High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge

Brooks, Benjamin D.; Miles, Adam; Abdiche, Yasmina

doi:10.1016/j.drudis.2014.05.011

Cited by 25 publications

(25 citation statements)

References 11 publications

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“…This approach is designed to characterize the blocking profiles of mAbs in both directions (as analyte and ligand), thus allowing for the validation of relationships within and across our original groups [44, 45]. A major advantage is that cross-competition can easily be carried out between large numbers of mAbs (analytes) and ligands (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…One of the four forms of gD (Fig 1A) was then injected across the chip surface, followed by each of the 39 mAbs injected in series (Fig 1C) [44]. After being tested in this pair-wise, combinatorial manner, the mAbs were arranged by the software into bins to reflect competition vs. no competition [43–45]. Our assay, using 39 mAbs and the 4 forms of gD, generated approximately 6,000 sensorgrams (Fig 1D) which are then algorithmically analyzed to generate a heat map (Fig 2A) and dendrogram (Fig 2B) detailing the relationships of the mAb bins for each form of gD tested.…”

Section: Resultsmentioning

confidence: 99%

“…Given the relevance of gD-specific antibodies to virus neutralization and protection from disease, our goal in the current study was to generate a comprehensive examination of the antigenic structure of gD using an epitope binning (antibody competition) and mapping analysis that employs high-throughput array-based surface plasmon resonance imaging (SPRi) [43–45]. This assay provides a major technological advance in the tools to study the topological antigenic structure of the viral proteins.…”

Section: Introductionmentioning

confidence: 99%

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Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging

et al. 2017

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While HSV-2 typically causes genital lesions, HSV-1 is increasingly the cause of genital herpes. In addition, neonatal HSV infections are associated with a high rate of mortality and HSV-2 may increase the risk for HIV or Zika infections, reinforcing the need to develop an effective vaccine. In the GSK Herpevac trial, doubly sero-negative women were vaccinated with a truncated form of gD2 [gD2(284t)], then examined for anti-gD serum titers and clinical manifestations of disease. Surprisingly, few vaccinees were protected against genital HSV-2 but 86% were protected from genital HSV-1. These observations suggest that subtle differences in gD structure might influence a protective response. To better understand the antigenic structure of gD and how it impacts a protective response, we previously utilized several key anti-gD monoclonal antibodies (mAbs) to dissect epitopes in vaccinee sera. Several correlations were observed but the methodology limited the number of sera and mAbs that could be tested. Here, we used array-based surface plasmon imaging (SPRi) to simultaneously measure a larger number of protein-protein interactions. We carried out cross-competition or “epitope binning” studies with 39 anti-gD mAbs and four soluble forms of gD, including a form [gD2(285t)] that resembles the Herpevac antigen. The results from these experiments allowed us to organize the mAbs into four epitope communities. Notably, relationships within and between communities differed depending on the form of gD, and off-rate analysis suggested differences in mAb-gD avidity depending on the gD serotype and length. Together, these results show that gD1 and gD2 differ in their structural topography. Consistent with the Herpevac results, several mAbs that bind both gD1 and gD2 neutralize only HSV-1. Thus, this technology provides new insights into the antigenic structure of gD and provides a rationale as to how vaccination with a gD2 subunit may lead to protection from HSV-1 infection.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging

et al. 2017

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“… 35 Our data on clonal lineages might simplify such antibody engineering development work, for example, by identifying highly constrained amino acid residues. Additionally, further development would require comprehensive epitope mapping 36 or epitope binning 37 on hundreds of our mAb candidates. Antibody development would also require larger-scale studies of specificity, for example with peptide arrays.…”

Section: Discussionmentioning

confidence: 99%

Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics

Adler

Mizrahi

Spindler

et al. 2017

mAbs

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Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with KD < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.

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“…The epitope is a characteristic of an antibody's functional capacity and is an innate property that cannot be engineered, unlike its affinity. [1][2][3] An antibody's epitope can be defined at the molecular level using techniques such as crystallography to characterize the antibody antigen complex. 4 Alternatively, epitopes can be also assessed using low-resolution but high-throughput techniques.…”

Section: Introductionmentioning

confidence: 99%

Flow Cytometry-Based Epitope Binning Using Competitive Binding Profiles for the Characterization of Monoclonal Antibodies against Cellular and Soluble Protein Targets

et al. 2018

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A key step in the therapeutic antibody drug discovery process is early identification of diverse candidate molecules. Information comparing antibody binding epitopes can be used to classify antibodies within a large panel, guiding rational lead molecule selection. We describe a novel epitope binning method utilizing high-throughput flow cytometry (HTFC) that leverages cellular barcoding or spectrally distinct beads to multiplex samples to characterize antibodies raised against cell membrane receptor or soluble protein targets. With no requirement for sample purification or direct labeling, the method is suited for early characterization of antibody candidates. This method generates competitive binding profiles of each antibody against a defined set of known or unknown reference antibodies for binding to epitopes of an antigen. Antibodies with closely related competitive binding profiles indicate similar epitopes and are classified in the same bin. These large, high-throughput, multiplexed experiments can yield epitope bins or clusters for the entire antibody panel, from which a conceptual map of the epitope space for each antibody can be created. Combining this valuable epitope information with other data, such as functional activity, sequence, and selectivity of binding to orthologs and paralogs, enables us to advance the best epitope-diverse candidates for further development.

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High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge

Cited by 25 publications

References 11 publications

Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging

Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging

Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics

Flow Cytometry-Based Epitope Binning Using Competitive Binding Profiles for the Characterization of Monoclonal Antibodies against Cellular and Soluble Protein Targets

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