WO2001084154A1 - Methode et compositions permettant de decouvrir de nouveaux medicaments - Google Patents

Methode et compositions permettant de decouvrir de nouveaux medicaments Download PDF

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Publication number
WO2001084154A1
WO2001084154A1 PCT/US2001/014091 US0114091W WO0184154A1 WO 2001084154 A1 WO2001084154 A1 WO 2001084154A1 US 0114091 W US0114091 W US 0114091W WO 0184154 A1 WO0184154 A1 WO 0184154A1
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drug
drag
binding
biological activity
substrate
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PCT/US2001/014091
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English (en)
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Charles Pidgeon
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Charles Pidgeon
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Priority to AU2001261116A priority Critical patent/AU2001261116A1/en
Priority to US10/275,142 priority patent/US20030211464A1/en
Priority to CA002407889A priority patent/CA2407889A1/fr
Publication of WO2001084154A1 publication Critical patent/WO2001084154A1/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • the present invention relates to a method of screening test compounds to identify those compounds exhibiting a potential biological activity.
  • the method is based on the specific binding of test compounds to drug-binding substrates formed or identified using drug substances known to exhibit the biological activity.
  • the antifungal compounds myostatin and amphotericin B were isolated from broths that inhibited growth of yeast in culture tests.
  • Most bioassays have now been replaced as primary screens with more sophisticated, mechanism- based assays, such as enzyme (biochemical) assays and binding assays.
  • enzyme (biochemical) assays and binding assays Such assays can be designed, and indeed today are designed with view ofthe need for high throughput capacity requiring that the components and protocols for such assays be robust, simple and amendable to automation.
  • Binding assays are particularly useful for screening soluble mixtures of test compounds for compounds that bind to, and thus potentially inhibit or activate, target endogenous molecules with resultant therapeutic effect.
  • the target endogenous molecule usually a macromolecule such as a protein or a nucleic acid, or a derivative or analog thereof, can be affixed or tethered to a solid substrate, such as the sides of microtiter wells, beads, or chromatographic supports. If the target molecule is a receptor, it can be expressed in the membrane of a cell, which can itself be attached to the solid support.
  • the test compounds typically in solution, are incubated with the immobilized targets, and bound ligands are detected, frequently through an associated colorimetric or fluorescent reaction.
  • test compounds may be mixed with soluble-phase target molecules that are captured using an anti-target antibody.
  • binding assays are advantageous as they facilitate the washing and isolation of target-ligand complexes.
  • they suffer from several disadvantages, not the least of which is the availability, isolation, and handling ofthe endogenous target molecules, each of which has unique chemical and physical properties that must be taken into account in assay development.
  • assays are focused on detecting but one quality ofthe test compounds, that is their ability to specifically bind to the target molecule.
  • Drug substances possess a plurality of properties that allow them to exhibit their therapeutic effect in the species to which they are administered.
  • Known drug substances not only exhibit binding to therapeutically valuable endogenous biologically functional molecules, they each also possess other properties necessary for in vivo drug functionality, including but not limited to, the so-called "ADME" properties (adsorption, distribution, metabolism and elimination).
  • ADME absorption, distribution, metabolism and elimination.
  • the set of drugs on the market are thus a unique set of compounds, subsets of which bind to the same target molecules, but all of which necessarily have in common certain properties that allow them to be efficacious therapeutics when administered to a species being treated.
  • One objective ofthe present invention is to use a set of drug-binding substrates, for example antibodies, each of said drug-binding substrates being formed using or identified by one or more known drug substances having the same or similar biological activity.
  • the set of drug-binding substrates is used for identifying and purifying test compounds having potential for biological activity.
  • a known drug or set of known drugs having the same or different biological activities can be used to prepare or identify a drug-binding substrate or a corresponding set of drug-binding substrates, each of which are capable of specific binding to the drug from which they are prepared.
  • One or more members ofthe set of drug-binding substrates can be used alone or in combination with other members ofthe set to identify and purify compounds from complex mixtures.
  • one or more drug binding substrates for example monoclonal antibodies, known to specifically bind to two or more drug substances having the same or similar biological activity is used to screen combinatorial libraries, natural product mixtures, or other sources of potential drug candidates, for compounds that exhibit specific binding to said substrates.
  • Test compounds that specifically bind to the drug-binding substrates will be more likely to exhibit the specific binding and ADME properties ofthe drug substance(s) from which the drug-binding substrates are prepared.
  • an anti-drug antibody or, more typically, a set of anti-drug antibodies can be used to find compounds capable of occupying the same or similar "chemical space" as the drag- substance(s) used to form the antibodies.
  • the drug-binding substrates employed in this invention are, therefore, not only predictive of specific binding of a test compound to the drug-targeted endogenous molecules, but they are, as well, selective for test compounds having ADME properties critical for drug functionality.
  • the present invention is directed to a method of screening a test compound or a set of test compounds for potential biological or chemical activity using a binding assay wherein the drug-binding substrate is other than the endogenous target molecule or a chemically modified form or analog thereof.
  • the drug-binding substrate is one that is prepared, or empirically determined, to bind specifically to a drug substance, that is, an exogenous compound that is known or has been shown to exhibit a systemic effect by specific binding to one or more structurally related endogenous target molecules in the species to which it is administered or applied.
  • known drug substances are used as a template for preparing drug-binding substrates, or for identifying non-endogenous drug-binding substrates, which exhibit specific binding affinity for said drug substance and often for other drug substances having the same or similar biological activity.
  • the drug-binding substrates useful in accordance with the present invention include antibodies raised against drug substances, particularly monoclonal antibodies, phage display libraries, and polymers imprinted molecularly with drug substances.
  • the drug-binding substrates are selected, in one aspect ofthe invention, to bind to at least a second drug substance having the same or similar biological activity as the drug substance used to prepare or identify the drug-binding substrate.
  • Such cross-reactivity favors the use of such substrates to identify, by specific binding interaction, test compounds that not only have the potential for specific binding to the endogenous target molecule, but have as well the pharmaceutically significant properties that are required for favorable drug absorption, distribution, metabolism and elimination.
  • a method for screening a test compound or a set of test compounds for potential biological activity includes the step of selecting a dr g- binding substrate exhibiting specific binding affinity for a drug substance having a biological activity known to derive from specific binding of that drug substance to one or more related endogenous molecules.
  • the drug-binding substrate is contacted with a test solution comprising a test compound, and the existence of specific binding ofthe test compound to the drug-binding substrate is determined.
  • the drug-binding substrate is a monoclonal antibody raised against a drug substance (or more precisely, a monoclonal antibody raised against an immunogenic drug hapten-carrier conjugate, formed using said drug substances).
  • the monoclonal antibody is preferably one that exhibits specific binding affinity for at least a second drug substance having the same or similar biological activity as the drug substance used to form the antibody.
  • Drug-binding substrate (antibody) cross-reactivity is key to enablement and implementation ofthe present invention. Typically the goal in preparing antibodies to specific binding partners to a ligand or analyte has been one of high specificity and minimum cross-reactivity.
  • the present invention relies on and indeed requires, drug-binding substrate cross-reactivity to identify test compounds having potential for exhibiting the biological activity ofthe drug substance used to form or identify the drug-binding substrate.
  • a method for identifying or selecting a drug-binding substrate for use in drug discovery testing comprises the steps of contacting potential drug-binding substrates with a solution of a drug substance having a known biological activity deriving from specific binding with one or more related endogenous target molecules.
  • the substrates exhibiting specific binding affinity to the drug substances are identified and contacted with a solution of a second drug substance exhibiting the same or similar biological activity as the first drug substance.
  • potential drug-binding substrates are contacted with a solution comprising a plurality of drug substances, each having a common biological activity deriving from specific binding to one or more related endogenous molecules. Those substrates which specifically bind to more than one ofthe drug substances are preferred for use in drug discovery testing in accordance with this invention.
  • kits and/or compositions for analysis or screening of compounds for biological activity comprises at least first and second drug-binding substrates wherein said first substrate exhibits a specific binding affinity to a first drug substance having a known biological activity deriving from its specific binding to one or more related endogenous molecules, and said second drug-binding substrate exhibits specific binding affinity to the same drag substance or to another drag substance having the same or similar biological activity as the first drug substance.
  • the drug-binding substrates are monoclonal antibodies raised against one or more drug substances, more particularly immunogenic hapten carrier conjugates formed with said drug substances.
  • the resultant monoclonal antidrug antibodies can be labeled, or they can be used unlabeled with the labeled or unlabeled drug substances to which they bind in standard testing protocols that have been developed to detect and/or quantify specific binding events with test compounds for predicting biological activity of specifically bound compounds.
  • anti-drug antibodies are substituted for endogenous target molecules for specific binding assays using, for example, capillary electrophoresis (Cetek Corporation, Marlborough, Massachusetts; U.S. Pat. No. 5,783,397) or surface plasma resonance (Biacore AB, Uppsala, SE; U.S. Pat. No. 5,965,456).
  • the present drug discovery methodology and the identification of useful drug-binding substrates can be carried out using anti-drug antibodies and drag substances without use of detectable labels.
  • supernatant test solutions comprising a test compound and a drug substrate in a well of a titer plate containing an immobilized monoclonal antibody raised against the drug substance can be analyzed qualitatively and quantitatively for specific binding events by mass spectral analysis ofthe supernatant or by chromatographic separation of an aliquot ofthe supernatant.
  • the relative concentration ofthe drug substance in the supernatant can be estimated by comparison with concentration calibration curves prepared for each drug substance.
  • the method enables high throughput screening without the cost and time investment required for preparation of labeled antibody and/or drug substance as a component.
  • a set of exogenous test compounds are screened for potential biological activity in a living species by contacting a test solution comprising said test compounds or a subset thereof with a drug-binding substrate exhibiting specific affinity for a drag substance having a biological activity known to derive from its specific binding to one or more related endogenous molecules in said species.
  • This method can be carried out by immobilizing the drug-binding substrate or substrates, for example, anti-drug monoclonal antibodies or drug molecule imprinted polymer substrates, in an affinity chromatography column and contacting the immobilized drug-binding substrate with the test solution.
  • test solution is separated from a drug-binding substrate, and test compounds specifically bound to the substrate are then separated from the substrate and from each other, for example, using liquid chromatography, and then characterized/identified by evaluation of their physical-chemical properties and other structure elucidation techniques.
  • drugs typically have unique ADME properties compared to the endogenous ligands they agonize or antagonize. Most therapeutically useful compounds are thus not endogenous ligands that bind to specific receptors or other endogenous biologically functional molecules, rather most useful therapeutics are compounds that not only specifically bind to an endogenous molecule, but they also have favorable absorption, distribution, metabolism, and other bio-properties.
  • ADME adsorption, distribution, metabolism, and elimination
  • the present invention is directed to a method and related embodiments for screening test compounds for potential biological activity using non-endogenous drug-binding substrates characterized by their specific binding affinity to drug substances.
  • anti- ligand antibodies or anti-drag antibodies may be used.
  • anti-drag antibodies or other drag-binding substrates formed or identified with known drag substances will enable identification of test compounds with more favorable ADME properties than those test compounds identified by their specific binding to anti-ligand antibodies.
  • Anti-drag antibodies have been used routinely for both drug-assays and models for studying receptor binding, and receptors and other endogenous biofunctional macromolecules have been used to screen test compounds for biological activity.
  • neither anti-drug antibodies nor other non-endogenous drug- binding substrates have been used as reagents for screening chemical libraries, or screening natural products to identify compounds having potential biological activity corresponding to that ofthe drug substance used to form or identify the drug-binding substrate.
  • Anti-drug antibodies have a binding site that mimics the topology ofthe drag(s) against which they are raised, drug(s) which has/have the ADME properties in addition to the ability to bind to a target molecule, necessary for drug efficacy.
  • anti-drug antibodies or other drug-binding substrates formed or identified using drug substance will, according to one premise of this invention, not only specifically bind compounds that may have affinity for the drug's target endogenous macromolecule, but also select for compounds having favorable bio-properties.
  • the drug discovery selection process has several steps that typically follow a sequence:
  • Step 1 identify a set of compounds that elicit activity in cell free receptor binding assays
  • Step 2 find the compounds in the set with activity in whole cell assays
  • Step 3 evaluate the activity in several in vitro screening assays to identify toxicity, cell penetration properties, metabolism, etc.;
  • Step 4 evaluate the in vivo activity ofthe compounds in many animal species,
  • Step 5 identify the compound with activity in humans.
  • This routine drug discovery process is in general a sequential process and is used to identify compounds that go into Phase I and Phase II clinical trials.
  • a compound that binds to a receptor and has favorable bio-properties will have a higher probability of success in the drag development process than one selected only for its receptor biding activity (Step 1 above).
  • the present method provides a test procedure for identifying test compounds that potentially exhibit a pre-determined biological activity.
  • the method comprises the steps of contacting a solution comprising one or more test compound(s) of unknown or undetermined biological activity, with a drug-binding substrate formed or identified using a drug substance known to exhibit a pre-determined biological activity.
  • the test compound(s) identified as potentially having the pre-determined biological activity are those that exhibit specific binding affinity with the drag-binding substrate.
  • the drug-binding substrate is selected based on its capacity to exhibit specific binding affinity, not only to the drug substance used to form or identify the drag-binding substrate, but as well to a second drag substance known to have the predetermined biological activity. It is the cross-reactive specific binding affinity ofthe drag-binding substrates used in accordance with this invention that enables the selection/identification of test compounds potentially having the pre-determined biological activity ofthe drug substance used to form or identify the drug-binding substrate.
  • the drug-binding substrates preferred for use in accordance with this invention are selected for their cross-reactivity with other drug substances having the pre-determined biological activity.
  • the test compounds can be drugs of known biological activity which are screened against drug-binding substrates formed or identified with a drug substance having a different biological activity whereby the test drags are assayed for potential toxicity.
  • the method ofthe invention may be used to screen for potential biological activity using well known immunoassay procedures, using polyclonal antidrug antibodies or fragments thereof, monoclonal anti-drag antibodies or fragments thereof, biosynthetic anti-drag antibody binding sites or other drug-binding proteins such as those presented in phage display libraries.
  • the method can be carried out using drug molecule imprinted polymer substrates such as the molecularly imprinted polymer substrate described in U.S. Pats. Nos. 5,872,198; 5,959,050; and 5,994,110.
  • Various specific assay protocols are known per se. See for example, U.S. Pats. Nos. 4,313,734; 4,366,241; 5,783,397; and 5,965,456.
  • endogenous molecule or "endogenous compound” for the purpose of defining this invention refers to a naturally occurring molecule/compound, exclusive of antibodies, typically genomically encoded or produced by natural biochemical processes, and having biological function relating to mediation of intracellular or intercellular biochemical processes.
  • the term includes, but is not limited to, enzymes, receptors, ion channels, carrier molecules, nucleic acids, chemical derivatives or analogs thereof, and metabolites of exogenous compounds, but it specifically excludes antibodies.
  • exogenous molecule or “exogenous compound” relevant to a given living species is a molecule or compound having origin external to the species and includes members of combinatorial chemical libraries, other synthetic compounds, and natural products, including extracts comprising endogenous molecules from other species.
  • a “drug substance” is an exogenous compound that is capable of exhibiting biological activity as a systemic effect by specific binding to one or more structurally related endogenous macromolecules in the species to which it is administered or applied.
  • the term contemplates effective therapeutics for human or animal use and, for example, growth regulators and pesticides, including herbicides and insecticides, for agricultural use.
  • a "drag-binding substrate” for the purpose of defining the present invention is a chemical entity other than an endogenous molecule that specifically binds to a drag substance.
  • the term includes antibodies raised against drug substances (or, more precisely, against their hapten-carrier conjugates), antibodies raised against other exogenous compounds that specifically bind to said anti-drug antibodies, phage display libraries and molecularly imprinted polymers that bind specifically to drug substances.
  • Drug-binding substrates of biological origin are typically immobilized on a solid carrier for use in this invention.
  • a "biologically functional molecule” is an endogenous macromolecule known to mediate one or more biochemical processes in a plant, animal or single cell species.
  • Drug substances having the "same or similar biological activity" are exogenous compounds that 1) specifically bind to one or more structurally related biologically functional molecules, and 2) can be administered or applied to produce a common or related biological effect. Examples of drug substances having the same or similar biological activity are listed for four drug substance categories below:
  • control compound as used herein is a drag substance or another non-endogenous compound which specifically binds to an antibody raised against a drag substance.
  • Specific binding or “specific binding affinity” is the affinity of a pair of molecules or a molecule and a chemical structure on a substrate surface deriving from a plurality of specific non-covalent interactions that depend on the three-dimensional structures ofthe binding entities involved.
  • Typical pairs of specific binding entities include antigen-antibody, hapten-antibody, hormone-receptor, nucleic acid strand-complementary nucleic acid strand, substrate-enzyme, inhibitor-enzyme, carbohydrate-lectin, biotin-avidin, virus-cellular receptor, and the interaction of certain molecules with molecularly imprinted polymer surfaces.
  • the strength or affinity of binding interactions can be expressed in terms ofthe dissociation constant (Kj) ofthe interaction, wherein a smaller K d represents a greater affinity.
  • Kj dissociation constant
  • Specific binding properties of various non-endogenous substrates for use in this invention can be qualified and quantified using methods well known in the art.
  • One such method entails measuring the rates of drag substance-recognition substrate complex formation and dissociation, wherein those rates depend on the concentrations ofthe complexing entities, the affinity ofthe interaction, and on parameters that equally influence the rate in both directions.
  • both the "on rate constant" (K ⁇ ) and the "off rate constant” (K off ) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation. The ratio of K ⁇ /K ⁇ .
  • immunoassay formats may be used to select antibodies specifically binding with one or more drag substance.
  • solid-phase ELISA immunoassays can be used to select monoclonal antibodies specifically immunoreactive with a drug substance or to a drug-carrier conjugate. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • antibody includes both intact antibody molecules ofthe appropriate specificity and antibody fragments (including Fab, F(ab'), and F(ab') 2 fragments) as well as chemically modified intact antibody molecules and antibody fragments, including hybrid antibodies assembled by in vitro reassociation of subunits.
  • antibody as used herein also refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • An antibody is typically a protein consisting of one or more polypeptide substantially or partially encoded by immuno globulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the term "antibody” includes monoclonal antibodies and antibody compositions with polyepitopic specificity (i.e., polyclonal antibodies).
  • the polyclonal antibodies are preferably "affinity purified" antibodies.
  • affinity purified means that the antibodies have been purified using the antigen or the hapten- carrier conjugate to selectively purify the polyclonal antibodies.
  • Affinity purification can be achieved by immobilizing the antigen on an affinity column (e.g., an agarose column) and passing the polyclonal antibodies through the column.
  • the affinity purified antibodies can be subsequently eluted from the column by changing the elution conditions or by adding a chaotropic agent, for example.
  • the antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies, and include antibody fragments such as, for example, Fab, F(ab') 2 , and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG.
  • An antibody has specific binding affinity for a ligand or is specific for a ligand if the antibody binds or is capable of binding the ligand as measured or determined by standard antibody-antigen or ligand-receptor assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA. This definition of specificity applies to single heavy and/or light chains, CDRS, fusion proteins or fragments of heavy and/or light chains, that are specific for the ligand if they bind the ligand alone or in combination.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are typically highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies for use in this invention include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of a selected antibody with a constant domain (e.g..
  • “humanized” antibodies or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv), so long as they exhibit the desired biological activity.
  • antibody fragments e.g., Fab, F(ab') 2 , and Fv
  • the modifier "monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production ofthe antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild chemical conditions to cleave the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part ofthe hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, NY (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Antibodies include single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • sFv single chain Fv
  • one of skill can easily make antibodies specific for drag substances using existing techniques, or modify those antibodies which are commercially or publicly available.
  • general methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art. See, e.g., Paul (ed) (1993) Fundamental nmunology, Third Edition Raven Press, Ltd., New York Cofigan
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 ⁇ M, preferably at least about 0.01 ⁇ M or better, and most typically and preferably, 0.001 ⁇ M or better.
  • Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Pat. Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference).
  • mRNA is isolated from a B cell population, and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the .lambda.]rnmunoZap(H) and .lambda.ImmunoZap(L) vectors.
  • vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al. supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E. coli.
  • drug-binding substrates for use in this invention may also be constracted utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encode a specifically binding antibody.
  • the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. Stratacyte (La Jolla, Calif.) sells primers for mouse and human variable regions including, among others, primers for V H3> Vm,, V Hc , V Hd , C H 1, V L and C L regions.
  • These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as -rnmunoZAP.TM H or -nrmunoZAP.TM L (Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion ofthe VH and VL domains may be produced (See Bird et al, Science 242:423-426, 1988). In addition, such techniques may be utilized to change a "murine" antibody to a "human” antibody, without altering the binding specificity ofthe antibody.
  • suitable antibodies or other drug-binding substrates may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.
  • label when used herein refers to a detectable compound or composition which can be conjugated directly or indirectly with a molecule such as an antibody or a drug-substance.
  • the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition which is detectable.
  • a phosphatase enzyme can be used as a label.
  • An example is a chemiluminescent enzyme-linked immunoassay, such as the so-called enzyme-linked immunosorbent assay or ELISA.
  • assays which contain colorimetric or radiometric indicators, are commonly used in manual format as well as on automated multi-test immunoassay systems.
  • the detectable labels that can be used in carrying out certain embodiments ofthe method ofthe present invention, which are attached to either the antibody or the drag substance, can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling).
  • primary labels where the label comprises an element that is detected directly or that produces a directly detectable element
  • secondary labels where the detected label binds to a primary label, e.g., as is common in immunological labeling.
  • Primary and secondary labels can include undetected elements as well as detected elements.
  • Useful primary and secondary labels can include spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green.TM, rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes.TM, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase, etc.), spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e
  • the label may be coupled directly or indirectly to a component ofthe specific binding assay (e.g., the detection reagent) according to methods well known in the art.
  • a component ofthe specific binding assay e.g., the detection reagent
  • a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Preferred labels include those that use: 1) chemiluminescence (using horseradish peroxidase and/or alkaline phosphatase with substrates that produce photons as breakdown products as described above) with kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) color production (using both horseradish peroxidase and/or alkaline phosphatase with substrates that produce a colored precipitate [kits available from Life Technologies/Gibco BRL, and Boehringer-Mannheim]); 3) hemifluorescence using, e.g., alkaline phosphatase and the substrate AttoPhos [Amersham] or other substrates that produce fluorescent products; 4) fluorescence (e.g., using Cy-5 [Amersham]), fluorescein, and other fluorescent tags]; 5) radioactivity.
  • Other methods for labeling and detection will be readily apparent to one skilled in the art.
  • Enzymes can also be conjugated to reagents used to detect and analyze binding events in carrying out the method ofthe invention include, e.g., beta-galactosidase, luciferase, horse radish peroxidase, and alkaline phosphatase.
  • the chemiluminescent substrate for luciferase is luciferin.
  • One embodiment of a chemiluminescent substrate for .beta.-galactosidase is 4-methylumbelliferyl-beta -D-galactoside.
  • Embodiments of alkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP), which is detected with a specfrophotometer; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCLP/NBT) and fast red/napthol AS-TR phosphate, which are detected visually; and
  • pNPP p-nitrophenyl phosphate
  • BCLP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
  • AS-TR phosphate fast red/napthol AS-TR phosphate
  • Embodiments of horse radish peroxidase substrates include 2,2'azino-bis(3-ethylbenzthiazoline-6 sulfonic acid)(ABTS), 5-aminosalicylic acid (5 AS), o-dianisidine, and o-phenylenediamine (OPD), which are detected with a spectrophotometer; and 3,3,5,5'-tetramethylbenzidine (TMB), 3,3'diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and 4-chloro-l-naphthol (4C1N), which are detected visually.
  • Other suitable substrates are known to those skilled in the art.
  • the enzyme-substrate reaction and product detection are performed according to standard procedures known to those skilled in the art and kits
  • a detector which monitors a particular probe or probe combination is used to detect the label.
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof.
  • Some embodiments of this invention can be implemented using a mass spectrometer optionally in combination with a liquid chromatograph. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • the method ofthe present invention can be carried out using standard immunoassay techniques.
  • a typical immunoassay the analyte hapten, antigen or antibody is assayed by detecting the presence or amount of an enzyme-labeled specific binding partner for the analyte or an enzyme label-analyte conjugate.
  • Various assay formats and the protocols for performing the immunochemical steps are well known in the art. These assays fall broadly into two categories.
  • Competitive assays feature an immunological binding of a specific antibody with the analyte and an analyte analog, e.g., a detectably labeled analyte molecule.
  • Sandwich assays result by the sequential or simultaneous binding of two antibodies, one of which is detectably labeled, with the analyte.
  • the detectably labeled binding pair so formed can be assayed with the compounds and methods ofthe present invention.
  • the detectable label is the enzyme, it is detected directly.
  • the detectable label is a member of another specific binding pair, e.g. , a hapten
  • a conjugate of its binding partner with an enzyme is reacted first and the enzyme then detected in accordance with the present methods.
  • Measurement can be performed with enzyme-labeled species attached to a solid surface or support including beads, tubes, microwells, magnetic particles, test strips, membranes and filters such as are in common use in the art.
  • the detectable enzyme-labeled species can also be present free in solution or enclosed within an organized assembly such as a liposome in which case a lytic agent is employed to lyse the liposome and free the detectable enzyme.
  • the antibodies are generally immobilized on a solid support having surface(s) which are amenable to attachment of such an antibody.
  • solid support is meant a non-aqueous matrix drag-binding substrate can adhere.
  • the solid phase can comprise a discontinuous solid phase of discrete particles.
  • the particles can be porous and formed from a number of different materials, e.g. , polysaccharides (e.g. , agarose), polyacrylamides, polystyrene, polyvinyl alcohol, silicones and glasses.
  • suitable particulate solid phases see U.S. Pat. No. 4,275,149.
  • suitable examples include without limitation microtiter plates; membrane material such as nitrocellulose, cellulose, cellulose acetate, polycarbonate, etc.; test tubes; particles such as beads, column packing materials, etc.; and derivatized surfaces having binding domains capable of binding the antibody attached thereon.
  • the antibody is a coating antibody which is fixed to a solid support.
  • Antibody assays may, in general, be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody, a labeled analyte, and the set of test compounds.
  • the antibody itself is labeled and in others the analyte (drag substance) is labeled, depending on the methodology employed for detecting specific binding events.
  • the signal arising from the label is modified, directly or indirectly, upon the binding ofthe antibody to the labeled analyte. Both the immunological reaction and detection ofthe extent thereof are typically carried out in a homogeneous solution.
  • Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • the reagents are usually the test compound or the set of test compounds, one or more anti-drag antibodies and a system or means for producing a detectable signal.
  • the antibody can be immobilized on a solid support, such as a bead, plate or slide, and contacted with the set of test compounds in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The location ofthe signal is related to the binding of one or more test compounds to the antibody.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzymes for use in colorimetric assays, and so forth.
  • the extent of retention ofthe detectable group on the solid support indicates the presence or absence of a competitively binding test compound in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence assays, enzyme-linked immuno adsorbant assays, and the like.
  • a drag-binding substrate formed or identified using a drag substance having a predetermined biological activity is used to screen or identify test compounds likely to exhibit the predetermined biological activity.
  • the potential biologically active test compounds are identified by their specific binding to the drag-binding substrate(s) as detected by any of a wide variety of specific binding assay techniques using labeled or unlabeled assay components.
  • a monoclonal antibody raised against a drug substance is used as a drag-binding substrate to identify and isolate test compounds in a natural product extract or other source of test compounds, for example, a chemical library for natural or synthetic sources.
  • the monoclonal antibody exhibits specific binding to at least one other drug substance having the same or a similar biological activity as the drag substance against which the antibody was raised.
  • the invention finds use inter alia in drug discovery protocols, in toxicity profiling of drug substances, and in assaying commercial natural products.
  • a method of identifying possible drug substances or lead compounds for drug substances having a predetermined biological activity in a living species comprising single cell organisms or multicellular organisms including, for example, plants and animals.
  • the method comprises the steps of contacting a test solution comprising an exogenous test compound with a drug-binding substrate capable of specific binding to a drag substance known to exhibit the predetermined biological activity in the living species.
  • the test compounds having potential for exlnbiting the predetermined biological activity are identified by detecting the existence of specific binding ofthe test compound to the drag-binding substrate.
  • the method further comprises the step of determining whether or not the test compounds that specifically bind to the drag-binding substrate exhibit the biological activity. That determination can be made by independent testing in known biological assays or animal tests designed to detect or confirm the predetermined biological activity.
  • the "drag-binding substrate” for use in accordance with the present invention is formed or identified using one or more drag substances having a predetermined biological activity.
  • the drag-binding substrate is an antibody, more preferably a monoclonal antibody, raised against a drug substance having the predetermined biological activity.
  • the drug- binding substrate can be a drug molecule imprinted polymer substrate.
  • the preparation of molecularly imprinted polymer substrates is known in the art. See, for example, U.S. Pats. Nos. 5,872,198; 5,959,050; and 5,994,110.
  • the drug-binding substrates can be identified empirically by exposing sources of substrates of high diversity, for example, those available as phage display libraries, to labeled drag substances and identifying those substrates that exhibit specific binding affinity to the drag substance.
  • drug-binding substrates for use in the drug discovery testing embodiments ofthe invention are selected based on their cross-reactivity with at least one other drug substance having the same biological activity as the drug substance used to form or identify the drug-binding substrate.
  • a method for defining a drag-binding substrate for use in drug discovery testing comprises the step of contacting potential drug-binding substrates with a solution of a drug substance having a known biological activity deriving from its specific binding to one or more related endogenous molecules.
  • the substrates exhibiting specific binding to the drag substance are identified and thereafter at least those substrates are contacted with a solution of a second drag substance exhibiting the predetermined biological activity.
  • drag-binding substrates for use in the present invention are those substrates that exhibit specific binding to each ofthe first and second drug substances.
  • drag-binding substrates for use in the present invention can be identified by contacting potential drag-binding substrates with a solution comprising a plurality of drag substances, each having a common biological activity deriving from specific binding to one or more related endogenous molecules.
  • Those substrates identified for their specific binding to one or more ofthe drug substances can be employed for use in the screening/analysis aspects ofthe present invention.
  • the potential "drag-binding substrates” can be contacted with a solution of a third drag substance exhibiting the same or similar biological activity as the first and second drug substances used to identify the drag-binding substrates, and those substrates that exhibit specific binding to at least two of said drag substances are employed for use in drag screening/analysis aspects ofthe invention.
  • One procedure for implementing the method for defining the drug-binding substrates for use in the present invention is to contact a plurality of potential drug- binding substrates with a solution comprising a known concentration of a drug substance, for example, each of a series of potential drag-binding substrates can be located/immobilized in a well of a microtiter plate and contacted with a solution comprising a known concentration of a drag substance.
  • the concentration ofthe drag substance in the solution after contact with the potential drag-binding substrate can be determined, for example, by mass spectrographic analysis of a volume ofthe test solution. Mass peak intensities correspond to the relative concentration ofthe drag substance in the supernatant test solution.
  • the drag-binding substrates located in those wells where the supernatants are determined to have a reduced concentration of the drug substance are indicated for specific binding to that drug substance.
  • Other methods of analyzing drag substance concentration in the supernatant can be employed, for example, quantitative chromatographic analysis.
  • labeled analogs ofthe drug substance can be used to detect specific binding using standard specific binding assay protocols.
  • a composition for analysis or screening of compounds for biological activity is provided.
  • the composition comprises at least first and second drag-binding substrates wherein the first substrate exhibits specific binding to a first drag substance having a known biological activity deriving from its specific binding to one or more related endogenous molecules and the second drag bonding substrate exhibits specific binding to the same drag substance or to another drug substance having the same or similar biological activity to the first drag substance.
  • at least one ofthe first or second drag binding substrates exhibits specific binding to another drag substance other than the first or second drug substance but one having the same or similar biological activity as the first and second drag substances.
  • the drag-binding substrates can be antibodies, most typically monoclonal antibodies, which are optionally immobilized on a solid support, ready for use in the analysis or screening embodiments of this invention.
  • the drag-binding substrates formed or identified using drags of known biological activity can also be used as components of a kit for analysis or screening of test compounds for potential biological activity.
  • the kit comprises at least first and second monoclonal antibodies.
  • the first monoclonal antibody is raised against a first drug substance having a predetermined biological activity
  • the second monoclonal antibody is raised against either the first drag substance or a second drug substance having the same or similar biological activity.
  • the monoclonal antibody components exhibit specific binding to another drug substance exhibiting the same biological activity, but a drag substance other than the drug substance against which each of said monoclonal antibodies was raised.
  • the kit comprises at least first and second monoclonal antibodies wherein the first monoclonal antibody is raised against a first drag substance having a predetermined biological activity and the second monoclonal antibody is raised against another monoclonal antibody having the same or a different biological activity.
  • the kit can be used to screen test compounds for more than one potential biological activity in a parallel processing protocol.
  • each of said first or second antibodies exhibit specific binding to another drag substance having the same biological activity as the drug substance against which the respective antibodies were raised.
  • the antibodies are preferably presented in the kit already immobilized on a solid substrate for use in screening test compounds for biological activity, for qualitative analysis of natural products, or for toxicity profiling of biologically active compounds in accordance with those respective embodiments of this invention.
  • test compounds can be components of a natural product comprising an extract of tissue or plant, fungal, microbial or marine origin, members of a combinatorial chemical library, or one or more compounds that have a biological activity different than that for which the compounds are being screened.
  • the method comprises the steps of selecting a drug-binding substrate exhibiting specific binding to a drug substance having the biological activity for which the test compounds are being screened, a biological activity typically derived from specific binding to one or more related endogenous molecules, and contacting the drug-binding substrate with a test solution comprising (1) a test compound and (2) a known amount ofthe drug substance or a second drag substance known to bind to said substrate competitively with said drug substance, for a period of time sufficient to allow specific binding of the drug substance to the drag-binding substrate. Finally, the supernatant test solution in contact with the drug-binding substrate is analyzed to determine the extent, if any, to which a test compound blocks binding ofthe substrate with the first or second drug substance.
  • the extent to which that test compound blocks binding ofthe drug-binding substrate with the drag substance can be assayed simply by mass spectrographic analysis of a fraction ofthe test solution after the period of time for specific binding.
  • the relative amount ofthe drug substance in the test solution can be determined simply by comparing mass peak intensities obtained with a known volume ofthe test solution with a pre-established quantitative calibration curve for the drag substance in the mass spectrograph.
  • the method can be carried out with a labeled drag substance and the extent that the test compound blocks binding ofthe substrate with the drag substance can be determined using art-recognized label-dependent procedures.
  • a set of exogenous test compounds is screened for potential biological activity using a monoclonal antibody exhibiting specific binding for a control compound known to exhibit specific binding to a biologically functional endogenous molecule in a living species.
  • the method comprises the steps of contacting the set of test compounds, typically in solution, with a drug-binding substrate comprising the monoclonal antibody under conditions conducive to specific binding between the antibody and at least one ofthe test compounds to form an antibody-test compound complex.
  • At least a portion ofthe complex is separated from at least a portion ofthe non-complex test compounds, and the complex test compound is then identified using art-recognized procedures including the steps of subjecting the complex to conditions effective to separate the complex test compounds from the monoclonal antibody and thereafter isolating the test compound or test compounds by, for example, chromatographic separation techniques.
  • the potential biological activity ofthe test compounds can then be further confirmed by measuring the affinity ofthe test compound forming the antibody-test compound complex for the biologically functional endogenous molecule, such as an enzyme, a receptor, a transport protein, for example an ion transporter or a carrier molecule or a nucleic acid.
  • test compounds forming antibody-test compound complexes can be subjected to further screening by contacting those compounds with a second antibody exhibiting specific binding to a second control compound known to exhibit specific binding to the same biologically functional endogenous molecule, and identify those test compounds that form antibody-test compound complexes with each antibody.
  • a method for screening a set of test compounds for potential biological activity including the steps of contacting a solution ofthe set of test compounds or a subset thereof with a substrate comprising an antibody, a phage display library, or a molecularly imprinted polymer exhibiting specific binding to a control compound known to exhibit specific binding to a biologically functional molecule. That contacting step is carried out under conditions conducive to specific binding between the substrate and any test compounds to which it specifically binds to form one or more complexes ofthe substrate and the respective test compounds.
  • the substrate- test compound complex is separated from at least a portion ofthe test solution and the test compounds forming complexes with the substrate are identified.
  • control compound is a drag substance and the substrate comprising a monoclonal antibody raised against that drug substance or a phage display library determined to bind specifically to said drug substance.
  • the complex test compounds can then be separated from the binding substrate and from each other, optionally further assayed by determining specific binding for the biologically functional molecule, and identified by art-recognized structure elucidation techniques.
  • the solution of test compounds can be contacted with at least first and second substrates, the first substrate exhibiting specific binding to a first control compound known to exhibit specific binding to the biologically functional molecule and the second substrate exhibiting specific binding either to said first control compound or to a second control compound known to exhibit specific binding to the biologically functional molecule.
  • the complexes formed between the substrate and a test compound are useful for predicting the biological activity ofthe test compound.
  • the test compound is not known to exhibit the predicted biological activity or to exhibit specific binding to an endogenous molecule associated with said biological activity.
  • the complex comprises said test compound specifically bound to a substrate known to exliibit specific binding to a drag substance known to exhibit said biological activity.
  • the substrate component of such complexes can be, for example, an antibody, a member of a phage display library, or a molecularly imprinted polymer substrate formed from or identified by a drug substance known to exhibit the biological activity.
  • the substrate is a monoclonal antibody raised against the drag substance and, more preferably, one that exhibits specific binding to at least one other drug substance known to exhibit the biological activity.
  • a method of using a monoclonal antibody for screening compounds for probable biological activity comprises the step of selecting a monoclonal antibody raised against a drag substance exhibiting the biological activity and capable of specific binding to said drug substance and at least one other drug substance known to exhibit said biological activity.
  • the monoclonal antibody is contacted with a solution comprising the test compounds and an antibody-test compound complex is formed, and specific binding ofthe antibody to a test compound is detected.
  • the test compound forming the antibody-test compound complex is thereafter identified.
  • the method includes the additional step of separating at least a portion of the antibody-test compound complex from at least a portion ofthe test compound solution.
  • the monoclonal antibody is preferably immobilized on a solid support, for example, a microtiter plate.
  • the test solution can further include the drug substance used to form the drag substance covalently linked to a detectable label.
  • the formation ofthe complex can be detected by a method comprising mass spectroscopic analysis of an aliquot of a test solution or by a quantitative chromatographic separation/analysis thereof.
  • assay instruments based on measuring the change in surface optical characteristics of a sensor chip using surface plasmon resonance is available from Biacore AB.
  • Biological functional molecules are immobilized on the sensor surface and changes in surface optical characteristics are measured as a solution of a test compound or compounds is flowed across the sensor surface.
  • Surface plasmon resonance is an optical phenomenon that is used to measure changes in the solution concentration of molecules at a biospecific surface, for example, that ofthe sensor chip having immobilized biologically functional molecules. The resonance signal depends on the refractive index of solutions in contact with the surface.
  • Molecules and solutions exhibit changes in refractive index and thus give rise to a measurable resonance signal if a biospecific interaction occurs.
  • protein or DNA is immobilized by one of several possible methods onto a carboxy methylated dextran-gold surface.
  • the interacting compound of interest is injected over the surface and the kinetics of binding is measured in real time.
  • an antibody preferably a monoclonal antibody
  • the antibody can be substituted for the potential target molecule or biologically functional molecule, the antibody being selected for its specific binding affinity to a drug substance known to exhibit a predetermined bioogical activity.
  • toxicity profiling refers to the use of a panel of anti-drag antibodies or other drug-binding substrates, each capable of specific binding to a unique drug- binding substrate to screen a putative drug substance for cross-reactivity with one or more drag-binding substrates for unrelated drag substances, the existence of which cross-reactivity is suggestive of undesired biological activity and consequent potential toxicity.
  • the toxicity profiling of a compound identified as a new drug substance against a panel of anti-drag antibodies to a plurality of drug substances having biological activities unrelated to that ofthe new drug substance should provide useful information predictive of potential side effects deriving from one or more detected secondary biological activities.
  • each of at least a portion ofthe wells in a 96-well titer plate is coated with a unique but known anti-drag monoclonal antibody.
  • At least a portion ofthe anti-drag antibodies are selected for their capacity to bind specifically to at least two drug substances exhibiting the same or similar biological activity. Some wells are left uncoated and others are coated with a monoclonal antibody to the test drag substance.
  • Each ofthe wells is loaded with an equal volume of a solution of the test drag substance. After an incubation period, adequate to allow specific binding events to reach equilibrium in the respective wells, a volume ofthe supernatant from each well is injected into a mass spectrometer (previously calibrated with varying amounts ofthe test drag substance). The relative concentration ofthe test drug substance in the supernatant of each well provides a measure ofthe specific binding ofthe drag substance by the respective anti-drag antibodies. Specific binding ofthe test drag with antibodies other than the antibody raised from a test-drag hapten conjugate, or other than antibodies raised from immunogenic conjugates of a drug substance having the same or similar biological activity as the test drag substance, is predictive of unwanted biological activity and concomitantly potential toxicity.
  • natural products for example, extracts of plant, microbial, fungal, yeast or marine species are subjected to evaluation for specific binding of certain components to drag-binding substrates, themselves selected for their capacity to bind specifically to drag substances having the same or similar activity as one or more biologically significant components ofthe natural product.
  • the method can be used to identify the presence of previously unknown biologically active natural products in complex natural product mixtures or it can be used, for example, as a means for enabling quality control testing on commercially available extracts being sold for human or animal use.
  • the method can be implemented using any of a wide variety of testing protocols heretofore developed and described generally hereinabove for detecting specific binding events using either labeled drug substances (corresponding to the anti-drug antibody or other drag- binding substrate) in competitive binding-based assays or labeled anti-drug antibodies for use in capillary zone electrophoresis-based assays (See, e.g., U.S. Pat. No. 5,783,397).
  • Other competitive or non-competitive binding assay protocols can be employed alone or in combination with chromatography and/or, for example, mass spectral detection/analysis without use of labeled drug substances or labeled antibodies.
  • unknown, potentially biologically active components of a natural product extract can be separated from the extract and ultimately from each other by first employing affinity chromatography techniques using immobilized anti-drag antibodies and subsequent non-affinity chromatographic separation techniques using mass spectrometry or other suitable detectors allowing identification o the bound components.
  • a natural product mixture detailed for a defined biological activity can be analyzed for such activity by using, for example, a microtiter plate having at least a portion ofthe wells coated with one or more antibodies, preferably monoclonal antibodies, raised to one or more drug substances having the same or similar biological activity as the component of natural product said to have such activity.
  • a solution ofthe natural product mixture and a known amount ofthe drug substance against which the respective antibodies were raised are added to each well.
  • the first step is the development an HPLC assay for separating up to 50 drags (drug substances) at a time using a reference mass spectrum (MS) library (a MS of each compound), a liquid chromatograph/mass spectrometer (LC/MS) automated peak picking method to find each drug, and a data analysis system to detect each compound.
  • MS mass spectrum
  • LC/MS liquid chromatograph/mass spectrometer
  • Step 1 Create a reference mass spectra of each drug (determine which compounds are + Vs. - mode).
  • Step 2 Create a standard curve for each drag, determine the limits of detection, sensitivity, and any other parameter needed.
  • Step 3 Create a chromatographic condition that allows all compounds to be separated on a chromatography column.
  • Step 1 An anti-drug antibody is located in each well or container.
  • Step 2 Immobilization method (e.g., ELISA plate).
  • Step 3 Incubate drag(s) (corresponding to the antibody) and test compound(s) in each well (e.g. , ELISA plate with 96 different anti-drug antibodies). The same sample may be applied to each well. This set up will work for individual compounds, and synthetic and natural product libraries.
  • Step 4 Pool aliquots from each well and quantitate the amount of free drag in solution using the LC/MS library established method. Knowing the mass of each ofthe respective drags for quantitation and where to look for the compounds during the chromatographic separation, one is able to quantitate the compound of drag in the supernatant in each well. If a natural product is screened, the chance for an overlapping molecular weight exactly equal to the elution ofthe drag is minimal. The amount ofthe drag in the supernatant is proportional to the amount, if any, of test compound(s) that compete with the drag for the immobilized anti-drug antibody in the well.
  • the need for a reference library is decreased as the resolution ofthe detector increases. In other words, by using a high resolution mass spectrometer with accurate mass out to 3-4 decimals, need for a reference library is not as great.

Abstract

Cette invention concerne un substrat se liant à des médicaments, formé ou identifié à partir d'une substance médicamenteuse à l'activité biologique déterminée, qui s'utilise pour le criblage et l'identification de composés tests susceptibles de présenter une activité biologique donnée. Ces composés tests potentiellement actifs au plan biologique s'identifient à la façon spécifique dont ils se lient aux substrats qui fixent des médicaments, façon que permettent de détecter l'une quelconque de multiples techniques faisant intervenir des composés tests marqués ou non. Selon un mode de réalisation, un anticorps monoclonal dirigé contre une substance médicamenteuse est utilisé comme substrat se liant à des médicaments pour identifier et isoler des composés tests dans un extrait de produit naturel ou dans une banque combinatoire de composés chimiques. Ledit anticorps monoclonal est caractérisé de préférence par son aptitude à se lier spécifiquement à au moins une autre substance médicamenteuse présentant une activité biologique semblable ou identique à celle de la substance médicamenteuse contre laquelle il a été dirigé. Cette invention peut s'utiliser entre autres choses dans des protocoles de découverte de médicaments, dans l'établissement de profils de toxicité pour des substances médicamenteuses et pour des analyses de produits naturels destinés à la vente.
PCT/US2001/014091 2000-05-03 2001-05-02 Methode et compositions permettant de decouvrir de nouveaux medicaments WO2001084154A1 (fr)

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