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WO1993013201A1 - Inhibiteur d'opsonine liant les lipopolyholosides et procedes d'utilisation - Google Patents

Inhibiteur d'opsonine liant les lipopolyholosides et procedes d'utilisation Download PDF

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Publication number
WO1993013201A1
WO1993013201A1 PCT/US1992/011192 US9211192W WO9313201A1 WO 1993013201 A1 WO1993013201 A1 WO 1993013201A1 US 9211192 W US9211192 W US 9211192W WO 9313201 A1 WO9313201 A1 WO 9313201A1
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WIPO (PCT)
Prior art keywords
agent
opsonin
antibody
binding
lipopolysaccharide
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Application number
PCT/US1992/011192
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English (en)
Inventor
Samuel D. Wright
Original Assignee
The Rockefeller University
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Filing date
Publication date
Application filed by The Rockefeller University filed Critical The Rockefeller University
Publication of WO1993013201A1 publication Critical patent/WO1993013201A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to newly discovered and isolated proteins and to methods and compositions including such proteins for preventing or treating sepsis. More particularly, the present invention relates to the inhibition of the binding of lipopolysaccharide (LPS) complexes by CD14-expressing cells, and the concomitant inhibition of the onset of endotoxic shock.
  • LPS lipopolysaccharide
  • Sepsis is morbid condition induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma.
  • the initial symptoms of sepsis typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, adult respiratory distress syndrome and multiple organ failure.
  • Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms.
  • bacterial toxins include the endotoxins or lipopolysaccharides (LPS) of the gram-negative bacteria. These molecules are glycolipids that are ubiquitous in the outer membrane of all gram-negative bacteria. While the chemical structure of most of the LPS molecule is complex and diverse, a common feature is the lipid A region of LPS [E. Th. Rietschel et al., in Handbook of Endotoxins. 1:187-214 eds., R.A. Proctor and E. Th.
  • LPS is believed to be a primary cause of death in humans during gram-negative sepsis, particularly when the symptoms include adult respiratory distress syndrome (ARDS) [van Deventer et al. , Lancet, .1:605 (1988); Ziegler et al. , J. Infect. Dis. f 136:19-28 (1987)].
  • ARDS adult respiratory distress syndrome
  • TNF tumor necrosis factor alpha/cachectin
  • LBP lipopolysaccharide binding protein
  • LBP recognizes the lipid A region of LPS and forms high affinity, 1:1 stoichiometric complexes with both rough and smooth form LPS [Tobias et al., J. Biol. Chem.. 26.4:10867-10871 (1989)].
  • LBP bears N-terminal sequence hemology with the LPS-binding protein known as bactericidal permeability-increasing factor, (BPI) [Tobias et al., J. Biol. Chem.. 263:13479-13481. (1988)].
  • BPI is stored in the specific granules of PMN [Weiss et al.. Blood.
  • the cells of the monocyte/macrophage lineage perform diverse immune function including the phagocytosis of micro-organisms, the uptake of antigenic material and its presentation in a form which is stimulatory to helper T cells. They are probably also involved in the immune surveillance against tumors and they secrete some complement components and cytokines. Surface membrane antigens play a critical role in regulating these activities. Several monocyte/macrophage surface antigens have been identified and their molecular weight has been determined. One such antigen, CD14, is a 55-kD glycoprotein expressed by monocytes, macrophages, and activated granulocytes.
  • LBP lipopolysaccharide binding protein
  • an agent has been discovered that appears to function as a mediator or inhibitor of the lipopolysaccharide binding opsonin known as "septin".
  • the agent comprises a protein in purified form that has the following characteristics: a. it is capable of inhibiting the binding to monocytes, macrophage cells and polymorphonuclear cells, of particles coated with said opsonin; and b. it inactivates lipopolysaccharide (LPS) that is in a complex with said opsonin, whereby the ability of said complexed LPS to bind to macrophage cells is substantially completely suppressed and cannot be reactivated.
  • LPS lipopolysaccharide
  • the agent possesses additional affirmative characteristics, among them that: a. it can be mimicked j-n vitro by a protease such as trypsin; and b. its activity can be blocked by an anti-protease exhibiting a broad spectrum of such activity.
  • the agent is found in plasma, and its preparation in a first instance can proceed by isolation and purification from body fluid and particularly serum or plasma.
  • the serum, plasma or other fluid may be subjected to a series of known isolation techniques, whereupon the agent may be recovered.
  • the present invention naturally contemplates alternate means for preparation of the agent, including where applicable known genetic replicative techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the isolation of the cDNA amino acid sequence will facilitate the reproduction of the agent by recombinant genetic techniques as discussed in detail hereinafter.
  • the DNA sequence encoding the agent or analogs thereof can be used to construct vectors for expression in host systems by recombinant DNA techniques.
  • the agent like the opsonin the activity of which it mediates, possesses the potential for use as a diagnostic tool to identify and perhaps differentiate between various stimuli whether invasive or idiopathic, by the activation of the agent that such stimuli may promote.
  • the agent of the present invention is believed to comprise an enzyme and more particularly, appears to be a protease. This is interesting in view of the observations that have been made earlier regarding the origin and action of the opsonin, that it comprises a complex of a protease and a substrate for the protease. It is therefore possible that the activity of the agent is likewise related to the protease cascade in which the opsonin has been theorized to participate in conjunction with its interaction with LPS, as the agent is believed to inactivate the opsonin by cleavage thereof.
  • the agent may likewise be a product of the protease cascade, and may participate therein by its interaction with a component precursor of the opsonin to interrupt the synthesis and/or activation of the latter. This mechanism of action may exist in tandem with the mechanism of cleavage of the already formed opsonin molecule.
  • the present invention likewise extends to antibodies developed to the agent.
  • antibodies developed to the agent.
  • Such antibodies could be used for both diagnostic and therapeutic purposes, to further monitor and control the host response to infection by mediating the activity of the agent.
  • Such antibodies may interact with those components in the protease cascade that promote the synthesis and activation of the agent, as well as directly interacting with the agent.
  • Both monoclonal and polyclonal antibodies to the agent are contemplated and includeable herein.
  • an assay system for screening of potential drugs effective to counteract the effect of the agent on the opsonin may be prepared.
  • the test drug could be administered to a macrophage sample with the opsonin and a quantity of lipopolysaccharide present, to determine its effect upon the binding activity of the opsonin to either the LPS or the macrophage.
  • the agent may be introduced into a cellular test system in which both the agent and the opsonin are known to be active, and the prospective drug may also be introduced to the same cell culture and the culture may thereafter be examined to observe any changes in the activity of the agent and/or the opsonin in comparison with the addition of the prospective drug alone, or the effect of added quantities of the known materials.
  • the present invention also relates to a method for detecting the presence of stimulated, spontaneous, or idiopathic pathological states in mammals, by measuring the activity and presence of the agent of the present invention. More particularly, the activity of the agent may be followed directly by the assay techniques discussed later on, through the use of an appropriately labeled quantity of the agent. Alternately, the agent can be used to raise binding partners or antibodies that could in turn, be labeled and introduced into a medium such as serum, to test for the presence of the agent therein, and to thereby assess the state of the host from which the medium was drawn.
  • both the agent and any antibodies that may be raised thereto are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the agent that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • immunoassays such as a radioimmunoassay, using for example, an antibody to the agent that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • a control quantity of the agent, its antibody, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a blood sample of a mammal believed to be undergoing invasion. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • radioactive label such as the isotopes ,4 C, ,31 I, 3 H, m I and 35 S
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro- photometric, amperometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the agent.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the agent; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the agent, antibodies to the agent, or upon agents or other drugs determined to possess the same or an antagonistic activity.
  • a first therapeutic method is associated with the prevention of the manifestations of conditions following from infection with a gram negative organism, such as inflammation, fever and shock, and comprises administering either the agent alone, or the agent in combination with a material selected from the group consisting of an antibody or antagonist to the opsonin other than the present agent, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • the therapeutic method generally referred to herein could include the method for the treatment of shock, sepsis, inflammation and/or fever by the administration of pharmaceutical compositions that may comprise effective quantities of the agent and/or the aforementioned materials, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • a variant embodiment of this therapeutic method could include initially detecting the presence and activity of the agent and/or the opsonin and thereafter administering the appropriate pharmaceutical composition.
  • compositions for use in therapeutic methods which comprise or are based upon the agent or its binding partner(s) , or upon drugs that control the production, or that mimic or antagonize the activities of the agent.
  • FIGURE 1 is a graph depicting the initial assessment of the activity of the agent of the present invention, by reference to the inhibitory effect of prolonged exposure to plasma on the binding of complexes of the opsonin and LPS to macrophage (MO) .
  • FIGURE 2 is a graph further illustrating the effect of the serum-derived agent on the activity of the opsonin, septin, by measurement of the attachment index of substrate-bound LPS on the stimulation of PMN as a function of sample incubation with either human plasma or human LBP under equivalent conditions. It was found that LBP generates stable complexes with LPS that activate CR3 on PMN, but the complexes with septin formed upon incubation with plasma are lost upon prolonged incubation.
  • FIGURE 3 is a graph depicting how prolonged exposure of tissue culture surfaces to plasma (containing the agent) inhibited the activity of septin/LPS to cause monocyte activation, which activation was measured as a function of TNF production.
  • FIGURE 4 is a graph illustrating the dose dependence of the activity of the agent. The ability of erythrocytes to bind to MO was measured as a function of the prolonged presence of plasma (containing the agent) .
  • FIGURE 5 is a graph depicting how the loss of septin from ELPS was caused by the agent of the present invention and that this inhibitory activity can be mimicked by the protease, trypsin. Specifically, ELPS coated with septin (prep A in the Figure) were incubated with trypsin, and the abolition of binding to MO was observed, indicating that the trypsin protease had destroyed septin.
  • FIGURE 6 is a graph depicting how the activity of the present agent, septinase, can be inhibited by the protease inhibitor ⁇ 2 macroglobulin.
  • stimulation and its plural as used herein are intended to apply to invasive events such as infection, as well as conditions caused by wounding, and to idiopathic or spontaneous states that may for example, originate from cellular or metabolic derangements or other causes.
  • agent and "opsonin” as used throughout the present application and claims refer to the respective protein material(s) having the profiles of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the opsonin. Also, the terms “agent” and “opsonin” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e.. capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5 ' to 3 ' direction along the nontranscribed strand of DNA (i.e.. the strand having a sequence homologous to the mRNA) .
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy1) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3 to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N- terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell.
  • Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. For instance, alpha-factor, a native yeast protein, is secreted from yeast, and its signal sequence can be attached to heterologous proteins to be secreted into the media (See U.S. Patent 4,546,082, EPO 0 116201, publication date 12 January 1983; U.S. Patent Application Serial No.
  • alpha-factor leader and its analogs have been found to secrete heterologous proteins from a variety of yeast, such as Saccharomyces and Kluyveromyces, (EPO 88312306.9 filed 23 December 1988; U.S. Patent Application Serial No. 139,682, filed 30 December 1987, and EPO Publication No. 0 301 669, publication date 1 February 1989) .
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses, inter alia, polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F( b') 2 and F(V) , which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. (The disclosures of the art cited herein are hereby incorporated by reference.).
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • substantially simultaneously is used herein to mean within a time period sufficient to produce concurrent results; e.g., bacterial lysis as a result of antibiotic administration and amelioration or prevention of symptoms of shock and/or sepsis that may occur as a result of that lysis by administration of the present agent, an anti-opsonin antibody, peptide analogs of either, or a subcombination or combination thereof, as described herein.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the plasma level of TNF, or other feature of septic shock such as elevated blood pressure, fever or white cell count.
  • the present invention concerns the isolation and identification of a newly discovered particular factor hereinafter referred to the agent, that has been found to be present in serum or plasma, and that participates in the inhibition of the binding of stimulator materials that characteristically accompany an invasive stimulus such as lipopolysaccharide (LPS) , to monocytes, macrophage cells and polymorphonuclear leukocytes such as bacteria, virus, certain tumors, protozoa and other toxins such as endotoxin, or an idiopathic state.
  • LPS lipopolysaccharide
  • the agent appears to function by cleaving the molecule referred to alternately herein as "septin” or the “(lipopolysaccharide-binding) opsonin", which latter molecule was previously discovered and described by the applicant herein as primarily responsible for promoting and facilitating the binding activity of LPS and like stimuli by forming a complex therewith.
  • the agent destroys the ability of such complexes to interact with CD14 and which thus blunts responses of cells to endotoxin. Consequently, septic shock may thus be treated by administering this serum factor to patients in need of such therapy.
  • the agent has been partially purified by ammonium sulfate precipitation.
  • Addition of 40% ammonium sulfate results in precipitation of approximately 90% of the plasma proteins, but agent activity remains in solution.
  • Further purification on a Mono Q column results in a single peak with agent activity.
  • the agent comprises a protein in purified form that as its primary affirmative characteristics, is capable of inhibiting the activity of the opsonin that is capable of binding to lipopolysaccharide to form a complex recognized by a receptor on monocytes, macrophage cells and polymorphonuclear cells; and that possesses an apparent molecular weight of about 90 kD as determined by SDS-PAGE analysis.
  • the agent has the following characteristics:
  • LPS lipopolysaccharide
  • the agent possesses additional characteristics, among them that: a. it can be mimicked in vitro by a protease such as trypsin; and b. its activity can be blocked by an anti-protease exhibiting a broad spectrum of such activity.
  • the agent of the present invention is be.lieved to comprise an enzyme and more particularly, appears to be a protease. Accordingly, as the opsonin is theorized to comprise a complex of a protease and a substrate for the protease, it is possible that the agent may also be active in the same protease cascade as that of the opsonin, as the agent is believed to inactivate the opsonin by cleavage thereof.
  • the agent may likewise be a product of the protease cascade, and may participate therein by its interaction with a component precursor of the opsonin to interrupt the synthesis and/or activation of the latter. This mechanism of action may proceed conjointly or alternately with the mechanism of cleavage of the already formed opsonin molecule.
  • the present invention contemplates methods of treating and/or preventing one or more of the symptoms of sepsis. particularly those associated with a transient increase in the blood level of TNF, such as fever, hypotension, neutropenia, leukopenia, thrombocytopenia, shock and multiple organ failure.
  • Patients in need of such treatment include those at risk for or suffering toxemia, such as endotoxemia resulting from a gram-negative bacterial infection, serpent venom poisoning, hepatic failure, and the like.
  • some patients having a gram-positive bacterial, viral or fungal infection display the symptoms of sepsis and may benefit from a therapeutic method of this invention.
  • Patients particularly able to benefit from the present invention are those suffering infection by E. coli.
  • Haemophilus influenza B Neisseria meninqitides. staphylococci, or pneumococci.
  • Patients at risk for sepsis include those suffering burns, gunshot wounds, renal or hepatic failure due to chemical poisoning or abuse, and the like.
  • the present invention contemplates a method of ameliorating one or more of the symptoms of sepsis by administering to a patient in need of such therapy a therapeutically effective amount of the agent.
  • Preferred therapeutically effective amounts for the agents used herein as active ingredients include those described hereinafter.
  • the agent or its binding partner(s) or other ligands or agents exhibiting either mimicry or antagonism .to the agent or control over its production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient having a tissue infection or other pathological derangement, for the treatment thereof.
  • a suitable carrier and at a strength effective for administration by various means to a patient having a tissue infection or other pathological derangement, for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them topical applications as in ointments or on surgical and other topical appliances such as, surgical sponges, bandages. gauze pads, and the like.
  • such compositions may be administered by parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, including delivery in an irrigation fluid used to wash body wound areas, catheterizations and the like. Average quantities of the agent may vary and should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the agent may possess certain therapeutic applications and may thus be utilized for the purpose of treating the effects of infection attributable to the activity of the agent.
  • the agent may be used to produce both polyclonal and monoclonal antibodies to itself in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • Suitable antibodies would also extend to antibodies that may be raised against a component of the protease cascade that gives rise to the opsonin of which the agent is believed to be a part. Accordingly, antibodies to any of these antigens would interrupt the synthesis and/or activity of the agent and would thereby inhibit or block its action.
  • Panels of monoclonal antibodies produced against agent peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of the agent. Such monoclonals can be readily identified in agent activity assays. High affinity antibodies are also useful in immunoaffinity purification of native or recombinant agent.
  • the anti-agent antibody used in a therapeutic method of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb) .
  • mAb monoclonal antibody
  • the anti-CD14 antibody molecules used herein be in the form of Fab, Fab', F(ab , ) 2 or F(V) portions of whole antibody molecules.
  • immunoreactivity in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of the antigen. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both B 0 and B have been adjusted for background. See Robard, Clin. Chem.. 20:1255-1270 (1974).
  • the therapeutic method of the present invention comprises administering a therapeutically effective amount of an anti-agent antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • an anti-agent antibody preferably an affinity-purified polyclonal antibody
  • the anti-agent antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • the amount of anti-agent antibody administered is sufficient to reduce by at least about 30 percent, preferably by at least 80 percent, the inhibition of an opsonin-LPS complex-induced, clinically significant increase in the blood level of TNF in a patient displaying at least one of the symptoms of sepsis.
  • patients capable of benefiting from this method include those suffering endotoxemia as a result of a gram- negative bacterial infection, where the activity of the opsonin and the agent must be carefully modulated.
  • Methods for isolating the agent and inducing anti-agent antibodies and for determining and optimizing the ability of an anti-agent antibody to limit the inhibition of the binding of opsonin-LPS complexes to CD14 and thereby inhibit opsonin-induced TNF secretions are all well-known in the art.
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with opsonin or an agent-binding portion thereof, or agent or an opsonin-binding portion thereof.
  • the myeloma cell line be from the same species as the lymphocytes.
  • a mouse of the strain 129 G1X + is the preferred mammal.
  • Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American Type Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively.
  • HAT hypoxanthine-aminopterin-thymidine-sensitive
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the agent and their ability to intervene in the inhibition of opsonin/LPS complex-induced TNF secretion.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • DMEM Dulbecco's minimal essential medium
  • the present invention contemplates administering a therapeutically effective amount of the agent, an anti-opsonin antibody, opsonin peptide analog, a subcombination or combination thereof, substantially simultaneously with therapeutic administration of a modality known to prevent or treat the symptoms of sepsis.
  • TNF TNF-induced fibroblast growth factor
  • levels of TNF in normal healthy humans or in laboratory animals are estimated to be no more than about 10 pg/ml, a value that is at the limit of detection by the most sensitive assays for TNF [Michie et al.. New En ⁇ . J. Med. 118:1481-1486
  • a clinically significant increase in the plasma level of TNF is an increase to at least about 25 pg/ml.
  • Methods for determining the plasma TNF levels are well-known in the art, particularly preferred methods being those described herein.
  • the levels of TNF have been shown to rise 10-20 fold to levels of up to 400 pg/ml (vide supra) .
  • a therapeutic method of this invention can further include substantially simultaneous treatment with a steroid, such as cortisol, hydrocortisone and the like.
  • a patient exhibiting the symptoms of sepsis is usually treated with an antibiotic, typically an aminoglycoside such as gentamicin or a beta-lactim such as penicillin, cephalosporin and the like.
  • an antibiotic typically an aminoglycoside such as gentamicin or a beta-lactim such as penicillin, cephalosporin and the like.
  • a preferred therapeutic method includes administering a therapeutically effective amount of the agent, an anti- opsonin antibody, a peptide analog or subcombination thereof as described herein, substantially simultaneously with administering a bactericidal amount of an antibiotic.
  • the phrase "bactericidal amount” is used herein to mean an amount of the bacteriocide sufficient to achieve a bacteria-killing blood concentration in the patient receiving the treatment.
  • the bactericidal amount of antibiotics generally recognized as safe for administration to humans is an amount well-known in the art and varies, as is also well-known, with the antibiotic and the type of bacterial infection being treated.
  • administration of the agent, an anti-opsonin antibody, peptide analog or combination thereof as described herein occurs within about 48 hours, preferably within about 12-36 hours, more preferably within about 2-8 hours and most preferably substantially concurrently with administration of the antibiotic.
  • Antibiotics useful in practicing the present invention include those antibiotic, antibacterial and antiseptic agents having formulations described in the Physicians' Desk Reference, Huff, B.B. ed. , Medical Economics Company, Inc., Oradell, N.J. (1989).
  • the present invention contemplates administering a therapeutically effective amount of the agent or its antibody, preferably a soluble portion thereof that binds LPS-opsonin complexes, alone or in subcombination or combination with a therapeutically effective amount of an anti-TNF antibody, an anti-CD14 antibody, an anti-opsonin antibody, and an antibiotic.
  • the cDNA coding for CD14 and its deduced amino acid residue sequence are well-known in the art. See Goyert et al.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of the agent, an anti-opsonin antibody, or polypeptide analog thereof, as described herein as an active ingredient.
  • the composition comprises an antibody or antigen capable of modulating either the binding of LPS to the opsonin, or the binding of LPS-opsonin complexes to CD14.
  • compositions comprise an agent, an anti-opsonin antibody, preferably a mAb, that modulates the binding of LPS-opsonin complexes to CD14.
  • Preferred therapeutic compositions further include an effective amount of the agent and/or the anti- agent antibody of the invention and one or more of the following active ingredients: an antibiotic, a steroid, and anti-TNF antibody an a TNF antagonist. Exemplary formulations are given below:
  • gentamicin (sulfate) 40 agent/anti-agent antibody 10 sodium bisulfite USP 3.2 disodium EDTA USP 0.1 water for injection q.s.a.d. 1.0 ml
  • the present invention contemplates a therapeutic composition useful in treating sepsis comprising the present agent or an opsonin-inhibiting soluble portion thereof in a pharmaceutically acceptable carrier.
  • the composition further includes a therapeutically effective concentration of one or more of an anti-TNF antibody, an anti-CD14 antibody, an anti- opsonin antibody and an antibiotic.
  • compositions which contain polypeptides or antibody molecules as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. 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 which enhance the effectiveness of the active ingredient.
  • a polypeptide or antibody can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,' tartaric, mandelic, and the like.
  • Salts formed from the free carboxy1 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.
  • the therapeutic polypeptide- or antibody-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of promotion or inhibition LPS-opsonin complex binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nano molair to ten micromolar in the blood are contemplated.
  • agent analogs may be prepared from nucleotide sequences of the agent derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of agent. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of agent coding sequences. Analogs exhibiting "agent activity" may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding agent can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the agent amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g. , Edge Nature. 292:756 (1981); Nambair et al. Science. 22.3:1299 (1984); Jay et al. , J. Biol. Chem. 259:6311 (1984).
  • DNA sequences allow convenient construction of genes which will express agent analogs or "muteins".
  • DNA encoding muteins can be made by site- directed mutagenesis of native agent genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • Site-directed mutagenesis is generally used to create analogs from a complete coding sequence. Site-directed mutagenesis is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.
  • 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence.
  • the resulting plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of invasive stimuli by reference to their ability to elicit the activities which are affected by the present agent.
  • the agent can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence or activity of the agent in suspect mammalian hosts.
  • Antibody(ies) to the agent can be produced and isolated by standard methods including the well known hybridoma techniques. For convenience, the antibody(ies) to the agent will be referred to herein as Ab, and antibody(ies) raised in another species as Abj.
  • agent activity in mammals can be ascertained by the usual immunological procedures applicable to such determinations.
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the agent labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Abj labeled with a detectable label.
  • the agent forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • Abj a characteristic property of Abj is that it will react with Ab,.
  • Ab raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens. Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a primary or anti-agent antibody, and Ab 2 will be referred to as a secondary or anti-Ab, antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the agent or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from l4 C, 13, I, 3 H, ,25 I and 35 S.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the agent may be radiolabeled and combined, for example, with LPS, after which binding studies would be carried out using for example, recently purified neutrophils. Solutions would then be prepared that contain various quantities of labeled and unlabeled agent and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed.and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of agent in a suspected mammalian host.
  • one class of such kits will contain at least the labeled agent or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive", “sandwich”, “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the reaction of a mammalian host to invasive stimuli, comprising:
  • the diagnostic test kit may comprise:
  • test kit (b) if necessary, other reagents; and (c) directions for use of said test kit.
  • test kit may be prepared and used for the purposes stated above, which operates according ,to a predetermined protocol (e.g. "competitive”, “sandwich”, “double antibody”, etc.), and comprises:
  • a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the agent and a specific binding partner thereto.
  • an assay system for screening potential drugs effective to modulate the activity of the agent may be prepared.
  • the agent may be introduced into a cellular test system such as neutrophils with 100 pg/ml LPS, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the activity of the agent, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent.
  • a drug assay could be conducted by culturing a colony of test cells such as the cell line THPl, which has a receptor for the complex of the opsonin and LPS, in a medium containing the opsonin, LPS and the agent.
  • the drug under test could be added to the resulting culture and the reactivity of the opsonin with the receptor on the test cells could thereafter be measured to determine whether the prospective drug possessed any activity in the inhibition of the activity of the agent as against the binding of the opsonin to either LPS or to the receptor.
  • ELPS LPS-coated erythrocytes
  • ELPS were prepared as previously described (Wright, S.D., et al., 1989 J. EXP. Med. 170:1231-1241) and incubated at 21°C with 0.5% human plasma for the intervals shown. The resulting coated erythrocytes were then washed and incubated for 30 min at 21°C with monolayers of human MO as described (Wright et al. , supra) . Attachment of erythrocytes to MO was enumerated by phase contrast microscopy and the results appearing in Fig. 1, are presented as attachment index, the number of erythrocytes per 100 MO.
  • ELPS were treated with 0.5% plasma for 10 min at 21°C to deposit septin on their surfaces.
  • the cells were washed and fresh plasma at the indicated doses was added.
  • the erythrocytes were washed and their binding to MO was determined in a 30 min incubation as described in Wright, S.D. , et al., 1989 J. EXP. Med. 170:1231-1241).
  • Septin is stable on the surface of ELPS, but addition of plasma (containing the agent) causes a dose-dependent loss in binding to MO.
  • Figure 5 illustrates that the loss of septin from ELPS requires plasma and can be mimicked by the protease, trypsin.
  • the binding of erythrocytes to monocytes was determined as described by Wright et al. supra.
  • septin-coated erythrocytes caused a dose-dependent and time-dependent loss of binding to MO, again indicating that plasma contains a factor that destroys septin.
  • This factor has the characteristics of an enzyme.
  • FIG. 6 demonstrated that septinase can be inhibited by the protease inhibitor ⁇ 2 macroglobulin.
  • ELPS were coated with septin in a 10 min incubation with 0.5% plasma at
  • Septinase detoxifies the LPS in septin/LPS complexes After the action of septinase on septin/LPS complexes, the LPS becomes unavailable for further interactions with septin (Fig 5) .
  • ELPS were coated with septin then incubated with plasma as a source of septinase. The resulting cells bound poorly to MO indicating the destruction of septin/LPS complexes, and binding could not be restored by incubating with fresh plasma as a source of -septin (Fig 5) or with purified lipopolysaccharide binding protein (not shown) .
  • Septinase appears to be a proteolytic enzyme. Addition of ⁇ 2 Macroglobulin, a broad spectrum anti-protease, strongly blocked the destruction of septin/LPS complexes by plasma (Fig 6) .
  • plasma contains a proteolytic enzyme that can inactivate septin/LPS complexes and thereby blocks stimulation of both PMN and monocytes. Since this enzyme leaves the LPS in an inactive state, it serves to "detoxify" endotoxin.

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Abstract

Un agent inhibant l'activité d'une opsonine liant les lipopolyholosides est décrit. L'agent a été isolé du plasma, et l'on présume qu'il se compose d'une protéase qui inactive l'opsonine en la segmentant. L'agent peut être utilisé pour traiter le syndrome de choc et d'autres états susceptibles d'être induits par l'activité de l'opsonine en réponse à l'invasion d'un hôte. Des utilisations diagnostiques et thérapeutiques son décrites, ainsi que des procédés d'essai, des substances sous forme de kit, des substances recombinées et les procédés associés, et des compositions pharmaceutiques.
PCT/US1992/011192 1991-12-30 1992-12-23 Inhibiteur d'opsonine liant les lipopolyholosides et procedes d'utilisation WO1993013201A1 (fr)

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US5928624A (en) * 1994-06-14 1999-07-27 The Rockefeller University Compositions for neutralization of lipopolysaccharides

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WO1991011464A1 (fr) * 1990-02-01 1991-08-08 The Rockefeller University Opsonine liant les lipopolysaccharides et ses procedes d'utilisation

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WO1991011464A1 (fr) * 1990-02-01 1991-08-08 The Rockefeller University Opsonine liant les lipopolysaccharides et ses procedes d'utilisation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928624A (en) * 1994-06-14 1999-07-27 The Rockefeller University Compositions for neutralization of lipopolysaccharides
US5932536A (en) * 1994-06-14 1999-08-03 The Rockefeller University Compositions for neutralization of lipopolysaccharides

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