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Definitions

• the invention relates to methods for the antibody-mediated generation of superoxide free radical from singlet oxygen.
• the invention also relates to the generation of hydrogen peroxide from singlet oxygen.
• Therapeutic methods are based upon both enhancing and inhibiting these processes. Screening methods relate to identifying modulators of antibody-mediated generation of hydrogen peroxide and superoxide free radical through the respective increase or decrease in detectable hydrogen peroxide or superoxide.
• the invention further relates to a simplified immunoassay based on detecting hydrogen peroxide.
• the invention also relates to therapeutic compositions that are engineered to increase the production of hydrogen peroxide and superoxide free radical as well as compositions that are engineered to prevent this production.
• reactive oxygen species resulting from incomplete reduction of oxygen include among others the free radicals, superoxide radical (O 2 " ) and hydroxyl radical (OH * ), that have one or more unpaired electrons.
• Superoxide spontaneously reacts with itself in a dismutation reaction to form hydrogen peroxide (H 2 O 2 ).
• the formed hydrogen peroxide while not being a free radical, under certain situations, e.g., in the absence of catalase, becomes a cytotoxic oxidant through the formation of hydroxyl radical and hypochlorous acid (HOCl) (McCord, Amer. J. Med.. 108:652-659 (2000)).
• Singlet oxygen results from irradiation by light of metal- free porphyrin precursors that are present in the skin of porphyria sufferers. Singlet oxygen is also generated by neutrophils and is thought to be responsible for damage created by phagocytes on their targets (Babior et al., Amer. J. Med.. 109:33-34 (2000)). Based on its high reactivity with biomolecules, singlet oxygen has generally been considered to be an endpoint in the cascade of oxygen-scavenging agents.
• the invention provides methods for utilizing the newly discovered abilities of an antibody to reduce singlet oxygen to superoxide. This catalytic reaction ultimately results in the formation of hydrogen peroxide.
• the invention also provides methods to utilize antibodies to produce hydrogen peroxide from singlet oxygen by the oxidation of water. Hydrogen peroxide, under certain biological conditions, itself generates reactive molecules. Thus, the invention generally provides methods to inhibit and facilitate these processes depending on the desired outcome.
• the invention further relates to screening methods to identify agents that modulate the newly discovered antibody-mediated processes.
• the invention further contemplates an improved immunoassay format based on the direct detection of hydrogen peroxide that is produced by antibody catalyzed oxidation of water.
• the invention also provides an improved immunoassay based on hydrogen peroxide produced from antibody-generated superoxide in the presence of singlet oxygen.
• the invention also contemplates therapeutic compositions, preferably antibody compositions, that are engineered to exhibit increased or decreased oxidative function.
• Figure 1 illustrates the oxygen-dependent microbicidal action of phagocytes.
• the interconversion of O 2 and O 2 *" is indicated and is an intrinsic ability to antibodies.
• Figure 2 illustrates the amplex red assay.
• Figure 3 shows the initial time course of H 2 O 2 production in PBS (pH 7.4) in the presence ( ⁇ ) or absence ( ⁇ ) of murine monoclonal IgG EP2-19G2 (20 ⁇ M). Error bars show the range of the data from the mean.
• Figure 4 shows the fluorescent micrograph of a single crystal of murine antibody 1D4 Fab fragment after UN irradiation and H 2 O 2 detection with the amplex red reagent.
• Figure 5 illustrates the (A) HP sensitization assay. Time course of H 2 O 2 formation in PBS (pH 7.4) with HP (40 ⁇ M) and visible light, in the presence (O) or absence ( ⁇ ) of 31127 (horse IgG, 20 ⁇ M).
• B Initial time course of H 2 O 2 production with HP (40 ⁇ M) and visible light, in the presence of 31127 (horse IgG, 6.7 ⁇ M) with no additive in PBS (pH 7.4) (D) or ⁇ a ⁇ 3 in PBS (pH 7.4) (O, 100 ⁇ M) or in a D 2 O solution of PBS (pH 7.4) (0).
• Figure 6 is a bar graph showing the measured initial rate of H 2 O 2 formation for a panel of proteins and comparison with antibodies (data from Table I). All points are mean values of at least duplicate experimental determinations. Error bars are the range of experimentally measured values form the mean. ONA, chick-egg ovalbumin; SOD, superoxide dismutase.
• Figure 8 shows H 2 O 2 production.
• A Production of H 2 O 2 by immunoglobulins and non-immunoglobulin proteins. Assays were performed by near-UV irradiation (312 nm, 800 ⁇ W cm “2 ) of individual protein samples (100 ⁇ L, 6.7 ⁇ M) in phosphate-buffered saline (PBS) [10 mM sodium phosphate, 150 mM ⁇ aCl (pH 7.4)] in a sealed glass vial on a transilluminator (Fischer Biotech) under ambient aerobic conditions at 20°C. Aliquots (10 ⁇ L) were removed throughout the assay. H 2 O 2 concentration was determined by the amplex red method.
• PBS phosphate-buffered saline
• D Determination of IC 50 of H 2 O 2 on the photo-production of H 2 O 2 by horse polylgG. A solution of horse IgG (6.7 ⁇ M) was incubated with varying concentrations of H 2 O 2 (0-450 ⁇ M) and the initial rate of H 2 O 2 formation measured as described in (A). The graph is a plot of rate of H 2 O 2 formation versus H 2 O 2 concentration and reveals an IC 50 of 225 ⁇ M.
• Figure 9 illustrates the superposition of native 4C6 Fab (light blue and pink in a color photograph) and 4C6 Fab in the presence of H 2 O 2 (dark blue and red in a color photograph) (A).
• the native 4C6 crystals were soaked for 3 minutes in 4 mM H 2 O 2 , and immediately flash frozen for data collection at SSRL BL 9-1.
• the RMSD was calculated in C ⁇ S.
• B High resolution x-ray structures show that Fab 4C6 is cross-reactive with benzoic acid.
• Figure 10 shows the absorbance spectra of horse polyclonal IgG measured on a diode array HP8452A spectrophotometer, Abs max 280 nm (A).
• the assay was performed in duplicate and involved addition of an antibody solution [6.7 ⁇ M in PBS (pH 7.4)] to a quartz tube that was then placed in a light beam produced by a xenon arc lamp and monochromator of an SLM specfrofluorimeter for 1 hour. H 2 O 2 concentration was measured by the amplex red assay.
• Figure 11 shows the production of H 2 O 2 .
• A Production of H 2 O 2 by tryptophan (20 ⁇ M). The conditions and assay procedures were as described in Figure 8 A.
• B Effect of chloride ion on antibody-mediated photo-production of H 2 O 2 A solution of sheep polylgG ⁇ (6.7 ⁇ M, 200 ⁇ L) or horse polylgG * (6.7 ⁇ M, 200 ⁇ L) was lyophilized to dryness and then dissolved in either deionized water or NaCl (aq.) such that the final concentration of chloride ion were (0-160 mM).
• Figure 12 shows ESI (negative polarity) mass spectra of TCEP [(M-H) " 249] and its oxide [(M-H) " 265 ( 16 O) and (M-H) “ 267 ( 18 O)] produced by oxidation with H 2 O 2 .
• A MS of TCEP and its oxides after irradiation of sheep polylgG (6.7/ ⁇ M) under 16 O 2 aerobic conditions in H 2 18 O (98 % 18 O) PB.
• B MS of TCEP and its oxides after irradiation of sheep polylgG (6.7 ⁇ M) under enriched 18 O 2 (90 % 18 O) aerobic conditions in H 2 16 O PB.
• Figure 13 shows the Xe binding sites in antibody 4C6 as described in materials and methods (Example II).
• A Standard side view of the Ca trace of Fab 4C6 with the light chain in pink and the heavy chain in blue in a color photograph. Three bound xenon atoms (green in a color photograph) are shown with the initial F 0 -F c electron density map contoured at 5 ⁇ .
• B Overlay of Fab 4C6 and the 2C ⁇ TCR (PDB/TCR) around the conserved xenon site 1.
• the present invention concerns the discovery that antibodies, as a class of molecules, have an inherent capability to intercept singlet oxygen and convert it to either superoxide or hydrogen peroxide. This process acts to rescue and recycle oxygen, particularly during phagocyte-mediated processes, thereby contributing to microbicidal action of the immune system. These properties are common to all antibodies and were not known prior to the present invention. The common ability to convert singlet oxygen to superoxide or hydrogen peroxide, regardless of source or antigenic specificity, is thought to link the previously appreciated recognition properties of antibodies with killing events.
• the present invention provides methods that relate to the ability of an antibody to reduce singlet oxygen ( ! O 2 ) to superoxide radical (O 2 " ) and hydrogen peroxide. In view of the critical nature and role of oxygen metabolism in an aerobic organism, the identification of this biological process provides multiple and varied methods as described herein. The detailed determination and characterization of the antibody-mediated reduction of singlet oxygen is described in examples I and II.
• the ability to produce superoxide from singlet oxygen is present in both intact immunoglobulins and well as Fab and F(ab') 2 fragments (see examples).
• the activity does not reside in molecules, including R ⁇ aseA, superoxide dismutase, and Bowman-Birk inhibitor protein, that can be oxidized (example I and Table 1). Also, the activity is not associated with the presence of disulfides in a molecule, even though they are sufficiently electron rich that they can be oxidized (Bent et al. J. Am. Chem. Soc. 87:2612-2619 (1975T).
• the activity resides in an aromatic amino acid such as tryptophan that can be oxidized by singlet molecular oxygen via electron transfer (Grossweiner, Curr. Top. Radiat. Res. P..11:141-199 (1976)).
• the activity is further attributed to the indole component of the tryptophan residue.
• the indole acts as a reductive center in connection with the redox reaction.
• the indole portion becomes oxidized to form a radical cation in the course of reducing singlet molecule oxygen to superoxide free radical.
• the antibody is called a reductant because it is oxidized in providing an electron to singlet molecular oxygen.
• TRP-36 and TRP-47 two aromatic tryptophan resides are conserved, referred to as TRP-36 and TRP-47, and are both deeply buried (Kabat, et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
• the ability of antibodies as a class of proteins to reduce singlet molecular oxygen to superoxide anion is thus based on the presence of the conserved buried aromatic tryptophan residues.
• the ability to produce hydrogen peroxide in an efficient and long term manner from singlet oxygen is present in immunoglobulins and in the T-cell receptor (example II, Figure IF).
• the T-cell receptor shares a similar arrangement of its immunoglobulin fold domains with antibodies (Garcia et al., Science, 274:209 (1996)). However, possession of this structural motif does not appear necessary to confer a hydrogen peroxide-generating ability on proteins.
• ⁇ 2 -macroglobulin a member of the immunoglobulin superfamily having this structural motif, does not generate hydrogen peroxide (Welinder et al., Mol. Immunol.. 28:177 (1991)).
• Information relating the structure to the function of immunoglobulins and the T-cell receptor allows molecules to be designed that will catalyze the oxidation of water. This information also provides many new methods and treatment schemes that may be utilized based on existing molecules.
• agent herein is used to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal
• Agents are evaluated for potential activity as antibody modulatory agents by inclusion in screening assays as described herein.
• antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding an epitope. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
• Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
• Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
• Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
• Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
• the preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green, et al., Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1 (1992), which are hereby incorporated by reference.
• Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
• Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• Fv fragments comprise an association of N H and N L chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
• the Fv fragments comprise N H and N L chains connected by a peptide linker.
• These single-chain antigen binding proteins are prepared by constructing a structural gene comprising D ⁇ A sequences encoding the N H and N L domains connected by an oligonucleotide.
• the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
• the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two N domains.
• Methods for producing sFvs are described, for example, by Whitlow, et al., Methods: a Companion to Methods in ⁇ nzymology. Vol. 2, page 97 (1991); Bird, et al, Science.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al., Methods: a Companion to Methods in ⁇ nzymology. Nol. 2, page 106 (1991).
• an effective amount is an amount that results in reducing, reversing, ameliorating, inhibiting, and the like improving directions, the effects of an oxidant generated by an antibody.
• an “engineered molecule” is a polypeptide that has been produced through recombinant techniques. Such molecules can include a reactive center that can catalyze the production of superoxide or hydrogen peroxide from singlet oxygen. Such engineered molecules may have a reactive indole contained within a polypeptide structure. The indole of such a molecule may be present as a tryptophan residue. Engineered molecules may also contain non- natural amino acids and linkages as well as peptidomimetics. Engineered molecules also include antibodies that are modified to eliminate the reaction center such that they are no longer able to generate superoxide or hydrogen peroxide.
• epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• Antigens can include polypeptides, fatty acids, lipoproteins, lipids, chemicals, hormones and the like.
• antigens include, but are not limited to, proteins from viruses such as human immunodeficiency virus, influenza virus, herpesvirus, papillomavirus, human T-cell leukemia virus and the like.
• antigens include, but are not limited to, proteins expressed on cancer cells such as lung cancer, prostate cancer, colon cancer, cervical cancer, endometrial cancer, bladder cancer, bone cancer, leukemia, lymphoma, brain cancer and the like.
• Antigens of the invention also include chemicals such as ethanol, tetrahydrocanabinol, LSD, heroin, cocaine and the like.
• modulate refers to the capacity to either enhance or inhibit a functional property of an antibody or engineered molecule of the invention, such as production of superoxide or hydrogen peroxide.
• a "non-natural” amino acid includes D-amino acids as well as amino acids that do not occur in nature, as exemplified by 4-hydroxyproline, ⁇ - carboxyglutamate, O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine and other such amino acids and imino acids.
• the term "peptidomimetic” or “peptide mimetic” describes a peptide analog, such as those commonly used in the pharmaceutical industry as non- peptide drugs, with properties analogous to those of the template peptide. (Fauchere, J., Adv. Drug Res.. 15: 29 (1986) and Evans et al., J. Med. Chem..
• Advantages of peptide mimetics over natural polypeptide embodiments may include more economical production, greater chemical stability, altered specificity, reduced antigenicity, and enhanced pharmacological properties such as half-life, absorption, potency and efficacy.
• the terms “phamiaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
• protein and “polypeptide” are used to describe a native protein, fragments, or analogs of a polypeptide sequence. These terms may be used interchangeably.
• Antibodies are used to describe a native protein, fragments, or analogs of a polypeptide sequence. These terms may be used interchangeably.
• the invention provides therapeutic antibodies. All antibody molecules belong to a family of plasma proteins called immunoglobulins. Their basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems.
• a typical immunoglobulin has four polypeptide chains, contains an antigen binding region known as a variable region, and contains a non- varying region known as the constant region.
• An antibody contemplated for use in the present invention can be in any of a variety of forms, including a whole immunoglobulin, Fv, Fab, other fragments, and a single chain antibody that includes the variable domain complementarity determining regions (CDR), or other forms. All of these terms fall under the broad term "antibody” as used herein.
• the present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and immunoreact with a specific antigen.
• an antibody or fragment thereof is used that is immunospecific for an antigen.
• the preparation of a therapeutic antibody of this invention can be accomplished by recombinant expression techniques as well as protein synthesis, methods of which are well known to one of ordinary skill in the art.
• mutation of a nucleic acid that encodes an antibody or fragment thereof can be conducted by a variety of means, but is most conveniently conducted using mutagenized oligonucleotides that are designed to introduce mutations at predetermined sites that then encode an altered amino acid sequence in the expressed molecule.
• Such alterations include substitutions, additions, and/or deletions of particular nucleotide sequences that similarly encode substitutions, additions, and/or deletions of the encoded amino acid residue sequence.
• Site-directed mutagenesis also referred to as oligonucleotide-directed mutagenesis and variations thereof, and the subsequent cloning of the altered genes are well known techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Chapter 15, Cold Spring Harbor Laboratory Press, (1989)).
• Another recombinant approach includes synthesizing the gene encoding a therapeutic molecule of this invention by combining long oligonucleotide strands that are subsequently annealed and converted to double-stranded DNA suitable for cloning and expression (Ausebel et al., Current Protocols in Molecular Biology. Units 10 and 15, Wiley and Sons, Inc. (2000)).
• Such techniques can be used to create engineered molecules that contain a reduction center and are able to generate hydrogen peroxide or superoxide from singlet oxygen. It is contemplated that such engineered molecules can be designed based on antibody structure and on the T-cell receptor, in the case of hydrogen peroxide.
• the present invention contemplates an antibody that has been engineered to generate more superoxide free radical or hydrogen peroxide in a desired location.
• the antibody is engineered to contain additional reductive centers, as described in examples I and II herein, that increase the reduction of singlet molecular oxygen to superoxide free radical or hydrogen peroxide.
• the invention also contemplates an antibody that has been engineered to have at least a diminished capacity to generate superoxide free radical or hydrogen peroxide from singlet oxygen. In that context, the antibody lacks at least one of its reductive centers and preferably is substantially free of a reductive center.
• Such antibody compositions are readily prepared with methods well known to one of ordinary skill in the art.
• polyclonal or monoclonal antibodies prepared for use as therapeutic compositions or in the methods of invention can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
• a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
• Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (Coligan, et al., Unit 9, Current Protocols in Immunology. Wiley Interscience, (1991)).
• the free radicals that result from oxygen activation are by definition chemical species that possess one or several mismatched electrons. Free radicals are generated when a single electron is removed from the molecule. This results in a molecule that has at least one of its electrons unpaired to another electron. The resultant free radical is reactive since it seeks out available electrons from other molecules, the process of which can create a second reactive molecule thereby setting off a chain reaction. Free radicals, also referred to as oxidants herein include superoxide, hydroxyl radical, halogenated oxygens and nitrogen containing molecules. Superoxide radical generated from the antibody-mediated reduction of singlet oxygen is itself an oxidant and also provides for the production of hydrogen peroxide.
• the latter which while not itself an oxidant or reactive molecule, can generate reactive oxygen species that include hydroxyl radical, its secondary products such as carbon, oxygen, nitrogen or sulfur, which can react with other compounds to produce yet other free radicals creating a free radical chain reaction.
• reactive oxygen species that include hydroxyl radical, its secondary products such as carbon, oxygen, nitrogen or sulfur, which can react with other compounds to produce yet other free radicals creating a free radical chain reaction.
• Other reactive species that are a consequence of the oxygen cascade include oxidized halogens, such as hypochlorous acid (HOCl), the HOCl-generated reactive species chloramine (NH 2 C1) and aldehydes, and reactive nitrogen species.
• a potential consequence of uncontrolled reactivity of free radicals is damage to DNA, RNA, membrane lipids, lipoproteins or enzymes, ultimately affecting the body.
• An end result is poor cell function leading to disease and even tissue death.
• free radicals aid the process of riding the body of unwanted bacteria or viruses.
• acute and chronic cellular and tissue injury can occur.
• Neutralizing processes include 1) enyzmes such as superoxide dismutase and catalase that together produce peroxidases and 2) molecules such as tocopherols, carotenoids, ubiquinones, flavonoids, ascorbic acid, uric acid and similar molecules that serve as a source of electrons that are provided to free radicals without damaging cellular components. Such processes are considered beneficial to the well being of an organism.
• any molecule that inhibits the antibody mediated generation of hydrogen peroxide or superoxide that ultimately leads to hydrogen peroxide formation is referred to as an antioxidant.
• an antioxidant any molecule that inhibits the antibody mediated generation of hydrogen peroxide or superoxide that ultimately leads to hydrogen peroxide formation.
• oxidative stress When the balance of oxidants to antioxidants tips in favor of the former, the oxidative state is generally referred to as "oxidative stress". This situation occurs in the presence of an excess production of oxidants or free radicals and a diminishing of the control antioxidant mechanisms.
• Advantages of the present invention are that the discovery of the role an antibody plays in the generation of oxidants in the oxygen cascade provides the basis for therapeutic methods that are useful in maintaining oxygen balance and control of oxygen metabolism, depending on the desired outcome.
• the methods of this invention provide 1) for the production of oxidants when their production is warranted, such as in promoting wound healing, lysing bacteria, eliminating viruses, targeting cancer cells for oxidant-induced lysis and the like processes, and 2) for the inhibition of antibody generated oxidants by exposure of antioxidants when the inhibition of antibody generated oxidants is warranted, such as in inflammation, heart conditions, diabetes and unwanted cellular proliferation.
• oxidants when their production is warranted, such as in promoting wound healing, lysing bacteria, eliminating viruses, targeting cancer cells for oxidant-induced lysis and the like processes
• the inhibition of antibody generated oxidants by exposure of antioxidants when the inhibition of antibody generated oxidants is warranted, such as in inflammation, heart conditions, diabetes and unwanted cellular proliferation.
• one may want to use antibody mediated generation of superoxide or hydrogen peroxide to supplement the local concentration of superoxide concentration generated by phagocytic neutrophils to combat a bacterial infection in a wound.
• the superoxide in effect acts as bactericidal agent destroying the bacteria and ultimately the neutrophil in the process.
• the method of this invention to provide an antibody composition to the area to cause an increase in the local concentration of superoxide.
• neutrophil-generated superoxide is deleterious in inflamed joints such as in patients with rheumatoid arthritis who are concomitantly undergoing intensive humoral antibody-mediated immune responses.
• the decision to use the methods of this invention to inhibit or promote the antibody- mediated generation of superoxide and hydrogen peroxide and their derivatives (i.e., molecules derived therefrom) products and/or their effects is thus dependent on the desired outcome.
• antibody mediated production of hydrogen peroxide encompasses the reactive species that are both precursor and derivative to the generation of hydrogen peroxide.
• antioxidants include 1) inhibitors of an antibody thereby inhibiting superoxide generation, 2) inhibitors of hydrogen peroxide generation, 3) inhibitors of the reactions converting hydrogen peroxide into derivative reactive oxidants; and 4) inhibitors of the reactive oxidants themselves.
• Preferred antioxidants include those that inhibit the activation of oxygen producing reactive oxidants as well as those neutralizing those already formed. The antioxidant effect can occur by any mechanism, including catalysis.
• Antioxidants as a category include oxygen scavengers or free radical scavengers. Antioxidants may be of different types so they are available if and when they are needed. In view of the presence of oxygen throughout an aerobic organism, antioxidants may be available in different cellular, tissue, organ and extracellular compartments. The latter include extracellular fluid spaces, intraocular fluids, synovial fluid, cerebrospinal fluid, gastrointestinal secretions, interstitial fluid, blood and lymphatic fluid. Antioxidants are present within an organism but are also provided by supplementing the diet and in the methods of this invention.
• antioxidants include but are not limited to ascorbic acid, ⁇ -tocopherol, ⁇ -glutamylcysteinylglycine, ⁇ -glutamyl transpeptidase, ⁇ -lipoic acid, dihydrolipoate, -acetyl-5-methoxytryptamine, flavones, flavonenes, flavanols, catalase, peroxidase, superoxide dismutase, metallothionein, and butylated hydroxytoluene.
• a further preferred molecule that has the capacity to function as an antioxidant in the context of the methods of this invention is an engineered antibody in which the ability to generate superoxide free radical from reducing singlet oxygen is diminished or preferably absent altogether. Such antibody molecules are described herein.
• antioxidants are directed to situations in which an antioxidant is required to prevent, control, minimize, reduce, or inhibit the damage of an oxidant.
• the invention contemplates the use of an antioxidant for reducing the antibody mediated production of hydrogen peroxide in a cell.
• the cellular damage may be so extensive that tissue injury results, for example, in inflammatory conditions, in trauma conditions, in organ transplantation and the like.
• the antibody having diminished or substantially no ability to generate superoxide or hydrogen peroxide since it lacks the reductive centers that reduce singlet oxygen, provides a therapeutic benefit in promoting a desired immune response without inducing additional tissue damage resulting from excess superoxide production.
• Preferred engineered therapeutic antibody compositions retain their antigen binding site so that targeting to a particular antigen is achieved in concert with the desired therapeutic benefits.
• the present invention further contemplates a method of ameliorating oxidative stress in a subject as well as alleviating a symptom in a subject where the symptom is associated with production of oxidant.
• Exemplary of conditions in which the therapeutic methods of inhibiting the antibody mediated production of hydrogen peroxide with an antioxidant of the present invention include but are not limited to inhibiting aberrant smooth muscle disorder, inhibiting liver disease, proliferation of cancer cells, inhibiting inflammation in cancer patients receiving radiotherapy, inflammatory diseases (arthritis, vasculitis, glomerulonephritis, systemic lupus erythematosus, and adult respiratory distress syndrome), ischemic diseases (heart disease, stroke, intestinal ischemia, and reperfusion injury), hemochromatosis, acquired immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers, hypertension, preeclampsia, neurological diseases (multiple sclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and muscular dystrophy) alcoholism and smoking-related diseases.
• inflammatory diseases arthritis, vasculitis, glomerulonephritis, systemic lupus erythematosus, and
• Cells in which oxidative stress is deleterious include but are not limited to endothelial, interstitial, epithelial, muscle (smooth, skeletal or cardiac), phagocytic (including neutrophils and macrophages), white blood cells, dendritic, connective tissue and nervous system cells.
• Effected tissues include but are not limited to muscle, nervous, skin, glandular, mesenchymal, splenic, sclerous, epithelial and endothelial tissues.
• the oxidants and oxygen scavengers of the invention may be formulated into a variety of acceptable compositions. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.
• pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, - ascorbate, ⁇ -ketoglutarate, and ⁇ -glycerophosphate.
• Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
• salts are obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
• a sufficiently basic compound such as an amine
• a suitable acid affording a physiologically acceptable anion.
• Alkali metal for example, sodium, potassium or lithium
• alkaline earth metal for example calcium
• the oxidants and oxygen scavengers may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
• the present compounds may be systemically administered, e.g., . orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
• a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
• the oxidants and oxygen scavengers may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should contain at least 0.1% of active compound.
• compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
• amount of oxidants and oxygen scavengers in such therapeutically useful compositions is such that an effective dosage level will be obtained.
• the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
• a liquid carrier such as a vegetable oil or a polyethylene glycol.
• any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
• the active compound may be incorporated into sustained-release preparations and devices.
• the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
• Solutions of the active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the oxidants and oxygen scavengers in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
• the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the oxidants and oxygen scavengers plus any additional desired ingredient present in the previously sterile-filtered solutions.
• the oxidants and oxygen scavengers may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
• a dermatologically acceptable carrier which may be a solid or a liquid.
• Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
• Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
• Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
• the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
• Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
• Examples of useful dermatological compositions that can be used to deliver the oxidants and oxygen scavengers of the present invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
• Useful dosages of the oxidants and oxygen scavengers of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
• the concentration of the oxidants and oxygen scavengers of the present invention in a liquid composition will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
• concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%), preferably about 0.5-2.5 wt-%.
• the amount of the oxidants and oxygen scavengers, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
• a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
• the oxidants and oxygen scavengers are conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
• the oxidants and oxygen scavengers should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 ⁇ M, preferably, about 1 to 50 ⁇ M, most preferably, about 2 to about 30 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the oxidants and oxygen scavengers, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the oxidants and oxygen scavengers.
• Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the oxidants and oxygen scavengers.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
• an antioxidant enters the cell and reacts with the hydrogen peroxide or its precursor oxygen molecules thereby reducing the hydrogen peroxide concentration in the cell.
• an antioxidant enters the cell or is present in the surrounding extracellular milieu and reacts with the oxidants generated from hydrogen peroxide.
• compositions of this invention are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount .
• the quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual. However, suitable dosage ranges for various types of applications depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at intervals to result in the desired outcome of the therapeutic treatment.
• Antioxidants contemplated for use in the present invention are delivered to the site of interest to mediate the desired outcome in a composition such as a liposome, the preparation of which is well known to one of ordinary skill in the art of liposome-mediated delivery.
• Alternative delivery means include but are not limited to administration intravenously, topically, orally, by inhalation, by cannulation, intracavitally, intramuscularly, transdermally, and subcutaneously.
• Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with an antioxidant as described herein or an antibody as described herein for providing antibody activity, dissolved or dispersed therein as an active ingredient.
• the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
• compositions that contains active ingredients dissolved or dispersed therein are well understood in the art and need not be limited based on formulation.
• compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
• the preparation can also be emulsified.
• the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
• Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
• the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
• compositions of the present invention can include pharmaceutically acceptable salts of the components therein.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
• Physiologically tolerable carriers are well known in the art.
• Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
• aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
• Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
• the present invention also generally contemplates the use of any antibody to generate superoxide radical or hydrogen peroxide in a situation where the production of superoxide or hydrogen peroxide is warranted.
• the present invention also contemplates the use of engineered molecules including engineered antibodies that have been altered to contain a reductive center, the presence of which provides for the capability to generate superoxide or hydrogen peroxide from singlet oxygen when such production is desired.
• engineered molecules having more than two reductive centers compared to a non-engineered antibody having the two conserved tryptophan residues is wa ⁇ anted when enhanced production of superoxide is needed.
• the present invention contemplates the use of antibodies as defined above that contain the naturally occurring buried tryptophan residues as well as the engineered antibodies and other molecules described herein.
• superoxide free radical also called superoxide
• the use of engineered molecules having additional reductive centers is warranted when enhanced production of hydrogen peroxide is needed.
• the present invention contemplates the use of antibodies as defined above that contain naturally occurring tryptophan residue as well as the engineered antibodies and other molecules described herein.
• the present invention contemplates the therapeutic use of an antibody to create an superoxide or hydrogen peroxide environment where one does not exist or enhance an already existing one.
• Such conditions are well known to practitioners in the art of oxygen cascade chemistry and the generation of oxidants to provide a desired beneficial outcome such as those described herein.
• the invention contemplates a method for exposing an antigen to superoxide and hydrogen peroxide where the antigen is contacted with a composition including an antibody able to generate hydrogen peroxide or superoxide from singlet oxygen.
• a composition including an antibody able to generate hydrogen peroxide or superoxide from singlet oxygen As previously discussed, the method is successful with either nonspecific or immunospecific (antigen directed ) intact antibody, fragments derived therefrom and further including single chain antibodies as well as the engineered molecules and antibodies described herein.
• Exemplary concentrations of antibody at the cell surface range from 1 to 5 micromolar. However, the concentration may vary depending on the desired outcome where the amount of antibody provided is that amount of antibody that is sufficient to obtain the desired physiological effect, i.e, the generation of hydrogen peroxide or superoxide radical and its derivative oxidants to generate oxidative stress.
• the antigen is preferably presented on a cell but need not be so limited.
• the antigen can be any antigen that is present in a cell, tissue or organ including extracellular fluids where the presence of superoxide and the antibody mediated process of producing it is warranted.
• the antigen is a fatty acid, a low density lipoprotein, an antigen associated with inflammation, a cancer cell antigen, a bacterial antigen or a similar molecule.
• Cells on which antigens are associated include but are not limited to endothelial, interstitial, epithelial, muscle, phagocytic, blood, dendritic, connective tissue and nervous system cells. Particularly preferred target cells for the present therapeutic approach are neutrophils or macrophages.
• the invention further contemplates exposing a target cell to irradiation with either ultraviolet, infrared or visible light in the method of generating antibody superoxide or hydrogen peroxide.
• a superoxide or hydrogen peroxide generating amount of a photosensitizer is utilized in the therapeutic methods described herein.
• a sensitizer is any molecule that induces or increases the concentration of singlet oxygen.
• Sensitizers are generally used in the presence of irradiation, the process of which includes exposure to ultraviolet, infrared or visible light for a period sufficient to activate the sensitizer. Exemplary exposures are described in examples I and II.
• a superoxide or hydrogen peroxide generating amount of sensitizer is the amount of sensitizer that is sufficient to obtain the desired physiological effect, e.g., generation of superoxide or hydrogen peroxide from singlet oxygen mediated by an antibody in any situation where superoxide or hydrogen peroxide presence and the derivatives thereof is warranted.
• a sensitizer is conjugated to the antibody.
• a sensitizer conjugated antibody is capable of binding to a antigen, i.e., retains an active antigen binding site, allowing for antigen recognition and complexing to occur.
• sensitizers include but are not limited to pterins, flavins, hematoporphyrin, tetrakis(4-sulfonatophenyl)porphyrin, bipyridyl ruthemium(II) complexes, rose bengal dye, quinones, rhodamine dyes, phtalocyanine, and hypocrellins.
• the generation of superoxide or hydrogen peroxide is enhanced by administering a means to enhance the production of singlet oxygen. Reduced singlet oxygen is the source of superoxide or hydrogen peroxide as previously discussed. Such reduction can occur through the action of an antibody or molecule containing greater than two reductive centers.
• prodrug that is any molecule, compound, reagent and the like that is useful in generating singlet oxygen.
• a preferred prodrug is endoperoxide, that is administered at a time subsequent to the administering or contacting of an antibody with a desired target cell, tissue or organ as described below.
• endoperoxide is preferably delivered after a superoxide or hydrogen peroxide producing antibody or molecule has immunoreacted with its target antigen forming an antibody- antigen complex.
• a preferred concentration of endoperoxide to achieve at the antibody-antigen complex site is about 10 micromolar.
• Preferred therapeutic methods based on the use of an antibody including an engineered antibody or molecule having reductive centers to generate superoxide or hydrogen peroxide from singlet oxygen includes a method for killing a cancer cell where the cancer cell is contacted with a composition including an antibody capable of generating superoxide or hydrogen peroxide from singlet oxygen.
• the antibody recognizes and immunoreacts with an antigen expressed on the cancer cell.
• Such methods are therapeutically useful for a subject with lung cancer, prostate cancer, colon cancer, cervical cancer, endometrial cancer, bladder cancer, bone cancer, leukemia, lymphoma, or brain cancer.
• the cancer cell is removed from a subject with cancer and cultured ex vivo for exposing to an antibody, and can further be exposed to ultraviolet light, infrared light or visible light for the cell to then be returned to the subject.
• the antibody composition is delivered in vivo to a subject with cancer. Preferred in vivo delivery methods include administration intravenously, topically, by inhalation, by cannulation, intracavitally, intramuscularly, transdermally, subcutaneously or by liposome containing the antibody.
• the antibody is a recombinant antibody, that is provided as above or alternatively is expressed from an expression vector delivered to the cell.
• the expression vector in this context can also express a sensitizer molecule.
• Therapeutic compositions in pharmaceutically acceptable excipients and pharmaceutically effective amounts as described for antioxidant containing compositions are applicable to the use of antibody containing compositions.
• Additional therapeutic methods based on using an antibody that is able to generate superoxide or hydrogen peroxide from singlet oxygen are 1) for inhibiting proliferation of a cancer cell, 2) for targeting and killing a cancer cell in a patient where the antibody recognizes and immunoreacts with an antigen expressed on the cancer cell, 3) for inhibiting tissue injury associated with neutrophil mediated inflammation in a subject, for example where the inflammation results from a bacterial infection or when the subject has an autoimmune disease, 4) for enhancing the bactericidal effectiveness of a phagocyte in a subject, 5) for promoting wound healing in a subject having a open wound where the superoxide or hydrogen peroxide stimulates fibroblast proliferation and/or the immune response that further includes lymphocyte proliferation, 6) for stimulating cell proliferation, such as stimulating fibroblast proliferation in a wound in a subject, and the like situations.
• topical application to a wound on a subject is a preferable delivery approach such as with a bandage containing an antibody.
• Other therapeutic conditions that would benefit from the creation or enhancement of superoxide or hydrogen peroxide in a cell, tissue, organ or extracellular compartment are well known to those of ordinary skill in the art and have been reviewed by
• the invention further contemplates screening methods that are based on the newly discovered antibody reduction of singlet oxygen to hydrogen peroxide or superoxide radical.
• the invention contemplates a method for identifying an agent that modulates antibody mediated production of hydrogen peroxide or superoxide.
• a modulator is a molecule that either inhibits or promotes the production of superoxide or hydrogen peroxide. Either type of modulator is identifiable with the same method.
• the method includes the steps of: a) contacting a composition comprising an antibody capable of generating superoxide or hydrogen peroxide with an agent to form an admixture in an assay solution in the presence of molecular oxygen; b) irradiating the admixture to generate singlet oxygen from molecular oxygen, wherein the singlet oxygen is reduced to hydrogen peroxide or superoxide by the antibody, wherein the superoxide dismutates to form hydrogen peroxide; c) detecting the formed hydrogen peroxide; and d) comparing the detected hydrogen peroxide with a suitable control, thereby determining how the agent modulates the production of hydrogen peroxide or superoxide.
• the irradiating step is performed with either ultraviolet light or visible light.
• a sensitizer as previously described can be added with the antibody composition.
• the formed hydrogen peroxide is detected through reaction directly with a hydrogen peroxide where the reacted substrate is detected with a fluorescent means, such as with fluorescent microscopy or fluorescent spectrometry.
• a fluorescent means such as with fluorescent microscopy or fluorescent spectrometry.
• detection is ELISA based or with done with a standard cuvette. Exemplary assay methods are performed as described in examples I and II.
• a method for performing an immunoassay to detect antibody immunoreactivity with an antigen is also contemplated based on the discovery of antibody generated superoxide or hydrogen peroxide.
• the method comprises the steps of: a) contacting in a singlet oxygen-generating medium a substrate having immobilized thereon a composition comprising a first reagent comprising an antigen or an antibody, with a second composition comprising an antigen or an antibody that is reactive with the first reagent to form an immobilized antigen-antibody complex, wherein the antibody generates superoxide or hydrogen peroxide from singlet oxygen in the presence of oxygen; and b) detecting the antibody-generated superoxide or hydrogen peroxide, thereby detecting the antibody immunoreactivity with the antigen.
• the reaction and detection means are those as described herein.
• the first composition is an antigen and the second composition is an antibody.
• the first composition is an antibody and the second composition is an antigen.
• the invention further contemplates a similar method for performing an immunoassay to detect antibody immunoreactivity with an antigen where an antigen is immobilized and contacted with an antibody composition.
• Such immunoassay methods are an improvement over those that are well known as methods to assess antigen-antibody immunoreactivity and to identify antigens and/or antibodies.
• the advantage of the present method over previous other immunoassay methods lies in the present elimination of at least one method step and/or the incorporation of a secondary labeled immunoreactive molecule, the labeling either being a radioactive or enzymatic compound.
• the minimum requirements are oxygen, an antibody reagent, an antigen reagent, and a detectable reactant that reacts with hydrogen peroxide generated from the antibody.
• a preferred reactant is a fluorogenic substrate.
• One such reactant used as described in examples I and II is called
• AMPLEXTM Red It is a commercially available reagent sold by Molecular Probes (Eugene, Oregon) for reacting antibody generated hydrogen peroxide in the immunoassay. It is sold in a kit that provides a one-step fluorometric method for measuring hydrogen peroxide using a fluorescent microplate or fluorimeter for detection. The assay is based on the detection of hydrogen peroxide using 10-acetyl- 3,7-dihyroxyphenoxazine, a highly sensitive and stable probe for hydrogen peroxide.
• the AMPLEXTM Red reagent reacts with hydrogen peroxide in a 1 :1 stoichiometry to produce highly fluorescent resorufin, that provides a detection mechanism to detect as little as 10 picomoles of hydrogen peroxide in a 200 microliter volume.
• prior immunoassay techniques including radioimmunoassays
• RIA radioactively labeled immunoreactive molecule
• EIA enzyme-immunoassays
• ELISA enzyme-linked immunosorbent assay
• the antibody mediated generation of hydrogen peroxide is detected with high detection capacity without radioactive agents, without requiring an additional reagent and or admixing step such as those practiced in US Patents 3,905,767; 4,016,043; USRE032696; and 4,376,110, the disclosures of which are hereby incorporated by reference. 3.
• Therapeutic Compositions are described in detail below.
• the present invention contemplates therapeutic compositions useful in practicing the therapeutic methods as described above.
• Antibodies as a class of proteins, are now known to act as reductants in reducing singlet molecular oxygen (also referred to herein as singlet oxygen) to generate superoxide free radical (also referred to herein as superoxide).
• singlet oxygen also referred to herein as singlet oxygen
• superoxide free radical also referred to herein as superoxide
• the indole in view of the redox reaction where the indole portion becomes oxidized forming a radical cation in the reaction of reducing singlet molecule oxygen to superoxide free radical, the indole is referred to as a reductive center.
• a reductive center is more efficient if it is not solvent-exposed, i.e., is buried within the therapeutic composition defined herein.
• compositions may also be produced and used according to the therapeutic methods described above with antibodies and engineered molecules that produce hydrogen peroxide through oxidation of water.
• Antibodies as a class of proteins, are now known to catalyze the oxidation of water to produce hydrogen peroxide. The activity is ascribed to a conserved tryptophan residue.
• compositions that are useful in either acting to reduce the local concentration of hydrogen peroxide or superoxide production or in the alternative useful in acting to enhance it.
• Such compositions contain reagents referred to generally as being "engineered”, defined herein to connote a reagent, such as an antibody or fragment thereof as defined herein, or other molecule, that has been altered in some form to either increase or decrease the number of reductive centers as defined herein.
• the invention thus contemplates an antibody that has been engineered to have at least a diminished capacity to generate hydrogen peroxide or superoxide free radical from singlet oxygen.
• the antibody lacks at least one of its reductive centers and preferably is substantially free of a reductive center.
• Such antibody compositions are readily prepared with recombinant expression methods well known to one of ordinary skill in the art.
• the antibody retains the same amino acid residue number but the reductive center has been replaced or substituted with a component that lacks the ability to reduce singlet oxygen.
• the reductive center comprises a buried indole and preferably, in the case of superoxide, two buried indoles.
• the reductive center comprises an indole on a tryptophan residue that is substituted by another amino acid that does not have reductive capacity.
• Such preferred substitutions includes the amino acids phenylalanine and alanine.
• the present invention also contemplates deletion of the tryptophan without replacement or substitution thereof as long as the desired antibody activity, particularly antigen binding activity, is not adversely affected.
• an engineered antibody having reduced or absent reductive centers while retaining antigen targeting ability provides the therapeutic advantage of providing an antibody to stimulate a desired immune response in particular situations while reducing or eliminating altogether the undesirable production of hydrogen peroxide or superoxide and its byproducts that can further damage cells and tissues.
• Methods for making an engineered antibody that functions as an antioxidant in the context of the therapeutic methods described herein are well known in the art, such as site- directed mutagenesis of a nucleotide sequence encoding the antibody of interest as previously discussed.
• Engineered antibodies that function as an antioxidant according to the methods of the invention are contemplated for any of the methods as described herein.
• the present invention also contemplates engineered therapeutic molecules including engineered antibodies that have been altered to contain a reductive center where they were in an insufficient amount to effect adequate production of superoxide or hydrogen peroxide, or where they are needed to increase the number of reductive centers to a number in excess of those that were naturally occurring in the molecule or antibody.
• Introduction of a reductive center in a engineered molecule or antibody is accomplished by methods well known to one of ordinary skill in the art. Preferred means including recombinant expression methods and well as direct protein synthesis methods have been previously described. The choice of method is necessarily dependent on the length of the molecule being engineered.
• the positioning, i.e., the location, of the engineered reductive center is based upon the ability of the engineered molecule to exhibit reducing activity on singlet oxygen.
• the incorporation of reductive centers are positioned such that they are deeply buried in the folded molecule allowing a retention of structural ability without comprising superoxide or hydrogen peroxide production.
• the location of an engineered reductive center is adjacent to a variable binding domain.
• one reductive center is contemplated.
• two reductive centers are contemplated.
• more than three reductive centers are contemplated.
• the reductive centers comprise indole.
• reductive centers comprising indole present in tryptophan residue. Any technique to engineer such reductive centers in a molecule or antibody is contemplated for use in the present invention.
• the reductive centers are introduced by site-directed mutagenesis of nucleotide sequences encoding the engineered antibody such that the substituted nucleotides encode tryptophan residues at predeteraiined locations in the encoded molecule.
• an engineered molecule such as an antibody to include desired reductive centers
• such molecule that is produced by recombinant technology is also contemplated to be in the form of a fusion conjugate, where the conjugate provides a sensitizer molecule as previously described for use in therapeutic methods as described herein.
• Antibodies have the intrinsic capacity to destroy antigens Materials and Methods Antibodies: The following whole antibodies were obtained from PharMingen: 49.2 (mouse IgG 2b K), G155-178 (mouse IgG 2a K), 107.3 (mouse IgG, K), A95-1 (rat IgG 2b ), G235-2356 (hamster IgG), R3-34 (rat IgG K), R35-95 (rat IgG 2a K), 27-74 (mouse IgE), Al 10-1 (rat IgG, X), 145-2C11 (hamster IgG groupl K), Ml 8-254 (mouse IgA K), and MOPC-315 (mouse IgA ⁇ ). The following were obtained from Pierce: 31243 (sheep IgG), 31154 (human IgG), 31127 (horse IgG), and 31146 (human IgM).
• F(ab') 2 fragments were obtained from Pierce: 31129 (rabbit IgG), 31189 (rabbit IgG), 31214 (goat IgG), 31165 (goat IgG), and 31203 (mouse IgG).
• Protein A protein G, trypsin-chymotrypsin inhibitor (Bowman-Birk inhibitor), ⁇ -lactoglobulin A, ⁇ -lactalbumin, myoglobin, ⁇ -galactosidase, chicken egg albumin, aprotinin, trypsinogen, lectin (peanut), lectin (Jacalin), BSA, superoxide dismutase, and catalase were obtained from Sigma.
• Ribonuclease I A was obtained from Amersham Pharmacia. The following immunoglobulins were obtained in-house using hybridoma technology: OB2-34C12 (mouse IgG, ⁇ ) 5 SHO1-41G9 (mouse IgG, K), OB3-14F1 (mouse IgG 2a K), DMP-15G12 (mouse IgG 2a K), AD1-19G1 (mouse IgG 2b K), NTJ-92C12 (mouse IgG, K), NBA-5G9 (mouse IgG, K), SPF-12H8 (mouse IgG 2a K), TIN-6C11 (mouse IgG 2a K), PRX-1B7 (mouse IgG 2a K), HA5-19A11 (mouse IgG 2a K), EP2-19G2 (mouse IgG, K), GNC- 92H2 (mouse IgG, K), WD1-6G6 (
• DRB polyclonal (human IgG) and DRB-M2 (human IgG) were supplied by Dennis R. Burton (The Scripps Research Institute).
• 1D4 Fab (crystallized) was supplied by Ian A. Wilson (The Scripps Research Institute).
• IgG 19G12 (100 ⁇ l, 6.7 ⁇ M) was heated to 100°C in an Eppendorf tube for 2 min. The resultant solution was transferred to a glass, screw- cap vial and irradiated with UV light for 30 min. The concentration of H 2 O 2 was determined after 30 min.
• the antibody is a remarkable adaptor molecule, having evolved both targeting and effector functions that place it at the frontline of vertebrate defense against foreign invaders (Burton, D. R., Trends Biochem. Sci.. 15. 64-69 (1990)).
• the central idea is that antibodies themselves do not possess destructive ability but mark foreign substances for removal by the complement cascade and/or phagocytosis (Arlaud et al., Immunol. Today. 8, 106- 111 (1987): Sim & Reid. Immunol. Today. 12. 307-311 (1991)).
• the advent of antibody catalysis has demonstrated that antibodies are capable of much more complex chemistry than simple binding (Wentworth & Janda, Curr. Opin. Chem. Biol..
• the preliminary step in the phagocytic oxidative burst is the single electron reduction of ground-state molecular oxygen ( 3 O 2 ) by the NADPH-dependent transmembrane phagocyte oxidase enzyme system that generates superoxide anion (O 2 * ⁇ ) (Figure 1) (Klebanoff, S. J. in Encyclopedia of Immunology, eds. Delves, P. J. & Roitt, I. M. (Academic, San Diego), pp. 1713-1718 (1998); Rosen, H. & Klebanoff, S. J., J. Biol. Chem.. 252. 4803-4810 (1997)).
• O 2 * ⁇ is a vital reducing agent that regenerates Fe 2+ , thus facilitating the iron-catalyzed Haber- Weiss reaction, or the so-called superoxide-driven Fenton reaction that produces HO * (Esq. 1 and 2). Therefore, processes that facilitate the generation of O 2 * ⁇ will ultimately perpetuate and potentiate oxygen-dependent microbicidal action.
• Another key component of the oxygen-scavenging cascade is singlet molecular oxygen ( ] O 2 ).
• This particularly reactive species is an excited state of molecular oxygen in which both outer shell electrons are spin-paired (Kearns, D. R., Chem. Rev.. 71. 395-427 (1971)). It is important in pathological biological systems and has a very short life-time (ca. 4 ⁇ s) in vivo (Foote, C. S. in Free Radicals in Biology, ed. Pryor, W. A. (Academic, New York), pp. 85-133 (1976)).
• Generation of ' O 2 during microbicidal processes is either direct, via the action of flavoprotein oxidases (Allen, R.
• O 2 * + 2HO * 2 ⁇ O 2 + H 2 O 2
• the high reactivity of O 2 with biomolecules has meant that it is generally considered to be an endpoint in the cascade of oxygen-scavenging agents.
• antibodies as a class of proteins, have the intrinsic ability to intercept O 2 and efficiently reduce it to O 2 * ⁇ , thus offering a mechanism by which oxygen can be rescued and recycled during phagocyte action, thereby potentiating the microbial action of the immune system.
• the rates of hydrogen peroxide formation were linear for more than 10% of the reaction, with respect to the oxygen concentration in PBS under ambient conditions (275 ⁇ M). With sufficient oxygen availability, the antibodies can generate at least 40 equivalents of H 2 O 2 per protein molecule without either a significant reduction in activity or structural fragmentation.
• An example of the initial time course of hydrogen peroxide formation in the presence or absence of antibody 19G2 is shown in Figure 3 A. This activity is lost following denaturation of the protein by heating.
• the data in Table 1 reveal a universal ability of antibodies to generate H 2 O 2 from O 2 . This function seems to be shared across a range of species and is independent of the heavy and light chain compositions investigated or antigen specificity.
• the initial rates of hydrogen peroxide formation for the intact antibodies is highly conserved, varying from 0.15 nmol/min/mg [clone A95-l(rat IgG2b)] to 0.97 nmol/min/mg (clone PCP-21H3, a murine monoclonal IgG) across the whole panel.
• the information available is more limited for the component antibody fragments, the activity seems to reside in both the Fab and F(ab') 2 fragments.
• the rate of hydrogen peroxide formation is proportional to IgG concentration between 0.5 and 20 ⁇ M but starts to curve at higher concentrations (Figure 5C).
• the lifetime of O 2 in protein solution is expected to be lower than in pure water due to the opportunity for reaction. It is therefore thought that the observed curvature may be due to a reduction in the lifetime of 1 0 2 due to reaction with the antibody.
• the effect of oxygen concentration on the observed rate of H 2 O 2 production shows a significant saturation about 200 ⁇ M of oxygen (Figure 5D). Therefore, the mechanism of reduction may involve either one or more oxygen binding sites within the antibody molecule.
• a - " m app(O 2 ) of 187 ⁇ M and a max app of 0.4 nmol/min/mg are obtained.
• This antibody rate is equivalent to that observed for mitochondrial enzymes that reduce molecular oxygen in vivo. The mechanism by which antibodies reduce ] O 2 is still being determined.
• Aromatic amino acids such as tryptophan (T ⁇ ) can be oxidized by O 2 via electron transfer (Grossweiner, L. I., Curr. Top. Radiat. Res. P.. JLi, 141-199 (1976)).
• disulfides are sufficiently electron rich that they can also be oxidized (Bent. D. V. & Hayon, E., J. Am. Chem. Soc. 87, 2612-2619 (1975)).
• chick ovalbumin which has only 2 T ⁇ residues (Feldhoff, R. & Peters, T. J., Biochem. J.. 159. 529-533 (1976)), is one of the most efficient proteins at reducing J O 2 .
• Aromatic amino acids in proteins are modified by the abso ⁇ tion of ultraviolet light, especially in the presence of sensitizing agents such as molecular oxygen or ozone (Foote, C. S., Science. 162. 963-970 (1968); Foote, C. S., Free Radicals Biol..
• T ⁇ reacts with ! O 2 via a [2 + 2] cycloaddition to generale N-formylkynurenine or kynurenine, which are both known to significantly quench the emission of buried T ⁇ residues (Mach, H., Burke, C. J., Sanyal, G., Tsai, P.-K, Volkin, D. B. & Middaugh, C. R. in Formulation and Delivery of Proteins and Peptides. eds. Cleland, J. L. & Langer, R. (American Chemical Society, Denver, CO) (1994)).
• IgG 4C6 was digested with papain and the Fab' fragment purified using standard protocols (Harlow and Lane). The Fab' was crystallized from 13-18%) PEG 8 K, 0.2 M ZnAc, 0.1 M cacodylate, pH 6.5. Crystals were pressurized under xenon gas at 200 psi for two minutes (Soltis et al., J. Appl. Crvst.. 30. 190, (1997)) and then flash cooled in liquid nitrogen. Data were collected to 2.0 A resolution at SSRL BL9-2. The structure was solved by molecular replacement using coordinates from the native 4C6 structure, and xenon atom sites were identified from strong peaks in the difference Fourier map.
• Inductively coupled plasma atomic emission spectroscopy ICP-AES
• ICP-AES Inductively coupled plasma atomic emission spectroscopy
• PCP21H3 Mouse monoclonal antibody (PCP21 H3) was exhaustively dialyzed into sodium phosphate buffered saline (PBS, 50 mM pH 7.4) with 20 mM EDTA.
• PBS sodium phosphate buffered saline
• EDTA sodium phosphate buffered saline
• 300 ⁇ L of a 10.5 % HNO 3 solution was added to 100 ⁇ L of a 10 mg/mL antibody solution and was incubated at 70°C for 14 hours.
• Oxygen isotope experiments In a typical experiment, a solution of antibody (6.7 ⁇ M, 100 ⁇ L) or non-immunoglobulin protein (50 ⁇ M, 100 ⁇ L) in PB (160 mM phosphate; pH 7.4) was lyophilized to dryness and then dissolved in H 2 0 2 (100 ⁇ L, 98 %). Sodium chloride was excluded to minimize signal suppression in the MS. The higher concentration of non-immunoglobulin protein was necessary to generate a detectable amount of H 2 0 2 for the MS assay. This protein solution was irradiated on a UV-transilluminator under saturating 16 0 2 aerobic conditions in a sealed quartz cuvette for 8 hours at 20°C.
• the H 2 0 2 concentration was determined after 8 hours using the Amplex Red assay (Zhou et al., Anal. Biochem.. 253. 162 (1997)). The sample was then filtered by centrifugation through a microcon (size-exclusion filter) to remove the protein and the H 2 0 2 concentration re-measured.
• TCEP freshly prepared 20 mM stock in H 2 18 0
• TCEP was added (ca. 2 mol eq relative to H 2 0 2 ) and the solution was left to stand at 37°C for 15 minutes, after which time all the H 2 0 2 had reacted.
• the TCEP solution in H 2 18 0 was prepared fresh prior to every assay because 18 0 is slowly inco ⁇ orated into the carboxylic acids of TCEP (over days). During the time course of the assay, no inco ⁇ oration of ls O occurs due to this pathway. Furthermore, there is no inco ⁇ oration of 18 0 from H 2 18 0 into the 16 0 phosphine oxide.
• the peak at 249 m/z is the (M-H) " of TCEP. The peak at 249 is observed in all the MS because an excess of TCEP (twofold) relative to H 2 0 2 is used in the assay.
• Antibodies from different species give similar ratios within the experimental constraints detailed below: 16 O: ]8 O: WD1-6G6 mlgG (murine) 2.1:1; polylgG (horse) 2.2:1; polylgG(sheep) 2.2:1; EP2-19G2 mlgG (murine) 2.1: 1; CH2-5H7 mlgG (murine) 2.0:1; polylgG (human) 2.1:1. Ratios are based on the mean value of duplicate determinations except for polylgG (horse) which is the mean value often measurements. All assays and conditions are as described above.
• the assay is a modification of a procedure developed by H. Sakai and co-workers, Proc. SPIE-Int. Soc. Opt. Eng.. 2371. 264 (1995).
• the horse polylgG (1 mg/mL) in PBS (50 mM, pH 7.4) and hematopo ⁇ hyrin IX (40 ⁇ M) is irradiated with white light from a transilluminator. Aliquots are removed (50 ⁇ L) and the concentration of H 2 O 2 and 3-aminophthalic acid measured simultaneously. H 2 O 2 concentration was measured by the amplex red assay (Zhou et al., Anal. Biochem.. 253. 162 (1997)).
• the concentrations of luminol and 3-aminophthalic acid were determined by comparison of peak height and area to control samples.
• the experimental data yields the amount of ] O 2 formed by hematopo ⁇ hyrin IX (being directly proportional to the amount of 3-aminophthalic acid formed) and the amount of H 2 O 2 formed by the antibody. ⁇ .B. There is no significant amount of ] O 2 formed by antibodies without hematopo ⁇ hyrin IX in white light.
• amplex red assay may be detecting protein- hydroperoxide derivatives in addition to H 2 O 2 have been discounted because the apparent H 2 O 2 concentration measured using this method is independent of whether irradiated protein is removed from the sample (by size-exclusion filtration).
• Antibodies are capable of generating hydrogen peroxide (H 2 O 2 ) from singlet molecular oxygen ( ] O 2 ). However, it was not known until now, as reported herein, that the process was catalytic and the source of electrons. It is now shown that antibodies are unique as a class of proteins in that they can produce up to 500 mole equivalents of H 2 O 2 from ] O 2 , without a reduction in rate, in the absence of any discernible cofactor and electron donor. Based on isotope inco ⁇ oration experiments and kinetic data, it is proposed that antibodies are capable of facilitating an unprecedented addition of H 2 O to O 2 to form H 2 O 3 as the first intermediate in a reaction cascade that eventually leads to H 2 O 2 .
• Antibodies regardless of source or antigenic specificity, generate hydrogen peroxide (H 2 O 2 ) from singlet molecular oxygen ( ] O 2 ) thereby potentially aligning recognition and killing within the same molecule (Wentworth et al., Proc. Natl. Acad. Sci. U.S.A.. 97, 10930 (2000)). Given the potential chemical and biological significance of this discovery, the mechanistic basis and structural location within the antibody of this process has been investigated. These combined studies reveal that, in contrast to other proteins, antibodies may catalyze an unprecedented set of chemical reactions between water and singlet oxygen.
• the ⁇ TCR shares a similar arrangement of its immunoglobulin fold domains with antibodies (Garcia et al., Science. 274. 209 (1996)). However, possession of this structural motif seems not necessarily to confer an H 2 O 2 -generating ability on proteins as demonstrated by ⁇ 2 -microglobulin which does not generate H 2 O 2 even though it is a member of the immunoglobulin superfamily (Welinder et al., Mol. Immunol., 28. 177 (1991)).
• the antibody structure is remarkably inert against the oxidizing effects of H 2 O 2 .
• a polyclonal horse IgG antibody sample becomes fully active once the inhibitory H 2 O 2 has been destroyed by catalase ( Figure 8E).
• Figure 8E The ability to continue H 2 O 2 production for long periods at a constant rate, even after exposure to H 2 O 2 , reveals a remarkable, and hitherto unnoticed, resistance of the antibody structural fold to both chemical and photo-oxidative modifications suffered by other proteins.
• RMSD root mean square difference
• the mechanism problem posed by the antibody-mediated H 2 O 2 production from singlet oxygen has to be sha ⁇ ly divided into two sub-problems: one referring to the electron source for the process and the other concerning the chemical mechanism of the process.
• the fact that antibodies can generate > 500 equivalents of H 2 O 2 per equivalent of antibody molecule raises an acute electron inventory problem.
• the search for this electron source began with the most distinct possibilities. Since electron transfer through proteins can occur with remarkable facility and over notably large distances (Winkler et al., Pure & Appl. Chem.. 71, 1753 (1999); Winkler, Curr. Opin. Chem. Biol..
• ROS reactive oxygen species
• the photo-production of H 2 O 2 by T ⁇ and molecular oxygen is a well-characterized process that involves, at least in part, the fo ⁇ nation and reduction of O 2 to O 2 * that spontaneously dismutates into H 2 O 2 and 3 O 2 (McMormick and Thompson, J. Am. Chem. Soc. 100. 312 (1978)).
• Tryptophan both as an individual amino-acid and as a constituent of proteins, is particularly sensitive to near-UN irradiation (300-375 nm) under aerobic conditions, owing to its conversion to N'-formylkynurenine ( ⁇ FK) that is a particularly effective near-UN ( ⁇ max 320 nm) photosensitizer (Walrant and Santus, Photochem.
• the antibody's function as a catalyst would have to be the supply of a specific molecular environment that would stabilize the critical intermediate relative to its reversible formation and, or, would accelerate the consumption of the intermediate by channeling its conversion to H 2 O 2 .
• An essential feature of such an environment might consist of a special constellation of organized water molecules at an active site conditioned by an antibody-specific surrounding.
• Plesnicar and co-workers have shown that H 2 O 3 , reductively generated from ozone, decomposes into H 2 O and O 2 (Koller and Plesnicar, J. Am. Chem. Soc. 118, 2470 (1996)).
• H 2 O 3 has a barrier of only 15.5 or 0 kcal/mol respectively, suggesting that H 2 O 3 is not stable in bulk water or water rich systems.
• the best site within the antibody structure for producing and utilizing H 2 O 3 is expected to be one in which there are localized waters and water dimers next to hydrophobic regions without such waters.
• the 16 O/ 18 O ratio in the phosphine oxide derived from the antibody-catalyzed photo-oxidation of water poses a significant constraint to the selection of reaction paths by which this primary intermediate H 2 O 3 would to convert to the final product H 2 O 2 .
• the ratio is primarily determined by the number of 1 0 2 molecules that chemically participate in the production of two moles of H 2 O 2 from two moles of H 2 O as well as by mechanistic details of this process.
• a ratio of 2.2:1 would coincide exactly with the value predicted for certain mechanisms in which two molecules of ] O 2 and two molecules of H 2 O are transformed into two molecules of H 2 O 2 and one molecule of molecular oxygen (which would have to be 3 O 2 for thermodynamic reasons).
• the xenon I binding site (Xel site) has been analyzed here in more detail because it is conserved in all antibodies and the ⁇ TCR ( Figure 13B).
• Xel is in the middle of a highly conserved region between the ⁇ -sheets of V L , 7 A from an invariant T ⁇ .
• the Xel site is sandwiched between the two ⁇ -sheets that comprise the immunoglobulin fold of the N L , approximately 5 A from the outside molecular surface.
• Xenon site two (Xe2) sits at the base of the antigen binding pocket directly above several highly conserved residues that form the structurally conserved interface between the heavy and light chains of an antibody ( Figure 13 A).
• the residues in the N L N H interface are primarily hydrophobic and include conserved aromatic side chains, such as T ⁇ H109 .
• the contacting side chains for Xel in Fab 4C6 are Ala L19 , Ile L21 , Leu L73 , and Ile L75 , which are highly conserved aliphatic side chains in all antibodies (Kabat et al., Sequences of Proteins of Immunological Interest (US Department of Health and Human Services, Public Health Service, ⁇ IH, ed. 5th, 1991)). Additionally, only slight structural variation was observed in this region in all antibodies surveyed. Notably, several other highly conserved and invariant residues are in the immediate vicinity of this xenon site, including T ⁇ L35 , Phe L62 , Tyr L86 , Leu L104 , and the disulfide-bridge between Cys L23 and Cys L88 .
• T ⁇ L35 stacks against the disulfide-bridge and is only 7 ⁇ from the xenon atom.
• T ⁇ L35 may be a putative molecular oxygen sensitizer, since it is the closest T ⁇ to Xel. Comparison with the 2C ⁇ TCR structure and all available TCR sequences shows that this Xel hydrophobic pocket is also highly conserved in TCRs ( Figure 5B) (Garcia, Science, 274. 209 (1996)).
• Human ⁇ 2 -microglobulin which does not generate H 2 O 2 , does not have the same detailed structural characteristics that define the antibody Xel binding pocket, despite its overall immunoglobulin fold. Also, ⁇ 2 -microglobulin does not contain the conserved T ⁇ residue that occurs there in both antibodies and TCRs. If T ⁇ L35 (antibodies) or T ⁇ ⁇ 34 (TCR) is the oxygen sensitizer, the lack of a corresponding T ⁇ in ⁇ 2 -microglobulin may relate to the finding that it does not catalyze the oxidation of water.
• the xenon experiments have identified at least one site that is both accessible to molecular oxygen and is in a conserved region (N L ) in close proximity to an invariant T ⁇ ; an equivalent conserved site is also possible in the fold of N H
• N L conserved region
• the structure and sequence around the Xel site is almost exactly reproduced in the N H domain by the pseudo two-fold rotation axis that relates N L to N H .
• Antibodies are unique among proteins in their ability to catalytically convert O 2 into H 2 O 2 . It is thought that this process participates in killing by event-related production of H 2 O 2 . Alternatively, antibodies can fulfill the function of defending an organism against ! O 2 . This would require the further processing of hydrogen peroxide into water and triplet oxygen by catalase.

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