WO1997039722A2 - Procede pour isoler les regulateurs de l'activation des lymphocytes t - Google Patents

Procede pour isoler les regulateurs de l'activation des lymphocytes t Download PDF

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WO1997039722A2
WO1997039722A2 PCT/US1997/007052 US9707052W WO9739722A2 WO 1997039722 A2 WO1997039722 A2 WO 1997039722A2 US 9707052 W US9707052 W US 9707052W WO 9739722 A2 WO9739722 A2 WO 9739722A2
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assay
compound
cells
composition
reporter gene
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PCT/US1997/007052
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WO1997039722A3 (fr
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Augustine Y.-T. Lin
Scott W. Umlauf
Andreas G. Batzer
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T Cell Sciences, Inc.
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Priority to AU28148/97A priority Critical patent/AU2814897A/en
Publication of WO1997039722A2 publication Critical patent/WO1997039722A2/fr
Publication of WO1997039722A3 publication Critical patent/WO1997039722A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • This invention relates generally to methods for identifying immunosuppressive and immunoactivating compounds.
  • described herein is a multi-step, high throughput screening assay which provides a means to identify and isolate from mixtures or libraries of compounds of known or unknown structure and activity individual compounds that are capable of inhibiting or stimulating transcription of genes regulated by the activation of T cells which occurs upon binding or cross-linking ofthe T cell antigen receptor expressed on the surface of T cells.
  • T cells i.e., helper T cells
  • T cells T cell antigen receptor
  • MHC major histocompatibility complex
  • the binding of a TCR to a cognate peptide associated with an MHC on an antigen-presenting cell triggers a signaling cascade that stimulates transcription of cytokines, such as interleukin-2 (IL-2), that are essential for early activation of T cells as well as the subsequent activation of a variety of other cells ofthe immune system including B cells, macrophages, and eosinophils.
  • cytokines such as interleukin-2 (IL-2)
  • IL-2 interleukin-2
  • the successful transduction ofthe activation signal from the binding ofthe TCR on the surface of a T cell to the nucleus where cytokine gene transcription occurs involves a complex network of intracellular reactions (see, for example, Figures 1 and 2).
  • cytokines Early activation cytokines, such as IL-2, which are released from the T ceil, activate the T cell itself to proliferate (autocrine proliferation) and also activate other cells ofthe immune system, such as B cells, macrophages, eosinophils.
  • the expression of these early activation cytokines is regulated by several transcription control factors that form a number of complexes which bind at specific (enhancer) transcription control sequences associated with each cytokine gene (Schwartz, Cell, 7: 1065 - 1068 (1992)).
  • the intracellular transduction of a signal for T cell activation leads to the activation of transcription factors which bind at specific transcription control sequences to stimulate cytokine gene expression in the T cell.
  • NF-AT Nuclear Factor of Activated T Cells
  • NF-AT nuclear Factor of Activated T Cells
  • interleukin-2 see, for example, Durand et al., Mol. Cell. Biol., 8: 1715 - 1724 (1988); Shaw et al., Science,
  • calcineurin a calmodulin-dependent phosphatase
  • dephosphorylates cytoplasmic NF-AT which then can pass into the nucleus where it associates with at least one other factor (such as, AP-1 or CREB) to form the NF-AT transcription control complex.
  • the NF-AT transcription control factor then can bind to the NF-AT transcription control sequence in the IL-2 enhancer region to activate transcription of the IL-2 gene.
  • transcription factors involved in the stimulation of transcription of the IL-2 gene include fos and jun proteins that form an AP-1 transcription factor complex, NF- ⁇ B, and an octamer-binding complex transcription factor (Oct) (Schwartz, Cell, 71: 1065 - 1068 (1992)).
  • Such transcription control factors do not act independently of one another as evidenced by the fact that AP-1 must bind in the IL-2 enhancer region in order for NF-AT to bind at its cognate sequence.
  • the exact composition and binding associations ofthe various transcription factors involved in cytokine expression during T cell activation remain to be fully elucidated (see, for example, Schwartz, Cell, 71: 1065 - 1068 (1992); Rao, 1994).
  • a compound could act to modulate (that is, either to stimulate or to inhibit) the T cell activation process (see, for example, Figures 1 and 2).
  • Therapeutic regulation ofthe immune system can be divided into two major clinical categories.
  • the first is immune suppression (or inhibition), which is useful, for example, in the prevention of organ and tissue transplant rejection, allergies, and autoimmune diseases.
  • Current therapies for transplant rejection employ either cyclosporin A (CSA) or FK-506, in combination with steroids and azathioprine (so-called "triple therapy”).
  • a target at which cyclosporin A and FK-506 act is the Ca ++ -dependent phosphatase calcineurin (see, Liu et al., Immunol. Today, 291: 290 - 295 (1993); Figures 1 and 2).
  • the second clinical category of immune system regulation is immune activation (or stimulation), which is useful, for example, to assist in tumor rejection or to boost immune responses to infectious agents such as bacteria, parasites, and viruses, in particular human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • a multi-step, primary screen is described that provides the means and methods for identifying and isolating compositions or compounds that act as immune regulators to inhibit or stimulate T cell activation.
  • the primary screen ofthis invention can be used to screen any composition or compound that can be added to and dissolved or dispersed in cell cultures.
  • compositions and compounds screened by the primary screen ofthis invention may range from relatively crude preparations such as extracts of microbiological cultures, which may contain multiple unknown components, to relatively pure substances such as synthetically produced organic compounds of known chemical structure.
  • the individual steps of the primary screen utilize T cells transfected with recombinant plasmid constructs containing a reporter gene, such as the luciferase gene (LUC), encoding an easily detected protein and which is operatively linked to, i.e., under the transcriptional control of, either specific transcription control sequences from cytokine genes, such as the interleukin-2 (IL-2) gene, which are normally involved in the early activation of T cells, a transcription control sequence of a ubiquitously expressed gene which is important for cell viability, or a sequence which is specifically bound by a transcription factor involved in the regulation of one or more genes involved in early activation of T cells.
  • a reporter gene such as the luciferase gene (LUC)
  • IL-2 interleukin-2
  • the initial step determines whether a composition or compound can inhibit or stimulate T cell activation by screening for the ability to inhibit or stimulate expression of a reporter gene operatively linked to the promoter/enhancer region of a gene involved in early activation of T cells. Then, a second assay step (Toxicity Assay) is used to determine whether the composition or compound is likely to have an activity that is generally toxic to all or many cells.
  • Toxicity Assay the transcription of a reporter gene is under the control of a promoter sequence from a gene, such as ⁇ -actin, which is constitutively expressed in all or most cells ofthe human body and which is important for cell viability.
  • compositions or compounds that inhibit or stimulate expression ofthe reporter gene construct in the Toxicity Assay are discarded as having a general toxicity, except for anti-apoptotic compounds which stimulate reporter gene expression in activated T cells when the Toxicity Assay is run for greater than 8 hours.
  • such compositions or compounds can be identified as anti-apoptotic agents if they stimulate reporter gene expression in activated but not in unactivated T cells used in the Toxicity Assay.
  • an assay is employed that indicates whether the composition or compound is capable of affecting a particular transcription factor's control over cytokine gene expression.
  • each step ofthe primary screen ofthis invention uses Jurkat cells transfected with an appropriate recombinant plasmid containing the luciferase reporter gene operatively linked to either an IL-2 promoter/enhancer sequence (IL-1)
  • ⁇ -actinLUC ⁇ -actin promoter region
  • NFATLUC NFKB transcription factor binding sequences from the IL-2 promoter region
  • composition or compound that affects (stimulates or inhibits) reporter gene expression in the assays ofthe first (Activation Assay) and third (Specificity Assay) steps ofthe primary screen and which also exhibits no significant effect on reporter gene expression in the assay ofthe second step (Toxicity Assay) when run for less than 8 hours is considered an immune regulator based on the primary screen described herein.
  • Subsequent secondary assays are provided to further characterize the mode of action ofthe immune regulator as well as the likely identity ofthe molecular target(s) acted on by the immune regulator.
  • the cell-based format of each assay step ofthe primary screen requires that a compound, a composition, or a component of the composition be capable of binding to or passing into the cell. Accordingly, the primary screen described herein provides an especially efficient means of screening for compositions and compounds that have immune regulatory activity by affecting a target molecule that plays a role at some point in the pathway for transducing the signal for T cell activation from TCR or the associated CD3 molecule on the surface of T cells to genes involved in early T cell activation in the nucleus ofthe cells.
  • each cell-based assay is carried out under conditions of T cell activation initiated by binding or cross-linking the TCR (or the associated CD3) on the surface ofthe T cell, thereby providing a condition resembling the physiological conditions of normal T cell activation.
  • Each step ofthe primary screen also utilizes a reporter gene the expression of which is easily detected and quantified, such as the luciferase gene, thereby providing a high degree of selectivity and sensitivity for detecting a composition or compound which has either an immune stimulatory or inhibitory activity.
  • the efficiency, selectivity, and sensitivity ofthe primary screen described herein has been demonstrated by screening libraries, some containing thousands of synthetic compounds, and identifying candidate immune regulators from such libraries that act at target molecules in the T cell activation signaling cascade.
  • the primary screen of this invention permits the identification of immune regulators that act at any of a variety of possible sites (target molecules) along the signal transduction pathway for T cell activation, including those target molecules that are distinct from the site of action ofthe well known immune inhibitors cyclosporin A and FK506.
  • the primary screen ofthe invention has also been used to screen libraries consisting of thousands of compositions of fungal culture extracts containing unknown components, leading to the identification of compositions of fungal culture extracts that exhibit immune regulatory activity.
  • the primary screen can also be used to identify active fractions during purification of compounds from mixtures or compositions.
  • This invention also provides a cell-based assay for identifying immune regulators which are inhibitors of apoptosis (anti-apoptotic agents).
  • Each step ofthis assay for inhibitors of apoptosis involves incubation of T cells with a composition or compound for greater than 8 hours.
  • the first step of the assay determines whether a composition or compound stimulates expression of a reporter gene in an activated T cell transfected with a reporter gene construct in which a promoter/enhancer region of a cytokine gene involved in early T cell activation, such as IL-2, is operatively linked to the structural coding sequence of the reporter gene.
  • a composition or compound that stimulates reporter gene expression in the first step is then assayed in the second step to determine whether the composition or compound stimulates expression of a reporter gene in both activated and unactivated T cells transfected with a reporter gene construct, wherein the transcriptional control sequence of a ubiquitously expressed gene, such ⁇ -actin, is operatively linked to the structural coding sequence ofthe reporter gene.
  • a composition or compound that stimulates reporter gene expression in the activated T cells in the first step, in the activated T cells of the second step, but not in the unactivated T cells of the second step is selected as an immune regulator which is an anti-apoptotic agent.
  • the reporter gene is the luciferase gene.
  • Immune regulators identified by the methods ofthis invention are potential therapeutic agents for regulating T cell activation and the various clinically significant phenomena dependent on T cell activation.
  • FIG. 1 depicts a general scheme for certain portions of the signaling cascade involved in the intracellular transduction of a T cell activation signal initiated by the interaction of a T cell antigen receptor (TCR) on the surface of a T cell to a peptide antigen in association with a class II major histocompatibility complex (MHC) on an antigen presenting cell leading to the expression of IL-2, a cytokine involved in early T cell activation.
  • TCR T cell antigen receptor
  • MHC major histocompatibility complex
  • IL-2 a cytokine involved in early T cell activation.
  • the "+” sign indicates an activating event.
  • the "-” sign indicates an inhibiting event.
  • a "?” indicates that further signal transduction pathways, effectors, or the mode of action in the T cell are currently not known.
  • the protein ppi 20 (phosphoprotein 120) and cdc2 were identified during the secondary assays as targets for cyclosporin A and FK506. Numbers indicate sites in the signaling cascade for which a secondary assay is suggested for further characterization of compositions or compounds identified as immune regulators in the primary screen.
  • Figure 2 depicts another scheme ofthe intracellular transduction ofthe signal for T cell activation illustrating the envisioned transcriptional control mechanisms ofthe IL-2, jun, c-fos, and Fra genes.
  • Solid arrows depict various events in activation (for example, DG/Ca f2 - PKC), translocation (e.g., NFAT-PPP ⁇ NFAT; ERK - ERK-PP), phosphorylation (for example, FRK - PP-fos), dephosphorylation (for example, JunPase - jun-PP), and gene transcription (see hooked arrows, IL-2, jun, c-fos, Fra genes).
  • activation for example, DG/Ca f2 - PKC
  • translocation e.g., NFAT-PPP ⁇ NFAT; ERK - ERK-PP
  • phosphorylation for example, FRK - PP-fos
  • dephosphorylation for
  • Thick dashed arrow shows point of presumptive interaction ("crosstalk") between Ras and JNK pathways. Thin dashed arrows show activation-dependent events reported from other cell lines. "XTcell” adjacent to calcineurin indicates a T cell specific, but unknown, activation event.
  • Figure 3 shows a flow chart of a preferred embodiment ofthe Primary Screen for Immune Regulators according to this invention.
  • Figure 4 shows a flow chart of suggested secondary screening assays for further characterization of compounds identified in a primary screen for immune regulators.
  • Figure 5 shows a flow chart for a primary screen for immune stimulators, a screen for anti-apoptotic agents, and suggested secondary screening assays.
  • Figures 6A and 6B show the structures of eight arylidene diamide (oxazolone) derivative compounds identified as immune inhibitors from a library of approximately 8,000 molecules using the Primary Screen ofthis invention.
  • the structures depicted in Figures 6A and 6B are intended to cover all isomeric forms implied by the structures as drawn.
  • FIGS. 6A and 6B Compounds in Figures 6A and 6B are designated by "AT” numbers and corresponding "AQ” numbers which can be used to obtain the compounds from a commercial supplier (ArQule, Inc., Medford, MA): AT-1 is AQ32778, AT-2 is AQ32705, AT-3 is AQ32785, AT-4 is AQ32809, AT-5 is AQ32687, AT-6 is AQ29268, AT-7 is AQ25958, and AT-8 is AQ588.
  • Figure 7 shows the results of an Activation Assay for one plate in the Primary Screen for immune regulator compounds.
  • Figure 8 shows the results of a Toxicity Assay for compound AT-7 identified as a putative immune suppressor from the Activation Assay ofthe Primary Screen. Results are plotted as light units from luciferase activity (vertical axis) versus stimulation conditions
  • Figure 9 shows the results of a Specificity Assay for compounds at indicated concentrations in the Primary Screen. Results are plotted as Per Cent Inhibition of luciferase activity (vertical axis) versus compound designation (horizontal axis).
  • Figure 10 shows the results of a Ca ++ Ionophore/PM A activation assay for compounds identified in the Primary Screen. Results are plotted as Per Cent Inhibition of luciferase activity (vertical axis) versus compound designation (horizontal axis).
  • Figure 1 1 shows the results of a Splenocyte Proliferation Assay for splenocytes from naive mice stimulated in vitro with Concanavilin A (Con A) at indicated concentrations in the presence of compounds identified in Primary Screen. Results are plotted as Per Cent Inhibition of 3 H-thymidine incorporation (vertical axis) versus compound designation (horizontal axis).
  • Figure 12 shows the in vitro proliferation of splenocytes from Sperm Whale Myoglobin (SWM)-primed Balb-c mice injected with indicated amounts of AT-2 compound identified in the Primary Screen or with cyclosporin A. Results are plotted as stimulation Indices (see text) (vertical axis) versus SWM concentration (horizontal axis).
  • SWM Sperm Whale Myoglobin
  • Figure 13 shows a Delayed-Typed Hypersensitivity (DTH) response in Sperm Whale Myoglobin-primed Balb-c mice injected with indicated amounts of compound AT-8 identified in the Primary Screen or cyclosporin A. Results are plotted as edema (mm, vertical axis) versus group (N, horizontal axis). All groups (N) received SWM, except the "no antigen group”.
  • DTH Delayed-Typed Hypersensitivity
  • Figure 14 shows results of repeated assays of immune activators identified in the Primary Screen for immune regulator compounds (see Example 2). Results are plotted as Per Cent Activation of luciferase activity (vertical axis) versus compound designation (horizontal axis).
  • Figure 15 shows results of a Toxicity Assay of immune activators identified in the Primary Screen for immune regulator compounds (see Example 2). Results are plotted as Per Cent Activation of luciferase activity (vertical axis) versus compound designation (horizontal axis).
  • Figure 16 shows results of Figure 15 but omitting data of compound designated "O".
  • Figure 17 shows results of a Specificity Assay of immune activators identified in the
  • Results are plotted as Per Cent Activation of luciferase activity (vertical axis) versus compound designation (horizontal axis).
  • Figures 18A - 18F show the results of an Apoptosis Assay using Flow Cytometry to detect fluorescein isothiocyanate (FITC)-labeled Annexin V binding (staining) to exposed phosphatidylserine (PS) on the surface of Jurkat cells. Data in Figures 18A - 18F are plotted as cell counts (Counts) versus fluorescence (FLl -Height).
  • FITC fluorescein isothiocyanate
  • PS phosphatidylserine
  • M3.5H8 and M4.6H5 are compounds identified as immune regulators by screening a library of 3,840 organic compounds synthesized by combinatorial chemistry using the primary screen as described in Example 4. Ml indicates the total range of fluorescent signal surveyed as an indication ofthe total number of cells in the assay.
  • M2 indicates the range of fluorescent signal surveyed to detect apoptotic cells in the assay.
  • Figure 19 shows production of IL-2 from anti-CD3 treated splenocytes in the presence of immune stimulator composition XG08 identified by a primary screen in Example 7.
  • Figure 20 shows expression of luciferase (light units) versus stimulation conditions versus concentration of cyclosporin A (CsA, ng/ml) in cells transfected with ⁇ -actinLUC and incubated for 18 hours under the indicated conditions.
  • Figure 21 shows stimulation of expression of luciferase in activated and unactivated
  • compositions or compounds that stimulate or inhibit T cell activation are any substances that can be dissolved or dispersed in the cell cultures of each step ofthe primary screen ofthis invention.
  • composition encompasses a wide range of substances that may be screened according to the protocol ofthis invention, ranging from relatively crude and complex mixtures, such as crude extracts of microbiological cultures having potentially multiple, undefined components, to pure, synthetically produced, individual organic compounds of known chemical structure, such as members of an array or library of compounds produced by combinatorial chemistry.
  • compositions screened according to the methods of this invention may contain one or more compounds. Any assay described herein may be used to screen or further characterize compositions or individual compounds. Compositions or compounds that are identified by the primary screen ofthis invention as having an inhibitory or stimulatory effect on T cell activation may be used to regulate immune system activity in humans and animals or to promote or suppress T cell activity not only in vivo, but also in vitro or ex vivo.
  • compositions or compounds are called immune regulators and, more specifically, are further termed immune stimulators/activators or immune inhibitors/suppressors depending on whether they effectively increase or suppress T cell activation, respectively, as indicated by the primary screen ofthis invention.
  • the primary screen described herein is designed to detect at least one of these activities of such an immune regulator.
  • immune regulatory compositions or compounds identified and isolated according to this invention include, but are not limited to, small organic compounds, proteins, polypeptides, and multi-component preparations or extracts from natural sources, such as bacterial or fungal cultures.
  • the primary screen ofthis invention also indicates whether a composition or compound is likely to have the undesirable property of being generally toxic to all or most cells, and, thus, should be screened out as not therapeutically useful even though it exhibits an immune stimulatory or inhibitory activity. Furthermore, once a particular composition or compound has been identified and characterized by the primary screen of this invention as a candidate immune regulator, a more detailed characterization ofthe composition or compound and its target molecule on which the composition or compound exerts its activity can be made using any of a variety of secondary assays described herein.
  • T cell activation can be mimicked in a variety of ways, including by cross-linking or binding the TCR with an anti- TCR antibody, by cross-linking or binding CD3 which becomes associated with TCR during activation, by exposing T cells to phorbol myristate acetate (PMA) which stimulates the activity of two groups ofthe ofthe protein kinase C (PKC) family, and/or by exposing cells to a calcium ionophore (such as ionomycin or A23187) which increases intracellular Ca +2 which is required for calcineurin to de-phosphorylate NF-AT (see, for example, Flanagan et al., Nature, 352: 803 - 807 (1991)).
  • PMA phorbol myristate acetate
  • PKC protein kinase C
  • T cells without the necessity of either using antigen presenting cells or providing soluble class II MHC peptide complexes is an attractive feature for designing a rapid screen for compounds that affect T cell activation.
  • one drawback of using the chemical mimics PMA and a calcium ionophore, such as ionomycin, to activate T cells is that these compounds activate T cells by stimulating intracellular enzymes that are involved in relatively late steps along the signaling cascade.
  • such "late step activation” bypasses the portion ofthe signaling cascade upstream of PKC and the calcium released from intracellular storage which is more closely associated with the transmembrane a and ⁇ chains of TCR and the associated CD3 complex and which includes the participation of a variety of other enzymes such as the src family of kinases (such as fyn, lck) and the ZAP-70 kinase involved in early steps ofthe T cell activation (see, Figures 1 and 2).
  • the src family of kinases such as fyn, lck
  • ZAP-70 kinase involved in early steps ofthe T cell activation
  • activation is preferably initiated by cross-linking TCR, or the associated CD3 complex present on the surface of T cells, thereby retaining more ofthe physiological components of the signaling cascade as potential targets at which an immune regulator may act.
  • T cell activation is initiated by using an antibody to the ⁇ and/or ⁇ chains of TCR.
  • an antibody to the ⁇ and/or ⁇ chains of TCR Either a V ⁇ or V ⁇ -specific antibody or a
  • Pan- ⁇ antibody that binds to the constant regions ofthe ⁇ and/or ⁇ proteins of all human TCR molecules would be especially convenient for initiating T cell activation in the primary screen described herein.
  • the anti-TCR monoclonal antibody (MAb) 16G8 (Endogen, Cambridge, MA), which specifically binds the V ⁇ 8.1 region of the TCR expressed on Jurkat cells (ATCC Accession Number TIB 152), is used to initiate T cell activation.
  • Whether a particular anti-TCR or anti-CD3 antibody is capable of activating T cells can be readily determined by using any of a variety of assays for T cell activation. For example, after incubating T cells with a particular anti-TCR or anti-CD3 antibody, the T cells can be assayed for secretion of a cytokine, such as IL-2, into the surrounding medium which is indicative of T cell activation.
  • a cytokine such as IL-2
  • ELISA ELISA
  • antibodies specific for IL-2 and for IL-4 are commercially available (Endogen, Cambridge, MA; Boehringer Mannheim, Indianapolis, IN).
  • T cells whether a particular anti-TCR or anti-CD3 antibody can activate T cells can be determined using a T cell line transfected with a recombinant reporter gene, such as the luciferase (LUC) or ⁇ -galactosidase (LAC) genes, placed under the control of an enhancer sequence from a cytokine gene involved in T cell activation.
  • a recombinant reporter gene such as the luciferase (LUC) or ⁇ -galactosidase (LAC) genes
  • Examples of such a recombinant reporter gene construct is found on the IL-2LUC plasmid in which transcription ofthe luciferase gene is under the control of the IL-2 promoter/enhancer sequence or the NFATLUC plasmid in which transcription of the luciferase gene is under the control of NF- AT binding sequences ofthe IL-2 gene (Northrop et al., J. Biol. Chem., 268: 2917 - 2923
  • PBLs peripheral blood lymphocytes
  • the antibody which specifically reacts with T cells in the PBL mixture
  • the cells are pulse-labeled with 3 H-thymidine for an additional 24 hours.
  • Cells are then harvested on glass filters and counted in a scintillation counter for incorporated 3 H-thymidine as evidence of growth ofthe activated T cells in the PBL mixture.
  • Proliferation assays are most useful and convenient for determining the ability to activate T cells when the antibody is capable of binding TCR molecules on a broad range of T cells, such as "pan- ⁇ " antibodies mentioned above, which are capable of activating enough T cells to proliferate and be detected in the PBL mixture.
  • the primary screen for immune regulators of T cell activation described herein comprises a multi-step, high-throughput screening procedure which utilizes at each step a cell-based assay so that candidate compositions or compounds that exhibit an immune regulatory activity are likely to bind to extracellular targets on the surface of a T cell or be capable of traversing the T cell's membrane to reach intracellular targets.
  • the T cells used in each step ofthe primary screen are transfected with recombinant plasmids containing a reporter gene, such as the luciferase gene (LUC).
  • a reporter gene such as the luciferase gene (LUC)
  • LUC luciferase gene
  • a reporter gene such as LUC
  • luciferase is well characterized and easily detected with a high signal-to-noise ratio, making the assay steps ofthe screen highly sensitive.
  • a luciferase assay can be performed routinely in standard 96-well plates, which permits the efficiency of multi-channel pipetting and robotics.
  • the screen may be used for detecting both stimulators and inhibitors of T cell activation.
  • the steps ofthe screen In each ofthe steps ofthis screen, activation ofthe T cells usually takes 4 to 8 hours to reach plateau levels ofthe reporter gene activity, and therefore, the steps ofthe screen can be performed in a single day or with an overnight incubation. However, upon incubations of greater than 8 hours, luciferase levels begin to decline due to apoptosis. Therefore, as explained below, the immune stimulators identified in the primary screen should be further assayed for anti-apoptotic activity. In the primary screen, the sensitivity of the screen for compositions or compounds that are immune stimulators can be increased by using a sub- optimal TCR stimulus so that an increased reporter gene expression is more readily detected compared to control cells that are not exposed to the composition or compound being tested.
  • a sub-optimal T cell activation can be achieved, for example, by using a more dilute concentration of an anti-TCR antibody than is normally used to obtain a maximal activation ofthe T cells used in the screen.
  • Such sub-optimal T cell activation conditions not only increase the sensitivity ofthe screen to stimulatory compositions or compounds, but still provides a sufficient level of reporter gene expression to screen for compositions or compounds having inhibitory activity.
  • the primary screen for immune regulators of T cell activation comprises three assays (steps).
  • a diagram of an example of a primary screen ofthis invention is depicted in Figure 3.
  • Each assay is a cell-based assay using a T cell line transfected with a recombinant plasmid containing a reporter gene, more particularly the structural coding sequence from the reporter gene, operatively linked to a promoter/enhancer sequence of a cytokine gene involved in T cell activation, such that reporter gene expression is under the direct control ofthe cytokine gene promoter/enhancer sequence.
  • the reporter gene When a signal for T cell activation is transduced to the transcription control factors that bind to the cytokine promoter/enhancer sequence, the reporter gene is expressed.
  • the reporter gene is the luciferase gene and the T cells are the human lymphoma T cell line Jurkat which express a TCR having a V ⁇ 8.1 protein.
  • the exact order in which the three assays ofthis primary screen are performed is not critical, because the pooling of the data from all three assays enables one to determine whether a composition or compound has a significant immune inhibitory or stimulatory activity.
  • the assays ofthe primary screen are performed in the order listed below which permits a large number of compositions or compounds to be routinely and efficiently screened (high-throughput) with an economical use of resources (for example, time, reagents, cells).
  • Step 1. Activation Assay
  • the first step ofthis multi-step primary screen for immune regulators uses a human T cell line transfected with a plasmid containing the structural coding sequence of a reporter gene, such as the luciferase gene (LUC) (see, De Wet et al., Mol. Cell.
  • a reporter gene such as the luciferase gene (LUC)
  • luciferase cDNA and 5' and 3' flanking sequences at Figure 1, page 728 see also GenBank/EMBL Accession Number X65323, luciferase structural coding sequence at nucleotides 76 to 1725) operatively linked to the promoter/enhancer transcription control region of a cytokine gene involved with early activation of T cells, such as interleukin-2 (IL-1)
  • the IL-2LUC plasmid has been deposited in the American Type Culture Collection (ATCC, Rockville, MD) in accordance with the Budapest Treaty (ATCC Accession Number CRL- 12091 ).
  • ATCC Accession Number CRL- 12091 ATCC Accession Number CRL- 12091 .
  • the luciferase reporter gene has such a property when placed under the transcriptional control of the IL-2 promoter/enhancer region. Upon activation via TCR binding or cross-linking, the luciferase activity typically increases 50- to 100-fold, consistent with the level of expression ofthe native IL-2 gene typically observed in activated T cells.
  • transfected T cells are preferably activated by binding TCR, for example using an anti-TCR antibody such as the 16G8 anti-TCR monoclonal antibody (MAb)
  • an anti-TCR antibody such as the 16G8 anti-TCR monoclonal antibody (MAb)
  • this assay is run by immobilizing the antibody on the bottom of wells of a 96-well assay plate or added as a soluble component ofthe culture medium, then adding T cells, which have been transfected with a plasmid containing the reporter gene under the control of a cytokine promoter/enhancer region, in the presence or, for control cells, the absence of a compound being screened.
  • Other controls recommended for this step include non-activated cells, for example, cells in wells not containing antibody, and validation controls, such as cells activated in the presence of a compound with known inhibitor activity such as cyclosporin A or FK506.
  • Activation of T cells by antibody typically takes 4 to 8 hours, after which time the plates can be assayed for reporter gene expression, although the plates may also be incubated longer, for example overnight particularly to identify anti-apoptotic agents (see below), and assayed the next day.
  • the reporter gene is luciferase
  • standard luciferase measurements can be efficiently run using multi-pipetting (manual or automated) and an automated plate reader such as a luminometer (Packard Top- Count, Canberra, Australia).
  • the level of luciferase expression relative to control cells is a direct indication of each compound's ability to further stimulate or to inhibit cytokine gene expression and, therefore, T cell activation.
  • the sensitivity ofthis step for identifying and quantifying a composition's or compound's immune stimulatory activity can be enhanced by using conditions that provide a sub-optimal level of T cell activation so that an increase in the expression of a reporter gene is more easily detected.
  • a sub-optimal level of T cell activation can be effected by using, for example, a lower concentration of anti-TCR antibody than required for maximal reporter gene expression.
  • a sub-optimal level of T cell activation still permits the assay to be used for identifying compositions or compounds with inhibitory activity since a decrease in reporter gene expression can still be detected.
  • this first assay step distinguishes candidate immune stimulators and inhibitors from compositions or compounds that have neither activity. Because the level of expression of reporter genes, such as the luciferase gene, can be easily quantitated, this first step can also be used to distinguish between immune regulators of varying strengths of stimulatory or inhibitory activity.
  • Step 2. Toxicitv Assay The Activation Assay, described above, provides the first indication that a composition or compound may be capable of regulating (either stimulating or inhibiting) cytokine gene expression. However, alone, the first step cannot indicate whether the observed regulatory activity of a composition or compound is specific for genes involved in T cell activation or whether the activity ofthe composition or compound is non-specific so that stimulation or inhibition of the expression of other genes can occur.
  • a Toxicity Assay step is run, in the same manner as the Activity Assay, but using T cells transfected with a plasmid containing a reporter gene, such as luciferase, operatively linked to the promoter or promoter/enhancer sequence from a gene that is ubiquitously expressed, or at least expressed in a majority of cells of the human body.
  • a reporter gene such as luciferase
  • a preferred promoter/enhancer sequence is from the ⁇ -actin gene, an example of a so-called "house-keeping gene” which is expressed in most cells (see, for example, Ng et al., Mol. Cell. Biol., 5: 2720 - 2732 (1985) (sequences from ⁇ -actin promoter/enhancer); GenBank
  • the transfected T cells are activated, but the level of expression of the reporter gene in the activated T cells now reflects the expression of genes, such as ⁇ -actin, that are expressed in most or all cells, not the expression of cytokine genes involved in early T cell activation.
  • composition or compound that affects (stimulates or inhibits) the expression of the reporter gene in both the Activation Assay and Toxicity Assay steps performed with a relatively short (i.e., less than 8 hours) incubation period has a high probability of similarly affecting the expression of many genes in the body, including several whose functions are required ubiquitously.
  • stimulators of the Activation and Toxicity Assays can be further screened for anti-apoptotic activity as explained below.
  • the ⁇ -actin promoter/enhancer sequence is operatively linked to the luciferase structural coding sequence, such as in plasmid ⁇ -actinLUC deposited in the American Type Culture Collection (Rockville, MD) in accordance with the Budapest Treaty (ATCC Accession Number 98036).
  • the Toxicity Assay step ofthis primary screen provides a sensitive assay for screening out those compositions or compounds that have a high probability of being generally toxic to cells, owing to their relatively non-specific ability to inhibit or stimulate gene expression and, therefore, as not having a desirable specific T cell regulatory activity.
  • this cell-based Toxicity Assay to accurately detect a cytotoxic composition or compound is generally comparable to those cytotoxicity assays which detect cell death based on the loss of an ability to exclude dyes (such as trypan blue) or to reduce the dye MTT (see, for example, Burres et al., J. Antibiotics, 48: 380 - 386 (1995)). Comparable results have been obtained in monitoring cytotoxicity using a commercial MTT dye reduction-based assay (Promega, Madison, WI) and a Toxicity Assay ofthe primary screen of this invention. Unlike cytotoxicity assays based on dye exclusion or reduction, the Toxicity
  • Assay of the primary screen of this invention specifically indicates whether a composition or compound capable of activating or inhibiting cytokine gene expression (Activation Assay) also has the undesirable ability to interfere with the transcription of genes necessary for cell viability.
  • compositions or compounds that appear to regulate cytokine gene expression can be assayed to determine whether a candidate immune regulator specifically affects the action of a particular cytokine transcription factor, such as NF-AT, NF- ⁇ B, AP- 1 , or octamer-binding complex (Oct), by using a particular regulatory region ofthe IL-2 enhancer which is known to be specifically bound or otherwise critical for the activity of each of the individual transcription factors.
  • a candidate immune regulator specifically affects the action of a particular cytokine transcription factor, such as NF-AT, NF- ⁇ B, AP- 1 , or octamer-binding complex (Oct)
  • the sequences specifically bound by a number of key transcription control factors are known: for the NFAT transcription control factor, the sequence is at nucleotides 146 to 175 (Verweij et al., J. Biol. Chem., 265;: 15788 - 15795 (1990)) for the NFKB transcription control factor, the sequence is at nucleotides 221 to 244; for the AP-1 transcription control factor, the sequence is at nucleotides 270 to 294; and for the Octamer binding complex (Oct) transcription control factor, the sequence is at nucleotides 337 to 365.
  • the upstream transcription control region of a reporter gene such as the luciferase gene, is replaced with multiple repeats ofthe individual factor binding sites. For example, for determining whether a candidate composition or compound specifically affects
  • NFAT-mediated transcription of genes involved in T cell activation three repeats ofthe NFAT binding sequence from the IL-2 promoter/enhancer region can be operatively linked to the luciferase structural coding sequence and used to determine whether a candidate composition or compound specifically affects NFAT (see, Northrop et al., J. Biol. Chem., 268: 2917 - 2923 (1993) (NFATLUC plasmid)).
  • Some transcription factors may work cooperatively to stimulate cytokine gene transcription.
  • This assay alone, does not distinguish between a candidate immune regulatory composition or compound that affects a cooperatively acting transcription factor and a candidate immune regulatory composition or compound that affects a transcription factor that is not involved in cooperative binding. Nevertheless, because this Specificity Assay step employs discrete sequences known to be involved in transcription regulation of a cytokine gene, such as IL-2, if the candidate composition or compound causes an effect (stimulatory or inhibitory) on reporter gene expression, then the possible targets at which the candidate regulatory composition or compound acts will be rapidly narrowed to those factors whose control over cytokine gene transcription requires the particular sequence used in the plasmid construct.
  • this assay step can be conducted under sub-optimal conditions for T cell activation to increase the sensitivity of the assay for detecting compositions or compounds that stimulate T cell activation.
  • Overnight T Cell Activation and Primary Screen Assays The use of overnight incubations permits additional samples to be assayed overnight in the absence of supervising personnel.
  • the Jurkat cell line is susceptible to T cell antigen receptor activation-dependent apoptosis and this fact must be taken into consideration for at least some applications ofthe primary screen of this invention.
  • Apoptosis during activation of T cells probably occurs by means ofthe fas - fas ligand (fasL) pathway in which Jurkat cells, which constitutively express fas, upregulate fasL upon activation via the T cell antigen receptor (Dhein et al., Nature, 373: 438 - 441 (1995); Brunner et al., Nature, 373: 441 .444 (1995); Ju et al., Nature, 373: 444 - 448 (1995)).
  • fasL fas - fas ligand
  • luciferase levels will rise for the first 4 to 8 hours and thereafter begin to decline thereafter.
  • compositions or compounds which inhibit luciferase expression from the IL-2LUC reporter gene construct are generally unaffected by longer periods of activation, i.e., even if apoptosis occurs, an inhibition of luciferase expression can still be detected as a failure to induce expression ofthe reporter gene product, such as luciferase.
  • the use of the primary screen of the invention for inhibitors of T cell activation may be performed using incubation periods longer than 8 hours.
  • apoptosis in Jurkat cells that are activated for greater than 8 hours (as in overnight incubations periods which are typically 12 to 16 hours) will affect the screen for compositions or compounds that stimulate T cell activation.
  • compositions or compounds which are identified as putative stimulators of T cell activation in the Activation Assay ofthe primary screen using activation periods of greater than 8 hours should be further screened in a modified Toxicity Assay in which the compositions or compounds are tested for the ability to stimulate reporter gene expression in resting (no T cell activation) cells and in cells activated for greater than 8 hours.
  • This combination of Activation Assay and modified Toxicity Assay can be used to identify anti-apoptotic agents in arrays or libraries of various compositions or compounds.
  • compositions or compounds should be tested for the ability to stimulate luciferase activity in the transfected Jurkat cells in the absence (i.e., no activation) and presence of prolonged (greater than 8 hours) activation conditions.
  • Compositions or compounds which do not stimulate luciferase expression in both resting and activated Jurkat cells are classified as specific immune activators or stimulators of IL-2 transcription and should be screened in the Specificity Assay and further characterized using one or more Secondary Assays described below.
  • compositions or compounds which stimulate luciferase expression in the Jurkat cells under non-activating conditions are classified as non-specific stimulators or activators of gene transcription (positive for the Toxicity Assay) and are discarded.
  • Compositions or compounds which stimulate luciferase expression in the Jurkat cells containing the ⁇ -actinLUC reporter gene construct which were activated overnight (i.e., greater than 8 hours), but not in non-activated resting cells are likely to be inhibitors of apoptosis, i.e., anti-apoptotic agents.
  • Anti-apoptotic activity of such compositions or compounds can be confirmed and further characterized using one or more additional apoptosis assays (see Secondary Assays, below).
  • Anti-apoptotic agents identified by the screening protocol described herein may find therapeutic utility in treating infectious diseases, including acquired immune deficiency syndrome (AIDS) where apoptosis of responding T cells has been correlated with a weakened immune response (Thompson, Science, 267: 1456 - 1462 (1995)).
  • AIDS acquired immune deficiency syndrome
  • This assay is designed to determine whether the target of an immune inhibitor identified in the primary screen is located in the signaling cascade upstream or downstream of the PKC (1 in Figure 1) and calcium ion (2 in Figure 1) and calcium-dependent (such as calcineurin, 3 in Figure 1) steps ofthe signaling cascade in the T cell.
  • This assay uses T cells transfected with the same plasmid constructs used in step 3 (Specificity Assay) ofthe primary screen, but uses chemical mimics for T cell activation, such as PMA and a calcium ionophore, such as ionomycin, to activate the T cells.
  • PMA acts on two groups of protein kinase C (PKC) to initiate a signal for T cell activation, and ionomycin acts to increase the calcium ion concentration thereby stimulating calcineurin activity in the signaling cascade
  • This assay measures increases in intracellular calcium ion upon crosslinking of the TCR.
  • a composition or compound identified in the primary screen as a stimulator of T cell activation may potentiate calcium ion flux under conditions of sub-optimal TCR signal.
  • Compositions or compounds which augment calcium ion flux can be identified using the fluorescence calcium ion stain Indo 1 (Molecular Probes, Eugene, Oregon).
  • fluorescence calcium ion stain Indo 1 Molecular Probes, Eugene, Oregon
  • This in vitro assay is used to determine whether a composition or compound is capable of inhibiting the actual binding of a transcription control factor to its cognate binding sequence in the promoter/enhancer region of a cytokine gene involved in early activation of T cells.
  • the promoter/enhancer region is from the IL-2 gene (Holbrook, et al.,
  • compositions or compound identified in the primary screen as an immune stimulator may potentiate binding of a transcription factor under conditions of sub-optimal TCR signaling.
  • This assay is designed to identify the mode of action of an immune inhibitor identified in the primary screen with respect to the phosphorylation state of NFATp. In particular, this assay determines whether an immune inhibitor affects the dephosphorylation ofthe NFATp protein (5 in Figure 1), which is characteristic of an inhibitor acting at calcineurin (3 in Figure 1).
  • the immune regulators cyclosporin A and FK506, for instance, have such an activity (reviewed in Schreiber and Crabtree, Immunol. Today, 13: 136 - 142, 1992) and are generally toxic to many cells, particularly cells ofthe liver and kidneys. Accordingly, this NFATp dephosphorylation assay aims at excluding compositions or compounds that inhibit calcineurin and would therefore have a similar toxicity as cyclosporin A or FK506.
  • NFATp (5 in Figure 1) is a member of a family of transcription factors involved in T cell regulation (reviewed in Rao, Immunol. Today, 15: 21 A - 281 (1994)). NFATp consists of 890 amino acids and runs with an apparent molecular weight
  • Detergent lysates are prepared from activated or non-activated T cells in the absence or presence of compositions or compounds identified from the primary screen.
  • NFATp is immunoprecipitated from the lysates with a specific anti-NFATp-antibody, run on a SDS- polyacrylamide gel (standard SDS-polyacrylamide gel electrophoresis), and transferred to a nitrocellulose membrane for immunoblot (Western blot) analysis using the same anti-NFATp antibody.
  • Inhibition of calcineurin can be determined by examining the apparent mobility of
  • NFATp NFATp.
  • a composition or compound that inhibits calcineurin would prevent dephosphorylation of NFATp.
  • a composition or compound identified as an immune stimulator in the primary assay that activates calcineurin when calcium ion is limiting would potentiate the dephosphorylation of NFATp, augmenting its transcriptional activity. 6. Phosphotyrosine Proteins Assay
  • This secondary assay is recommended for compositions or compounds which inhibited reporter gene expression in the Specificity Assay (step 3) of the primary screen but did not inhibit reporter gene expression in PMA/ionophore-activated T cells in the Ca ⁇ 2 Ionophore/PMA Activation Assay (secondary assay 2, above).
  • This assay is designed to detect changes in the pattern of tyrosine phosphorylated proteins in T cells activated with an anti-TCR antibody and gives a better indication of the point in the signaling cascade at which a composition or compound identified in the primary screen asserts it affect. It is well established that protein tyrosine kinases are critically involved in a variety of signal transduction pathways. One group of protein kinases consists of receptor tyrosine kinases whereas the other group contains the non-receptor type kinases (reviewed in Hunter and
  • Protein tyrosine kinases are crucial for the signal transduction through lymphocyte antigen receptors (reviewed Weiss and Liftman, Cell, 76: 263 - 274 (1994)). Anti-phosphotyrosine antibodies have proved to be extremely valuable for the evaluation of signaling events (Friedman et al., Proc. Natl. Acad. Sci. USA, 81: 3034 - 3038 (1984);
  • detergent lysates are prepared from activated or non-activated T cells, in the absence or presence of lead compositions or compounds; the lysates electrophoresed on SDS-gels and transferred to a nitrocellulose membrane for Western blot analysis using anti- phosphotyrosine antibodies. Changes in the pattern of tyrosine phosphorylated proteins in the presence of a candidate composition or compound indicate a mode of action closer to the TCR and before the PKC and calcium-dependent steps of signal transduction (for example,
  • a composition or compound identified as an immune stimulator in the primary assay may augment tyrosine phosphorylation under conditions of sub-optimal TCR signaling.
  • Assay for ppi 20 Dephosphorylation
  • the purpose ofthis assay is to determine whether the mode of action of compositions or compounds identified in the primary screen affects tyrosine phosphorylation in HUT78 T cells stimulated with PMA and ionomycin.
  • a protein which has an apparent molecular weight (Mr) of 120,000 daltons, becomes phosphorylated on tyrosine upon activation with ionomycin.
  • Mr apparent molecular weight
  • this assay provides a comparison ofthe mode of action of a candidate composition or compound identified in the primary screen and cyclosporin A, which is known to inhibit calcineurin.
  • the assay is run in basically the same manner as the Phosphotyrosine Protein Assay described above, except the HUT78 T cells are activated with
  • This assay is to determine whether compositions or compounds identified in the primary screen affect the phosphorylation state of the cyclin-dependent kinases (cdks) cdc2 and cdk2 in the nucleus (see Figure 1).
  • the cyclin-dependent kinases are a class of serine/threonine kinases that regulate cell cycle checkpoints and progression through the cell cycle. Cdk activity is tightly regulated by several mechanisms, including an association with a specific cyclin regulatory subunit, activating and inhibiting phosphorylations, and binding of a class of small proteins termed the cyclin kinase inhibitors (see, Morgan, Nature, 374: 131- 134 (1995)).
  • the kinase activity of cdc2 is required for the
  • T cells typically approximately 100 x 10 ⁇ T cells are pre-incubated for 15 minutes at room temperature in the absence or presence of a composition or compound (for example, 30 ⁇ g/ml) identified in the primary screen, followed by activation either with 10 / Ug/ml anti-T cell receptor antibody (for example, 16G8, Endogen, Cambridge, MA) and
  • the pellet is washed with Cytb minus NP40 buffer (Cytb buffer as described above, but with NP-40 omitted) and then extracted with 420-buffer (Cytb- buffer plus 10% glycerol and 420 mM NaCI) for 30 minutes on a rocker at 4°C.
  • the supernatant after centrifugation (for example, 12 minutes at 14,000 ⁇ m in an Eppendorf tabletop centrifuge) yields the "nuclear extract".
  • ⁇ dc2 is immunoprecipitated from 1 mg of lysate protein with 5 ⁇ l of anti- cdc2-antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C.
  • cdk2 is immunoprecipitated using an anti- cdk2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Otherwise, the assays are identical. Standard immunoblotting (Western blot analysis) is performed with the appropriate antibody and anti-phosphotyrosine antibodies.
  • the lysates are then subjected to SDS-PAGE followed by Western immunoblot analysis using commercially available anti-Raf, -MEK or -ERK antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). All three kinases are activated by phosphorylation through upstream kinases.
  • Raf is activated by phosphorylation through an unidentified membrane-bound kinase on serine and threonine.
  • MEK is activated by phosphorylation by Raf on serine and threonine residues.
  • ERK is activated by phosphorylation by MEK on tyrosine and threonine residues.
  • JNKs jun N-terminal kinases
  • JNKs are important in the signal integration during T cell activation via TCR cross-linking (Su et al., Cell, 77: 727 - 736 (1994)) because they are responsible for the phosphorylation ofthe transcription factors c-jun, TCF, and ATF2, necessary steps in the formation ofthe active AP-1 transcription factor complex (Derijard et al., Cell, 76: 1025 - 1037 (1994)).
  • detergent lysates are prepared from activated or non- activated T cells in the absence or presence of a candidate immune regulator identified in the primary screen. Such lysates may then be analyzed by using any or all of a variety of assays for JNK activity, such as an in vitro kinase assay of immunoprecipitated JNK using GST-jun fusion proteins as substrates, "precipitation/kinase” assays using GST-jun fusion proteins, and "in-gel” assays which allow visualization of jun phosphorylation by JNK (see, Hibi et al., Genes Dev., 7: 2135 - 2148 (1993)).
  • compositions or compounds identified by the primary screen can also be analyzed for their ability to affect the translocation of proteins involved in the regulation of cytokine transcription from the cytosol into the nucleus, either upon activation (for example, the translocation of dephosphorylated NFAT, 5 and 6 in Figure 1) or after their synthesis in the cytosol (for example, c-fos).
  • proteins typically contain short stretches of basic amino acids which act as nuclear localization signals (NLS) (reviewed in Dingwall and Laskey, Trends Biochem. Sci., 16:478-481, 1991).
  • NPS nuclear localization signals
  • the transport mechanism mediated by nuclear pore complexes (NPC) is fairly well understood (reviewed in Gerace, Curr. Opin. Cell Biol., 4:637-645, 1992).
  • a composition or compound that blocks the translocation into the nucleus of a specific protein involved in T cell activation would be particularly desirable owing to the critical role NFAT plays in the expression of cytokines, such IL-2, in T cell activation.
  • a composition or compound that blocks or inhibits nuclear translocation in general is less desirable as a therapeutic agent, because such a general inhibitor of nuclear translocation may inhibit translocation of proteins that are unrelated to T cell activation but important for critical functions necessary to most or all cells.
  • cytosolic and nuclear extracts are prepared as in the Nuclear cdc2 Assay, described above.
  • T cells are extracted with a detergent and low salt (for example, 15 mM KCl in Cytb-buffer, see above) buffer to yield cytosolic extracts having intact nuclei, followed by extraction ofthe nuclei with a high salt (e.g., 420 mM NaCI in 420-buffer, see above) buffer to yield nuclear extracts.
  • a detergent and low salt for example, 15 mM KCl in Cytb-buffer, see above
  • a high salt e.g., 420 mM NaCI in 420-buffer, see above
  • Selected target proteins such as NFAT, ERK, c-fos, c-jun
  • cytokine genes such as IL-2
  • the Western blot analysis allows a comparison to be made in the pattern of the specific target proteins in the nuclear extracts of T cells activated in the presence and the absence of a composition or compound from the primary screen to determine whether the composition or compound affects the translocation of any ofthe specific target proteins involved in T cell activation.
  • PLC Protein Kinase C
  • PKC protein kinase C
  • detergent lysates are prepared from T cells, activated and non- activated, in the absence or presence of a composition or compound for the primary screen.
  • the lysates or species of PKC immunoprecipitated from the lysates are assayed for kinase activity.
  • Addition of a composition or compound from the primary screen to immunoprecipitated PKC from untreated T cells allows an assessment of a direct effect on a PKC kinase activity.
  • the pu ⁇ ose of this assay is to determine whether a composition or compound identified by the primary screen can affect (stimulate or inhibit) the expression of genes coding for proteins of the jun and fos families (8 and 9, respectively, in Figure 1). Jun and fos proteins are components ofthe dimeric AP-1 transcription factor (see, Angel and Karin, Biochim. Biophys. Acta., 1072: 129 - 157 (1991)).
  • AP-1 proteins for example, c- jun, junB, junD, c-fos, fosB, fra-1, and fra-2
  • detergent lysates are prepared from T cells that were activated (and those that were not activated) in the absence or presence of a composition or compound identified in the primary screen.
  • Whole cell lysates are prepared and specific antibodies used to immunoprecipitate one or more ofthe various fos/jun family members.
  • the immunoprecipitates are then assessed by Western blotting using the relevant antibodies (e.g., available from Santa Cruz Biotechnology, Santa Cruz, CA) to identify the pattern of proteins expressed in the T cells to determine whether a composition or compound can stimulate or inhibit fos or jun protein expression.
  • relevant antibodies e.g., available from Santa Cruz Biotechnology, Santa Cruz, CA
  • Mouse Splenocyte and Human Peripheral Blood Lymphocyte Proliferation Assay Compositions or compounds identified in the primary screen as having immune regulatory activity can be assayed for their ability to stimulate or inhibit proliferation of non- transformed cells by examining the effect of such stimulators or inhibitors have on the proliferation of mouse splenocytes or human peripheral blood lymphocytes (PBL).
  • PBL peripheral blood lymphocytes
  • the candidate immune regulatory compositions or compounds are incubated with splenocytes from naive DBA-2 mice, under conditions that activate T cells and thereby promote proliferation, such as incubation in the presence ofthe presence ofthe poly-clonal T cell stimulator Concanavilin A (ConA, Calbiochem, San Diego, CA) at one or more concentrations.
  • the cells are pulsed labeled with 3 H- thymidine and harvested onto glass fiber filters for scintillation counting (Packard, Meriden, CT). Activation or inhibition of proliferation is measured by the extent to which the labeled
  • 3 H-thymidine is inco ⁇ orated into cells in the presence and absence of candidate immune regulatory compositions or compounds.
  • a composition or compound ofthe primary screen As an indication ofthe ability of a composition or compound ofthe primary screen to stimulate or inhibit T cell activation, supernatants from T cell proliferation cultures, such as from mouse splenocytes or human peripheral blood lymphocytes, as described above, grown in the presence or absence of a candidate immune regulatory composition or compound can be assayed for the presence of cytokines secreted during T cell activation.
  • cytokines secreted during T cell activation can be tested by quantitation using a standard ELISA set up or commercial kit (for example, Endogen, Woburn, MA).
  • Other cytokines whose expression is stimulated by T cell activation such as gamma interferon, can be similarly quantitated by ELISA using available antibodies and reagents.
  • T cell culture conditions can be modified to yield cytokines such as IL-4 (Litton et al., J. Immunol. Methods, 175: Al - 58 (1994)), which can also be quantitated by ELISA. 16.
  • cytokines such as IL-4 (Litton et al., J. Immunol. Methods, 175: Al - 58 (1994)), which can also be quantitated by ELISA. 16.
  • cytokines such as IL-4 (Litton et al., J. Immunol. Methods, 175: Al - 58 (1994)), which can also be quantitated by ELISA. 16.
  • Apoptosis Assays Apoptosis or programmed cell death is an active, physiological form of cellular suicide to regulate cell growth and homeostasis. It is executed by a genetically regulated, evolutionarily highly conserved, cell death program that can react to a wide variety of different stimuli.
  • Apoptosis assays can be used to determine the mode of action of a composition or compound identified in the primary screen with respect to cytotoxicity to T cells. In contrast to the Toxicity Assay ofthe primary screen, however, these assays are designed to identify a particular form of cell death, i.e., apoptosis, that may be induced by compositions or compounds identified in the primary screen.
  • Apoptosis may be specifically induced in activated T cells by a composition or compound identified as an immune suppressor in the primary screen. Such activation- dependent apoptosis may be useful to achieve antigen-specific tolerance which would be particularly beneficial to prevent graft rejection in tissue and organ transplant recipients or to treat autoimmune disorders.
  • assays are performed on activated and unactivated T cells. Compositions or compounds that induce apoptosis in activated but not unactivated T cells are selected as useful immune suppressors.
  • the ability to induce apoptosis in resting cells is indicative of a condition under which an immune regulator of the primary screen may exert a toxic effect on cells. Induction of apoptosis in resting cells may not be a significant factor at the concentrations used in the primary screen and in therapeutic uses. However, the ability to induce apoptosis in resting cells may be an ever increasing concern for higher concentrations of such compositions or compounds, as may accumulate during relatively long-term (i.e., months or years) therapeutic uses.
  • Apoptosis assays can also be used to confirm anti-apoptotic activity of compositions or compounds that are identified as both immune stimulators and anti-apoptotic agents by an
  • a secondary assay for apoptosis involves inducing apoptosis by incubating T cells with a TCR- crosslinking agent in the presence or absence of a test composition or compound for greater than 8 hours.
  • This activation-dependent apoptosis is mediated by the induction of fasL and its interaction with fas.
  • compositions or compounds which inhibit either the induction of fasL or apoptotic pathways initiated by fas will be detected.
  • apoptosis secondary assays are based on the fact that apoptosis is defined by a characteristic pattern of mo ⁇ hological, biochemical, and molecular changes that are broadly assigned to three stages: early, intermediate, and late apoptosis.
  • Necrotic cell death is mo ⁇ hologically distinct from cell death by apoptosis.
  • necrotic cell death is characterized by early membrane disruption.
  • membrane integrity is maintained until the late stage of apoptosis.
  • the intermediate apoptotic stage is the internucleosomal cleavage of DNA into distinct fragments of 180 to 200 base pairs and multiples thereof by activated endonucleases.
  • Various assays for apoptosis are known (see, for example, McGahon et al., Meth.Cell
  • an apoptosis assay is based on labeling (i.e., "staining") ofthe surface of cells that are undergoing apoptosis compared to cells that are not.
  • labeling i.e., "staining”
  • a distinct feature of early apoptosis is the specific translocation ofthe membrane phospholipid phosphatidyl serine (PS) from the inner leaflet (i.e., the cytoplasmic side) ofthe plasma membrane to the outer surface ofthe membrane, thereby exposing PS on the cell surface.
  • PS membrane phospholipid phosphatidyl serine
  • DNA ladder DNA ladder which is observable after electrophoresis through a gel.
  • DNA isolated from cells exposed to known or putative apoptosis stimuli is electrophoresed through a gel, such as agarose or polyacrylamide and the like, and the gel examined for the presence or absence of a DNA ladder of cleavage products.
  • a library of approximately 8,000 arylidene diamide compounds was screened for immune regulators using the multi-step primary screen described herein.
  • a library of compounds in array form is commercially available from ArQule, Inc. (Medford, MA), under the product designation AN- 1001. The synthesis ofthe compounds ofthis library is described in the concurrently filed United States Patent Application (U.S. Serial No.08/641,986, filed April 25, 1996), inco ⁇ orated herein by reference. Plasmids
  • IL-2 Luciferase (IL-2LUC) recombinant reporter gene plasmid containing the structural coding sequence of a luciferase gene (GenBank Accession Number X65323 (nucleotides 76 to 1725); De Wet et al., Mol. Cell. Biol., 7: 725 - 737 (1987) (see, Figure 1 at page 728)) under the transcriptional control of (operatively linked to) the promoter/enhancer region ofthe IL-2 gene (GenBank Accession Number J00264) was obtained from Gerald
  • Plasmid IL-2LUC was used in the first step (Activation Assay) of a primary screen for immune regulators of T cell activation according to this invention.
  • a Jurkat cell line stably transfected with plasmid IL-2LUC was deposited with the American Type Culture Collection (ATCC, Rockville, MD) in accordance with the Budapest Treaty on April 26, 1996 and assigned the ATCC Accession Number CRL- 12091.
  • a ⁇ -actinLUC plasmid used in the second step (Toxicity Assay) ofthe primary screen was constructed by Randall Faircloth and Scott Umlauf (Umlauf et al., 1994).
  • the luciferase gene from the pGL2 plasmid was cloned into the Hindlll and BamHI sites of plasmid pH ⁇ Apr-1-neo (Gunning et al., 1987) to form plasmid ⁇ -actinLUC in which a promoter/enhancer region ofthe ⁇ -actin gene is operatively linked to the luciferase structural coding sequence so that transcription ofthe luciferase coding sequence is under the control ofthe promoter/enhancer region ofthe ⁇ -actin gene.
  • Plasmid ⁇ -actinLUC in an Escherichia coli bacterial host cell was deposited in accordance with the Budapest Treaty in the American Type Culture Collection (ATCC, Rockville, MD) on April 26, 1996 and assigned Accession Number 98036.
  • ATCC American Type Culture Collection
  • the ⁇ - actinLUC plasmid is most preferably used in transient transfected cell lines since in these lines the basal level of luciferase expression is significantly higher than in stable lines.
  • the levels of luciferase gene expression were typically 20-fold higher in transiently transfected cells than in stably transfected cells.
  • NFATLUC plasmid was obtained from Gerald Crabtree (Northrop et al., 1993) for use in the third step (Specificity Assay) ofthe primary screen.
  • the NFATLUC plasmid contains three copies ofthe NFAT binding sequence ofthe IL-2 promoter/enhancer region (NFAT binds to a sequence defined by nucleotides 146 to 175 of IL-2 promoter/enhancer sequence in GenBank Accession Number J00264) operatively linked to the luciferase structural coding sequence.
  • a Jurkat cell line stably transfected with plasmid NFATLUC was deposited with the American Type Culture Collection (ATCC, Rockville, MD) in accordance with the Budapest Treaty on April 26, 1996 and assigned the ATCC Accession Number CRL- 12092.
  • An AP-1 LUC plasmid was constructed that contained the AP-1 binding sequence of the IL-2 promoter/enhancer (AP-1 binds to a sequence defined by nucleotides 270 to 294 of
  • IL-2 promoter/enhancer sequence in GenBank Accession Number J00264) operatively linked to the luciferase structural coding sequence in a manner analogous to the other recombinant LUC plasmids.
  • complementary DNA strands containing the multimeric AP-1 binding sequences ofthe AP-1 LUC plasmid were synthesized by and purchased from Operon Technologies, Inc. (Alameda, CA).
  • the first strand ofthe synthetic AP-1 binding site containing the three tandem copies ofthe AP-1 transcription control sequence has the nucleotide sequence of SEQ ID NO:l : TCG AGA ATT CCA AAG AGT CAT CAG AAG AAA TTC CAA AGA GTC ATC AGA AGAAATTCCAAAGAGTCATCAGAAGAC.
  • the complementary second strand of the synthetic AP-1 binding site containing the three tandem copies ofthe AP-1 transcription control sequence has the nucleotide sequence of SEQ ID NO:2: TCG AGT CTT CTG ATG ACT CTT TGG AAT TTC TTC TGA TGA CTC TTT GGA ATTTCTTCTGATGACTCTTTGGAATTC.
  • the first strand (SEQ ID NO: 1) and second strand (SEQ ID NO:2) were annealed and cloned into the Xhol site of plasmid NFATLUC using standard methods (Maniatis et al., Molecular Cloning (A Laboratory Manual). (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982)). Digestion ofthe plasmid NFATLUC with Xhol permits the insertion of the synthetic oligonucleotide in place ofthe three tandem copies of the NFAT transcription control sequence. Transfections
  • Transfections were performed by electroporation: 250 volts, 960 ⁇ F in a 0.4 cm cuvette. Approximately 1 x IO 7 cells were transfected in 0.5 ml. To obtain stably transfected cell lines, the transfected cells were recovered from the electroporation cuvette and grown in suspension in selection medium: RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with fetal calf serum (10% v/v, Hyclone, Logan, Utah), L-glutamine (2 mM, GIBCO BRL), penicillin/streptamycin (100 U/ml and 100 ⁇ g/ml, respectively, GIBCO BRL), 2-merca ⁇ toethanol (55 ⁇ M, GIBCO BRL), and geneticin (G418, GIBCO BRL, Gaithersburg, MD) at 1 mg/ml.
  • selection medium RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with fetal
  • transiently transfected Jurkat cell lines for use in the second step (Toxicity Assay) ofthe primary screen
  • cells transfected with plasmid ⁇ -actinLUC were recovered from the electroporation cuvette and grown in suspension overnight in the liquid medium as described above for the stably transfected cells, but without selection (i.e., the medium was not supplemented with geneticin). Assays were performed within 72 hours of electroporation. Screening Compounds for Immune Regulatory Activity
  • Jurkat (human T cell lymphoma) cells were transfected with IL-2 LUC plasmid for use in the first step (Activation Assay), with ⁇ -actinLUC plasmid for use in the second step (Toxicity Assay), or with NFATLUC plasmid for use in the third step (Specificity Assay) of the primary screen for immune regulators.
  • IL-2 LUC plasmid for use in the first step
  • ⁇ -actinLUC plasmid for use in the second step
  • NFATLUC plasmid for use in the third step of the primary screen for immune regulators.
  • Jurkat T cells were activated using both the 16G8 anti-TCR monoclonal antibody
  • MAb Endogen, Cambridge, MA
  • PMA phorbol myristate acetate
  • the wells of a 96-well tissue culture plate were coated with the 16G8 MAb (which adhere to the surface ofthe wells) so that the TCR on the surface of T cells in each well would be crosslinked by the MAb on the surface of the well, thereby simultaneously immobilizing and activating the T cells.
  • the antibody concentration (titer) in the antibody coating solution should be carefully measured.
  • the level of activation reflected in luciferase gene expression is 50- to 100-fold greater in activated T cells than in non-activated T cells.
  • a sub-optimal 50% activation i.e., 25- to 50-fold stimulation. This can be accomplished by using an antibody concentration of approximately 1 ⁇ g/ml in PBS (GIBCO BRL). Titering each lot of antibody permits adjustments for lot-to-lot variation. A stock solution of antibody can be reused to coat several plates with antibody since each coating seems to deplete the antibody solution only slightly. Care should be taken, though, to monitor the fold-stimulation of luciferase expression so that potential inhibitors are less likely to be missed during the screening procedure due to insufficient activation of T cells in the wells.
  • the luciferase assay is best performed using a luminometer which can read 96-well culture plates, such as the Packard Top-Count (Packard, Meriden, CT). Plates can be obtained which are suitable for tissue culture yet are composed largely of opaque plastic which reduces the background luminescence.
  • the View-Plate from Packard is especially well-suited for this assay since the bottom ofthe well is clear allowing the operator to view the condition ofthe cultured cells prior to lysis.
  • a candidate composition or compound can then be added in an aqueous solution. Some concentration of organic solvents can be tolerated at this stage, however the antibody bound to the wells must not be denatured.
  • the following three-step primary screen was used to screen a library of approximately 8,000 synthesized oxazalone-derivative arylidene diamide compounds of the ArQule, Inc. AN-1001.
  • each compound from the library was added to a MAb-coated well in 25 ⁇ l of PBS/DMSO (98%/2%).
  • Cells (2 X 10 5 ) premixed with PMA (20 ng/ml) were added to a final volume of 100 ⁇ l.
  • the final concentration of each compound was approximately 20 ⁇ M.
  • non-activated cells wells with no antibody
  • activated cells with no compound and validation controls such as cells activated in the presence ofthe immunosuppressive agent cyclosporin A (CsA). The cells were incubated for several hours
  • luciferase is an intracellular protein, the cells were lysed to assay for luciferase activity.
  • the luciferase assay is compatible with many low- concentration detergent lysis buffers including Reporter Lysis Buffer supplied by Promega (Madison, WI).
  • a volume of 20 ⁇ l of Reporter Lysis Buffer was sufficient to achieve lysis in two minutes at room temperature.
  • the luciferase substrate luciferin was then added along with an energy source such as ATP or acetyl-CoA, routinely in a volume of 100 ⁇ l.
  • the luciferin-acetyl-CoA combination supplied by Promega (Madison, WI) is preferable since the half-life of decay of luminescence is 5 minutes as compared to one minute for ATP systems. Plates were counted in a luminometer (light units are counts per second) as quickly as possible since light emission decays logarithmically. Results are shown in Figure 7.
  • lysates of unstimulated cells exhibited an emission of about 200 light units (medium, Figure 7)
  • lysates of cells stimulated with antibody and PMA exhibited an emission of approximately 1200 light units
  • Compounds that exhibited inhibitory activity were further tested in the next step, the Toxicity Assay.
  • Toxicity Assay For the second step (Toxicity Assay) ofthis primary screen, Jurkat cells were transiently transfected (electroporated) with the ⁇ -actinLUC plasmid (see above). Eighteen hours after electroporation, viable cells were plated in the MAb-coated wells and in the presence ofthe compound to be tested (approximately 20 ⁇ M) as described above in the Activation Assay. In this Toxicity Assay, the expression of luciferase in the cells transfected with the ⁇ -actinLUC plasmid is constitutive and poorly stimulated by TCR cross-linking in the assay wells.
  • cyclosporin A CSA
  • the compounds that inhibited the Activation Assay significantly affected luciferase activity in non-activated cells ofthis Toxicity Assay.
  • Cyclosporin A (known to be generally toxic to many cells) inhibited luciferase expression by approximately 40% in activated cells.
  • Activation Assay inhibited luciferase activity in this Toxicity Assay.
  • This result suggested that the inhibition of luciferase expression under the regulation of the IL-2 promoter/enhancer region in the Activation Assay (first step) was specific and not of a general (toxic) nature. Accordingly, these compounds were next screened in a Specificity Assay (third step) for their ability to affect luciferase expression under the specific control ofthe NFAT transcription factor.
  • the third step (Specificity Assay) Jurkat cells transfected with the NFATLUC plasmid (containing three copies ofthe NFAT binding sequence operatively linked to the luciferase structural coding sequence, see above) were activated in the presence of each of the compounds that did not affect luciferase expression in the previous Toxicity Assay. The cells were activated in the presence of each compound (approximately 20 ⁇ M) as described for the Activation Assay above. Eight such compounds that did not affect the Toxicity Assay were found to inhibit luciferase expression under the regulation ofthe NFAT binding sequences ( Figure 9). This indicated that the inhibition of luciferase exhibited by these eight compounds in the Activation Assay (first step) was at least partly linked to NFAT-mediated transcriptional control, and not, for example, exclusively to other transcription factors such as NFKB.
  • the compounds were tested in a standard in vivo acute toxicity assay.
  • mice Each compound (100 ⁇ g in PBS/DMSO (75%/25%)) was injected intraperitoneally into a group of two mice (one male and one female). All ofthe mice that received a compound survived for the 9 day observation period, after which the mice were sacrificed. Necropsies were performed and no pathology observed. Furthermore, splenocytes from mice from four groups (those that received AT-6, AT-7, AT-5, and AT-3) were tested and found to show reduced proliferation to anti-CD3 antibody ex vivo, as expected if T cell activation is inhibited.
  • Non-activated T cells were also used as a control. The cells were incubated for a total of two hours to allow activation by PMA and ionomycin, where present. Controls included non-activated cells, cells activated without compound, and cells activated with a validation control such as cyclosporin A which inhibits luciferase expression in cells transfected with the IL-2LUC and NFATLUC plasmids.
  • Cells were harvested from culture medium, washed with IX PBS, and then lysed with a low- concentration detergent buffer (for example, NP-40 at 0.2%) containing a protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF).
  • a low- concentration detergent buffer for example, NP-40 at 0.2%) containing a protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF).
  • the detergent In order to prepare nuclei, the detergent must be ofthe right type and at the correct concentration
  • Nuclei in the cell lysates were then pelleted by low speed centrifugation, and the supernatant cytoplasm removed. The nuclei were then lysed in a high salt concentration (for example, 60 mM ammonium sulfate or 400 mM NaCI) buffer. Nuclear DNA-binding proteins (including nuclear transcription factors for IL-2 expression) were either collected in the supernatant ofthe nuclear debris pellet or were precipitated with an ammonium sulfate cut
  • Protein recoveries were quantitated by ultraviolet light absorbance (280 nm) or standard Bradford assay.
  • oligonucleotides were synthesized by and purchased from Operon Technologies, Inc. (Alameda, CA) for use in this assay to test the ability of a compound identified in the primary screen for its ability to inhibit the binding of a particular transcription control factor to its cognate DNA sequence.
  • the IL2 NFAT oligonucleotide was formed by annealing a DNA strand having the sequence of SEQ ID NO:3 and a complementary DNA strand having the sequence of SEQ ID NO:4, and used to test the ability of compounds identified in the primary screen to inhibit NFAT binding to its site in the IL-2 promoter/enhancer region.
  • the GMCSFNFAT oligonucleotide was formed by annealing a DNA strand having the sequence of SEQ ID NO:5 and a complementary sequence of SEQ ID NO:6, and used to test the ability of compounds identified in the primary screen to inhibit a
  • the AP- 1 oligonucleotide was formed by annealing a DNA strand having the sequence of SEQ ID NO:7 with a complementary DNA strand having the sequence of SEQ ID NO:8 and used to test the ability of a compound identified in the primary screen to inhibit AP-1 binding to this site from the IL-2 promoter/enhancer region.
  • the NFKB oligonucleotide was formed by annealing a DNA strand having the sequence of SEQ ID NO: 9 with a complementary DNA strand having the sequence of SEQ ID NO: 10 and used to test the ability of a compound identified in the primary screen to inhibit NFKB binding to its cognate DNA sequence in the IL-2 promoter/enhancer region. Binding reactions were generally performed with 2 ng of 32 P end-labeled DNA fragments or oligonucleotides containing relevant transcription factor binding sites.
  • Reaction volumes were typically around 10 to 20 ⁇ l in binding buffer (10 mM HEPES (pH 7.5), 100 mM NaCI, 20% glycerol, 1 mM DTT, and non-specific DNA such as poly-dl/dC at 200 ng per reaction).
  • binding buffer 10 mM HEPES (pH 7.5), 100 mM NaCI, 20% glycerol, 1 mM DTT, and non-specific DNA such as poly-dl/dC at 200 ng per reaction.
  • 2 - 5 ⁇ g of nuclear DNA-binding proteins were incubated with the labeled DNA in binding buffer for 15 minutes at room temperature.
  • Bound and unbound DNA were then separated by electrophoresing on non-denaturing poly ⁇ acrylamide gels (6% acrylamide in IX TBE buffer (GIBCO BRL)). Gels were dried and exposed to X-ray film.
  • Nuclei were extracted with ammonium sulfate, and crude nuclear protein extracts were prepared for gel shift assays as described above. Normalized amounts of extracts were incubated with labeled DNA fragments for 15 minutes at room temperature.
  • Binding reactions were loaded onto 6% acrylamide gels in IX TBE and were run at 120 volts at room temperature. The gels were dried on a vacuum drier and exposed to X-ray film. Activation in controls caused the induction of binding activities specific for both the NFAT and AP-1 sites resulting in slower mobility bands in the lanes of the gels containing
  • Three ofthe eight compounds (AT-2, AT-7, and AT-8) identified in the primary screen as immune inhibitors were assayed again using an improved gel-shift assay protocol.
  • the improved gel shift assays were run as described with the following changes. Nuclei were prepared from Jurkat cells as used in the primary screen. The nuclei were lysed using a high salt concentration (400 nM NaCI) buffer. The nuclear DNA-binding protein complexes (including nuclear transcription factors for IL-2 expression) were collected in the supernatants ofthe nuclear debris pellet.
  • Reaction volumes were 20 ⁇ l in binding buffer (20 mM HEPES (pH 7.5), 35 mM NaCI , 35 nM KCl, 15% glycerol, 1 mM EDTA, 1 mM DTT, 100 ⁇ g/ml BSA, and non-specific DNA such as poly-dl/dC at 1 ⁇ g per reaction).
  • binding buffer 20 mM HEPES (pH 7.5), 35 mM NaCI , 35 nM KCl, 15% glycerol, 1 mM EDTA, 1 mM DTT, 100 ⁇ g/ml BSA, and non-specific DNA such as poly-dl/dC at 1 ⁇ g per reaction).
  • 5 - 7.5 ⁇ g ofthe nuclear DNA-binding protein complexes were incubated with the labeled DNA in binding buffer for 15 minutes at room temperature.
  • HUT78 cells were incubated for 15 minutes at room temperature (22 °C) in the absence or presence a compound (25 ⁇ g/ml) identified as an immune inhibitor in the primary screen or, as a positive control, in the presence of cyclosporin A (Sigma Chemical Co., St. Louis, MO) or FK506 (100 ng/ml).
  • a compound 25 ⁇ g/ml identified as an immune inhibitor in the primary screen or, as a positive control, in the presence of cyclosporin A (Sigma Chemical Co., St. Louis, MO) or FK506 (100 ng/ml).
  • the cells were then activated in solution either with 10 ⁇ g/ml of the anti-TCR MAb 16G8 (Endogen) and 20 ⁇ g/ml of goat anti-mouse antibody or with 2 ⁇ M ionomycin (Calbiochem) in DMSO and 50 nM phorbol- 12-myristate-13-acetate (PMA, Calbiochem) in ethanol for 5 to 10 minutes at room temperature (22 °C).
  • the cells were washed in ice-cold phosphate buffered saline (PBS) and lysed in RIPA buffer (20 mM Tris[hydroxymethyl] aminomethane, pH 7.6; 300 mM NaCI; 5 mM EDTA ([ethylenedinitrilo] tetraacetic acid); 5 mM, EGTS (ethylene glycol- bis-[ ⁇ -aminoethyl ether]-N, N, N ⁇ N' -tetraacetic acid); 1 % (v/v) Triton X-100; 1 % (v/v) deoxycholate; 0.1 % (w/v) sodium dodecyl sulfate (SDS); 50 mM pyrophosphate, 50 mM NaF; 20 ⁇ M ZnCL; 500 ⁇ M orthovanadate; 1 mM phenylmethylsulfonyl fluoride (PMSF); 10 ⁇ g/ml aprotinin; 10
  • the immunoprecipitates were washed with RIPA buffer, boiled in SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 2% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, 10% glycerol, bromphenol blue), and electrophoresed on SDS-polyacrylamide gels.
  • the proteins were transferred from the gels to nitrocellulose membranes for detection of NFATp species by immunoblotting with anti-NFATp antibody (Western blot analysis) and horseradish peroxidase-protein A (Kirkegaard and Perry, Gaithersburg, MD).
  • Cells were lysed using RIPA buffer (see above) or lysis buffer (50 mM HEPES (4-[2-hydroxethyl]-l-piperazineethanesulfonic acid) (pH 7.5), 10% glycerol, 150 mM NaCI, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM pyrophosphate, 50 mM NaF, 20 ⁇ M ZnCl 2 , 500 ⁇ M orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 50 ⁇ g/ml pepstatin,
  • ERK activation was assessed by immunoblotting of 10 ⁇ g/ml of lysate protein with an anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or by immune complex kinase assay.
  • an anti-ERK2 antibody Santa Cruz Biotechnology, Santa Cruz, CA
  • immune complex kinase assay For the in vitro kinase assay, ERK2 was immunoprecipitated with 10 ⁇ l anti-ERK2 antibody from 500 ⁇ g of lysate protein as described above.
  • the immune complex was washed with 1 ml of preK-buffer (25 mM HEPES (pH7.5), 5 mM NaF, 5 ⁇ M ZnCl 2 , 20 mM ⁇ - glycerophosphate, 10 mM pNPP (p-nitrophenyl phosphate), 10 mM MgCl 2 , 2 mM DTT (Dithiothreitol), 200 ⁇ M orthovanadate).
  • preK-buffer 25 mM HEPES (pH7.5), 5 mM NaF, 5 ⁇ M ZnCl 2 , 20 mM ⁇ - glycerophosphate, 10 mM pNPP (p-nitrophenyl phosphate), 10 mM MgCl 2 , 2 mM DTT (Dithiothreitol), 200 ⁇ M orthovanadate).
  • kinase reaction was performed for 20 minutes at room temperature (22 °C) in kinase-buffer (preK-buffer plus 20 ⁇ M ATP, 10 ⁇ Ci of [ ⁇ - 32 P] ATP (NEN-Dupont)) in the presence of 2 ⁇ g MBP (myelin basic protein, Sigma Chemical
  • Raf-1, MEK1 and ERK2 were immunoprecipitated with 25, 10 or 5 ⁇ l antibody, respectively, from 1 mg of lysate protein as described above.
  • the kinase reaction was performed for 20 minutes at room temperature (22 °C) in kinase-buffer (preK-buffer plus 20 ⁇ M ATP, 10 ⁇ Ci of [ ⁇ - 32 P] ATP (NEN-Dupont)) in the presence of 3 ⁇ g His-MEK, 5 ⁇ g His-ERK or 5 ⁇ g MBP as substrates for Raf-1, MEK1 or ERK2, respectively, after which it was stopped by boiling in sample buffer.
  • This analysis showed that none ofthe three compounds tested, AT-2, AT-7 and AT-8, inhibited Raf-1, MEK1 or
  • JNK Activity Assay An in vitro kinase assay was used as a quantitative measurement of JNK activation as activated JNKs, in contrast to ERKs, do not appear to significantly shift in SDS-gels.
  • JNKl was immunoprecipitated with 10 ⁇ l antibody from 1 - 1.5 mg of lysates obtained from cells stimulated in the absence or presences of the immune inhibitors AT-2, AT-7 or AT-8 (30 ⁇ g/ml).
  • T cells Approximately 100 x IO 6 T cells were pre-incubated for 15 minutes at room temperature in the absence or presence of 30 ⁇ g/ml ofthe immune regulator AT-7 from the primary screen, then activated either with 10 ⁇ g/ml anti-T cell receptor antibody (16G8,
  • Cytb buffer (10 mM HEPES (pH 7.3), 15 mM KCl, 2.5 mM MgCl 2 , 0.25% NP-40, 0.1 mM EDTA, 20 ⁇ M ZnCl 2 , 25 mM NaF, 10 mM pyrophosphate, 500 ⁇ M orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 50 ⁇ g/ml pepstatin, 50 ⁇ g/ml trypsin inhibitor from soybean).
  • Cytb buffer (10 mM HEPES (pH 7.3), 15 mM KCl, 2.5 mM MgCl 2 , 0.25% NP-40, 0.1 mM EDTA, 20 ⁇ M ZnCl 2 , 25 mM NaF, 10 mM pyrophosphate, 500 ⁇ M orthovanadate, 1
  • the lysed cells were centrifuged at 5,000 ⁇ m for 1 minute. The supernatant was the "cytosolic extract". The pellet was washed with Cytb buffer without NP40 and then extracted with 420-buffer (Cytb buffer plus 10% glycerol and 420 mM NaCI) for 30 minutes with rocking at 4°C. The extract was centrifuged (for example, for 12 minutes. At 14,000 ⁇ m in an Eppendorf tabletop centrifuge). The supernatant was the "nuclear extract”.
  • NFATp was immunoprecipitated with anti-NFATp-antibodies from cytosolic and nuclear extracts of non-activated (control) and activated cells in the absence and presence of immune inhibitor.
  • Western blot with anti-NFATp-antibodies showed that NFATp in activated cells was dephosphorylated and ran faster on SDS-polyacrylamide gels.
  • phosphorylated NFATp remained in the cytosolic fraction whereas the dephosphorylated protein translocated into the nucleus (i.e., seen in lanes loaded with nuclear extract).
  • a preliminary result ofthis assay suggested that NFAT dephosphorylation and translocation to the nucleus was not inhibited by compound AT-7 at 30 ⁇ g/ml.
  • the ability ofthe immune inhibitors identified in the primary screen to inhibit proliferation of non-transformed cells was assessed.
  • the compounds were incubated with splenocytes from naive DBA-2 mice.
  • splenocytes were cultured in 96-well plates in the identical medium described above for growing Jurkat cells (RPMI 1640 with 10% fetal bovine serum) at 1 X IO 6 cells per well in the presence ofthe poly-clonal T cell stimulator Concanavilin A (ConA, Calbiochem, San Diego, CA) at 0.3, 1 or 3 ⁇ g/ml.
  • Cells were incubated in the presence ofthe eight compounds (25 ⁇ g/ml) for 72 hours.
  • the cultures were then pulse-labeled with 1 ⁇ Ci/well of 3 H-thymidine (Dupont NEN, Boston, MA) for another 24 hours and harvested onto glass fiber filters for scintillation counting (Packard, Meriden, CT).
  • SWM Myoglobin
  • SWM Myoglobin peptide
  • SEQ ID NO: 1 1 showing amino acids 110 to 121 of SWM, synthesized by and purchased from QCB, Hopkinton, MA; see also, Infante et al., J. Exp. Med, 154: 1342 -1356 (1981)
  • CFA complete Freund's adjuvant
  • emulsion were sacrificed on day 21 and splenocytes were cultured with either antigen (SWM peptide) or with anti-CD3 antibody (2C1 1, Pharmingen, San Diego, CA).
  • Cells were cultured as above except that the medium was HL- 1 (Biowhitaker, Walkersville, MD) supplemented with glutamine, penicillin, streptomycin, and 2-mercaptoethanol (GIBCO BRL, Gaithersburg, MD amounts as above) and with sodium pyruvate and non-essential amino acids (both 100 ⁇ M, GIBCO BRL).
  • Antigen-specific T cells were stimulated with SWM peptide between 350 and 0.3 ⁇ M for 72 hours. Cultures were then pulse-labeled and counted as above.
  • Control cultures were set up with the 2C11 antibody (2.5 ⁇ g/ml to 0.1 ng/ml) instead of SWM peptide.
  • the results were plotted as stimulation indices (CPM of thymidine inco ⁇ oration with antigen divided by CPM without antigen).
  • CPM stimulation indices
  • the results show that T cells from mice treated with the highest dose of AT-2 did not proliferate in response to the SWM antigen (see Figure 12), similar to unprimed mice (negative control).
  • cyclosporin A-treated mice responded similarly to primed mice without either cyclosporin A or AT-2 (positive control) since cyclosporin A does not induce long-term tolerance.
  • Control cultures with the anti-CD3 antibody 2C11 show no striking differences between groups.
  • DTH Delaved-Type Hypersensitivity
  • mice were injected subcutaneously at the base ofthe tail with Sperm Whale Myoglobin (SWM) peptide in complete Freund's adjuvant (CFA) in the presence of 10, 1, 0.1, or 0 mg of AT-8.
  • SWM Sperm Whale Myoglobin
  • CFA complete Freund's adjuvant
  • An unprimed control received CFA alone without SWM peptide.
  • a cyclosporin A control received 2 mg of cyclosporin A.
  • each mouse received a subcutaneous injection in one rear footpad of SWM peptide (50 ⁇ g per mouse) in 25 ⁇ l PBS.
  • the other rear footpad (uninjected) ofthe same mouse served as the control.
  • the footpads were examined for edema in the injected foot as compared to the control footpad.
  • the results indicate that CsA (2 mg) and AT-8 at 1 and 10 mg significantly suppressed edema due to antigen-specific T cell activation (p ⁇ 0.05) compared to controls ( Figure 13).
  • API -oligo yes less yes less
  • the primary screen identified 18 compounds which activated luciferase expression (i.e., greater than 125% activation relative to control (no compound) 100%, see Figure 14). Of those eighteen, eight (designated A, B, C, D, K, M, S, and Y in Figures 15 and 16) were determined in the Toxicity Assay to not be general transcription activators and, therefore, were next examined in the third step ofthe primary screen
  • the Toxicity Assay was able to identified a particularly potent general transcription activator (O in Figure 15) which was not studied further. All eight compounds also activated luciferase expression in the Specificity Assay using cells transfected with the NFATLUC plasmids (see Figure 17).
  • Another library consisting of 3,840 synthetic organic compounds synthesized by combinatorial chemistry (Hogan, Nature, 384 (Supp.): 17 - 19 (1996)) was screened using the primary screen and secondary assays as described in Examples 1 and 2.
  • the Activation Assay of the primary screen identified 62 compounds as having a potential immune regulatory activity. 11 of these 62 were eliminated by the Toxicity Assay and 42 ofthe remaining compounds had some specific effect on NFAT-mediated gene expression (Specificity Assay). These compounds were further characterized by secondary assays as described above.
  • the Activation Assay ofthe primary screen was also used to calculate the concentration at which 50 per cent (IC 50 ) and 90 per cent (IC 90 ) ofthe cells in the assay were inhibited from activation by compounds M3.5H8 and M4.6H5. These two compounds were then further assayed using flow cytometry for the ability to induce apoptosis.
  • a standard apoptotic assay ofthis example uses flow cytometry to detect the staining of cells with one or two molecules which emit a different fluorescent signal that can be detected simultaneously by a flow cytometer to determine whether cells are undergoing early apoptosis, are viable, or are necrotic.
  • FITC fluorescein isothiocyanate
  • PI DNA intercalating fluorescent dye propidium iodide
  • Viable cells not undergoing apoptosis will not be stained significantly by FITC-labeled Annexin V and will not be stained by PI.
  • Necrotic cells stain positive with both FITC-labeled Annexin V and PI.
  • the cells were pelleted in a microcentrifuge (1000 ⁇ m, 5 minutes, room temperature), washed once with 3 ml of phosphate buffered saline (PBS)/bovine serum albumin (BSA, 1 % w/v), pelleted again, and resuspended into 100 ⁇ l of IX binding buffer (Kamiya Biomedical Company, Seattle, WA).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • FITC-labeled-Annexin V (Kamiya Biomedical Company, Seattle, WA) alone, at a final concentration of 400 ng/ml, or were mixed simultaneously with both FITC -Annexin V (400 ng/ml) and propidium iodide (PI, 5 ⁇ g/ml), followed by incubation in the dark for 10 to 20 minutes at room temperature.
  • the cells were then removed from the dark and washed with 3 ml of PBS/1 % BSA, pelleted as described above, and resuspended into 500 ⁇ l of PBS/1 % BSA, and analyzed using a flow cytometer (FACSCANTM, Becton & Dickinson, San Jose, CA) equipped with a single laser emitting excitation light at 488 nm. Cells were incubated in the presence or absence of M3.5H8 and M4.6H5 for 4 hours at 37 °C. The results are shown in Figures 18A - 18F.
  • Figure 18A shows the results of incubating cells in the absence of compound (control).
  • Figure 18B shows the results of incubating cells in the presence of antibody to Fas (Kamiya Biomedical Company, Seattle, WA) available from which induces apoptosis as indicated as detection of fluorescently labeled cells in the M2 region ofthe graph (positive control).
  • Figures 18C and 18D show the results of incubating cells in the presence of M3.5H8 at the IC 50 and IC 90 concentrations of 60 ng/ml and 2 ⁇ g/ml, respectively.
  • Figures 18E and 18F show the results of incubating cells in the presence of M4.6H5 at the IC 50 and IC 90 concentrations of 60 ng/ml and 5 ⁇ g/ml, respectively. The results show that neither ofthe two immune inhibitor compounds induced apoptosis in the assay.
  • Example 5 Screening a Library of 17,920 Synthetic Compounds Another library consisting of 17,920 synthesized compounds was run through the primary screen as basically as described in Example 1 above. To reduce the amount of manual manipulation and time needed to read samples from the primary screen, it is desirable to use a luciferase substrate formulation which provides a light signal which is stable and has a half-life as long as possible. Such an improved luciferase formulation is LUC-LIGHTTM (Packard, Meriden, CT) which provides a light signal with a half-life of greater than 5 hours.
  • LUC-LIGHTTM Packard, Meriden, CT
  • This formulation permitted the use of stacking devices which sequentially feed a series of plates into the luminometer.
  • the Activation Assay ofthe primary screen identified 323 compounds as having a potential immune inhibitory activity. Of these 323 compounds, 94 were eliminated by the Toxicity Assay as having a non-specific effect on transcription.
  • NFATLUC as in Example 1 identified 130 compounds as having a specific effect on NFAT- mediated gene expression. These compounds are being further characterized by secondary assays as described above.
  • 323 compounds ofthe library were identified in the primary screen performed with an 18 hour incubation period as immune stimulatory compounds. These compounds were further screened for anti-apoptotic activity.
  • Jurkat cells transfected with ⁇ - actinLUC were incubated for 18 hours in the presence or absence of PMA, anti-TCR and PMA, or anti-fas antibody with or without cyclosporin A.
  • Cyclosporin A is known to inhibit T cell antigen receptor induced expression of fasL (Dhein et al., Nature, 377: 438 - 441 (1995)) and serves as a positive control.
  • Figure 20 shows expression of luciferase (light units) versus stimulation conditions versus concentration of cyclosporin A (CsA, ng/ml) in cells in the modified Toxicity Assay transfected with ⁇ -actinLUC and incubated for 18 hours under the indicated conditions.
  • CsA cyclosporin A
  • Another library of 216 fungal culture extract compositions prepared from 61 different fungal strains and 6 different media and growth conditions were screened using the primary screen described above.
  • Nine of these compositions exhibited inhibitory activity in the first step (Activation Assay) ofthe primary screen using the IL2-LUC construct.
  • Two of these nine compositions were eliminated in the second step (Toxicity Assay) using the ⁇ -actinLUC construct.
  • the remaining seven fungal extract compositions exhibited inhibitory activity in the third step (Specificity Assay) using the NFATLUC construct as described in Example 1.
  • Two of the seven fungal extracts that inhibited the NFATLUC showed particularly potent immune inhibitory in the Specificity Assay ofthe primary screen.
  • These seven compositions prepared from fungal cultures are being further characterized using various secondary assays as described above.
  • compositions were prepared from fungal cultures and screened by the primary screen as described in Example 1 above. 29 of these compositions showed a potential immune inhibitory activity by the Activation Assay (IL-2LUC construct) ofthe primary screened. 14 of these 29 were eliminated by the Toxicity Assay using Jurkat cells transfected with the ⁇ -actinLUC construct. 12 ofthe remaining 14 were identified by the Specificity Assay as regulating NFAT-mediated gene expression.
  • IL-2LUC construct Activation Assay
  • 14 of these 29 were eliminated by the Toxicity Assay using Jurkat cells transfected with the ⁇ -actinLUC construct. 12 ofthe remaining 14 were identified by the Specificity Assay as regulating NFAT-mediated gene expression.
  • These 12 fungal extract compositions from the primary screen are being further characterized by secondary assays as described above.
  • One other composition, XG08 showed strong immune stimulatory activity in the primary screen. This composition augmented IL-2 production from anti-CD3 stimulated splenocytes (see Figure 19).

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Abstract

Criblage préliminaire de haute capacité à étapes multiples pour identifier les régulateurs immunitaires de l'activation des lymphocytes T, à utiliser en tant qu'agents thérapeutiques. Des compositions ou des composés sont criblés. On analyse les capacités de compositions ou de composés de stimuler ou d'inhiber l'expression d'un gène reporter lié de manière fonctionnelle à des séquences spécifiques de régulation transcriptionnelle dans les lymphocytes T. On peut caractériser de manière plus aprofondie une composition ou un composé identifié comme un stimulant ou un inhibiteur immunitaire au moyen de criblage selon l'invention, afin de déterminer la molécule cible sur laquelle la composition ou le composé agir pour régler l'activation des lymphocytes T et les processus dépendant de cette activation, comme l'apoptose.
PCT/US1997/007052 1996-04-25 1997-04-25 Procede pour isoler les regulateurs de l'activation des lymphocytes t WO1997039722A2 (fr)

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Cited By (7)

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WO1998024918A1 (fr) * 1996-12-06 1998-06-11 HÖLZER, Dieter Piege a genes obtenu par recombinaison pour identifier et isoler des genes
US5994507A (en) * 1997-12-31 1999-11-30 Amersham Pharmacia Biotech Ab Method for binding albumin and means to be used in the method
EP1041144A1 (fr) * 1999-03-29 2000-10-04 Michael Blind Procédé pour l'identification et la validation des cibles au moyen des intramers ou de la selection in vivo
WO2001079555A2 (fr) * 2000-04-14 2001-10-25 Millennium Pharmaceuticals, Inc. Roles des membres de la famille jak/stat dans l'induction de la tolerance
WO2004029620A2 (fr) * 2002-09-26 2004-04-08 Novartis Ag Dosage biologique
WO2005030970A3 (fr) * 2003-09-26 2005-06-09 Internat Inst Of Molecular And Outils et procedes utilises pour caracteriser l'activite immunotoxique de substances xenobiotiques
WO2017132279A1 (fr) * 2016-01-25 2017-08-03 Genentech, Inc. Méthodes de dosage d'anticorps bispécifiques dépendants des lymphocytes t

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US20050084967A1 (en) 2002-06-28 2005-04-21 Xcyte Therapies, Inc. Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024918A1 (fr) * 1996-12-06 1998-06-11 HÖLZER, Dieter Piege a genes obtenu par recombinaison pour identifier et isoler des genes
US5994507A (en) * 1997-12-31 1999-11-30 Amersham Pharmacia Biotech Ab Method for binding albumin and means to be used in the method
EP1041144A1 (fr) * 1999-03-29 2000-10-04 Michael Blind Procédé pour l'identification et la validation des cibles au moyen des intramers ou de la selection in vivo
WO2000058455A2 (fr) * 1999-03-29 2000-10-05 Michael Blind Methodes d'identification et de validation de cibles intracellulaires fonctionnelles avec des acides nucleiques fonctionnels ou par selection in vivo
WO2000058455A3 (fr) * 1999-03-29 2001-01-04 Michael Blind Methodes d'identification et de validation de cibles intracellulaires fonctionnelles avec des acides nucleiques fonctionnels ou par selection in vivo
WO2001079555A2 (fr) * 2000-04-14 2001-10-25 Millennium Pharmaceuticals, Inc. Roles des membres de la famille jak/stat dans l'induction de la tolerance
WO2001079555A3 (fr) * 2000-04-14 2003-02-27 Millennium Pharm Inc Roles des membres de la famille jak/stat dans l'induction de la tolerance
WO2004029620A2 (fr) * 2002-09-26 2004-04-08 Novartis Ag Dosage biologique
WO2004029620A3 (fr) * 2002-09-26 2004-05-27 Novartis Ag Dosage biologique
WO2005030970A3 (fr) * 2003-09-26 2005-06-09 Internat Inst Of Molecular And Outils et procedes utilises pour caracteriser l'activite immunotoxique de substances xenobiotiques
US8361711B2 (en) 2003-09-26 2013-01-29 National Institute Of Public Health And The Environment, Laboratory For Pathology And Immunobiology Tools and methods useful in characterising the immunotoxic activity of xenobiotic substances
WO2017132279A1 (fr) * 2016-01-25 2017-08-03 Genentech, Inc. Méthodes de dosage d'anticorps bispécifiques dépendants des lymphocytes t
CN109073635A (zh) * 2016-01-25 2018-12-21 豪夫迈·罗氏有限公司 用于测定t细胞依赖性双特异性抗体的方法
US11513127B2 (en) 2016-01-25 2022-11-29 Genentech, Inc. Methods for assaying T-cell dependent bispecific antibodies

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