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Find video protocols related to scientific articles indexed in Pubmed.
Precise and efficient antibody epitope determination through library design, yeast display and next generation sequencing.
J. Mol. Biol.
PUBLISHED: 08-07-2014
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The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies which neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.
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Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes.
MAbs
PUBLISHED: 05-17-2014
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Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization.
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Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.
Cell Host Microbe
PUBLISHED: 03-17-2014
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B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
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High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.
PLoS ONE
PUBLISHED: 01-01-2014
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Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.
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The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.
J. Immunol.
PUBLISHED: 04-29-2013
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The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.
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Differential effects of smoking and COPD upon circulating myeloid derived suppressor cells.
Respir Med
PUBLISHED: 04-18-2013
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Chronic obstructive pulmonary disease (COPD) is characterized by an enhanced and persistent innate and acquired immune response to tobacco smoking. Myeloid-derived suppressor cells (MDSCs) modulate T-cell responses by down-modulating the T cell receptor ? chain (TCR ?) through the catabolism of l-arginine. The effects of smoking on MDSCs and their potential participation in COPD immunopathogenesis have not been explored so far.
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HLA distribution in COPD patients.
COPD
PUBLISHED: 03-20-2013
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Auto-immunity may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD), particularly to the presence of emphysema. Auto-immune diseases are characterized by an abnormal distribution of HLA class II alleles (DR and DQ). The distribution of DRB1 and DQB1 alleles has not been investigated in COPD.
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Mechanism of action and in vivo efficacy of a human-derived antibody against Staphylococcus aureus ?-hemolysin.
J. Mol. Biol.
PUBLISHED: 02-01-2013
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The emergence and spread of multi-drug-resistant strains of Staphylococcus aureus in hospitals and in the community emphasize the urgency for the development of novel therapeutic interventions. Our approach was to evaluate the potential of harnessing the human immune system to guide the development of novel therapeutics. We explored the role of preexisting antibodies against S. aureus ?-hemolysin in the serum of human individuals by isolating and characterizing one antibody with a remarkably high affinity to ?-hemolysin. The antibody provided protection in S. aureus pneumonia, skin, and bacteremia mouse models of infection and also showed therapeutic efficacy when dosed up to 18 h post-infection in the pneumonia model. Additionally, in pneumonia and bacteremia animal models, the therapeutic efficacy of the ?-hemolysin antibody appeared additive to the antibiotic linezolid. To better understand the mechanism of action of this isolated antibody, we solved the crystal structure of the ?-hemolysin:antibody complex. To our knowledge, this is the first report of the crystal structure of the ?-hemolysin monomer. The structure of the complex shows that the antibody binds ?-hemolysin between the cap and the rim domains. In combination with biochemical data, the structure suggests that the antibody neutralizes the activity of the toxin by preventing binding to the plasma membrane of susceptible host cells. The data presented here suggest that protective antibodies directed against S. aureus molecules exist in some individuals and that such antibodies have a therapeutic potential either alone or in combination with antibiotics.
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Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates.
Chem. Biol.
PUBLISHED: 01-02-2013
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Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
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Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.
PLoS ONE
PUBLISHED: 01-01-2013
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Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigens endogenous concentration, by combining the kinetic exclusion assay and Biacores calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.
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Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-28-2011
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A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V(H) and D(H) segment use is strongly determined by an individuals germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.
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Proprotein convertase substilisin/kexin type 9 antagonism reduces low-density lipoprotein cholesterol in statin-treated hypercholesterolemic nonhuman primates.
J. Pharmacol. Exp. Ther.
PUBLISHED: 10-21-2011
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Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.
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Structural basis of C-terminal ?-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimers disease.
J. Mol. Biol.
PUBLISHED: 09-29-2011
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Alzheimers disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of ?-amyloid (A?) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of A?40. Ponezumab can label A? that is deposited in brain parenchyma found in sections from Alzheimers disease casualties and in transgenic mouse models that overexpress A?. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble A? present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma A?40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human A?. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to A?40, we determined the X-ray crystal structure of ponezumab in complex with A?40 and found that the A?40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of A?V40 in the A?-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of A?40 and the brain A?-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.
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Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-08-2011
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Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.
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Anti-amyloid therapy protects against retinal pigmented epithelium damage and vision loss in a model of age-related macular degeneration.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-20-2011
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Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid ? (A?) peptides, A?1-40 (A?40) and A?1-42 (A?42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for A?, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with A?-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of A?40 and A?42. Concomitant reduction in the levels of A? and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-A?40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimers disease treated with anti-A? antibodies. They also implicate A? in the pathogenesis of AMD and identify A? as a viable therapeutic target for its treatment.
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Synthetic antibodies designed on natural sequence landscapes.
J. Mol. Biol.
PUBLISHED: 04-28-2011
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We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(?)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(?) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.
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Intestinal lymphangiectasia: an undescribed cause of malabsorption and incomplete immunological recovery in HIV-infected patients.
Enferm. Infecc. Microbiol. Clin.
PUBLISHED: 02-25-2011
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Although paradoxical virological and immunological response after HAART has been well studied, intestinal lymphangiectasia (IL) in HIV-1 infected patients has not previously described.
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Expanding the ProteOn XPR36 biosensor into a 36-ligand array expedites protein interaction analysis.
Anal. Biochem.
PUBLISHED: 10-29-2010
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Here we demonstrate methods to expand the throughput of the ProteOn XPR36 biosensor allowing for the simultaneous kinetic characterization of several multiplexed formats, such as 36 disparate antibodies targeting the same antigen, and facilitating detailed epitope binning and mapping studies. The kinetic rate constants determined by these methods correlated with those obtained on Biacore 2000 and the absolute parameter values obtained on the ProteOns alginate-based GLC chip agreed closer with those from Biacores flat C1 chip than Biacores dextran-based CM4 chip. Pairwise epitope binning data from the ProteOn 36-ligand array format and those generated on an orthogonal array-based biosensor, the Octet QK384, gave similar results. In an epitope mapping study using biotinylated peptides, all three biosensor platforms were similar in their ability to identify antibodies that bound to linear epitopes. We apply alternative formats of the ProteOn array that enable a significantly higher number of assays to be conducted simultaneously than previously anticipated on this platform.
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Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-29-2009
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Antibody repertoire diversity, potentially as high as 10(11) unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 x 10(6) reads, including more than 1.9 x 10(5) high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 x 10(4) sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 x 10(10). The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.
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Suppression of amyloid deposition leads to long-term reductions in Alzheimers pathologies in Tg2576 mice.
J. Neurosci.
PUBLISHED: 04-17-2009
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In amyloid precursor protein (APP) models of amyloid deposition, the amount of amyloid deposits increase with mouse age. At a first approximation, the extent of amyloid accumulation may either reflect small excesses of production over clearance that accumulate over time or, alternatively, indicate a steady-state equilibrium at that age, reflecting the instantaneous excess of production over clearance, which increases as the organism ages. To discriminate between these options, we reversibly suppressed amyloid deposition in Tg2576 mice with the anti-Abeta antibody 2H6, starting at 8 months, just before the first histological deposits can be discerned. Six months later, we stopped the suppression and monitored the progression of amyloid accumulation in control APP mice and suppressed APP mice over the next 3 months. The accumulation hypothesis would predict that the rate of amyloid from 14 to 17 months would be similar in the suppressed and control mice, while the equilibrium hypothesis would predict that the increase would be faster in the suppressed group, possibly catching up completely with the control mice. The results strongly support the accumulation hypothesis, with no evidence of the suppressed mice catching up with the control mice as predicted by equilibrium models. If anything, there was a slower rate of increase in the suppressed APP mice than the control mice, suggesting that a slow seeding mechanism likely precedes a rapid fibrillogenesis in determining the extent of amyloid deposition.
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Exploring blocking assays using Octet, ProteOn, and Biacore biosensors.
Anal. Biochem.
PUBLISHED: 04-07-2009
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We demonstrate the use of label-free real-time optical biosensors in competitive binding assays by epitope binning a panel of antibodies. We describe three assay orientations that we term in tandem, premix, and classical sandwich blocking, and we perform each of them on three platforms: ForteBios Octet QK, Bio-Rads ProteOn XPR36, and GE Healthcares Biacore 3000. By testing whether antibodies block one anothers binding to their antigen in a pairwise fashion, we establish a blocking profile for each antibody relative to the others in the panel. The blocking information is then used to create "bins" of antibodies with similar epitopes. The advantages and disadvantages of each biosensor, factors to consider when deciding on the most appropriate blocking assay orientation for a particular interaction system, and tips for dealing with ambiguous data are discussed. The data from our different assay orientations and biosensors agree very well, establishing these machines as valuable tools for characterizing antibody epitopes and multiprotein complexes of biological significance.
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Impact of mutations on the allosteric conformational equilibrium.
J. Mol. Biol.
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Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and effector-unbound protein structures. These simulations can be performed using our web server (http://salilab.org/allosmod/). We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a data set of 10 proteins and 179 mutations, we predict both the magnitude and the sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1k(B)T. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction.
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Effects of chronic sildenafil use on pulmonary hemodynamics and clinical outcomes in heart transplantation.
J. Heart Lung Transplant.
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Elevated pulmonary vascular resistance (PVR) in heart transplant (HT) candidates is associated with poor survival after HT. This study assessed the effect of peri-operative sildenafil administration on pulmonary hemodynamics and clinical outcomes in patients with advanced heart failure who were considered high-risk for HT because of elevated PVR and transpulmonary gradient (TPG).
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A method for integrative structure determination of protein-protein complexes.
Bioinformatics
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Structural characterization of protein interactions is necessary for understanding and modulating biological processes. On one hand, X-ray crystallography or NMR spectroscopy provide atomic resolution structures but the data collection process is typically long and the success rate is low. On the other hand, computational methods for modeling assembly structures from individual components frequently suffer from high false-positive rate, rarely resulting in a unique solution.
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Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity.
J. Immunol. Methods
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Label-free biosensors are often used in the discovery of therapeutic antibodies to characterize the epitope binding regions of a panel of monoclonal antibodies that target a specific antigen, thus facilitating their organization into epitope groups or "bins". When tested in a pairwise combinatorial manner, two antibodies that compete with one another for binding to a specific antigen may be grouped into the same epitope bin - that is, they recognize similar or overlapping epitopes - whereas two antibodies that bind simultaneously to the antigen are placed into different epitope bins. However, depending on the assay format used, results from such experiments can sometimes contradict one another. Here, we provide two examples that illustrate how antigen heterogeneity, either inherent in an antigen sample, or induced by the assay conditions, can confound the interpretation of epitope binning results and, in some cases, lead to erroneous conclusions. We highlight why assays that employ solution antigen are often more reliable than those that employ immobilized antigen and, by corroborating our binning results with assays that utilize native antigen, we determine which subpopulations of our heterogeneous antigen samples are biologically relevant and thus improve the correlation between epitope bins and functional activity. Furthermore, we provide recommendations for performing definitive binning assays and a diagnostic assay procedure that can be followed when antigen heterogeneity is suspected.
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Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.
PLoS ONE
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Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.
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Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair.
J. Mol. Biol.
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Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.
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Structure-based model of allostery predicts coupling between distant sites.
Proc. Natl. Acad. Sci. U.S.A.
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Allostery is a phenomenon that couples effector ligand binding at an allosteric site to a structural and/or dynamic change at a distant regulated site. To study an allosteric transition, we vary the size of the allosteric site and its interactions to construct a series of energy landscapes with pronounced minima corresponding to both the effector bound and unbound crystal structures. We use molecular dynamics to sample these landscapes. The degree of perturbation by the effector, modeled by the size of the allosteric site, provides an order parameter for allostery that allows us to determine how microscopic motions give rise to commonly discussed macroscopic mechanisms: (i) induced fit, (ii) population shift, and (iii) entropy driven. These mechanisms involve decreasing structural differences between the effector bound and unbound populations. A metric (ligand-induced cooperativity) can measure how cooperatively a given regulated site responds to effector binding and therefore what kind of allosteric mechanism is involved. We apply the model to three proteins with experimentally characterized transitions: (i) calmodulin-GFP Ca(2+) sensor protein, (ii) maltose binding protein, and (iii) CSL transcription factor. Remarkably, the model is able to reproduce allosteric motion and predict coupling in a manner consistent with experiment.
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Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9.
J. Biol. Chem.
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Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.
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Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates.
Bioconjug. Chem.
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Antibody drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical conjugation methods that yield heterogeneous mixtures of varying number of drugs attached at different positions. The potential benefits of site-specific drug conjugation in terms of stability, manufacturing, and improved therapeutic index has recently lead to the development of several new site specific conjugation technologies. However, detail characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme based site-specific antibody drug conjugation technology that we recently developed. We found that in addition to conjugation of the engineered site, a small amount of aglycosylated antibody present in starting material led to conjugation at position Q295, resulting in approximately 1.3% of off-target conjugation. Based on our detection limits, we show that Q295N mutant eliminates the off-target conjugation yielding highly homogeneous conjugates that are better than 99.8% site specific. Our study demonstrates the importance of detail characterization of ADCs and the described methods can be utilized not only to characterize our enzyme based conjugates, but also ADCs generated by other site specific conjugation technologies.
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