Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies.
ABSTRACT: Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in beta branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-beta structures and fibrils. Mapping on the available Fab-receptor/antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.
Project description:Enhanced protein aggregation and/or impaired clearance of aggregates can lead to neurodegenerative disorders such as Alzheimer's Disease, Huntington's Disease, and prion diseases. Therefore, many protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Prions, which generally result from self-propagating protein aggregates, must therefore evade or outcompete these quality control systems in order to form and propagate in a cellular context. We developed a genetic screen in yeast that allowed us to explore the sequence features that promote degradation versus aggregation of a model glutamine/asparagine (Q/N)-rich prion domain from the yeast prion protein, Sup35, and two model glycine (G)-rich prion-like domains from the human proteins hnRNPA1 and hnRNPA2. Unexpectedly, we found that aggregation propensity and degradation propensity could be uncoupled in multiple ways. First, only a subset of classically aggregation-promoting amino acids elicited a strong degradation response in the G-rich prion-like domains. Specifically, large aliphatic residues enhanced degradation of the prion-like domains, whereas aromatic residues promoted prion aggregation without enhancing degradation. Second, the degradation-promoting effect of aliphatic residues was suppressed in the context of the Q/N-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation. Degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated activity for these residues in yeast prion domains. Collectively, these results provide key insights into how certain aggregation-prone proteins may evade protein quality control degradation systems.
Project description:PURPOSE:To analyze contribution of short aggregation-prone regions (APRs), which may self-associate via cross-beta motif and were earlier identified in therapeutic mAbs, towards antigen recognition via structural analyses of antibody-antigen complexes. METHODS:A dataset of 29 publically available high-resolution crystal structures of Fab-antigen complexes was collected. Contribution of APRs towards the surface areas of the Fabs buried by the cognate antigens was computed. Propensities of amino acids to occur in APRs and to be involved in antigen binding were compared. Coincidence between APRs and individual CDR loops was examined. RESULTS:All Fabs in the dataset contain at least one APR in CDR loops and adjacent framework beta-strands. The average contribution of APRs towards buried surface area of Fabs is 16.0 +/- 10.7%. Aggregation and antigen recognition may be coupled via aromatic residues (Tyr, Trp), which occur with high propensities in both APRs and antigen binding sites. APRs are infrequent in the heavy chain CDR 3 (H3) loops (7%), but are frequent in H2 loops (45%). CONCLUSIONS:Co-incidence of APRs with antigen recognition sites can potentially lead to the loss of function upon aggregation. Rational structure-based design or selection strategies are suggested for biotherapeutics with improved druggability while maintaining potency.
Project description:The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.
Project description:We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V(H)) domain framework (FR) residues. Substitution of the FR cavity V(H) Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and V(H) FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V(H)1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.
Project description:Der p 1 and Der f 1 are major allergens from Dermatophagoides pteronyssinus and D. farinae, respectively. An analysis of antigenic determinants on both allergens was performed by site-directed mutagenesis. The analysis was based on the x-ray crystal structures of the allergens in complex with Fab fragments of three murine mAbs that interfere with IgE Ab binding: the two Der p 1-specific mAbs 5H8 and 10B9, and the cross-reactive mAb 4C1. On one hand, selected residues in the epitopes for mAb 5H8 and mAb 4C1 were substituted with amino acids that resulted in impaired Ab binding to Der p 1. On the other hand, an epitope for the Der p 1-specific mAb 10B9, which partially overlaps with mAb 4C1, was created in Der f 1. The mutation of 1-3 aa residues in Der f 1 was sufficient to bind mAb 10B9. These residues form hydrogen bonds with CDRs of the Ab other than H CDR3. This observation unveils an exception to the dominant role of H CDR3 commonly observed in Ag recognition. Overall, this study resulted in the identification of important residues for mAb and IgE Ab recognition in group 1 mite allergens. This information can be used to engineer allergen mutants with reduced IgE Ab binding for immunotherapy.
Project description:Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.
Project description:PURPOSE:Tryptophan's (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. METHODS:Selective Trp oxidation was induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. RESULTS:Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. CONCLUSIONS:Trp oxidation in CDRs can adversely impact mAbs' conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.
Project description:Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.
Project description:Monoclonal antibodies (mAbs) have become a major class of protein therapeutics that target a spectrum of diseases ranging from cancers to infectious diseases. Similar to any protein molecule, mAbs are susceptible to chemical modifications during the manufacturing process, long-term storage, and in vivo circulation that can impair their potency. One such modification is the oxidation of methionine residues. Chemical modifications that occur in the complementarity-determining regions (CDRs) of mAbs can lead to the abrogation of antigen binding and reduce the drug's potency and efficacy. Thus, it is highly desirable to identify and eliminate any chemically unstable residues in the CDRs during the therapeutic antibody discovery process. To provide increased throughput over experimental methods, we extracted features from the mAbs' sequences, structures, and dynamics, used random forests to identify important features and develop a quantitative and highly predictive in silico methionine oxidation model.
Project description:This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.