In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Powerful selection technologies have made in vitro evolution of protein binders more efficient and paved the way for the use of tailor-made antibodies in therapy. After initial selections of antibody candidates with desired specificity, lead antibodies are typically improved by affinity maturation in multiple rounds of randomization and selection (1) to reach the subnanomolar affinities ideally required for targeting soluble ligands (2–4). This is usually attempted by introduction of point substitutions, either at random positions across the entire V-gene (5, 6) or in the complementary-determining regions (CDRs; e.g., by CDR walking mutagenesis) (7).In Nature, diversification of the primary antibody repertoire occurs by several mechanisms that generate variation in the regions forming the antigen-binding site, the CDRs, including considerable length variation (8–11) that is initially introduced by recombination of V(D)J gene segments. Length variations are concentrated in the CDR3 region (12), at the junctions of the joined segments, where additional diversity is produced by N- or P-nucleotide additions that can further extend the CDR3. The length of the CDRs considerably affects the topography of the combining site, as different shapes brought about by extension or shortening can form pockets, grooves, or fill space (13, 14).Following B cell stimulation by the antigen, further diversification of the antigen-binding interface is generated through somatic hypermutation (SHM) (15), involving mainly point mutagenesis that preferentially targets hotspots in the CDRs (16, 17). This process is initiated through deamination of cytosine to uracil by activation-induced cytidine deaminase (AID), leading to uracil:guanine mismatches (16). Upon removal of these uracil bases by base excision-repair enzymes, error-prone DNA polymerases are then recruited to fill in the gaps and introduce mutations around the position of the deaminated cytosines. Interestingly, up to 6% of the mutations generated by SHM are insertions and deletions (InDels) (18), which occur due to misalignment of repeated DNA sequences (19, 20). Thus, insertions occur by duplication, while deletions are brought about by removal of repeated sequences (21, 22).A small percentage of antibodies selected by in vivo SHM contain InDels in the CDRs 1 and 2 (1.6 to 6.5%) (21–24), while junctional diversity by N- or P-nucleotide additions in the CDR3 confounds the analysis of SHM-derived InDels, leading to an underestimation of the total percentage of affinity-improving InDels. In vitro-directed evolution has been unsuitable for introduction of InDels at random positions into an antibody gene, because of restrictions in the diversity of InDels that could be introduced (i.e., insertions by duplication in in vitro SHM) (22, 25). Rational (26) or computational (27) strategies have been successful at introducing InDels in a few, carefully chosen positions instead of random sampling. In contrast, an unusually high percentage of InDels with a functional role among in vivo affinity matured broadly neutralizing antibodies (bnAbs) to HIV-1 (28–30): ∼40% of the reported anti–HIV-1 bnAbs contain InDels that accumulate during in vivo SHM (28). Based on the frequent occurrence of InDels among multispecific, cross-reactive antibodies, one could infer that they provide a molecular solution for recognizing multiple targets by providing an altered interface (enlarged or tightened), possibly even involving conformational diversity (31). The accumulation of InDels in bnAbs has been attributed to extensive in vivo SHM, so that even positions that are rarely modified by SHM are also altered (17, 28).Insertions in the V-genes occur only by duplication of adjacent sequences (21, 22), so that the actual sequence diversity of the resulting insertions is limited because they repeat existing modules. To introduce more diversity in the inserted sequences, point mutations are required in subsequent rounds of SHM. However, since the CDRs can tolerate considerable length variation, it is likely that the antibody fold can accommodate a larger number of affinity-enhancing InDels compared to those observed in antibodies affinity-matured by SHM.Affinity gains by introduction of InDels have indeed been recognized (22, 25, 26, 32, 33) in in vitro-directed evolution, but often were by-products of campaigns focused on point mutations and not elicited systematically (32, 33). Only in mammalian cell surface display does the action of AID lead to InDels, just as AID brings about InDels in SHM in vivo (22, 25). In a seminal study by Bowers et al. (22), overexpression of AID enabled in vitro SHM of 53 antibodies against 21 antigens to identify InDels in multiple regions likely to improve binding, in particular to variable heavy domain (V_H) and variable light domain (V_L) CDR1, where 9 of 53 antibodies contained InDels. Despite the comprehensive nature of this study, AID-enabled insertions mirrored in vivo SHM and were therefore limited to direct duplication of adjacent sequences, not allowing the full exploration of length and sequence diversity in the insertions, and the low frequency of incorporation of in-frame InDels by AID (<0.1%) limited the combinatorial diversity explored. Finally, InDels have been introduced rationally based on structural analysis and natural length variation (26, 27). Taken together, only limited diversity of InDels in terms of length, position, and insert sequence across the variable domains has been explored thus far.Here we address this omission and explore libraries with in-frame InDels of different lengths and high diversity of inserted sequences at random positions across the entire antibody variable regions (Fig. 1). We applied a new transposon-based mutagenesis approach, dubbed TRIAD (transposition-based random insertion and deletion mutagenesis) (34) that introduces short in-frame insertions and deletions randomly across a gene (in sequences of steps following transposition that excise the transposon, religate the plasmid, and insert designed cassettes) (SI Appendix, Figs. S1 and S2). TRIAD was used here to build libraries with InDels at random positions across an entire single-chain variable fragment (scFv) gene. The antibody chosen for this campaign was the anti–IL-13 antibody BAK1 (35), a derivative of which, tralokinumab, is under clinical investigation for asthma (36). In addition, we built libraries that explore diversity in the different lengths of insertions in a semirandom approach, insertional-scanning mutagenesis (InScaM). These InDel libraries were starting points for antibody affinity evolution in vitro, leading to insertions in two loops that, together with two previously known point mutations, brought about a 256-fold affinity improvement. The observation of alternative routes to affinity maturation validate our strategy and suggest that InDel mutagenesis can complement existing approaches.Open in a separate windowFig. 1.Overview of the affinity maturation of the antibody BAK1 by transposon-based TRIAD and subsequent insertional scanning mutagenesis. TRIAD (Left) was applied to make libraries with deletions of one to three amino acids (step 1a) or single amino acid insertions (step 1b) at random positions across the scFv gene. These libraries were recombined (step 2) and four rounds of ribosome display selections for improved affinity to IL-13 were carried out by panning (step 3). The best binder was carrying an insertion in the V_L FWR3 (BAK1-INS1). Scanning (Right) was used to guide the design of libraries with different lengths of insertions at targeted positions. A fraction of the insertion library generated in step 1b (5,632 variants) was screened by HTRF to identify variants with insertions that retained binding to IL-13 (step 4). Based on sequencing analysis, regions able to tolerate single amino acid insertions were identified (Fig. 4) and the V_L CDR3 was chosen for targeted insertional mutagenesis. Libraries with zero to five amino acid insertions in targeted positions in the V_L CDR3 were constructed (step 5), followed by four rounds of phage display selections for improved affinity to IL-13 (step 6).