Antigenic integral membrane glycoprotein Antibody

What defines an antigenic integral membrane glycoprotein and why are they challenging targets for antibody development?

Integral membrane glycoproteins are proteins embedded within the cell membrane that contain carbohydrate components. They perform most of the specific functions of membranes, while the lipid bilayer provides the basic structure . These proteins include important therapeutic target classes such as G protein-coupled receptors (GPCRs), ion channels, transporters, enzymes, and adhesion molecules .

These targets present several significant challenges for antibody development:

• Conformational Dependence: Their native structure depends on the lipid bilayer environment, making them difficult to extract and purify while maintaining proper conformation

• Detergent Sensitivity: Traditional extraction methods using detergents often cause these proteins to become insoluble, unstable, or display altered structure and activity

• Expression Difficulties: Recombinant expression of integral membrane proteins frequently encounters problems with trafficking, toxicity, and poor yields

• Limited Accessibility: Many epitopes are occluded by the membrane or only transiently exposed during conformational changes

Despite representing targets for approximately half of all small molecule drugs, integral membrane proteins are the targets of only three therapeutic monoclonal antibodies, highlighting their underrepresentation as successful antibody targets despite their therapeutic importance .

How do the structural features of integral membrane glycoproteins affect antibody recognition and binding?

The structural complexity of membrane glycoproteins significantly impacts antibody recognition through several key features:

• Transmembrane Domains: These hydrophobic regions embedded in the lipid bilayer are generally inaccessible to antibodies in native conditions . For example, glycophorin exists as a homodimer with interactions between transmembrane α-helices forming part of its structure.

• Glycosylation Patterns: The majority of transmembrane proteins in animal cells are glycosylated, with oligosaccharide chains always present on the non-cytosolic side of the membrane . These glycans can:

  • Create or mask potential epitopes

  • Contribute to protein stability and folding

  • Change over time due to evolutionary pressures (as seen with RSV surface glycoproteins)

• Disulfide Bonds: These form on the non-cytosolic side of membranes and play important roles in stabilizing folded structures or mediating interactions between polypeptide chains , creating conformational epitopes that antibodies may recognize.

• Extracellular Domains: These regions, like the four external domains (D1-D4) of CD4 that show homology to immunoglobulin superfamily members, are the primary targets for antibody recognition .

• Domain Mobility: Many membrane proteins undergo significant conformational changes during their functional cycle, creating state-specific epitopes that can be recognized differentially by antibodies .

Antibody-antigen recognition involves "myriad non-covalent interactions between the epitope – the binding site on the Ag, and the paratopes – the binding site on the Ab" . The complex architecture of membrane glycoproteins means these interactions are highly dependent on maintaining native protein structure.

What are the key differences between antibodies targeting integral membrane proteins versus soluble proteins?

Developing antibodies against integral membrane proteins involves distinct challenges and considerations compared to soluble protein targets:

The synthetic antibody technologies typically used for soluble proteins "depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function" . This has driven the development of specialized approaches like CellectSeq for "targeting integral membrane proteins on native cells in complex environment" and the MPS platform that delivers antibodies against challenging membrane protein targets with "high-affinity, high specificity, and documented developability" .

What are the current methodologies for generating antibodies against native conformations of integral membrane glycoproteins?

Modern approaches for generating antibodies against membrane glycoproteins focus on preserving native conformations throughout the discovery process:

• Specialized Antigen Preparation Methods:

  • SMALPs Technology: Styrene Maleic Acid Lipid Particles encapsulate membrane proteins in their native lipid environment without detergents

  • Lipoparticles: Virus-like particles that display membrane proteins in their native orientation and conformation

  • Cell-surface Expression: Using intact cells expressing the target protein as both immunogen and screening substrate

• Advanced Immunization Strategies:

  • Divergent Host Species: Using chickens which produce "robust immune responses and offering excellent opportunities for MAb diversity while overcoming homology challenges associated with mammalian hosts"

  • Multi-format Immunization: Combining "DNA, mRNA, and Lipoparticles (VLPs) to preserve the structure of the antigen, proprietary adjuvants, and phage protocols optimized for membrane protein challenges"

• Innovative Selection Methodologies:

  • CellectSeq: Combines "in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders" with next-generation sequencing

  • MPS Platform: Specifically designed to "generate the most diverse antibodies against even the most difficult targets, with a success rate of >95%"

• Computational Analysis:

  • Motif-Based Scoring: Algorithms that identify patterns associated with specific binding properties

  • NGS Analysis: "Comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances"

These methods collectively overcome the limitations of traditional approaches that often fail to produce antibodies recognizing the native conformation of membrane glycoproteins.

How can researchers effectively validate the specificity of antibodies targeting integral membrane glycoproteins?

Validating antibody specificity for membrane glycoproteins requires comprehensive characterization using multiple complementary approaches:

• Biochemical Methods:

  • Vectorial Labeling: Using "a covalent labeling reagent (such as a radioactive or fluorescent marker) that is water-soluble and therefore cannot penetrate the lipid bilayer" to determine protein orientation

  • Protease Protection Assays: Exposing "either the external or internal surface to proteolytic enzymes, which are membrane-impermeant" to determine protein topology

  • Immunoblotting: Validating recognition of denatured protein at the expected molecular weight (e.g., confirming a 65-kD glycoprotein in Trypanosoma vivax)

• Imaging Techniques:

  • Immunofluorescence Microscopy: On fixed, permeabilized cells to determine subcellular localization

  • Immunoelectron Microscopy: On thin sections of embedded cells for high-resolution localization

  • Double-Labeling: Simultaneously localizing multiple antigens to confirm distinct distribution patterns

• Binding Analysis:

  • Surface Plasmon Resonance: Determining binding kinetics and specificity using purified protein or SMALPs

  • Grating-Coupled Interferometry: Alternative biosensor platform for kinetic analysis of antibody-membrane protein interactions

• Functional Assays:

  • Ligand Competition: Testing if antibodies compete with or alter binding of natural ligands

  • Activity Modulation: Assessing if antibodies affect protein function

A robust validation workflow might include:

• Initial characterization using immunoblotting to confirm target recognition

• Cell-based imaging to verify subcellular localization consistent with the target

• Binding analysis to determine specificity and affinity

• Functional assays to assess biological activity

For example, in validating a monoclonal antibody against a Trypanosoma vivax membrane glycoprotein, researchers used immunofluorescence, immunoelectron microscopy, and immunoblot analyses, finding that "some mAb-labeled vesicles contained endocytosed 10 nm BSA-gold after incubation of the parasites with the marker for 5-30 min at 37°C" , confirming the antibody's specificity for proteins in the endocytic compartment.

What role does the immunization strategy play in developing diverse antibody panels against conserved membrane protein epitopes?

Immunization strategy is critical for generating diverse, high-quality antibodies against membrane proteins, particularly those with highly conserved structures:

• Host Selection Considerations:

  • Evolutionary Distance: Using "divergent species (chickens)" overcomes "homology challenges associated with mammalian hosts" when targeting conserved epitopes

  • Immune System Differences: Avian immune systems generate antibody diversity through different mechanisms than mammals, potentially recognizing epitopes that are immunologically silent in traditional hosts

• Antigen Format Diversity:

  • Multiple Presentation Formats: Combining "DNA, mRNA, and Lipoparticles (VLPs)" exposes the immune system to the antigen in different contexts

  • Native Conformation Preservation: Using formats that "preserve the structure of the antigen" ensures antibodies recognize physiologically relevant epitopes

  • Specialized Adjuvants: "Proprietary adjuvants optimized for membrane protein challenges" enhance immune responses against difficult targets

• Immunization Protocol Design:

  • Prime-Boost Strategies: Sequential use of different antigen formats

  • Extended Schedules: Longer immunization protocols allow affinity maturation

  • Site-Directed Approaches: Focusing immune responses on specific domains

• Antibody Library Creation:

  • Technology Integration: The MPS platform integrates optimized immunization with "Humanized Chicken Antibody Discovery technology (hCAT)" where "antibody CDR's from responsive chickens are humanized prior to the creation of immune libraries"

  • Library Diversity: Ensuring broad representation of the immune response

The effectiveness of appropriate immunization strategies is demonstrated by the ability to isolate "high-affinity, humanized MAbs against highly conserved membrane protein targets, with unique characteristics including state-specific binding and functional activity" . This represents a significant advancement over traditional approaches that often struggle to generate diverse antibody panels against conserved membrane protein epitopes.

How do post-translational modifications, particularly glycosylation, impact antibody recognition of integral membrane glycoproteins?

Glycosylation and other post-translational modifications (PTMs) significantly influence antibody recognition through multiple mechanisms:

• Epitope Formation and Masking:

  • Glycans can form part of composite epitopes recognized by antibodies

  • Large glycan structures may physically mask potential protein epitopes

  • Changes in glycosylation patterns can create or destroy antigenic sites

• Structural Impacts:

  • N-linked and O-linked glycans stabilize protein conformations

  • As observed in Trypanosoma vivax, membrane glycoproteins often possess "carbohydrate linkages cleaved by both endoglycosidase H and O-glycosidase, suggesting the presence of N- and O-linked glycans"

  • These structural contributions influence which epitopes are presented to antibodies

• Evolutionary Considerations:

  • Glycoproteins "are continuously evolving" with "sequence diversities in the G protein second hypervariable region" and other modifications

  • These changes affect antibody recognition, requiring "continued surveillance...for the clinical evaluation of immunoprophylactic products"

• Antibody Development Implications:

  • Immunization strategies must account for native glycosylation patterns

  • Expression systems for antigen production should maintain appropriate glycosylation

  • Screening procedures should identify antibodies that recognize or are independent of glycan structures, depending on desired properties

The CD4 glycoprotein illustrates this complexity – a "58 kDa integral membrane glycoprotein" with "four external domains (D1 to D4)" where glycosylation contributes to both structure and antibody recognition . Antibodies recognizing CD4 must accommodate its complex glycosylation pattern to maintain specificity for target cell populations.

Researchers must carefully consider these factors when developing antibodies against glycosylated membrane proteins to ensure recognition of physiologically relevant forms of the target.

What approaches can preserve the native conformation of integral membrane proteins during antibody discovery and screening?

Maintaining native membrane protein conformations throughout the antibody discovery process is critical for developing antibodies with relevant biological activity. Several innovative approaches have been developed:

• SMALP Technology:

  • Styrene Maleic Acid Lipid Particles allow "encapsulation of target membrane protein complexes" without detergents

  • This approach maintains proteins "in their native lipid environment" while making them water-soluble

  • SMALPs have been demonstrated as "a precise and efficient procedure for label-free kinetic analysis of diverse antibody binding interactions"

  • They work in both "purified and complex mixture environment" settings

• Lipoparticle Technology:

  • Uses virus-like particles to display membrane proteins in their native orientation

  • The MPS platform begins with "the highest-quality antigen available for immunization and discovery"

  • This technology preserves "the structure of the antigen" while making it accessible for antibody binding

• Cell-Based Approaches:

  • Direct use of cells expressing target proteins maintains them in their natural membrane environment

  • CellectSeq performs "in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders"

  • This enables targeting "integral membrane proteins on native cells in complex environment"

• Integrated Analysis Platforms:

  • The "integrated SMALP-Biosensor platform" combines native-like presentation with advanced binding analysis

  • This "demonstrated the strength of the two techniques and provides a robust label-free approach to quantify membrane target binding directly with diverse ligands"

• Specialized Expression Systems:

  • "Transposase-based systems (e.g., PiggyBac)" can "generate inducible cell lines for membrane protein expression"

  • These systems provide control over expression levels and timing

The effectiveness of these approaches is demonstrated by successful antibody discovery against challenging targets like "tetraspanin CD151, carbonic anhydrase 9, and integrin-α11" and the ability to determine precise binding parameters using biosensor platforms with SMALPs .

How do researchers address epitope accessibility challenges when targeting multi-spanning membrane proteins?

Multi-spanning membrane proteins present unique epitope accessibility challenges that require specialized strategies:

• Structural Analysis for Epitope Mapping:

  • Understanding that "the individual transmembrane segments of a multipass membrane protein occupy defined positions in the folded protein structure"

  • These positions are "determined by interactions between neighboring transmembrane α helices"

  • This knowledge helps identify accessible regions for antibody targeting

• Domain-Specific Targeting Strategies:

  • Extracellular Loop Focus: Designing immunogens that present extracellular loops between transmembrane segments

  • N-terminal/C-terminal Domains: Targeting soluble domains that extend from the membrane

  • Conformation-Specific Epitopes: Identifying epitopes that form only in specific functional states

• Advanced Display Technologies:

  • Lipoparticles that display membrane proteins in native orientation and density

  • Cell-based selections that naturally present only accessible epitopes

  • SMALPs that maintain native lipid environment while making proteins water-soluble

• Computational Approaches:

  • Predicting accessible epitopes based on protein topology models

  • Using "motif-based scoring and sequencing error-filtering algorithms" to identify antibodies against rare or less accessible epitopes

• Diverse Antibody Sampling:

  • Next-generation sequencing to identify antibodies "even at extremely low abundances, which are very difficult to identify using manual sampling"

  • This increases the likelihood of finding antibodies against challenging epitopes

The experimental approach might involve:

• Initial structural analysis to identify potentially accessible regions

• Design of targeted immunization strategies focusing on these regions

• Cell-based screening to select antibodies binding to native protein

• Comprehensive NGS analysis to identify rare binders to challenging epitopes

• Functional characterization to identify antibodies with desired properties

This multi-faceted approach has enabled the development of antibodies against complex membrane proteins that were previously considered "undruggable" .

What methods are most effective for characterizing the functional effects of antibodies binding to integral membrane glycoproteins?

Characterizing functional effects requires a multi-dimensional approach combining biophysical, cellular, and biochemical methods:

• Biosensor-Based Kinetic Analysis:

  • Surface Plasmon Resonance: Determines binding kinetics with purified proteins or SMALPs

  • Grating-Coupled Interferometry: Alternative platform for detailed binding analysis

  • These approaches provide "both qualitative and quantitative analysis of membrane protein interactions with a variety of antibodies in specific manner"

• Cell-Based Functional Assays:

  • Signaling Pathway Analysis: Measuring downstream effects on cellular signaling

  • Receptor Internalization: Quantifying changes in surface expression following antibody binding

  • Ligand Competition: Assessing antibody impact on natural ligand binding

  • Functional Readouts: Measuring specific activities (ion flux, enzyme activity, etc.)

• Structural Characterization:

  • Epitope Mapping: Identifying the precise binding site to correlate with functional effects

  • Conformational Analysis: Determining if antibodies stabilize specific protein conformations

• Advanced Imaging Approaches:

  • Immunolocalization: Using techniques like those applied to the 65-kD glycoprotein in Trypanosoma, combining immunofluorescence and immunoelectron microscopy

  • Double Labeling: "Simultaneously localize" multiple antigens to understand relationships

  • Dynamic Imaging: Tracking changes in protein localization or trafficking after antibody binding

• Biochemical Characterization:

  • Protease Protection: Determining if antibody binding alters accessibility to proteolytic enzymes

  • Crosslinking Studies: Identifying if antibodies affect protein-protein interactions

A comprehensive workflow might include:

• Initial characterization of binding kinetics and affinity using biosensor platforms

• Cell-based assays to determine functional effects in a physiological context

• Detailed mechanistic studies to understand the molecular basis of observed effects

• Advanced imaging to visualize changes in protein localization or trafficking

For example, researchers studying membrane glycoproteins in Trypanosoma combined multiple approaches to demonstrate that their antibody recognized "vesicles and tubules in the posterior portion of the parasite" involved in endocytosis , providing functional context for antibody binding.

How can researchers distinguish between antibodies that recognize different conformational states of the same membrane protein?

Distinguishing conformation-specific antibodies requires specialized approaches that can detect subtle differences in epitope recognition:

• State-Stabilizing Conditions:

  • Using ligands, ions, or pH conditions that stabilize specific conformational states

  • Testing antibody binding under these varying conditions to identify state preferences

  • The MPS platform can isolate antibodies with "state-specific binding" characteristics

• Functional Correlation Analysis:

  • Determining if antibodies affect specific functions associated with distinct conformational states

  • Correlating binding affinity with functional effects in different conditions

  • For example, testing if antibodies affect only the active or inactive state of a GPCR

• Competitive Binding Studies:

  • Using ligands known to stabilize specific conformations as competitors

  • Determining if antibodies compete differentially with these ligands

  • Measuring binding kinetics in the presence of conformation-stabilizing compounds

• Advanced Structural Approaches:

  • Using structural biology techniques to visualize antibody-protein complexes

  • As noted in search result , "the availability of increasing amounts of structural data in recent years now allows for a much better understanding of the structural basis of Ab function"

  • This can directly reveal which conformation an antibody recognizes

• Biosensor Analysis with Conformation Controls:

  • Comparing binding kinetics to protein prepared under conditions favoring different states

  • The SMALP-biosensor platform can characterize "diverse antibody binding interactions" and could be adapted to this purpose

A typical experimental workflow might include:

• Initial screening in multiple conditions that favor different conformational states

• Detailed kinetic analysis in the presence/absence of conformation-stabilizing ligands

• Functional assays to determine if antibodies selectively modulate state-specific activities

• Structural studies to confirm binding to specific conformational epitopes

The ability to isolate antibodies with "state-specific binding and functional activity" demonstrates that this approach is feasible for complex membrane proteins, offering powerful tools for studying protein dynamics and developing therapeutics that target specific functional states.

What strategies exist for developing antibodies that can modulate the function of integral membrane proteins?

Developing function-modulating antibodies requires strategic approaches targeting mechanistically relevant epitopes:

• Structure-Guided Epitope Selection:

  • Using structural information to identify regions involved in protein function

  • Targeting "determinants within the Ab structure that contribute to Ag binding"

  • This knowledge can be "exploited to identify residues that are important for the function of the Ab in general and for Ag recognition in particular"

• Diverse Antibody Panel Generation:

  • Creating "large, diverse panels" of antibodies to increase chances of finding functional modulators

  • The MPS platform generates antibodies with "unique characteristics including state-specific binding and functional activity"

• Conformation-Specific Targeting:

  • Developing antibodies that stabilize active or inactive conformations

  • Targeting epitopes that control transitions between functional states

  • Exploiting "allosteric effects in Ab function" where binding at one site affects function at another

• Rational Engineering Approaches:

  • Optimizing antibodies through "affinity maturation" to improve binding properties

  • Fine-tuning specificity through "antibody engineering" to target specific epitopes

  • Exploiting knowledge of "the role each structural element in the Ab plays in Ag recognition"

• Integrated Functional Screening:

  • Implementing primary screens that directly measure functional modulation

  • Using cell-based assays that report on target protein activity

  • Combining binding data with functional readouts to identify correlations

The experimental approach might involve:

• Initial generation of diverse antibody panels using specialized platforms

• Primary screening for binding to the target protein

• Secondary screening for functional effects in cellular assays

• Detailed characterization of mechanism for promising candidates

• Optimization of lead antibodies through engineering approaches

As noted in search result , "understanding of the underpinnings of Ab-Ag recognition" provides a foundation for developing antibodies with specific functional properties, whether for research tools or therapeutic applications.

How do allosteric effects influence antibody binding and function when targeting integral membrane glycoproteins?

Allosteric effects play crucial but often overlooked roles in antibody-antigen interactions with membrane glycoproteins:

• Bidirectional Allosteric Communication:

  • "Structural changes in the variable region caused by Ag binding may be transferred into the constant domains, potentially influencing effector activation and cellular response"

  • This creates a mechanism where target binding can directly affect immune function

  • For example, studies showed that "the binding of staphylococcal protein A (SPA) or streptococcal protein G (SPG) to the constant region was inhibited by hapten binding in several Abs"

• Isotype-Dependent Effects:

  • "Differences in affinity and specificity of Abs with the same variable region but different isotypes" can significantly impact function

  • A striking example from lupus research showed that "a set of anti-PL9–11 Abs sharing the same variable domain but different isotypes" bound to targets "with different affinities that were associated with significant differences in renal pathogenicity in vivo and survival"

  • These differences were observed "binding DNA and chromatin, as well as the renal Ags"

• Conformational Propagation in Membrane Proteins:

  • Antibody binding to one domain of a membrane protein can affect conformation of distant domains

  • This may alter function even when the antibody binds away from the functional site

  • For multi-spanning membrane proteins, this is particularly relevant as "transmembrane segments of a multipass membrane protein occupy defined positions" with interactions "between neighboring transmembrane α helices"

• Therapeutic Implications:

  • Understanding allosteric mechanisms can guide development of therapeutic antibodies

  • Different isotypes may be selected based on desired functional outcomes

  • Targeting specific epitopes may allow fine-tuned modulation of protein function

These allosteric mechanisms create complex relationships between antibody binding and functional outcomes that must be considered when developing and characterizing antibodies against membrane glycoproteins.

What are the challenges and solutions for studying antibody-membrane protein interactions using structural biology techniques?

Structural biology of antibody-membrane protein complexes presents unique challenges that require specialized approaches:

• Key Challenges:

  • Native Conformation Preservation: "The main challenge associated with biophysical analysis of membrane protein is the need for detergent extraction from the bilayer environment, which in many cases causes the proteins to become insoluble, unstable or display altered structure or activity"

  • Conformational Heterogeneity: Membrane proteins often adopt multiple conformational states

  • Complex Formation Stability: Antibody-membrane protein complexes may be unstable outside the membrane environment

  • Size Limitations: Large membrane protein-antibody complexes may exceed size limits for some techniques

• Innovative Solutions:

  • SMALP Technology: "The amphipathic co-polymer of styrene and maleic acid (SMA)...can release lipids and integral membrane proteins into water soluble native particles (or vesicles)"

  • Nanodiscs and Lipid Nanodiscs: Alternative membrane mimetics that maintain native-like environment

  • Cryo-EM Advances: Allowing visualization of membrane proteins in various states without crystallization

  • Integrated Biosensor Approaches: "Integrated SMALP-Biosensor platform" provides "a robust label-free approach to quantify membrane target binding"

• Method-Specific Strategies:

  • X-ray Crystallography: Using antibody fragments (Fab, scFv) to facilitate crystallization

  • Cryo-Electron Microscopy: Preparation in detergent-free systems like SMALPs

  • NMR Spectroscopy: Selective labeling strategies for specific domains

• Expression System Optimization:

  • "Transposase-based systems (e.g., PiggyBac)" to "generate inducible cell lines for membrane protein expression"

  • Baculovirus expression systems for complex eukaryotic membrane proteins

  • Cell-free expression systems with defined membrane environments

The "SMALP-Biosensor platform developed for multiple membrane proteins of interest demonstrated the strength of the two techniques and provides a robust label-free approach to quantify membrane target binding directly with diverse ligands" . This integration of native-like presentation with advanced analysis techniques represents a promising direction for studying antibody-membrane protein interactions at the structural level.

How can next-generation sequencing technologies improve the discovery of antibodies against poorly immunogenic membrane protein epitopes?

Next-generation sequencing (NGS) technologies offer transformative approaches for antibody discovery against challenging membrane protein targets:

• Comprehensive Repertoire Analysis:

  • NGS enables "comprehensive interrogation of next-generation sequencing pools"

  • This allows identification of "Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances"

  • Traditional approaches likely miss rare antibodies against poorly immunogenic epitopes

• Advanced Computational Approaches:

  • "Motif-based scoring" algorithms identify patterns associated with specific binding properties

  • "Sequencing error-filtering algorithms" improve accuracy of antibody sequence identification

  • These computational tools extract meaningful signals from complex NGS datasets

• Integration with Specialized Selection Methods:

  • The CellectSeq approach combines cell-based selections with NGS analysis

  • First, researchers perform "in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders"

  • Then, they apply NGS analysis to identify antibodies that would be missed by traditional methods

• Practical Implementation Strategy:

  • Initial Enrichment: Use cell-based or other selection methods to create enriched antibody pools

  • Deep Sequencing: Apply NGS to exhaustively characterize these pools

  • Computational Analysis: Use specialized algorithms to identify promising candidates

  • Validation: Express and characterize selected antibodies experimentally

This approach has been validated by successfully targeting "three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11" - all challenging membrane protein targets.

The integration of NGS with specialized selection methods represents a powerful approach for discovering antibodies against membrane protein epitopes that have traditionally been considered "undruggable" .