Recent advances in next-generation sequencing NGS have revolutionized the analysis of antibody repertoires by dramatically increasing sample depth compared to previous low-throughput methods 5. In addition, new methods have been developed for single-cell sequencing, which allow large-scale determination of paired L and H chains.

These advances, in conjunction with computational tools for reconstituting antibody clonal lineages, can provide a genetic record for the evolutionary processes of recombination and somatic hypermutation in immune responses to specific microbial pathogens. Crystal structures of affinity-matured and germ line antibodies from such lineages in complex with their target antigens have produced new insights into the molecular basis of affinity maturation.

Here, we first review our understanding of the basic biophysical principles underlying affinity maturation before the arrival of NGS. We then summarize what studies of bulk B cell populations by NGS have taught us about the general features of antibody repertoire selection.

Finally, we discuss structural studies of reconstructed antibody clonal lineages with special emphasis on the immune response to HIV-1, which has so far benefited most from the application of NGS and single-cell analysis to better understanding affinity maturation 6 Table 1. Table 1. Structural studies of antibody clonal lineages reconstructed using next-generation sequencing.

Before the advent of NGS, a number of structural studies were carried out comparing affinity-matured antibodies and their putative germ line precursors bound to the same antigen. In studies involving small molecules haptens such as phenyloxazolone and nitrophenyl phosphonate, rather than proteins, it was found that somatic mutations in complementarity-determining region CDR residues directly or indirectly implicated in binding hapten permit the formation of additional hydrogen bonds, electrostatic interactions, and van der Waals contacts 23 — Large changes in the conformation of the antigen-binding site paratope of 48G7g were observed upon hapten engagement by this germ line antibody, whereas the free and hapten-bound forms of affinity-matured 48G7 showed few structural differences.

Thus, affinity maturation in this case appeared to be driven largely by a mechanism of preorganizing the paratope into a conformation favorable for binding its hapten ligand Such conformational preorganization was accompanied by a decrease in the flexibility of the paratope during the maturation process, which may increase specificity for the target antigen while reducing the possibility of cross-reactivity with other antigens, including self-antigens 27 — The antibody maturation process appears to simultaneously select for both higher binding affinity and increased thermodynamic stability.

In a study of matured antibody 93F3, which recognizes a small hapten, somatic mutations in the paratope that increased affinity were found to reduce the melting temperature of 93F3 compared to its germ line precursor.

The first structural study of the maturation of an antibody response to a protein antigen, instead of a hapten, involved a set of closely related antibodies specific for hen egg white lysozyme HEL These antibodies represented different stages of affinity maturation, whereby the number of somatic mutations correlated with increasing affinity.

Surprisingly, improved affinity could not be attributed to the formation of additional hydrogen bonds or salt bridges or to an increase in total buried surface area. Instead, affinity maturation resulted mainly from burial of increasing amounts of hydrophobic surface at the expense of polar surface, accompanied by improved shape complementarity at the V H —HEL interface. The increase in hydrophobic interactions resulted from highly correlated structural rearrangements in antibody residues at the periphery of the interface with antigen, adjacent to the central energetic hot spot Indeed, the periphery may offer more suitable sites for optimization because these regions are typically more flexible and tolerant to mutations than central sites 12 , in agreement with the finding that somatic hypermutation spreads structural diversity generated by V D J recombination from central to peripheral regions of the antibody binding site Collectively, these structural studies showed that increased antibody affinity for small haptens or model protein antigens such as HEL can arise from any one or any combination of several variables, including additional interfacial hydrogen bonds or van der Waals contacts, conformational preorganization of the paratope, improved shape complementarity at the interface with antigen, or increased burial of total or hydrophobic surface area.

These same basic strategies, as well as others, govern affinity maturation of antibody responses to biological antigens such as the envelope glycoproteins of HIV-1 and other viral pathogens, as discussed below. We first summarize what NGS of bulk B cell populations has taught us about antibody repertoire selection.

We then discuss recent insights into affinity maturation gained from structural studies of antibody clonal lineages that were reconstructed using NGS Table 1. Next-generation sequencing of paired antibody L and H chains combined with computational modeling of antibody structures has been used to profile human antibody repertoire selection and maturation at the population level 5.

Repertoire-wide computational structure prediction was carried out to characterize the physiochemical properties of the antibody paratopes. Overall, however, the evolutionary processes of somatic hypermutation and affinity selection that occur in periphery blood did not leave a distinctive physiochemical imprint on the antigen-experienced antibody repertoire, even at the level of CDR3s, which are a major focus for somatic hypermutation.

By contrast, bone marrow B cells expressing antibodies with positively charged CDR3 loops undergo preferential elimination at discrete developmental checkpoints before entering the periphery, possibly as a mechanism for reducing the risk of self-reactivity In a study to determine whether NGS could be used to detect antigen-specific sequences in bulk B cell populations, an analysis of identical CDR3 sequences that were shared by individuals previously vaccinated against Haemophilus influenzae type b identified a number of sequences known to be specific for this bacterium Conserved CDR3 sequences were also observed in patients recovering from acute dengue infection 35 , indicating convergent antibody evolution in different individuals exposed to the same antigens.

In another study, NGS and single-cell sorting of peripheral blood plasmablasts were used to profile the acute antibody response to influenza A vaccination Antibodies able to neutralize the virus were selected bioinformatically from clonal families.

Next-generation sequencing coupled with bioinformatics analysis has allowed, for the first time, the reconstruction of antibody clonal lineages and inference of germ line progenitor sequences, neither of which was possible in earlier studies of affinity maturation However, an important caveat is that germ line sequences are predicted sequences that may differ from the true unmutated ancestor sequences.

In particular, it is impossible to know if insertions or deletions in these sequences took place during V D J recombination or were introduced during B cell affinity maturation. As a consequence, statistical methods must be used to infer the most likely unmutated common ancestor for an aligned set of sequences that are taken to be clonally related Although seemingly low, such affinities are nevertheless sufficient to trigger affinity maturation of unmutated B cells in vivo 40 , Moreover, this failure was observed even though the amino acid sequences of the V D J junctions of the affinity-matured antibodies were left unchanged in the reconstructed germ line versions.

One possibility is that maturation of anti-CD4 binding site bNAb precursors was triggered by non-HIV antigens and that the resultant antibody intermediates serendipitously cross-reacted with Env.

More likely, however, interactions between proteins in solution 3D affinity , as measured by SPR or related techniques, differ from those at contacts between two cells or between a cell and a virus 2D affinity Therefore, under physiological conditions, germ line precursors of anti-CD4 binding site bNAbs, expressed on the surface of B cells, might be engaged by membrane-anchored Env on the virion surface or on the surface of infected cells with sufficient affinity to trigger B cell maturation.

In support of this idea, the germ line precursors of several anti-influenza HA bNAbs were found to bind to HA only when presented on membranes in the form of cell surface IgMs; as soluble IgGs, these precursors had no detectable affinity for HA In the following sections, we have selected representative examples of affinity maturation in order to illustrate the multiple structural strategies that the antibodies employ to increase potency and breadth of pathogen neutralization.

The structure of affinity-matured CH58 in complex with an Env V2 peptide showed that this bNAb targets V2 residue Lys, which is a site of vaccine-induced immune pressure. Structures have also been determined for the putative germ line precursor of CH58 in unliganded form and bound to the V2 peptide 8. Such preorganization of the CH58 paratope into a configuration more suitable for binding V2, accompanied by rigidification of V L CDR3 to lower the entropic cost of complex formation, further contributes to the 2,fold affinity increase during maturation.

Paratope preorganization and rigidification have also been described for the CH65 lineage of anti-influenza virus HA antibodies 21 , underscoring the general utility of these mechanisms for improving affinity 26 — Figure 1. Affinity maturation through formation of additional interactions with antigen. V L is green; V2 is magenta.

V L is teal. V H is green; V2 is magenta. V H is teal. This residue was not visible in C due to disorder in the C-terminus of the V2 peptide. Importantly, the high variability and constantly evolving nature of HIV-1 Env distinguish this conformationally dynamic glycoprotein from the static model antigens used in studies of affinity maturation prior to NGS 23 — 26 , Dynamic regions of a protein take up deuterium more rapidly than stable regions.

In both cases, the paratopes of the matured bNAbs were less dynamic overall that those of their germ line counterparts, in agreement with previous evidence that paratopes become more rigid during the maturation process 28 , 30 , Surprisingly, however, the largest decreases in dynamics occurred at the periphery of the paratopes, at sites adjacent to Env glycans, rather than at primary Env-contacting sites.

This stabilization of the paratope periphery may serve to minimize potential clashes with nearby Env glycans, while maintaining critical binding interactions mediated by the center of the paratope.

It is probably not coincidental that a similar focus of affinity maturation on sites peripheral rather than central to the interface with antigen was also observed for anti-HEL antibodies Affinity maturation of CH is associated with mutations in both contacting and non-contacting residues, including framework FR residues distant from the interface with Env.

Displacement of V L away from V5 allowed accommodation of insertions in V5 without steric hindrance In addition, the conformation of V H CDR3 in the germ line precursor of CH is incompatible with gp binding, at least as observed in the CH—gp complex 13 , thereby necessitating rearrangement of this loop during the maturation process Figure 2.

Reorientation of V L and V H domains in response to viral escape mutations. During affinity maturation, a shift occurred in the orientation of V L with respect to V H. The shift is an adaptation to insertions in the gp V5 loop during infection. Movement of V L away from gp enables accommodation of the V5 insertion. In sharp contrast to bNAbs against HIV-1, which are highly mutated, the potent human anti-Middle East respiratory syndrome coronavirus antibody m is almost germ line, with only one somatic mutation in the H chain To systematically dissect the contribution of mutations in FR residues to affinity maturation, deep mutational scanning was applied to the anti-vascular endothelial cell growth factor antibody G6.

Besides high sequence variability, another feature of HIV-1 Env that distinguishes it from model antigens such as HEL used in previous studies of affinity maturation is glycosylation.

Indeed, extensive N-glycosylation masks much of the Env protein surface from antibody recognition. Nevertheless, a number of potent bNAbs have been discovered that penetrate this glycan shield and engage both carbohydrate and protein antigenic determinants 51 , In the most thoroughly studied example to date, the PGT family of bNAbs was shown to bind to N-glycans located in a high-mannose patch centered on the highly conserved Asn glycan and to protein elements at the base of the V3 loop 15 , As revealed by NGS and X-ray crystallography, the putative germ line precursor of the PGT family splits into two evolutionary branches that differ considerably in how they interact with Env glycans Figures 3 A,B.

This conservation suggests that a critical event in triggering the antibody response is simultaneous recognition of both carbohydrate and protein determinants.

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Affinity maturation is the process to improve antibody affinity for an antigen. In vivo , natural affinity maturation by the immune system takes place by somatic hypermutation and clonal selection. In vitro , in the laboratory affinity maturation, can be obtained by mutation and selection. Creative Biolabs has gained extensive experience in antibody affinity maturation.

We usually take scFv as the antibody format in affinity maturation. Also, a monovalent display phagemid system is used to reduce the avidity effects during antigen-binding screening. We also provide affinity maturation services for single domain antibodies. Two methods, untargeted mutagenesis and oligonucleotide-directed mutagenesis, are employed to construct random or defined sub-libraries to introduce a large number of mutants of the original antibody.

Antibody binders of higher affinity are then selected by increasing the screening stringency. We have successfully obtained a scFv antibody that has an extremely high affinity of 10 , whose binding to the antigen is essentially irreversible.

If the potential of introducing immunogenic mutations to framework positions is not a concern, we usually use this approach to create mutations at completely random positions across the entire V H and V L fragments. We are able to create any sub-libraries to incorporate the defined mutations using trimer codon technology. Most of the time, we need study the AA sequences of the antibody to find out the conserved sequences in comparison with the germ-line and antibody subfamily sequences.

We may then introduce mutations to the positions in the frame work regions that are not conserved. Supposedly, these regions will be antigen-specific and change in these regions may not increase immunogenicity. Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. Indeed, our results suggested that mutations to CDRH3 of the antibody, which formed a large part of the hydrophobic cleft, were not tolerated and were rapidly eliminated during selections of the unbiased recombination libraries.

There is existing evidence to suggest that feasible regions for affinity maturation are often not involved in key contacts, but lie in positions that provide indirect effects or establish fresh new interactions with its antigen [ 13—15 ].

Moreover, improved affinities can arise from multiple diverse mechanisms which are often unpredictable [ 15—17 ]. In vitro selections provide a way to probe the vast number of binding possibilities, with the potential to find the best available binding solution without requiring prior assumptions or dictation.

Given its utility, it is important that efforts are made to find the most suitable affinity maturation strategy for any given application. A diagram highlighting several features and considerations in the use of different technologies is shown in Figure 2. Generally, a key objective of affinity maturation is to maximise the sequence diversity of the initial library repertoire, as this would be translated to a higher structural diversity from which superior binders may be selected.

However, we are often limited by the display capacity of the technology used. Methods with a cellular requirement, such as phage and yeast display, have a lower display size typically 10 8 —10 9 due to limitations in transformation efficiency [ 18—23 ]. Besides the common compromise of limiting mutations to certain CDRs, there have been reports of alternative strategies to overcome this obstacle.

Tiller et. To accommodate early re-combinatorial changes, they used alanine scanning to narrow down the permissive sites for mutagenesis, before generating libraries with restricted amino acid variants based on natural CDR diversity. Based on amino acid usage analyses, Gonzalez-Munoz et al. This tailored diversification approach allowed them to cover more positional ground with fewer library builds, with comparable effectiveness to full amino acid randomisation.

A variety of technologies and methods with different strengths and limitations; features which may guide the design of an effective affinity maturation strategy. Cell-free systems such as ribosome and mRNA display have a much higher display capacity in the range of 10 12 —10 13 [ 26—29 ], and are favoured methods for the exploration of larger sequence space.

Direct comparisons of phage and ribosome display methods have suggested higher diversity and affinity gains in the outputs of the latter [ 30 , 31 ], which lends itself to a broader range of applications. A recent study described the use of insertion and deletion InDel mutagenesis to create large diversified libraries in the affinity maturation of an anti-IL antibody [ 32 ].

Random in-frame InDels were introduced using a transposon-based system and selected using ribosome display, which uncovered positions of tolerance and allowed for the exploration of loop length variation on maturation outcomes.

This is a particularly interesting application as increasing evidence have shown the importance of InDels in antibody maturation, both in vivo and in vitro [ 33—35 ]. Lengths of CDRs, particularly that of CDR3, vary considerably in nature [ 36—39 ], yet much of our focus during antibody engineering have tended to remain on point substitutions. There is evidence that unconventional loop lengths may confer advantages for antibodies against challenging antigens, such as G-protein-coupled receptors GPCRs [ 40 , 41 ] and rapidly evolving pathogens influenza and HIV-1 [ 35 , 42 ].

The likely expansion of structural or conformational diversity that may be attained through length diversification makes it an attractive strategy for affinity maturation. Ribosome display is also ideal for the selection of libraries diversified through DNA shuffling. In one example, this was achieved through the random digestion by DNAse I followed by enzymatic ligation to recombine point mutations accumulated from error-prone selections [ 43 ], with interesting parallels to our approach.

Such recombination and shuffling methods have the potential to eliminate deleterious mutations, as a result of backcrossing with original template DNA segments in the sequence pool.

Moreover, spontaneous mutations which occur through the numerous amplification steps through the selection and recovery cycles further adds to the diversity, promoting the simultaneous evolution of non-targeted regions, with favourable implications for directed evolution. Progressive or continuous diversification at the same time as selections is a clear advantage for in vitro maturation, as it allows for the gradual emergence of epistatic and synergistic mutations, lessening the demand on the diversity of the initial library repertoire.

This is exemplified in the case of mammalian cell display, which, despite having a small display size, can be induced to diversify in vitro through the addition of activation-induced cytidine deaminase AID [ 44—47 ].

Full-length IgG or Fabs are typically expressed on the mammalian cell surface, and between rounds of selections and sorting, AID can be introduced to induce somatic hypermutation in situ , without further requirements for reformatting or library builds. This can be viewed as an evolving library; with diversification concurrent with selections. Advancements in the use of gene editing techniques have also allowed for a more directed approach. A recent report describes the use of TALE nucleases and CRISPR-Cas9 to promote site-specific integration of antibody gene populations; allowing for the creation of large diversified libraries which were successfully used to affinity mature a PD1-blocking antibody using mammalian cell display [ 48 ].

The increased capacity for diversification is particularly advantageous for optimisation strategies seeking to cover a broader ground. While it is widely established that CDRs are major determinants of antigen recognition, there is evidence to suggest that framework FR regions may also play an important role.

Other FR regions in the VH and VL domains may also contribute to antigen binding, through direct antigen contact or distal effects [ 53—55 ]. Pairings of different VH and VL frameworks has been shown to affect antigen and Fc receptor FcR engagement [ 56 ], and conversely isotype selection for the constant region can also influence antigen binding [ 57—59 ]. Such observations remind us to think of an antibody as a whole protein during engineering, with interconnected domains that can exert influence on each other [ 60 ].

The numerous display and maturation strategies, each with their own unique features, provide us with an extensive toolkit to fulfil our antibody engineering needs. Exploration of a larger experimental and sequence space has the clear ability to provide a wider range of conceivable binding solutions, with the potential to deliver greater improvements.

On the other hand it can also lead to increased functional divergence or thermostability trade-offs, which may require additional screening steps or compensatory mechanisms [ 61 ]. As exemplified by the many different approaches devised in response to different challenges, it is about choosing the right strategy for the antibody lineage under examination.

Assessment of antigenic properties, choice of an optimal diversification strategy paired with appropriate display and screening methods, adaptation of selection strategy as the protein evolves; these are all important considerations which are key to the success of affinity maturation.

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Author Contribution. Article Navigation. Perspective March 04 Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies Denice T.