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Metabolites produced in the human gut are known modulators of host immunity. However, large-scale identification of metabolite–host receptor interactions remains a daunting challenge. Here, we employed computational approaches to identify 983 potential metabolite–target interactions using the Inflammatory Bowel Disease (IBD) cohort dataset of the Human Microbiome Project 2 (HMP2). Using a consensus of multiple machine learning methods, we ranked metabolites based on importance to IBD, followed by virtual ligand-based screening to identify possible human targets and adding evidence from compound assay, differential gene expression, pathway enrichment, and genome-wide association studies. We confirmed known metabolite–target pairs such as nicotinic acid–GPR109a or linoleoyl ethanolamide–GPR119 and inferred interactions of interest including oleanolic acid–GABRG2 and alpha-CEHC–THRB. Eleven metabolites were tested for bioactivity in vitro using human primary cell-types. By expanding the universe of possible microbial metabolite–host protein interactions, we provide multiple drug targets for potential immune-therapies., Using computational approaches, Nuzzo et al. identify 983 potential metabolite–human target interactions from the Inflammatory Bowel Disease (IBD) cohort dataset of the Human Microbiome Project 2 (HMP2) and public databases. These predicted interactions can further the understanding of host–microbiome interactions and assist in drug discovery for IBD and other diseases.

Screening in phenotypic assays is an important strategy for the discovery of innovative drugs and novel targets. Here we present key strategies for developing successful phenotypic screens and prosecuting phenotypic drug discovery (PDD) programs. Successful screens incorporate physiological relevance through the use of human cell types and assay designs that have (1) strong mechanistic connection to clinical outcomes and (2) strong biological justification for both efficacy and safety. In addition to guidance for designing successful screens, we also propose incorporation of specific counterscreens at an early point in the program. The suggested counterscreens are based on analysis of 1000s of drugs and drug candidates profiled through a large set of human-based phenotypic assays. These assays include cytotoxicity in human primary vascular endothelial cells, proliferation of endothelial cells, and proliferation of lymphocytes, all under specific activation conditions. These counterscreens form a generic screening funnel to triage a large fraction of early-stage hits, binning compounds into those with undesirable mechanisms (associated with acute toxicity), mechanisms with utility for oncology indications, and mechanisms useful for autoimmune indications. The application of this screening funnel offers a standardized and more predictable path for prosecuting PDD programs, reducing the risk of failure, and improving program timelines.

Ensuring the safety of new drugs is critically important to regulators, pharmaceutical researchers and patients alike. Even so, unexpected toxicities still account for 20–30% of clinical trial failures, in part due to the persistence of animal testing as the primary approach for de-risking new drugs. Clearly, improved methods for safety attrition that incorporate human-relevant biology are needed. This recognition has spurred interest in non-animal alternatives or new approach methodologies (NAMs) including in vitro models that utilize advances in the culture of human cell types to provide greater clinical relevance for assessing risk. These phenotypic assay systems use human primary and induced pluripotent stem cell-derived cells in various formats, including co-cultures and advanced cellular systems such as organoids, bioprinted tissues, and organs-on-a-chip. Despite the promise of these human-based phenotypic approaches, adoption of these platforms into drug discovery programs for reducing safety-related attrition has been slow. Here we discuss the value of large-scale human cell-based phenotypic profiling for incorporating human-specific biology into the de-risking process. We describe learnings from our experiences with human primary cell-based assays and analysis of clinically relevant reference datasets in developing in vitro-based toxicity signatures. We also describe how Adverse Outcome Pathway (AOP) frameworks can be used to integrate results from diverse platforms congruent with weight-of-evidence approaches from risk assessment to improve safety-related decisions in early discovery.

Excipients, considered “inactive ingredients,” are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant Kd values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

Nonclinical tests are considered crucial for understanding the safety of investigational medicines. However, the effective translation from nonclinical to human application is limited and must be improved. Drug development stakeholders are working to advance human-based in vitro and in silico methods that may be more predictive of human efficacy and safety in vivo because they enable scientists to model the direct interaction of drugs with human cells, tissues, and biological processes. Here, we recommend test-neutral regulations; increased funding for development and integration of human-based approaches; support for existing initiatives that advance human-based approaches; evaluation of new approaches using human data; establishment of guidelines for procuring human cells and tissues for research; and additional training and educational opportunities in human-based approaches.

Background The BioMAP® platform is a complex human primary cell-based system for modelling tissue and disease states that is used to characterise drug activities based on the analyses of 148 clinically relevant biomarker readouts. Objectives To confirm that the BioMAP phenotypic signatures of two originator etanercept (ETN) samples remained comparable over time. Methods Two different ETN samples (ETN_1 and ETN_2) were independently profiled with a 5 year interval across a panel of 12 disease-relevant systems (3C and 4 hour [endothelial inflammation]; LPS [monocyte activation]; SAg [T cell activation]; BT [B cell activation]; BF4T and BE3C [epithelial inflammation]; CASM3C [vascular inflammation]; HDF3CGF, KF3CT, and MyoF [tissue remodelling, fibrosis]; lMphg [macrophage activation]). BioMAP systems consist of human primary cells or cocultures from healthy donors cultured in the presence of cytokines and growth factors. Protein levels, measured using immune-based methods or functional assays for cell viability and proliferation, were used to generate a BioMAP activity profile for each sample. ETN activities were annotated if they differed from the vehicle control and had an effect size >20%. The profiles of the 2 samples were compared using Pearson’s correlation. Results BioMAP phenotypic profiling of ETN_1 versus ETN_2 samples at 10 µg/mL revealed similar signatures across 148 biomarkers in 12 disease-relevant systems. The Pearson’s correlation coefficient was 0.781, which is above the determined threshold for mechanistic similarity (r≥0.7). Key efficacy-related anti-inflammatory and immunomodulatory activities were commonly inhibited in multiple systems including tumour necrosis factor alpha (LPS and BT), interleukin (IL)−2 (BT), vascular cell adhesion molecule 1 (MyoF and lMphg), IL-8 (SAg and MyoF), and E-Selectin (SAg and lMphg). The profiles of ETN samples at 1 µg/mL in the SAg system modelling T cell activation responses also revealed statistically significant similarity in signatures (p Conclusions The BioMAP phenotypic signatures of the ETN_1 and ETN_2 samples profiled in independent experiments using different primary cell pools remained comparable, which was consistent with conserved ETN mechanisms of action. The BioMAP platform represents a useful orthogonal approach for assessing ETN activity. Disclosure of Interest A. O’Mahony Employee of: Eurofins DiscoverX, E. L. Berg Employee of: Eurofins DiscoverX, H. Jones Shareholder of: Pfizer, Employee of: Pfizer, B. Fitzpatrick Shareholder of: Pfizer, Employee of: Pfizer, B. Hassett Shareholder of: Pfizer, Employee of: Pfizer, S. Vicik Shareholder of: Pfizer, Employee of: Pfizer, L. Marshall Shareholder of: Pfizer, Employee of: Pfizer, K. Roshak: None declared, E. Choy Grant/research support from: Novimmune, Pfizer, Roche, UCB, Consultant for: Abbott Laboratories, Amgen, Biogen, BMS, Celgene, Chugai Pharma, Eli Lilly, GSK, Hospira, Janssen, MedImmune, Napp, Novimmune, Novartis, Pfizer, Regeneron, Roche, R-Pharm, Sanofi

Introduction Unexpected drug activities account for many of the failures of new chemical entities in clinical trials. These activities can be target-dependent, resulting from feedback mechanisms downstream of the primary target, or they can occur as a result of unanticipated secondary target(s). Methods that would provide rapid and efficient characterization of compounds with respect to a broad range of biological pathways and mechanisms relevant to human disease have the potential to improve preclinical and clinical success rates. Methods BioMAP assays containing primary human cells (endothelial cells and co-cultures with peripheral blood leukocytes) were stimulated in complex formats (specific combinations of inflammatory mediators) for 24 h in the presence or absence of test agents (drugs, experimental compounds, etc.). The levels of selected protein readouts (adhesion receptors, cytokines, enzymes, etc.) were measured and activity profiles (normalized data sets comprising BioMAP profiles) were generated for each test agent. The resulting profiles were compared by statistical methods to identify similarities and mechanistic insights. Results Compounds with known mechanisms including inhibitors of histamine H1 receptor, angiotensin converting enzyme, IκB kinase-2, β2 adrenergic receptor and others were shown to generate reproducible and distinguishable BioMAP activity profiles. Similarities were observed between compounds targeting components within the same signal transduction pathway (e.g. NFκB), and also between compounds that share secondary targets (e.g. ibuprofen and FMOC-L-leucine, a PPARγ agonist). Discussion Complex primary cell-based assays can be applied for detecting and distinguishing unexpected activities that may be of relevance to drug action in vivo. The ability to rapidly test compounds prior to animal or clinical studies may reduce the number of compounds that unexpectedly fail in preclinical or clinical studies.

Rapid, quantitative methods for characterizing the biological activities of kinase inhibitors in complex human cell systems could allow the biological consequences of differential target selectivity to be monitored early in development, improving the selection of drug candidates. We have previously shown that Biologically Multiplexed Activity Profiling (BioMAP) permits rapid characterization of drug function based on statistical analysis of protein expression data sets from complex primary human cellbased models of disease biology. Here, using four such model systems containing primary human endothelial cells and peripheral blood mononuclear cells in which multiple signaling pathways relevant to inflammation and immune responses are simultaneously activated, we demonstrate that BioMAP analysis can detect and distinguish a wide range of inhibitors directed against different kinase targets. Using a panel of p38 mitogen-activated protein kinase antagonists as a test set, we show further that related compounds can be distinguished by unique features of the biological responses they induce in complex systems, and can be classified according to their induction of shared (on-target) and secondary activities. Statistical comparisons of quantitative BioMAP profiles and analysis of profile features allow correlation of induced biological effects with chemical structure and mapping of biological responses to chemical series or substituents on a common scaffold. Integration of automated BioMAP analysis for prioritization of hits and for structure-activity relationship studies may improve and accelerate the design and selection of optimal therapeutic candidates.

Phenotypic or biofunctional assays play an important role in drug discovery by helping to bridge the gap between high-throughput, target-based screening assays used for compound identification and more physiologically relevant in vivo disease models used for preclinical development. We have developed a standardised panel of phenotypic assays using primary human cells and co-cultures that model tissue and disease biology for characterization of drug leads. Here we show application of these assays for characterisation of clinical stage kinase inhibitors for rheumatoid arthritis, the recently approved JAK kinase inhibitor, tofacitinib, and the SYK kinase inhibitor, fostamatinib. We demonstrate how profiling in this assay panel can relate to clinical effects, both efficacy and safety related.

The heterogeneous distribution of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of vascular endothelium provides an opportunity to deliver drugs to select tissues. The targeting could be achieved by using carriers whose outer surface has a ligand for a selectively expressed ECAM. The carriers would interact with the endothelium in a fluid dynamic environment and in many of these schemes nanoparticles would be used. It is unclear what role various parameters (e.g., ligand-ECAM chemistry, fluid shear) will have on the adhesion of the nanoparticles to the endothelium. To facilitate studies in this area, we have developed a prototypical in vitro model that allows investigation of nanoparticle adhesion. We coated polystyrene nanospheres with a humanized mAb (HuEP5C7.g2) that recognizes the ECAMs E- and P-selectin. Adhesion assays revealed that HuEP5C7.g2 nanospheres exhibit augmented, specific adhesion to selectin presenting cellular monolayers and that the adhesion can be affected by the fluid shear. These results; (i) strongly suggest that HuEP5C7.g2 could be used to target nanoparticles to selectin presenting endothelium; (ii) demonstrate that fluid shear can affect nanoparticle adhesion; and (iii) define a system which can be used to study the effects of various system parameters on nanoparticle adhesion.

Objective To determine the role of the endothelial cell adhesion molecules E- and P-selectin in the development and severity of adjuvant-induced arthritis (AIA) in the rat. Methods Lewis rats were immunized subcutaneously with Mycobacterium butyricum (Mb), and blocking monoclonal antibodies (mAb) to rat E- and P-selectin were administered. Clinical score, radiolabeled (51Cr and 111In) blood polymorphonuclear leukocyte (PMN) and monocyte migration to joints, and histologic features were monitored. Results When mAb treatment was started on day 5 postimmunization with Mb (preclinical stage), development of AIA was significantly (P < 0.01) inhibited by mAb to E- but not to P-selectin (mean score on day 14 control 10.2, anti-E 2.8, anti-P 9.1). This was associated with markedly decreased migration (by 66–94%) of PMN and monocytes to arthritic joints and diminished cartilage degradation. When treatment was delayed until animals showed signs of arthritis (day 10 postimmunization), only a marginal and variable effect was observed as compared with blockade during the preclinical (day 5) stage. E-selectin blockade on day 5 and day 7 postimmunization resulted in inhibition of antigen-dependent T cell–mediated inflammation, since it decreased T cell migration to sites of dermal-delayed hypersensitivity induced by Mb without affecting migration to concanavalin A or cytokines. The proliferative response of T cells to Mb in vitro was not altered. Conclusion E-selectin plays an important role early in the development of AIA. This adhesion molecule may contribute to the migration of antigen-reactive T cells to peripheral tissues, including the joints where T cells initiate the arthritis.

Background —After activation, platelets adhere to neutrophils via P-selectin and β 2 -integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. Methods and Results —Whole blood was sheared at ≈100 s −1 . The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (≈30%) and neutrophil aggregation (≈70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. Conclusions —The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

The epitope of mouse monoclonal antibody (mAb) EP-5C7, which binds to and blocks both human E- and P-selectin, was mapped onto the protein structure of E-selectin. Analyses with E- and L-selectin chimeric proteins and randomly mutagenized E-selectins demonstrated that the EP-5C7 epitope consists of the amino acid residues at positions 21, 22, 23, 119 and 120 of E-selectin. The binding of three neutralizing anti-E-selectin mAb's (E-1E4, H18/7 and CL2), whose epitopes were found to overlap with the E-selectin binding site for carbohydrate ligands, was not affected by the amino acid substitutions at these five positions. Inspection of the three-dimensional structure of E-selectin indicated that the EP-5C7 epitope is located near the junction between the lectin and EGF-like domains. The ligand binding site was distant from the EP-5C7 epitope, suggesting that the amino acid residues in the EP-5C7 epitope play an important role other than ligand binding in selectin-mediated cell adhesion.

Blocking the function of l -selectin with a monoclonal antibody (mAb) is a promising way to prevent neutrophils from causing tissue damage during inflammation. HuDREG-55 and HuDREG-200 are humanized mAb which bind to human l -selectin and block its function as an adhesion molecule. To understand the mechanism of the action of HuDREG-55 and HuDREG-200, we determined their epitopes on l -selectin at the amino acid level. The analysis of human e - and l -selectin chimeric proteins demonstrated that the lectin domain of l -selectin is necessary for the binding of HuDREG-55 and HuDREG-200. Mutational analysis of Escherichia coli -expressed l -selectin showed that HuDREG-55 binding is sensitive to amino acid changes at positions 11, 56, 87, 89, 105, 107 and 111 (counting from the amino-terminus of mature l -selectin) while HuDREG-200 binding is sensitive to amino acid changes at 45, 46 and 47. Both epitopes are located close to the predicted carbohydrate binding site, indicating that HuDREG-55 and HuDREG-200 block the function of l -selectin by directly inhibiting the binding to carbohydrate ligands.

Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1–dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and α4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

E- and P-selectin are inflammation-induced cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions. Monoclonal antibodies (MoAbs) specific for either E-selectin or P- selectin are protective in several animal models of inflammatory disease. To generate an MoAb with broader therapeutic potential, MoAbs that bind to both E- and P-selectin were generated by immunization of mice with mouse pre-B cell lines transfected with human E- and P- selectin. Interestingly, although the only selection criterion was the ability to bind both E- and P-selectin, all three antibodies obtained efficiently block both E- and P-selectin-mediated functions. The inhibited functions include neutrophil or HL-60 cell binding to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells, E- or P-selectin transfectant cell lines, and platelet-HL-60 rosetting. These antibodies, EP-5C7, EP-2C9, and EP-1D8, recognize the same or overlapping epitope within the lectin domains of E- and P-selectin. The data suggest that functionally important epitopes of homologous proteins can be targeted by selecting for antibodies with reactivity toward both proteins. Furthermore, a potent blocking antibody specific for both E- and P-selectin may provide a more effective and broadly useful reagent for treating acute and potentially certain chronic inflammatory conditions.

Introduction The ability to predict the health effects resulting from drug or chemical exposure has been challenging due to the complexity of human biology. Approaches that detect and discriminate a broad range of mechanisms in testing formats that are predictive and yet cost-effective are needed. Methods Here, we evaluated the performance of BioMAP systems, primary human cell-based disease models, as a platform for characterization of chemical toxicity mechanisms. For this we tested a set of compounds with known or well-studied mechanisms in a panel of 8 BioMAP assays relevant to human respiratory, skin, immune and vascular exposure sites. Results We evaluated the ability to detect and distinguish compounds based on mechanisms of action, comparing the BioMAP activity profiles generated in a reduced sample number format to reference database profiles derived from multiple experiments. We also studied the role of BioMAP assay panel size and concentration effects, both of which were found to contribute to the ability to discriminate chemicals and mechanisms. Discussion Compounds with diverse mechanisms, including modulators of the NFκB pathway, microtubule function and mitochondrial activity, could be discriminated and classified into target and pathway mechanisms in both assay formats. Certain inhibitors of mitochondrial function, such as rotenone and sodium azide, but not others, were classified with inducers of endoplasmic reticulum stress, providing insight into the toxicity mechanisms of these agents. This method may have utility in classifying novel agents with unknown modes of action according to their effects on toxicity pathways.

The emerging class of novel drugs, including targeted biologics, shows compelling efficacy in oncology. The ability to test new agents, alone or in combination, in predictive human disease models supports the identification, optimization and application of new therapeutic strategies. Human primary cell-based BioMAP® Systems are designed to recapitulate the complex signals and phenotypic responses of diseased tissues and thus provide broad biological coverage of multiple and diverse indications. The BioMAP Diversity Plus panel consists of 12 systems with combinations of human primary endothelial cells, blood leukocytes, macrophages, bronchial epithelial cells, smooth muscle cells, fibroblasts, and keratinocytes relevant for inflammation, oncology and fibrosis. BioMAP Systems are capable of detecting and distinguishing activities of a very broad range of mechanistically diverse compound classes. We have generated a proprietary reference database of BioMAP profiles of >4,500 bioactive agents (drugs, biologics, experimental and environmental chemicals) and employ proprietary software and data mining tools for classification and similarity analyses of bioactivity profiles. The activities captured in the BioMAP profile are consistent with biological activities observed in patients. We have recently expanded the platform to include tumor microenvironment model systems to interrogate the phenotypic impact of compounds on inflammation, immune-function, tissue-matrix remodeling/metastasis, vascular/angiogenesis and/or tumor biology. BioMAP oncology systems consist of co-cultures of human primary fibroblasts (stromal) or endothelial cells (vascular) and PBMC (immune) with the HT29 (CRC) or NCI-H1299 cell lines (NSCLC) to model the inflamed host-tumor stromal and vascular microenvironments, respectively. Profiling clinical and development checkpoint modulators including antibodies, small molecules and combinations resulted in increased immune-related activity as measured by the resulting cytokine profile and cell markers of immune modulation, consistent both with the levels of CTLA4 in the system and with clinical results from patients. BioMAP® Systems thus provide a highly useful platform to (1) identify anti-inflammatory and anti-cancer effects of various agents and combinations and (2) identify different phenotypic outcomes of these agents that influence disease progression and resolution. Together these BioMAP data will support the discovery and development of safer and more effective therapies. Citation Format: Alison O'Mahony, Jason Ptacek, Jennifer Melrose, Karen Matta, Ellen Berg. Profiling compounds in human primary cell BioMAP® systems for drug discovery and development in cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B134.

Unexpected drug activities discovered during clinical testing establish the need for better characterization of compounds in human disease-relevant conditions early in the discovery process. Here, we describe an approach to characterize drug function based on statistical analysis of protein expression datasets from multiple primary human cell-based models of inflammatory disease. This approach, termed Biologically Multiplexed Activity Profiling (BioMAP), provides rapid characterization of drug function, including mechanism of action, secondary or off-target activities, and insights into clinical phenomena. Using three model systems containing primary human endothelial cells and peripheral blood mononuclear cells in different environments relevant to vascular inflammation and immune activation, we show that BioMAP profiles detect and discriminate multiple functional drug classes, including glucocorticoids; TNF-alpha antagonists; and inhibitors of HMG-CoA reductase, calcineurin, IMPDH, PDE4, PI-3 kinase, hsp90, and p38 MAPK, among others. The ability of cholesterol lowering HMG-CoA reductase inhibitors (statins) to improve outcomes in rheumatic disease patients correlates with the activities of these compounds in our BioMAP assays. In addition, the activity profiles identified for the immunosuppressants mycophenolic acid, cyclosporin A, and FK-506 provide a potential explanation for a reduced incidence of posttransplant cardiovascular disease in patients receiving mycophenolic acid. BioMAP profiling can allow integration of meaningful human biology into drug development programs.

Background A significant advance in rheumatoid arthritis therapy has been the development of biologics targeting proinflammatory cytokines. Biologics are commonly used with methotrexate (MTX) because clinical trials of anti–tumor necrosis factor alpha (TNFα) antibodies, such as adalimumab (ADA), have shown that combination therapy is more effective than monotherapy based, in part, on the assumption that MTX reduces the immunogenicity of biologics. However, combining MTX with the anti–interleukin-6 (IL-6) receptor antibody tocilizumab (TCZ) does not demonstrate the same level of incremental benefit over monotherapy. Given the known biologic effects of TNFα, IL-6, and MTX, we hypothesized that because IL-6 has more diverse effects on the immune system than TNFα, MTX may have fewer supplementary effects than TNFα inhibitors when added to TCZ. Objectives Using the BioMAP platform, we previously reported that TCZ, ADA, and MTX have distinct activity profiles. Here we used BioMAP systems to compare the profiles of combinations (ADA+MTX and TCZ+MTX) with the profiles of the individual agents (ADA or TCZ or MTX) to distinguish adjuvant effects. Methods BioMAP systems model disease biology in early-passage human primary cells, cultured alone or with different stimulus combinations, and have been used extensively to characterize compounds based on phenotypic signatures.1,2 BioMAP systems were used to generate profiles of TCZ, ADA, and MTX alone and of TCZ+MTX and ADA+MTX combinations. Agents were profiled at concentrations that would include their clinical Cmax ranges for the respective approved dosing regimen. Changes in protein-based and clinically relevant endpoints (biomarkers) and other cellular events (eg, proliferation, cell cytotoxicity) were evaluated to determine whether activities detected with the combinations were significantly different from the individual agents profiled in parallel. Results BioMAP profiling of ADA+MTX revealed more statistically significant different activities (p Conclusions These data show that TCZ has diverse effects on the immune response; the addition of MTX elicits few additional activities other than antiproliferative effects compared with TCZ alone. In contrast, ADA+MTX has statistically significantly more nonoverlapping effects on immune function, inflammation markers, and matrix-remodeling endpoints in primary human cell disease models than ADA or MTX alone. This finding supports our hypothesis that pharmacodynamic interactions between TCZ and MTX are less pronounced than those between MTX and ADA and may explain the clinical observation that TCZ is more effective than ADA as monotherapy.3 References Berg EL. Drug Discov Today. 2013; doi: 10.1016/j.drudis.2013.10.003. Berg EL et al. J Biomol Screen. 2013; doi: 10.1177/1087057113505324. Gabay C et al. Lancet. 2013;381:1541-1550. Disclosure of Interest : A. O9Mahony Shareholder of: DiscoveRx, Employee of: DiscoveRx, E. Berg Employee of: DiscoveRx, M. John Employee of: Roche, K. Ganeshalingam Shareholder of: Roche, Employee of: Roche, E. Choy Grant/research support: Abbott, Boehringer Ingelheim, Chelsea Therapeutics, Chugai Pharma, GSK, Jazz Pharmaceuticals, MSD, Novartis, Pierre Fabre Medicament, Roche, UCB, Consultant for: Abbott, Allergan, AstraZeneca, Boehringer Ingelheim, Chelsea Therapeutics, Chugai Pharma, Daiichi Sankyo, Eli Lilly, Ferring Pharmaceutical, GSK, ISIS, Jazz Pharmaceuticals, MedImmune, Merrimack Pharmaceutical, MSD, Novartis, Pfizer, Pierre Fabre Medicament, Roche, Schering Plough, Synovate, UCB, Speakers bureau: Abbott, Boehringer Ingelheim, Chugai Pharma, Eli Lilly, Jazz Pharmaceuticals, MSD, Novartis, Pfizer, Pierre Fabre Medicament, Roche, Scheering Plough, Synovate, UCB DOI 10.1136/annrheumdis-2014-eular.3386

Despite recent successful treatment of murine autoimmune disease with anti-IL-12 mAb, it has not yet been addressed whether anti-IL-12 mAb can also be effective in late stages of disease and whether it can provide lasting protection against recurrence, especially during continued presence of autoantigen. We used a newly developed psoriasis model in scid/scid mice, which allows easy tracking of pathogenic T cells, to show that when anti-IL-12 mAb is given for 2 wk (1 mg/wk) in the late stage of severe disease, inflammation is greatly reduced, as measured by ear thickness and histology (scores, 1.1 ± 0.1 vs 2.0 ± 0.4). Moreover, prolonged treatment (4 wk) of chronic psoriatic mice with high doses of mAb (1 mg/wk; prolonged active anti-inflammatory treatment (PAAIT)) results in the almost complete resolution of lesions (scores, 0.3 ± 0.1 vs 2.7 ± 0.2). Surprisingly, however, despite these significant treatment results, the psoriasis-like lesions return soon after the anti-IL-12 mAb treatment is discontinued. This rapid relapse of disease may be attributed to large populations of activated CD4+ T cells present in the lymph nodes of PAAIT animals still expressing an effector/memory phenotype (CD45RBlow, L-selectinlow). Upon stimulation in vitro such PAAIT lymph node cells secrete high amounts of IFN-γ (129 ng/ml); when transferred into naive scid/scid animals they are able to rapidly induce disease without costimulation. Our data indicates an alternative IL-12-independent pathway for pathogenic Th-1-like cells in vivo during the chronic phase of disease that allows these cells to persist and maintain their pathogenicity in the draining lymph tissue of the autoimmune site.

Background: The participation of L-selectin in leukocyte recruitment during inflammation has suggested the use of L-selectin inhibitors as potential anti-inflammatory therapeutics. Blocking monoclonal antibodies could serve as such therapeutic agents, particularly if humanized to reduce their immunogenicity and improve their serum half-life. Objectives: For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized. Study design: The resulting humanized antibodies, HuDREG-55 and HuDREG-200, constructed with human IgG4 constant regions, were evaluated for their specificity, affinity and ability to block L-selectin-dependent adhesion in in vitro assays. Their pharmacokinetic behavior in rhesus monkeys was also studied. Results: HuDREG-55 and HuDREG-200 were found to retain the specificity and affinity, within 2-fold, of the parent murine antibodies. HuDREG-55 and HuDREG-200 block L-selectin-dependent adhesion of human lymphocytes to high endothelial venules in frozen sections of lymph nodes. In addition, HuDREG-55 and HuDREG-200 are inhibitory in a novel L-selectin-dependent adhesion assay. This assay utilizes flow cytometry to measure binding of polymerized liposomes containing an analog of sialyl Lewis X, sialyl Lewis X glycoliposomes, to peripheral blood neutrophils and lymphocytes. Studying the pharmacokinetics of HuDREG-55 and HuDREG-200 in rhesus monkeys showed terminal elimination half-lives at 12.0 and 20.3 days, respectively. Conclusion: The shorter terminal elimination half-life of HuDREG-55 in rhesus monkeys may be due to the ability of HuDREG-55 but not HuDREG-200 to bind rhesus monkey L-selectin.

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.

Background and Purpose E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia. Methods The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n=4) and 3 hours of occlusion with reperfusion (1 hour, n=4; 4 hours, n=6; 24 hours, n=6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy. Results Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin=0.32±0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61±0.17) compared with control and nonischemic tissues (2 P P =.0005). The latter were further confirmed by an E:P-selectin immunoprobe. Conclusions E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.

Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L-selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on co-immobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV.

It is now widely accepted that chronic inflammatory events are critical contributors to both the development and progression of cancer. While the complex interrelationship between immune events and their effects on the neoplastic process remain to be defined, targeting inflammation has now become a priority of drug discovery in oncology. A major factor propelling this strategy is that both cancer cells and immune cells share many common signaling pathways that can contribute to the disease promoting microenvironment of the tumor. Drugs that target these pathways now make up a significant portion of pharmaceutical pipelines but how these compounds affect cancer cells, normal tissues and immune processes remains poorly understood. In order to develop more effective therapeutic strategies it is imperative that we understand the biological impact of compounds that target key signaling intermediates on normal and immune cells as well as neoplastic cells. BioSeek has pioneered the development of in vitro human cell-based systems (BioMAP® Systems) that recapitulate the complex signals and phenotypic responses of inflamed tissues in vivo. In addition to providing broad coverage of inflammatory targets, BioMAP® Systems model modulation of many signaling pathways relevant to cancer. Characterization of pathway-associated kinase inhibitors, including JAK, PI3K, p38MAPK, ERK, JNK and NF-κB in BioMAP® Systems has revealed activities that differentially impact inflammatory, proliferative, angiogenic and tissue remodeling processes. Secondly, profiling of 1000s of off-patent compounds through BioMAP® Systems identified agents that selectively prevent the growth of smooth muscle cells and related cancers (leiomyosarcomas and rhabdomyosarcomas) with little impact on the activation of immune cells. BioMAP® Systems provide a highly useful platform to (1) distinguish anti-inflammatory and anti-proliferative effects of various kinase inhibitors and (2) identify different phenotypic outcomes of these agents that influence cancer progression and disease resolution. Together these data will help inform the selection of more effective therapeutic strategies to target different cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1093. doi:10.1158/1538-7445.AM2011-1093

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.

Interactions of lymphocytes, monocytes, and granulocytes with endothelial cells in vivo exhibit striking specificity. The mechanisms involved in these interactions are important potential sites for therapeutic intervention into diverse immune and inflammatory processes. The involvement of the CD11a-c/18 family of integrin-related molecules, and the intercellular adhesion molecule-1 (ICAM-1) ligand for lymphocyte function-associated antigen-1 (LFA-1), in certain leukocyte-endothelial cell binding events has been amply demonstrated in in vitro studies well represented in this publication. Employing primarily in vivo and ex vivo approaches, we have demonstrated the existence of another class of leukocyte-endothelial cell recognition or adhesion receptors and ligands—lymphocyte homing receptors and their complementary endothelial cell ligands, the tissue-specific vascular addressins. In this chapter, we briefly summarize the important features of these molecules and emphasize that they are important not only in lymphocyte extravasation, but also in the extravasation of other leukocytes. Thus, homing receptors and vascular addressins may offer important targets for therapeutic modulation not only of tissue-specific immune responses, but also of acute and subacute inflammatory reactions characterized by granulocyte and monocyte-induced tissue damage.

An endothelial cell surface molecule that is selectively expressed in mucosal organs is required for lymphocyte homing to mucosal lymphoid tissues. This 'vascular addressin' appears to function as a tissue-specific marker or address signal for recognition by lymphocytes circulating in the blood.

Tissue position-dependent or address-dependent expression of cell adhesion molecules has been proposed to play a part in cellular positioning in a variety of systems, for example during neural development, the metastasis of neoplasms, and the tissue-specific homing of lymphocytes. The extravasation of blood-borne lymphocytes is regulated by interactions with the endothelium of specialised venules, such as the high endothelial venules (HEY) in organized lymph nodes and mucosal lymphoid tissues1–4. At least three separate lymphocyte-HEV recognition systems have been described, one mediating tissue-selective lymphocyte interac-tions with HEV in peripheral lymph nodes, another in mucosal lymphoid organs, and a third in inflamed synovium4–8. We have previously identified a tissue-specific "vascular addressin’ in the mouse which is selectively expressed by venules mediating lym-phocyte-homing into mucosal tissues9. To determine whether this addressin is a specific adhesion molecule for lymphocytes, we have isolated it by monoclonal antibody-affinity chromatography and inserted it into supported phospholipid planar membranes10,11. Lymphocytes bind to membranes containing the addressin, but not to phospholipid bilayers or to control glycophorin-reconstituted membranes. Only those lymphocytes and lymphoma cell lines capable of binding to mucosal HEV adhere well to the isolated addressin; peripheral lymph node HEV-specific or HEV-non-binding cell lines do not bind. Binding is blocked by anti-addressin antibody MECA-367. We conclude that the mucosal vascular addressin is a tissue-specific endothelial cell-adhesion molecule for lymphocytes, and suggest that it could regulate lymphocyte traffic into mucosal tissues by mediating attachment of blood-borne cells to endothelium.

Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.

Flow cytometry, immunohistology, and SDS- PAGE Western blot analysis were used to characterize two novel anti-mouse mononuclear phagocyte differentiation antigens defined by monoclonal antibodies. One antibody, Monts-4, recognized an 80-100 kd cell-surface protein expressed on resident macrophages in the peritoneum and stained macrophages in the splenic white pulp and marginal zone, liver, lymph node paracortical regions, Peyers patches, and cortex of the thymus. Monts-4 did not stain blood monocytes or monocyte-derived inflammatory macrophages in the peritoneal cavity. Macrophages that were Monts-4 positive became Monts-4 negative within 24-72 h after culturing in vitro. Monoclonal antibody SK39 recognized a 180 kd cell-surface molecule expressed on inflamed peritoneal monocytes/macro- phages and stained macrophages in the splenic red pulp, cortex of the thymus, subcapsule and medullary regions of lymph nodes, and in sites of acute and chronic inflammation. No differences were seen in the expression of these antigens in the immunodeficient SCID versus normal BALB/c mouse. A comparison of the distribution and molecular weights of the Monts-4 and SK39 antigens with other described mononuclear phagocyte-specific antigens, including F4/80 and those defined by the Ml/70, ERTR9, MOMA1, MOMA2, and SER4 monoclonal antibodies, shows these to be novel mononuclear phagocyte-specific differentiation antigens.