As experts in flow cytometry, the FlowMetric team applies this technology in numerous ways to analyze a variety of samples in support of basic research and through all levels of clinical trials. Common techniques offered include: immunophenotyping, signaling and phophoflow, cell sorting, bead assays, transfectant gene expression as well as many other cytometry methods. One specialized application of our expertise involves the support of our hybridoma development group, who employ flow cytometry techniques in various ways to enhance the selection and characterization of hybridoma-expressed antibodies.
During the typical flow of hybridoma projects, after animal inoculation and basic titers are determined, the spleens are harvested for single-cell suspension preparation and subsequent fusion with any of a number of different immortal fusion partner cell lines. Fusions will ultimately be screened for:
- antigen-specific antibody (may be against soluble or cell-bound receptor, or another antibody for anti-idiotype monoclonal antibodies, or CAR-T cell receptors, for example)
- cross-reactivity (can be the removal of undesired, or the addition of desired, cross-reactivity)
- isotype (can be helpful for diagnostic development purposes)
- function (engagement of different epitopes on a receptor can affect the result of antibody binding)
- or various other characterization studies.
Enriching Cell Populations by FACS Sorting
One way we can improve our workflow using flow cytometry is by performing fluorescence- activated cell sorting (FACS) to screen and select cells in bulk format for those that express antibodies of interest which can enrich antigen-specific antibody-producing clones many hundred-fold (Dangl and Herzenberg 1982). Our scientists have used for this type of work the BD FACSAria Cell Sorter for bulk cell sorts to enrich a population for desired antibody expression. Subsequently, the enriched population of cells would then be plated for ELISA screening. This improves efficiency by reducing the overall number of ELISA plates to analyze for the desired characteristics as described above and increasing the “hit rate” of positive colonies on each plate.
Enhanced Screening with Flow Cytometry
While ELISAs are often a convenient and quick method of constructing a simple screen assay, we must also be aware that plate-based screening may not always present an antigen in its native conformation which may result in selection of an antibody that reacts with a distorted form of the antigen that is not actually presented in nature (O’Reilly et. al. 1998). This is an area where flow cytometric screening has an edge since hybridoma supernatants can be reacted with and screened against actual cellular expression of a target.
Additionally, for more complex screens we can also use multiplex bead assays combined with flow cytometric screens to allow for a more thorough analysis of binding or function (Uribe-Benninghoff et. al. 2014). By way of example, therapeutic antibody binding can be directed in various ways: to block or to stimulate a signal; to cluster receptors such as CD3 or CD40; or to deliver a drug via antibody-drug conjugate (ADC), as well as to label a foreign substance for immunogenic action. The specific epitopes for which the different hybridoma clones might express antibody can differentially affect these actions. By screening antibodies that have been expressed from positively sorted hybridomas for engagement of target cells (by flow cytometry), and up- or down-regulation of soluble cytokines secreted from those target cells (by bead assay), one can obtain an extremely valuable early characterization of the fusion pool.
As becomes clear from the examples above, the more in-depth screening that is desired, the more useful the performance of clonal sorts can be. This we also do via the FACSAria and can be coupled with the HyperCyt system from IntelliCyt to perform multiplexed screening assays at speeds of up to 40 wells per minute enabling the processing of a 96-well plats in as little as three minutes (Black et. al. 2011).
FACS screen for cell surface target binding
FACS separates a population of cells into sub-populations based on fluorescent labeling. FACS enables researchers to better understand the characteristics of a single cell population, without the influence of other cells. An innovative method for separating and cloning the hybridoma cells using monoclonal immunoglobulin as the selection marker has been reported earlier (Park et al. 1979). Flow cytometry-based methods for rapid and high-throughput screening of hybridoma cells have been reported previously. Akagi et al. (2018) has developed a highly reliable and efficient FCM-based screening and cloning system (membrane immunoglobulin directed hybridoma screening and cloning) that can be easily adapted in labs.
FACS is well-suited to hybridoma production for potential clinical applications as cell sorters, and staining protocols can be done in compliance with regulatory requirements such as GLP conditions. In this case, working with a GLP-compliant partner, such as FlowMetric, may be a worthwhile consideration to assure that any experimental monoclonal antibody that may eventually be tested in a clinical trial will satisfy appropriate regulatory criteria. Hybridoma technology and FACS are keeping the monoclonal antibody pipeline filled with many new and exciting biologic candidates.
Beyond Binding Assays- the Future Application of Flow Cytometry for Enhanced Biological Drug Development.
Binding alone is often not sufficient for effective hybridoma screening, particularly for the screening of therapeutic monoclonal antibodies, which must be screened for their ability to modulate a specific response or function. To overcome this limitation, researchers have developed several method platforms that incorporate a functional component into the screen. In some instances, this may still require the clonal expansion of the hybridomas, whereas others are able to screen without the need for expansion, and therefore opening the possibility of direct screening of non-immortal cells including B-cells prior to fusion.
One of these platforms, droplet-based microfluidics, holds great promise and flexibility in screening. Individual cells (clones) are encapsulated in microdroplets surrounded by an immiscible carrier phase (typically a lipid). There are different screening types based on the functionality being screened for, but these encapsulations can contain specific targets to assess binding by Fӧrster Resonance Energy Transfer (FRET) or assess the inhibition of a drug target based on a fluorescent signal. Alternatively, the microdroplets may be fused with a second reaction droplet containing the screening reagents. Based on the fluorescent read-out, microdroplets characterized with a fluorescent signal indicative of a positive functional profile, may then be charged (positive or negative) and diverted for collection by cell sorting, a process labelled Fluorescence Activated Droplet Sorting (FADS).
Combining FADS technology with lentivirus transduction systems enables the functional screening of huge numbers of clones for a range of functions, including bi-specific antibodies with targeted applications in precision medicine. Examples of targets screened this way include inhibitors of angiotensin converting enzyme 1 (ACE1) (Debs et. al. 2012), costimulatory receptor CD40 agonist antibodies and anti-Her2/anti-CD3 bispecific antibodies (Wang et. al. 2020).
Such platforms as FADS are highly efficient, particularly for screening heterogeneous hybridoma populations, and even non-immortalized cells such as B-cell from HIV patients. With unique chemistries available, functional screening can include screening for desired metabolic-, inhibitory-, stimulatory-, or enzymatic (Baret et. al. 2009) phenotypes (Pierzchalski et. al. 2011).
Phenotypic screening is a valuable tool in drug discovery since it does not require a complete understanding of the target. With many chemical libraries now holding 100s of thousands of unique compounds, the automation of the screening workflow is central to the success of the screening initiative. Flow cytometry by nature is not typically used in this way, however researchers at GNSF Novartis, have developed high throughput methods with flow cytometry to enable phenotypic drug discovery across many different programs (Joslin et. al. 2018). Their screening platform utilizes a multiparameter flow cytometer equipped with an Application Program Interface (API) and coupled with a powerful informatics system for automated data analysis. Fluorescently barcoded cells expressing the primary antigens of interest or fluorescently barcoded beads conjugated with the antigen of interest are used for hybridoma screening. The barcoded capture cell or bead is mixed and plated in wells containing hybridoma supernatants and fluorescently labelled secondary detection antibodies are used to identify and sort individual cells or bead populations of interest. This approach provides the ability to multiplex the primary screen and effectively reduce the number of hits and the time needed to elucidate the desired phenotype. Accomplishing this within the primary screen, greatly improves the efficiency of screening without the need for a full understanding of the underlying molecular mechanisms of action. The result is a means of novel drug discovery and insights into disease pathology.
Screening Monoclonal Antibodies for Functional Engagement
Following the discovery and characterization of interferon in 1957, over 90 inflammatory cytokines and their receptors have been described that are generated by various cell types for numerous immune responses. Although an appropriate inflammatory response is the cornerstone of the host defense mechanism, the production of excessive or persistent inflammatory cytokines can be highly damaging and potentially result in the development of autoimmune diseases. The therapeutic targeting of pro-inflammatory cytokines has to date focused on the use of monoclonal antibodies targeting TNF-α, IL-6, IL-23 and IL-17 for the highly effective management of multiple autoimmune diseases including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. With the heterogeneity and complexity of human autoimmune diseases, there is a profound need for monoclonal antibody-dependent biological drug development. Flow cytometry can provide a simple and effective means of hybridoma screening for these targets using multiplexed capture beads and secondary detection probes for screening on a high throughput system. Competition binding studies can be designed to screen for specific target epitope binding characteristics even within a primary screen to ensure high quality hits
are identified early in the screening process. The same principles can be applied to target other small molecular targets of hybridoma screening, and even within pooled populations. Using this approach, it is therefore possible to immunize animals will pools of antigen species or combine spleens from separate immunized animals for a more streamlined higher throughput screening process.
Overcoming the Unique Challenges of GPCR Drug Targeting
G-protein-coupled receptors (GPCRs) play important roles in numerous physiological processes and pathways, and GPCRs represent one of the largest families of drug targets. Despite this, the confirmational flexibility and low immunogenicity/antigenicity of GPCRs makes it challenging to generate target-specific monoclonal antibodies for therapeutic applications. Although, several studies have identified neutral binders or antagonists, only in rare circumstances have agonist clones been identified. As a result, agonistic activity is usually achieved using natural peptide ligands and ligand-mimics fused with Fc, however, pharmacokinetically this is less desirable (Verhelst et. al. 2004).
To enable more effective screening for anti-GPCR agonist monoclonals, a powerful cell-based system employing a reporter assay system independent of G protein downstream signaling pathways was developed. Using lentiviral expression system, libraries of clones utilizing GPI to anchor their unique antibodies to the cell surface were generated in a cell line expressing a β-arrestin recruitment-based reporter system. This links recombinant antibody genotype with phenotype, enables the sorting of cells based on the β-lactamase production and subsequent fluorescence fingerprint based on antagonistic or agnostic antibody binding. Importantly, this β-arrestin recruitment-based cell sorting and screening does not depend on knowledge of the G protein signaling specificity of the target receptor and can be used to identify antibody agonists and antagonists to any GPCR target receptor (Ren et. al. 2020).
Advances in flow cytometry instrumentation, along with related technologies and methods are advancing many areas of cell-based and bead-based screening initiatives. The combination of fast-sample loading platforms, with multiparameter flow cytometry and powerful data analytics has revolutionized the application of cytometry beyond the clinical lab and into mainstream drug development.
With the rapid development of biological drug pipelines, flow cytometry is now being applied to numerous screening campaigns including functional-, toxicology-, and hybridoma- high-throughput screens to full effect. The ability to screen compounds and monoclonal antibodies against druggable targets within specific cell types has become central for a wide range of primary and secondary drug-screening programs.
Our team of experts can help design multiplexed screening programs to support a wide range of biological drug development programs and provide innovative solutions to the complex profiling of these therapeutics for our clients.
Authored by: Stephen McCarthy
Stephen McCarthy leads the hybridoma effort at FlowMetric Life Sciences. He joined the company with a rich background in an array of scientific disciplines including protein design, molecular biology, immunology, cell biology, assay development, chromatography, and cytometry. After receiving his B.S. in Biology from The Pennsylvania State University, he worked in private, government, industrial, and contract laboratories, and has amassed over 30 years of experience in the pharmaceutical industry with 25 years in antibody R&D and 5 years in vaccine research. Throughout his years in research, he has designed, cloned, expressed, purified, validated, and developed numerous novel antibody variants, including multi-specifics, with enhanced functionality, pharmacokinetics, and reduced immunogenicity through amino acid substitution or carbohydrate modification. He has co-authored two awarded patents and several peer-reviewed publications and presented posters at national antibody meetings. His work has contributed to the successful research and development of several FDA-approved antibody therapeutics.
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