High-throughput Flow Cytometry Screening Research Articles

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP

2’-Fucosyllactose (2’-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2’-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2′-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

249 (PB029) - Identification of Nectin-4 Specific Monoclonal Antibodies Using a High-throughput Flow Cytometry Screening System

Background: Nectin-4 has recently emerged as a highly valuable therapeutic target. This is due to its overexpression in epithelial cancers, including ovarian, breast, colorectal, lung, and pancreatic, while exhibiting limited expression in normal adult tissue. This overexpression is functionally significant for tumor progression, making Nectin-4 an ideal target for potential cancer therapeutics. Using a high-throughput flow cytometry-based antibody screening procedure, wherein antibody binding to native cell-expressed Nectin-4 is assessed concomitantly with off-target binding, multiple antibodies were identified.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Gut-licensed IFNγ+ NK cells drive LAMP1+TRAIL+ anti-inflammatory astrocytes.

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome.

Abstract 6307: In vivo drug screening platform accelerates anti-hCD40 antibody drug discovery

Abstract The CD40 (also known as TNFRSF5) and its ligand CD40L (also known as CD154) belong to the TNF/TNFR family. CD40 is expressed on antigen presenting cells (APCs), including B cell, monocytes, macrophages and dendritic cells. CD40-CD40L is a pair of costimulatory molecules that, after interacting at the cell surface, promotes intracellular signaling by recruiting TNFR-associated factor (TRAF) in the cytosol, thereby activating multiple downstream signaling pathways. Therapeutic targeting of CD40 by agonistic monoclonal antibodies (mAbs) aims to activate CD40+ APCs to effectively boost tumor-specific cytotoxic T cells to eliminate tumor cells. In principle it has the advantage of greater tumor specificity over other T cell activating approaches, such as checkpoint inhibition or indiscriminate T cell activation. We are interested in developing a novel immune checkpoint monoclonal antibody that targets CD40 and can treat cancer patients. Using Biocytogen's antibody research platform, dozens of candidate antibodies were obtained by classical hybridoma technology as well as high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in humanized mouse models (B-hCD40) bearing a syngeneic cancer cell line. We discovered candidates that have potent anti-tumor activity in vivo. These candidate antibodies were further humanized, and we found an antibody called YH003 with potent antitumor activity in the MC38 tumor model. Fortunately, YH003 was able to inhibit tumor growth in combination with Pembrolizumab in the double humanized model of h-CD40 and h-PD1 bearing a syngeneic B16F10 cancer cell line. In vitro studies demonstrated that YH003 bound specifically and potently to human and monkey CD40 proteins, we used cynomolgus monkeys to further explore the safety evaluation. In conclusion, we use Biocytogen's in vivo drug screening platform to obtain more novel CD40 antibodies with anti-tumor activity in addition to YH003, accelerating the progress of preclinical antibody discovery. Citation Format: Yi Yang, Zhihong Li, Yu Zhu, Jingshu Xie, Yunyun Chen. In vivo drug screening platform accelerates anti-hCD40 antibody drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6307.

High-Content Immunophenotyping and Hierarchical Clustering Reveal Sources of Heterogeneity and New Surface Markers of Human Blood Monocyte Subsets

Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

Abstract 1212: Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models

Abstract Signal regulatory protein α (SIRPα) is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs which mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. SIRPα is especially abundant in myeloid cells such as macrophages and DCs, whereas it is expressed at very low levels in T, B, NK, and NK T cells. SIRPα inhibits phagocytosis in macrophages upon interacting with its ligand CD47, which is commonly upregulated on the surface of malignant cells. Thus, antibodies that block the CD47-SIRPα interaction should enhance macrophage phagocytosis in the tumor microenvironment and inhibit tumor growth, making anti-SIRPα antibodies promising as potential new tools for cancer immunotherapy. We are interested in subverting the negative feedback of the CD47-SIRPa axis, in order to treat cancer patients. Using Biocytogen’s antibody research platform, we obtained several high-affinity anti-human SIRPα mAb by high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in single or double humanized mice models (B-hSIRPα or B-hSIRPα/hCD47) bearing a syngeneic humanized cancer cell line (MC38-hCD47), where we discovered candidates that have potent anti-tumor activity in vivo. Chimeric antibodies and humanized antibodies based on these candidates were generated and further examined in vivo. In addition, we uncovered synergistic anti-tumor activity of anti-human SIRPα mAbs in combination with anti-mouse PD-1 mAb in B-hSIRPα/hCD47 mice. In brief, our existing studies demonstrate that SIRPα is an ideal target for preclinical cancer models. Clinical evaluation of the translational merits for these novel anti-SIRPα antibodies is pending. Citation Format: James Jin, Benny(Yi) Yang, Chaoshe Guo, Yuelei Shen, Jian Ni. Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1212.

Abstract 3054: Cell surface targets in head and neck cancer

Abstract HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient. Citation Format: Maria Kondratyev, Aleksandra Pesic, Azin Sayad, Troy Ketela, Natalie Stickle, Carl Virtanen, Jason Moffat, Laurie Ailles, Marianne Koritzinsky, Brad Wouters. Cell surface targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3054.

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Background:Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform.Methods:Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation.Results:A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment.Conclusions:Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. DisclosuresPrashad:Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells

Hepatocellular carcinoma (HCC) remains a common and lethal cancer. Cancer stem cells, or tumor-initiating cells (TICs), are thought to contribute to the pathogenesis of HCC, but remain to be fully characterized. Unbiased screens of primary human HCC cells for the identification of novel HCC TIC markers have not been reported. We conducted high-throughput flow cytometry (HT-FC) profiling to characterize the expression of 375 CD antigens on tumor cells from 10 different human HCC samples. We selected 91 of these for further analysis based on HT-FC data that showed consistent expression in discrete, rare, sortable populations of HCC cells. Nine of these CD antigens demonstrated significantly increased expression in the EpCAM+ stem/progenitor fraction of a human HCC cell line and were further evaluated in primary human HCC tissues from 30 different patients. Of the nine tested, only CD146 demonstrated significantly increased expression in HCC tumor tissue as compared with matched adjacent non-tumor liver tissue. CD146+CD31−CD45− cells purified from HCC tumors and cell lines demonstrated a unique phenotype distinct from mesenchymal stem cells. As compared with other tumor cell fractions, CD146+CD31−CD45− cells showed significantly increased colony-forming capacity in vitro, consistent with TICs. This study demonstrates that HT-FC screening can be successfully applied to primary human HCC and reveals CD146 to be a novel TIC marker in this disease.

Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry

Neutrophils are the most abundant white blood cell and are an essential component of the innate immune system. A complete cataloguing of cell surface markers has not been undertaken for neutrophils isolated from circulation as well as healthy and inflamed tissues. To identify cell-surface markers specific to human neutrophils, we used high-throughput flow cytometry to screen neutrophil populations isolated from blood and oral rinses from healthy and chronic periodontitis patients against a panel of 374 known cluster of differentiation (CD) antibodies. This screen identified CD11b, CD16, and CD66b as markers that are consistently expressed on neutrophils independent of the cell location, level of activation and disease state. Cell sorting against CD11b, CD16 and CD66b allowed for the enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These findings suggest an ideal surface marker set for isolating mature neutrophils from humans. The screen also demonstrated that tissue neutrophils from chronically inflamed tissue display a unique surface marker set compared to tissue neutrophils present in healthy, non-inflamed tissues.

Abstract 3476: Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC)

Abstract Accumulating evidence supports the cancer stem cell (CSC) hypothesis, which posits that CSCs are the source of many solid tumor types including hepatocellular carcinoma (HCC). In HCC, CSCs have been identified by a variety of CD antigens, including CD133, CD326 (EpCAM), CD90, CD44, CD24, CD13, which could serve as potential therapeutic targets. However, due to the heterogeneity of HCC, these markers do not account for CSC activity in all tumors, and there is thus a need to identify novel HCC CSC markers. We have developed a cell-based high-throughput screening flow cytometry (HTS-FC) platform to robustly characterize expression profiles of CD antigens on primary human HCC cells. We titrated all commercial fluorescence conjugated CD antibodies (CD1 to CD363) into four 96-well microplates, isolated and stained HCC cells with CD45 antibody then divided and incubated these cells with different CD antibodies in microplates (1X10^5 cells/well) followed by a LIVE/DEAD® reactive dye treatment. Stained plates were fixed and subjected to BD™ LSR II flow cytometry using High Throughput Sampler Unit. Each CD antigen expression percentage was analyzed within the CD45 negative subpopulation by FlowJo software. After investigating 10 HCC tumors, we performed heat-map and cluster analysis using Multi Experiment Viewer software. Our analysis of primary human HCC cells demonstrated that previously identified index markers of HCC CSC are expressed on a highly variable fraction of tumor cells as follows: CD13 (76.10 ± 8.72%), CD24 (7.79 ± 5.33%), CD90 (5.29 ± 0.84%), EpCAM (0.50 ± 0.46%), CD44 (0.041 ± 0.019%) and CD133 (0.0051 ± 0.0034%). Based on the heat-map, we classified all CD markers into five groups: 20 CD13-like high expressions (30%∼100%), 12 median expressions (10%∼30%), 32 CD90-like low expressions (1.0%∼10%), 60 EpCAM-like rare expressions (0.03%∼1.0%) and 249 nonsense expressions. We focused on the CD90- and EpCAM-like groups (92 CD candidates) for novel potential CSC markers. Using quantitative real-time RT-PCR, we determined the mRNA level of 92 antigens in reference to the EpCAM+/CD133+ CSC subpopulations sorted from 4 human HCC cell lines (HepG2, Hep3B, PLC/PRF/5 and Huh7) and identified 20% of these 92 candidates with stem/progenitor mRNA phenotype. We then measured the mRNA level of these remaining candidates in 40 pairs of primary human HCC tumor/adjacent normal tissues and identified two novel CD antigens showing high tumor specificity. We are now purifying these cells from primary HCC tumors in order to determine whether these small populations identify cells with CSC activity in xenograft models and sphere-forming assays. These studies demonstrate that cell-based high-throughput screening flow cytometry screening can be used in combination with other cell/molecular biological techniques to identify novel populations of tumor cells with unique functional characteristics. Citation Format: Kui Chen, Laurie Ailles, John E. Dick, Anand Ghanekar. Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2014-3476

High-Throughput Flow Cytometry Screening Reveals a Role for Junctional Adhesion Molecule A as a Cancer Stem Cell Maintenance Factor

Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability tointeract with its niche through adhesion mechanisms. To identify targets that disrupt cancer stemcell (CSC) adhesion, we performed a flow cytometryscreen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A(JAM-A) as a CSC adhesion mechanism essentialfor self-renewal and tumor growth. JAM-A wasdispensable for normal neural stem/progenitorcell (NPC) function, and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromised the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate thatGBM-targeting strategies can be identified through screening adhesion receptors and JAM-A represents a mechanism for niche-driven CSC maintenance.

Monoclonal antibodies to transmembrane proteins (P4022)

Abstract Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20. Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies.

Abstract 4325: Monoclonal antibodies to transmembrane proteins.

Abstract Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20 (a tetraspan membrane protein). Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Epitope mapping with mutant proteins identified diverse patterns of reactivity including known and novel specificities. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies Citation Format: Michael C. Brown, Ross Chambers, Dale V. Onisk, Tony R. Joaquim, Lewis J. Stafford, Klaus Lindpaintner, Daniel Keter, James W. Stave. Monoclonal antibodies to transmembrane proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2013-4325

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.

Profiling of Toxicity and Identification of Distinct Apoptosis Profiles Using a 384-Well High-Throughput Flow Cytometry Screening Platform

Methods and techniques used to detect apoptosis have benefited from advances in technologies such as flow cytometry. With a large arsenal of lasers, fluorescent labels, and readily accessible biological targets, it is possible to detect multiple targets with unique combinations of fluorescent spectral signatures from a single sample. Traditional flow cytometry has been limited as a screening tool as the sample throughput has been low, whereas the data analysis and generation of screening relevant results have been complex. The HTFC Screening System running ForeCyt software is an instrument platform designed to perform high-throughput, multiplexed screening with seamless transformation of flow cytometry data into screening hits. We report the results of a screen that simultaneously quantified caspase 3/7 activation, annexin V binding, cell viability, and mitochondrial integrity. Assay performance over 5 days demonstrated robustness, reliability, and performance of the assay. This system is high throughput in that a 384-well plate can be read and fully analyzed within 30 min and is sensitive with an assay window of at least 10-fold for all parameters and a Z′ factor of ≥0.75 for all endpoints and time points. From a screen of 231 compounds, 11 representative toxicity profiles highlighting differential activation of apoptotic pathways were identified.

Cell-Based Screening Using High-Throughput Flow Cytometry

This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

Recognition of decay accelerating factor and αvβ3 by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and αvβ3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (Kd∼26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral–cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.