Predicting deamidation and isomerization sites in therapeutic antibodies using structure-based in silico approaches.

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.

How can we discover developable antibody-based biotherapeutics?

Antibody-based biotherapeutics have emerged as a successful class of pharmaceuticals despite significant challenges and risks to their discovery and development. This review discusses the most frequently encountered hurdles in the research and development (R&D) of antibody-based biotherapeutics and proposes a conceptual framework called biopharmaceutical informatics. Our vision advocates for the syncretic use of computation and experimentation at every stage of biologic drug discovery, considering developability (manufacturability, safety, efficacy, and pharmacology) of potential drug candidates from the earliest stages of the drug discovery phase. The computational advances in recent years allow for more precise formulation of disease concepts, rapid identification, and validation of targets suitable for therapeutic intervention and discovery of potential biotherapeutics that can agonize or antagonize them. Furthermore, computational methods for de novo and epitope-specific antibody design are increasingly being developed, opening novel computationally driven opportunities for biologic drug discovery. Here, we review the opportunities and limitations of emerging computational approaches for optimizing antigens to generate robust immune responses, in silico generation of antibody sequences, discovery of potential antibody binders through virtual screening, assessment of hits, identification of lead drug candidates and their affinity maturation, and optimization for developability. The adoption of biopharmaceutical informatics across all aspects of drug discovery and development cycles should help bring affordable and effective biotherapeutics to patients more quickly.

Embedding Dynamics in Intrinsic Physicochemical Profiles of Market-Stage Antibody-Based Biotherapeutics.

Adequate stability, manufacturability, and safety are crucial to bringing an antibody-based biotherapeutic to the market. Following the concept of holistic in silico developability, we introduce a physicochemical description of 91 market-stage antibody-based biotherapeutics based on orthogonal molecular properties of variable regions (Fvs) embedded in different simulation environments, mimicking conditions experienced by antibodies during manufacturing, formulation, and in vivo. In this work, the evaluation of molecular properties includes conformational flexibility of the Fvs using molecular dynamics (MD) simulations. The comparison between static homology models and simulations shows that MD significantly affects certain molecular descriptors like surface molecular patches. Moreover, the structural stability of a subset of Fv regions is linked to changes in their specific molecular interactions with ions in different experimental conditions. This is supported by the observation of differences in protein melting temperatures upon addition of NaCl. A DEvelopability Navigator In Silico (DENIS) is proposed to compare mAb candidates for their similarity with market-stage biotherapeutics in terms of physicochemical properties and conformational stability. Expanding on our previous developability guidelines (Ahmed et al. Proc. Natl. Acad. Sci. 2021, 118 (37), e2020577118), the hydrodynamic radius and the protein strand ratio are introduced as two additional descriptors that enable a more comprehensive in silico characterization of biotherapeutic drug candidates. Test cases show how this approach can facilitate identification and optimization of intrinsically developable lead candidates. DENIS represents an advanced computational tool to progress biotherapeutic drug candidates from discovery into early development by predicting drug properties in different aqueous environments.

SARS-CoV-2 antibody kinetics in blood donors with a previously positive SARS-CoV-2 antibody test within a seroprevalence survey.

The persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is a matter of importance regarding the coronavirus disease 19 (COVID-19) pandemic. To observe antibody dynamics, 105 blood donors, positive for SARS-CoV-2 antibodies by a lateral flow test within a seroprevalence study, were included in this study. Thirty-nine (37%) of 105 the donors were confirmed positive by a total Ig Wantai enzyme-linked immunosorbent assay (ELISA). Three (8%) in this group of 39 reported severe and 26/39 (67%) mild to moderate COVID-19 symptoms. By further ELISA-testing, 33/39 (85%) donors were initially positive for IgG antibodies, 31/39 (79%) for IgA, and 32/39 (82%) for IgM, while 27/39 (69%) were positive for all three isotypes. Persistence of IgG, IgA, and IgM was observed in 73%, 79%, and 32% of donors, respectively, after 6-9 months of observation. For IgM antibodies, the decline in the proportion of positive donors was statistically significant (p = 0.002) during 12 months observation, for IgG only the decline at 3 months was statistically significant (p = 0.042). Four donors exhibited notable increases in antibody levels. In conclusion, persistent SARS-CoV-2 IgA antibodies and IgG antibodies at 6-9 months are present in approximately three of four individuals with previous mild to moderate COVID-19.

Stress-dependent Flexibility of a Full-length Human Monoclonal Antibody: Insights from Molecular Dynamics to Support Biopharmaceutical Development.

After several decades of advancements in drug discovery, product development of biopharmaceuticals remains a time- and resource-consuming endeavor. One of the main reasons is associated to the lack of fundamental understanding of conformational dynamics of such biologic entities, and how they respond to various stresses encountered during manufacturing, storage, and shipping. In this work, we have studied the conformational dynamics of human IgG1κ b12 monoclonal antibody (mAb) using molecular dynamics simulations. The hundreds of nanoseconds long trajectories reveal that b12 mAb is highly flexible. Its variable domains show greater conformational fluctuations than the constant domains. Additionally, it collapses towards a more globular shape in response to thermal stress, leading to decrease in the total solvent exposed surface area and radius of gyration. This behavior is more pronounced for the deglycosylated b12 mAb, and it appears to correlate with increase in inter-domain contacts between specific regions of the antibody. Conformational fluctuations also cause transient formation and disruption of hydrophobic and charged patches on the antibody surface, which is particularly important for the prediction of CMC properties during development phases of antibody-based biotherapeutics. The insights gained through these simulations may help the development of biologic drugs.

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography.

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.

Rational optimization of a monoclonal antibody improves the aggregation propensity and enhances the CMC properties along the entire pharmaceutical process chain.

The discovery of therapeutic monoclonal antibodies (mAbs) primarily focuses on their biological activity favoring the selection of highly potent drug candidates. These candidates, however, may have physical or chemical attributes that lead to unfavorable chemistry, manufacturing, and control (CMC) properties, such as low product titers, conformational and colloidal instabilities, or poor solubility, which can hamper or even prevent development and manufacturing. Hence, there is an urgent need to consider the developability of mAb candidates during lead identification and optimization. This work provides a comprehensive proof of concept study for the significantly improved developability of a mAb variant that was optimized with the help of sophisticated in silico tools relative to its difficult-to-develop parental counterpart. Interestingly, a single amino acid substitution in the variable domain of the light chain resulted in a three-fold increased product titer after stable expression in Chinese hamster ovary cells. Microscopic investigations revealed that wild type mAb-producing cells displayed potential antibody inclusions, while the in silico optimized variant-producing cells showed a rescued phenotype. Notably, the drug substance of the in silico optimized variant contained substantially reduced levels of aggregates and fragments after downstream process purification. Finally, formulation studies unraveled a significantly enhanced colloidal stability of the in silico optimized variant while its folding stability and potency were maintained. This study emphasizes that implementation of bioinformatics early in lead generation and optimization of biotherapeutics reduces failures during subsequent development activities and supports the reduction of project timelines and resources.

Adaption of human antibody λ and κ light chain architectures to CDR repertoires.

Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved ß-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.

Universal nanothin silk coatings via controlled spidroin self-assembly.

Robust, biocompatible, and facile coatings are promising for improving the in vivo performance of medical implants and devices. Here, we demonstrate the formation of nanothin silk coatings by leveraging the biomimetic self-assembly of eADF4(C16), an amphiphilic recombinant protein based on the Araneus diadematus dragline spidroin ADF4. These coatings result from concurrent adsorption and supramolecular assembly of eADF4(C16) induced by KH2PO4, thereby providing a mild one-pot coating strategy in which the coating rate can be controlled by protein and KH2PO4 concentration. The thickness of the coatings ranges from 2-30 nm depending on the time immersed in the aqueous coating solution. Coatings can be formed on hydrophobic and hydrophilic substrates regardless of surface chemistry and without requiring specialized surface activation. Moreover, coatings appear to be stable through vigorous rinsing and prolonged agitation in water. Grazing incidence wide angle X-ray scattering, single-molecule force spectroscopy, and Congo red staining techniques confirm the formation of ß-sheet nanocrystals within the eADF4(C16) coating, which contributes to the cohesive and adhesive stability of the material. Coatings are exceptionally smooth in the dry state and are hydrophilic regardless of substrate hydrophobicity. Under aqueous conditions, nanothin silk coatings exhibit the properties of a hydrogel material.

Dimerization of the Conserved N-Terminal Domain of a Spider Silk Protein Controls the Self-Assembly of the Repetitive Core Domain.

Spider silk proteins comprise a repetitive core domain with polyalanine and glycine/proline-rich stretches flanked by highly conserved nonrepetitive N- and C-terminal domains. The termini are responsive to assembly triggers, sensing changes in the ionic (H+, phosphate) and mechanical (shear stress) environment along the spinning duct. The N-terminal domain dimerizes in a pH-dependent manner induced by protonation of conserved acidic residues. To date, dimerization of N-terminal spider silk domains has been individually investigated in the absence of large core domains. In this work, the impact of an engineered 50 kDa (AQ) core domain was studied on N-terminal dimerization by circular dichroism, fluorescence and absorbance spectroscopy, multiangle light scattering, as well as scanning electron and transmission electron microscopy. Although the core domain showed no apparent influence on the dimerization behavior of the N-terminal domain, the N-terminal domain in contrast influenced the behavior of the core domain: the monomeric state enhanced (AQ)'s solubility, and dimer formation triggered self-assembly. The monomer-dimer equilibrium was influenced by using several previously established mutants, confirming these results. This work thereby provides molecular insights into how key residues of the N-terminal domain control the dimerization-mediated transformation of soluble spidroins into fibrillary assemblies.

Conformational Stability and Interplay of Helical N- and C-Terminal Domains with Implications on Major Ampullate Spidroin Assembly.

Major ampullate spidroin (MaSp) assembly starts in the abdomen of the spider, where spidroins are stored as a liquid dope at a high concentration. The dope is squeezed into the spinning duct, and assembly is finished upon drawing of fibers. Unwanted aggregation of the spidroin solution in the gland is suppressed by prestructuring of the spidroins in micelle-like assemblies, with their hydrophobic stretches being hidden from the solvent and the hydrophilic nonrepetitive amino (NRN) and carboxy (NRC) terminal domains being exposed on the micelle surface. Conversion of the fluid dope into a solid fiber is induced within the spinning duct by acidification and ion exchange (sodium chloride against potassium phosphate), with the impact on the structure of the NRN and NRC domains acting as a regulatory switch for fiber assembly. While NRN dimerizes pH-dependently in an antiparallel fashion (i.e. quaternary structural changes), the tertiary structure of dimeric NRC is changed by shear stress and a drop in pH, inducing the alignment of the intrinsically unstructured core domains accompanied by ß-sheet formation of motifs of the core domain. Here, the conformational stability of NRN1 and NRC1 of Latrodectus hesperus MaSp1 were studied using independent techniques such as circular dichroism, fluorescence and absorbance spectroscopy, and scanning electron, transmission electron, and atomic force microscopy. In this context, it could be shown that strong, non-natural acidification drives NRC1 to unfold and aggregate into ß-sheet-rich structures, preventing recombinant spidroins from assembling into aligned fibrils. Interestingly, NRN1 and NRC1 apparently do not interact with each other, making spidroin assembly easy to control step-by-step and straightforward due to missing unproductive side reactions.

Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders' Major Ampullate Spidroin 1.

Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

Resonance assignment of an engineered amino-terminal domain of a major ampullate spider silk with neutralized charge cluster.

Spider dragline fibers are predominantly made out of the major ampullate spidroins (MaSp) 1 and 2. The assembly of dissolved spidroin into a stable fiber is highly controlled for example by dimerization of its amino-terminal domain (NRN) upon acidification, as well as removal of sodium chloride along the spinning duct. Clustered residues D39, E76 and E81 are the most highly conserved residues of the five-helix bundle, and they are hypothesized to be key residues for switching between a monomeric and a dimeric conformation. Simultaneous replacement of these residues by their non-titratable analogues results in variant D39N/E76Q/E81Q, which is supposed to fold into an intermediate conformation between that of the monomeric and the dimeric state at neutral pH. Here we report the resonance assignment of Latrodectus hesperus NRN variant D39N/E76Q/E81Q at pH 7.2 obtained by high-resolution triple resonance NMR spectroscopy.