RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

492

824

Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotypematched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.monoclonal antibody | developability | biophysical properties | manufacturability | nonspecificity T arget binding is the predominant first concern in development of any drug. However, once a lead molecule attains the desired potency of biological modification, a suite of characteristics termed "developability" assumes critical importance. For monoclonal antibodies, these properties include high-level expression, high solubility, covalent integrity, conformational and colloidal stability, low polyspecificity, and low immunogenicity. The high cost of failing any of these criteria at a late stage in drug development has led to considerable efforts at predicting developability on the basis of sequence motifs and experimentally determined biophysical properties (1-15).In a landmark study of small-molecule drugs over 2,000 molecules with United States Adopted Names (USAN) designations and known to have oral availability were collected and computationally analyzed (16). A simple set of thresholds, encapsulated as the "Lipinski rule of fives," was formulated and has been used by many to prioritize small molecules for entry into clinical development. To date, analogous guiding principles for antibody drugs have not emerged-we therefore endeavor here to do so. By analogy to the Lipinski effort, we first collected the sequences of antibodies that had reached at least phase-2 trials and had USAN or WHO International Nonproprietary Names (INN) designations (137 in total as of the start of this project). As a common basis for comparison of intrinsic variable domain phenotypes we expressed each antibody as the human IgG1 isotype and formulated them in simple Hepes-buffered saline. Each antibody was then subjected to a battery of 12 different biophysical assays in common use for developability assessment.Unexpectedly, for many of the measures the distribution of values was not symmetrically Gaussian, but instead was lon...

Section: Significancesupporting

confidence: 78%

Biophysical properties of the clinical-stage antibody landscape

Jain

Sun

Durand

et al. 2017

492

824

“…ERs for all mutations were calculated by dividing the frequency of a given mutation at a given position in the sorted sample by the frequency of the same mutation in the unsorted sample, as described previously (25,26). Mutagenesis of one segment of V H (position [14][15][16][17][18][19][20][21][22][23][24][25] in the original V H library resulted in errors which led to low expression of the respective segment. This result was likely related to a mutational error in the original stop template used in the mutagenesis reaction for this V H segment 14-25, which affected expression of the variants.…”

Section: Methodsmentioning

confidence: 99%

“…Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).…”

mentioning

confidence: 99%

“…Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15)(16)(17)(18)(19)(20)(21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

“…In addition, we are still learning about how the V regions can undergo such high rates of mutation and still assemble their heavy and light chain V regions to produce a stable antigen-binding site that can undergo affinity maturation and changes in fine specificity. Although it is clear that mutations in the complementary determining regions (CDRs) play a critical role in antigen binding, recent studies reveal that some of these highly mutated sites in the CDRs do not directly interact with antigen but have an important role in generating protective broadly neutralizing antibodies to influenza and HIV (1,30,31).Taken together, these findings suggest that the DNA sequence of Ig V regions has evolved so that AID will mutate certain subregions while protecting those parts of the V region that are required to …”

mentioning

confidence: 99%

Overlapping hotspots in CDRs are critical sites for V region diversification

Wei

Chahwan

Wang

et al. 2015

Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.A fter an encounter with antigen and subsequent migration into the germinal centers of the secondary lymphoid organs, B cells undergo a regulated cascade of mutational events that occur at a very high frequency and are largely restricted to the variable (V) and switch (S) regions of the Ig heavy chain locus and the V region of the light chain locus. These mutagenic events are responsible for the somatic hypermutation (SHM) of the V regions and the class switch recombination of the constant (C) regions that are required for protective antibodies (1, 2). Both SHM and class switch recombination are initiated by activationinduced deaminase (AID) that preferentially deaminates the dC residues in WRC (W = A/T, R = A/G) hotspot motifs at frequencies 2-10-fold higher than SYC (S = G/C; Y = C/T) cold spots (3-7). During V region SHM, the resulting dU:G mismatch can then be replicated during S-phase to produce transition mutations, be processed by uracil-DNA glycosylase 2 and apurinic/ apyrimidinic endonucleases through the base excision repair pathway to produce both transitions and transversions (8-10), or be recognized by MutS homolog (MSH)2/MSH6 of the mismatch repair (MMR) complex that recruits the low-fidelity polymerase eta (Polη) to generate additional mutations at neighboring A:T residues (11).The specificity of AID targeting to the Ig gene has been under intense investigation. Studies have shown that AID deamination and mutagenesis targets single-stranded DNA substrates generated during ...