords: binding specificity; heavy-chain complementarity-determining region 3; inverse PCR; rearrangement; single-chain Fv display.

Figure 1

Yeast display sorting and analysis of CDK2-specific antibodies. (A) After two rounds of phage selection, the antibody population was displayed on yeast cells and enriched for CDK2-specific binders by two rounds of flow cytometry-assisted sorting performed at 100 nM. (B) Abundance distribution of the selected anti-CDK2 clones as identified by their heavy-chain complementarity-determining region 3 (HCDR3s) by next-generation sequencing.

Figure 2

Sequences and affinities of single-chain Fvs with an identical heavy-chain complementarity-determining region 3 (HCDR3). Inverse PCR of the most abundant clone of the selected population was analyzed by Sanger sequencing. (A) Sanger sequence analysis of the eight unique clones with identical HCDR3 obtained by inverse PCR. (B) VDJ gene usage and affinity values of the eight different clones.

Figure 3

Analysis of the naïve library for single-chain Fvs (scFvs) with identical heavy-chain complementarity-determining region 3 (HCDR3s). Yeast clones displaying scFvs from the HCDR3-specific mini-library obtained from the naïve library via inverse PCR were analyzed by flow cytometry. Cells expressing scFvs on the surface and detected by anti-SV5-PE antibody were gated and sorted (left panel). Once grown and analyzed, they showed an increase in the expressing population (middle panel) with a limited, but detectable, binding for biotinylated CDK2 detected by streptavidin-APC conjugation (right panel).

Figure 4

Analysis of antibody sequences obtained by inverse PCR. (A) The alignment of antibody sequences with the same heavy-chain complementarity-determining region 3 (HCDR3) from naïve and selected libraries shows differences in VH gene sequences. This representation, a so-called highlighter plot, indicates amino acid mutations in the sites that differ from the first sequence. The alignment was generated with the Highlighter Tool (hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter_top.html). (B) The sequences of the antibodies sharing the same HCDR3 obtained by inverse PCR from the population enriched for specific antigen binders were phylogenetically clustered to show relations among variants that share VH alleles. (C) Single-chain Fvs (scFvs) sharing the same HCDR3 but obtained from either the selected or the naïve population were displayed on yeast and their binding activity was measured by flow cytometry and reported in the histogram as percentage of binding. None of the scFvs isolated from the naïve library bound CDK2, as opposed to the ones from the selected population.