EP0679083A1 - Prevention et traitement de la septicemie - Google Patents

Prevention et traitement de la septicemie

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Publication number
EP0679083A1
EP0679083A1 EP94909422A EP94909422A EP0679083A1 EP 0679083 A1 EP0679083 A1 EP 0679083A1 EP 94909422 A EP94909422 A EP 94909422A EP 94909422 A EP94909422 A EP 94909422A EP 0679083 A1 EP0679083 A1 EP 0679083A1
Authority
EP
European Patent Office
Prior art keywords
conjugate
antibiotic
igg
pmb
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94909422A
Other languages
German (de)
English (en)
Other versions
EP0679083A4 (fr
Inventor
Sean B. Carroll
Joseph R. Firca
Charles S.G. Pugh
Nisha Vikas Padhye
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Promega Corp
Original Assignee
Ophidian Pharmaceuticals Inc
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Filing date
Publication date
Priority claimed from US08/169,701 external-priority patent/US5545721A/en
Application filed by Ophidian Pharmaceuticals Inc filed Critical Ophidian Pharmaceuticals Inc
Publication of EP0679083A1 publication Critical patent/EP0679083A1/fr
Publication of EP0679083A4 publication Critical patent/EP0679083A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C07K7/58—Bacitracins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to therapeutics for the prevention and treatment of blood- borne and toxin mediated diseases, and in particular the prevention and treatment of sepsis in humans as well as other animals.
  • Sepsis is a major cause of morbidity and mortality in humans and other animals. It is estimated that 400,000-500,000 episodes of sepsis resulted in 100,000-175,000 human deaths in the U.S. alone in 1991. Sepsis has become the leading cause of death in intensive care units among patients with non-traumatic illnesses. [G.W. Machiedo et al., Surg. Gyn. & Obstet. 152:757-759 (1981).] It is also the leading cause of death in young livestock, affecting 7.5-29% of neonatal calves [D.D. Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986)], and is a common medical problem in neonatal foals. [A.M.
  • Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), including bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxin in the blood).
  • bacteremia includes occult bacteremia observed in young febrile children with no apparent foci of infection.
  • fungemia i.e., fungi in the blood
  • viremia i.e., viruses or virus particles in the blood
  • parasitemia i.e., helminthic or protozoan parasites in the blood.
  • septicemia and septic shock acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate
  • septicemia and septic shock acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate
  • microorganisms The systemic invasion of microorganisms presents two distinct problems. First, the growth of the microorganisms can directly damage tissues, organs, and vascular function. Second, toxic components of the microorganisms can lead to rapid systemic inflammatory responses that can quickly damage vital organs and lead to circulatory collapse (i.e., septic shock) and oftentimes, death.
  • Gram-negative sepsis is the most common and has a case fatality rate of about 35%. The majority of these infections are caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such as the staphylococci and streptococci are the second major cause of sepsis.
  • the third major group includes the fungi, with fungal infections causing a relatively small percentage of sepsis cases, but with a high mortality rate.
  • LPS lipopolysaccharide
  • antibiotic-resistant organisms are facilitated by the high density of potentially infected patients and the extent of staff-to-staff and staff-to-patient contact.
  • those antibiotics that are the most economical, safest, and easiest to administer may not have a broad enough spectrum to suppress certain infections.
  • many antibiotics with broad spectra are not deliverable orally and physicians are reluctant to place patients on intravenous lines due to the enhanced risk of infection.
  • antibiotics can be toxic to varying degrees including causing allergy, untoward interactions with other drugs, and direct damage to major organs (e.g., kidneys, liver). Many potent antibiotics are eliminated from routine use due to the probability of adverse reactions at therapeutic doses.
  • polymyxin antibiotics most notably polymyxin B and polymyxin ⁇ (also known as colistin) are cyclic polypeptide compounds produced by certain strains of Bacillus polymyxa. These antibiotics bind to the lipid A portion of endotoxin [D.C. Morrison and D.M. Jacobs,
  • aeruginosa (l-2.5mg/kg body weight/day), there is a significant risk of renal impairment.
  • This is a major concern in patients already suffering from kidney disease.
  • neurotoxic reactions have been observed, the most severe being respiratory paralysis when given soon after anesthesia and/or muscle relaxants.
  • Polymyxin B in its intravenous form, is only given to hospitalized patients under constant supervision and monitoring of renal function. As such, polymyxins are not used routinely for systemic infections (but they are quite common as components of topical ointments).
  • Colistin exhibits a lower systemic toxicity, and when complexed as methanesulfonate salt, the locally severe pain experienced at intramuscular injection sites is diminished.
  • the toxicity of polymyxin B is also reduced by attachment to dextran, a high molecular weight carrier.
  • polymyxin B has a half-life of only a few hours [G. Brownlee et al., Brit. J. Pharmacol. 7:170-188 (1952)], while dextran (M.W. 70,000) has a half-life in humans of about a day, depending upon the dose infused.
  • Polymyxin B has been investigated as a specific therapy for gram-negative sepsis or endotoxemia over the past 20 years in both animal models and human trials but with mixed results.
  • endotoxin-induced disseminated intravascular coagulation (DIC) was not prevented in rabbits administered polymyxin B fifteen (15) minutes after endotoxin challenge.
  • DIC disseminated intravascular coagulation
  • Antibodies to endotoxin have two important functions. First, by binding free endotoxin, antibodies may block endotoxin activity or remove it from the circulation. Second, immunoglobulin effector functions such as complement fixation and binding to Fc receptors on phagocytes can mediate killing and opsonophagocytosis of bacteria. Thus, endotoxemia, bacteremia, and the onset of sepsis, may be thwarted by such antibodies. i) Active Immunization
  • endotoxin-binding antibodies that could be passively transferred to experimental animals or human subjects.
  • a large number of endotoxin antibodies have been prepared by: (i) immunization of animals or humans with bacteria, LPS, or derivatives thereof and collection of immune serum or plasma; or (ii) production of monoclonal murine or human antibodies and collection and purification of these antibodies by established methods.
  • IgM and IgG antibodies The two major antibody types elicited by either procedure are IgM and IgG antibodies. These antibodies differ in important aspects of their structure and effector functions as well as their titer in normal and hyperimmune plasma. Most studies suggest that IgM antibodies, by virtue of their greater avidity are more effective than IgG antibodies at protecting animals [W.R. McCabe et al., J. Infect. Dis. 158:291-300 (1988)] and humans [Id.; E.J. Ziegler et al. New. Eng. J. Med. 307:1225-1230 (1982)] from gram-negative bacteremia or endotoxin challenge.
  • IgG preparations from immunized animal donors have been developed and demonstrated to have some protective effect in experimental studies. [D.L. Dunn et al., Surgery 96:440-446 (1984); and S.J. Spier et al., Circulatory Shock 28:235-248 (1989).]
  • the advantage to IgG preparations is that IgG titers may increase in response to repeated immunization whereas IgM titers are relatively constant. No matter what the immunization course, however, the total amount of bacterially-reactive or endotoxin-reactive antibodies in hyperimmune plasma or serum is only a small fraction of total antibody and is highly variable from donor to donor.
  • each antibody was tested in humans after the onset of symptoms of sepsis and when the type of organism was uncertain. It is widely believed that anti-endotoxin antibody treatment administered after sepsis is established may yield little benefit because these antibodies cannot reverse the inflammatory cascade initiated by endotoxin and the attendant triggering of mediators such as TNF and IL- 1. In addition, the high cost of each antibody (Centoxin HA-IA was expected to cost $3700 per 100 mg dose) would limt physicians' use of aproduct where no clear benefit has been demonstrated. [K.A.
  • IL-1 receptor antagonist has been identified that occupies the same receptor site as IL-1, but mediates no biological effect. Blockage of the IL-1 receptor with this molecule can reduce mortality from endotoxin shock. [K. Ohlsson et al. Nature 348:550-552 (1990).] While the IL-1 receptor antagonist appears to be well-tolerated, the required dosage is extremely large (over 100 mg of recombinant protein per kg of body weight is infused over a period of hours to days).
  • TNF therapies target removal of this mediator from the circulation.
  • Monoclonal antibodies have been found to offer some protection in experimental animals [S.M. Opal et al. , J. Infect. Dis. 161 :1 148-1 152 (1990)] but studies in human patients with sepsis have not been conclusive. Once again, these antibodies are likely to be expensive therapeutic agents administered only when signs of sepsis are present.
  • Monoclonal antibodies have also been made. While these preparations should possess greater potency, their high cost, immunogenicity [S. Harkonen et al., Antimicrob. Agents Chemother. 32:710-716 (1988)] and unusually short circulating half-lives (less than 24 hr) [S. Harkonen et al., Antimicrob. Agents Chemother. 32:710-716 (1988); and C.J. Fisher et al., Clin. Care Med. 18:1311-1315 (1990)] make them unattractive candidates for prophylaxis.
  • agents capable of preventing and treating sepsis must be capable of neutralizing the effects of endotoxin in gram-negative sepsis as well as controlling and reducing bacteremia. It would be desirable if such agents could be administered prophylactically in a cost-effective fashion. Furthermore, approaches are needed to combat all forms of sepsis, not just gram-negative cases.
  • the present invention relates to therapeutics for the prevention and treatment of blood- borne and toxin-mediated diseases, and in particular the prevention and treatment of sepsis in humans as well as other animals.
  • the present invention relates to compositions and methods for preventing sepsis in high-risk patients (e.g.,
  • the present invention contemplates treatment of humans and animals having symptoms of a systemic septic reaction.
  • antibody-antibiotic conjugates a member from the class of compounds broadly described as antibody-antibiotic conjugates or "antibodiotics" is employed for intravenous, intramuscular, intrathecal or topical administration.
  • Antibodiotics are comprised of antibody (e.g., IgG, IgM, IgA) to which an antibiotic is covalently attached to make an antibody-antibiotic conjugate.
  • the antibody is non-specific IgG.
  • non-specific it is meant that no single specificity within the antibody population or pool is dominant.
  • the present invention contemplates an antibiotic-antibody conjugate, comprising antibiotic covalently bound to non-specific immunoglobulin. It is preferred that the immunoglobulin is IgG having an Fc region and is capable of binding to phagocytic cells via the Fc region.
  • the conjugate is capable of binding to bacteria via the antibiotic.
  • the conjugate may be bacteriostatic, bactericidal, neither, or both.
  • antibiotics contemplated are not limited to antibacterial agents; antifungal agents and antiviral agents are also contemplated. Where antibacterial antibiotics are used, agents effective against both gram-positive and gram-negative organisms are contemplated.
  • the present invention contemplates conjugates capable of binding lipopolysaccharide on gram negative bacteria as well as conjugates capable of binding free endotoxin and neutralizing free endotoxin.
  • Preferred antibiotics include polymyxins, specifically polymyxin B.
  • Polymyxin is a known endotoxin-binding compound capable of binding free endotoxin.
  • the present invention also contemplates a therapeutic preparation, comprising antibiotic covalently bound to non-specific immunoglobulin, wherein the preparation is bactericidal for both gram-positive and gram-negative organisms.
  • the antibiotic is selected from the group comprising cephalosporins and penicillins.
  • the therapeutic preparation further comprises: (i) a first conjugate consisting of a first antibiotic covalently bound to non-specific
  • immunoglobulin and (ii) a second conjugate consisting of a second antibiotic covalently bound to non-specific immunoglobulin (e.g., where the first antibiotic is polymyxin and the second antibiotic is vancomycin or bacitracin).
  • a second antibiotic covalently bound to non-specific immunoglobulin e.g., where the first antibiotic is polymyxin and the second antibiotic is vancomycin or bacitracin.
  • two different antibiotics are covalently bound to the same immunoglobulin molecule, one capable of binding to gram-positive organisms and the other capable of binding to gram-negative organisms.
  • the present invention contemplates a method of treatment, comprising: (a) providing a mammal for treatment; (b) providing a therapeutic preparation, comprising an endotoxin- binding compound covalently bound to protein; and (c) administering the preparation to the mammal (e.g., intravenous).
  • the endotoxin-binding compound may be polymyxin and the protein is preferably non-specific immunoglobulin such as IgG.
  • the treatment with the antibodiotic is expected to have many of the effects of the antibiotic alone - however, without the toxicity and short half-life typically associated with these agents. Furthermore, these conjugates are expected to possess the opsonizing function of immunoglobulin which may facilitate clearance of both the toxin and organism.
  • the present invention contemplates a method of treatment of mammals at risk for developing sepsis, in which a therapeutic preparation comprised of an antibiotic capable of binding to a microorganism covalently bound to a non-specific immunoglobulin is
  • the method of the present invention will be administered intravenously.
  • the present invention contemplates that the method will be used for such animals as neonatal calves and foals, as well as human and veterinary surgical patients, trauma, and burn victims. It is contemplated that the method will be used to treat immunocompromised patients.
  • the present invention will be useful for the treatment of mammals potentially exposed to gram-negative and/or gram-positive bacteria. It is contemplated that the therapeutic preparation used in the method of the present invention is capable of binding endotoxin.
  • the present invention further contemplates a method of treatment of mammals infected with a pathogenic organism, wherein a therapeutic preparation, comprising a surface-active antibiotic covalently bound to a non-specific immunoglobulin G having an Fc region capable of mediating opsonization of the pathogenic organism is administered.
  • a therapeutic preparation comprising a surface-active antibiotic covalently bound to a non-specific immunoglobulin G having an Fc region capable of mediating opsonization of the pathogenic organism is administered.
  • the infecting pathogen is a gram-negative or gram-positive bacterial organism.
  • the surface-active antibiotic used in the therapeutic preparation is a polymyxin (e.g., polymyxin B).
  • One embodiment of the present invention contemplates a method of diagnosis, comprising: (a) an antigen associated with the surface of a pathogenic organism immobilized to a solid support; (b) a conjugate comprising a surface-active antibiotic covalently bound to a non-specific immunoglobulin; and (c) a competitor comprising the surface antigen present in solution.
  • the immobilized antigen is incubated with the conjugate in the presence of the competitor, washed to remove unbound conjugate and competitor, followed by detection of the conjugate bound to the immobilized surface antigen.
  • the present method of diagnosis comprises immobilization of surface antigen in the well(s) of a microtiter plate. It is also contemplated that the surface antigen of the method is isolated from bacterial organisms. It is contemplated that the surface antigen be isolated from such gram-negative bacteria as Escherichia coli, (e.g.,
  • lipopolysaccharide the competitor in the present method of diagnosis is comprised of lipopolysaccharide from gram-negative bacteria. It is further contemplated that the competitor will be comprised of lipopolysaccharide from such gram- negative bacteria as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Vibrio cholerae, Shigella flexneri, Klebsiella pneumoniae, Salmonella enter itidit is, Serratia marcescens and Rhodobacter sphaeroides.
  • the present invention also contemplates a method of synthesizing a conjugate comprising the steps of: a) reacting an antibiotic with a crosslinking agent to form a derivatized antibiotic; and b) reacting the derivatized antibiotic with non-specific
  • the antibiotic will bind to the surface of microorganisms.
  • the antibiotic is a peptide.
  • the peptide is Limulus antilipopolysaccharide factor, in another, the peptide is a D-amino acid-containing peptide. It is also contemplated that the peptide binds endotoxin.
  • the antibiotic is a polymyxin such as polymyxin B.
  • the antibiotic used is bactericidal to gram- negative bacteria.
  • the antibiotic is bactericidal to gram-positive bacteria.
  • the antibiotic is vancomycin.
  • the non-specific progenitor is bactericidal to gram-negative bacteria.
  • the antibiotic is bactericidal to gram-positive bacteria.
  • the antibiotic is vancomycin.
  • the non-specific progenitor is bactericidal to gram-negative bacteria.
  • the antibiotic is bactericidal to gram-positive bacteria.
  • the antibiotic is vancomycin.
  • immunoglobulin consists of an Fc region.
  • the present invention also contemplates a method of synthesizing a conjugate comprising the steps of: a) reacting an antibiotic with a first bifunctional crosslinking agent to form a derivatized antibiotic; b) reacting non-specific immunoglobulin with a second bifunctional crosslinking agent to form a derivatized immunoglobulin; and c) reacting the derivatized antibiotic with the derivatized immunoglobulin to form a covalent bond between the derivatized antibiotic with the derivatized immunoglobulin to form a conjugate.
  • the antibiotic binds to the surface of
  • the antibiotic is a peptide, such as (but not limited to) Limulus antilipopolysaccharide factor
  • the peptide is a D-amino acid-containing peptide.
  • the peptide binds endotoxin.
  • the antibiotic is a polymyxin, such as (but not limited to) polymyxin B.
  • the antibiotic is bactericidal to gram-negative bacteria. In another embodiment, the antibiotic is bactericidal to gram-positive bacteria. In a preferred embodiment, the antibiotic is vancomycin.
  • the first and second bifunctional crosslinking agents are N-succinimidyl 3-(2-pyridyldithio) propionate. In another embodiment of the method, the first bifunctional crosslinking agent is S-acetylmercaptosuccinic anhydride. In an additional embodiment, the second bifunctional crosslinking agent is sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
  • the present invention also contemplates a method of synthesizing a conjugate comprising the steps of: a) providing in any order: i) a crosslinking agent having first and second reactive sites, with the first site being exposed, active, and reactive with primary amino groups, and the second site being blocked by a cleavable group; ii) an antibiotic having one or more primary amino groups; and iii) non-specific immunoglobulin having one or more primary amino groups; b) reacting in any order: i) the crosslinking agent with the antibiotic, forming a blocked derivatized antibiotic; and ii) the crosslinking agent with immunoglobulin, forming a blocked derivatized immunoglobulin; c) reacting in any order: i) the blocked derivatized antibiotic with a modifying reagent, forming a free derivatized antibiotic; ii) the blocked derivatized immunoglobulin with a modifying reagent, forming a free derivatized immunoglobulin;
  • the antibiotic binds to the surface of microorganisms.
  • the antibiotic is a peptide, such as (but not limited to) Limulus antilipopolysaccharide factor.
  • the peptide is a D- amino acid-containing peptide.
  • the peptide binds endotoxin.
  • the antibiotic is a polymyxin, including (but not limited to) polymyxin B.
  • an antibiotic bactericidal to gram-negative bacteria is used in the method of the present invention.
  • the antibiotic is bactericidal to gram-positive bacteria.
  • the antibiotic is vancomycin.
  • the non-specific immunoglobulin consists of an Fc region.
  • the modifying reagent is a reducing agent.
  • the reducing agent is dithiothreitol.
  • the present invention further contemplates a method of synthesizing a conjugate comprising the steps of: a) providing in any order: i) a first crosslinking agent having first and second reactive sites, with the first site being reactive with primary amino groups, and the second site being reactive with maleimide groups; ii) an antibiotic having one or more primary amino groups; iii) a second crosslinking agent having first and second reactive sites, with the first site being reactive with primary amino groups, and the second site being reactive with sulfhydryl groups; and iv) non-specific immunoglobulin having one or more primary amino groups; b) reacting in any order: i) the first crosslinking agent with the antibiotic, forming a derivatized antibiotic; and ii) the second crosslinking agent with the immunoglobulin, forming a derivatized immunoglobulin; and c) reacting the derivatized antibiotic with the derivatized immunoglobulin to form a conjugate.
  • the first crosslinking agent is bifunctional.
  • the first bifunctional crosslinking agent is S-acetylmercaptosuccinic anhydride.
  • the second crosslinking agent is bifunctional. In one embodiment, the second bifunctional crosslinking agent is
  • the present invention also contemplates a method of synthesizing a conjugate comprising the steps of: a) reacting a non-specific immunoglobulin with a first modifying reagent to form an oxidized immunoglobulin preparation;and b) reacting the oxidized immunoglobulin preparation with an antibiotic and a second modifying reagent to form an antibiotic-immunoglobulin conjugate.
  • the immunoglobulin consists of an Fc region.
  • the first modifying reagent is an oxidizing agent.
  • the oxidizing agent is periodate.
  • the method includes a second modifying reagent which is a reducing agent.
  • the reducing agent is sodium borohydride.
  • the present invention also contemplates a method of synthesizing a conjugate comprising the steps of: a) reacting an antibiotic precursor with a first crosslinking agent, the antibiotic precursor possessing limited bactericidal activity to form a derivatized antibiotic precursor, and a derivatized antibiotic precursor possessing increased bactericidal activity; b) reacting non-specific immunoglobulin with a second crosslinking agent, to form a derivatized immunoglobulin; and c) reacting the derivatized antibiotic precursor with derivatized immunoglobulin to form a covalent bond between the derivatized antibiotic precursor and derivatized immunoglobulin to form a conjugate.
  • the antibiotic precursor is selected from the group consisting of 7-aminocephalosporanic acid and 6-aminopenicillanic acid.
  • the first crosslinking agent is bifunctional. In one preferred embodiment, the first bifunctional crosslinking agent is m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester. In another embodiment the second crosslinking agent is bifunctional. In one preferred embodiment, the second bifunctional crosslinking agent is iminothiolane.
  • the present invention further contemplates a method of synthesizing a conjugate comprising the steps of: a) providing in any order: i) a first crosslinking agent having first and second reactive sites, the first site being reactive with primary amino groups, and the second site being reactive with sulfhydryl groups; ii) an antibiotic precursor having one or more primary amino groups, with the antibiotic precursor possessing limited bactericidal activity; iii) a second crosslinking agent having first and second reactive sites, the first site being reactive with primary amino groups, and the second site being reactive with maleimide groups; and iv) non-specific immunoglobulin having one or more primary amino groups; b) reacting in any order: i) the first crosslinking agent with the antibiotic precursor, forming a derivatized antibiotic precursor; ii) the second crosslinking agent with the immunoglobulin, forming a derivatized immunoglobulin; c) reacting the derivatized antibiotic precursor with the derivatized immunoglobulin to form
  • the antibiotic precursor is selected from the group consisting of 7-aminocephalosporanic acid and 6-aminopenicillanic acid.
  • the first crosslinking agent is bifunctional.
  • the first bifunctional crosslinking agent is m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester.
  • the second crosslinking agent is bifunctional.
  • the second bifunctional crosslinking agent is iminothiolane.
  • Figure IA schematically shows the design of an antibodiotic of the present invention.
  • Figure IB schematically shows the design of another antibodiotic of the present invention.
  • Figure 2 schematically shows a means of screening modified antibiotics for antibacterial activity.
  • Figure 3 outlines an alternative method by which new antibiotics can be screened for use as compounds for conjugation with immunoglobulins.
  • Figure 3A shows a means by which the minimum concentration for bacterial growth inhibition is established.
  • Figure 3B shows a means by which a new antibiotic can be assessed for bactericidal activity.
  • Figure 4 describes solid phase assays for determining the level of binding of antibodiotics of the present invention.
  • Step 1 shows toxin or organisms in a testing microwell.
  • Step 2 schematically represents the binding of antibodiotic.
  • Step 3 schematically shows the binding of secondary reagents.
  • Figure 5 shows conjugates of the present invention binding to LPS, as measured by ELISA.
  • Figure 6 shows additional conjugates of the present invention binding to LPS, as measured by ELISA.
  • Figure 7 shows inhibition of LPS binding of conjugates of the present invention using free polymyxin (PMB), as measured by ELISA.
  • PMB free polymyxin
  • Figure 8 shows periodate conjugates of the present invention binding to LPS, as measured by ELISA.
  • Figure 9 shows inhibition of LPS binding of conjugates of the present invention using LPS of various bacterial species, as measured by ELISA.
  • Figure 10 shows the binding of conjugates of the present invention to phagocytic cells in a radioactive competition assay.
  • Figure 1 1 shows the pharmacokinetic profile of intravenously administered PMB-HlgG and HIgG in rabbits expressed in absorbance at 410 nm.
  • Figure 12 shows the pharmacokinetic profile of intravenously administered PMB-HlgG and HIgG in rabbits expressed in IgG concentration.
  • Figure 13 shows the sequence of Limulus antilipopolysaccharide factor (LALF), a single chain peptide known to bind and neutralize endotoxin.
  • LALF Limulus antilipopolysaccharide factor
  • the present invention relates to therapeutics for the prevention and treatment of blood- borne and toxin mediated diseases, and in particular the prevention and treatment of sepsis caused by various types of organisms in humans as well as other animals.
  • the present invention is particularly suited for the in vivo neutralization of the effects of endotoxin.
  • the present invention will be used in the treatment of gram- negative and gram-positive sepsis.
  • the invention may be used for treatment of sepsis due to one organism, it may also be used to treat sepsis caused by multiple organisms (e.g., sepsis and/or bacteremia due to gram-negative and gram-positive organisms).
  • the present invention also contemplates treatment comprising multiple antibody-antibiotic conjugates used in combination. It is also contemplated that the present invention will be used to treat bacteremia, viremia or fungemia, by enhancing the removal of organisms by opsonization.
  • soluble antibody-antibiotic conjugates or "antibodiotics” are administered intravenously, intra-muscularly, subcutaneously,
  • the conjugate is water-soluble if it has a solubility in physiologic saline of at least 0.1 mg/ml, and preferably of at least 1.0 mg/ml, when measured at room temperature.
  • antibodiotics provide a low cost, reasonably effective and needed preventive as well as treatment. Antibodiotics can suppress fungal and viral infection.
  • antibodiotics suppress bacteremia as well as endotoxin-mediated effects.
  • Antibodiotics with long (e.g., days to weeks) duration of action are easily administered.
  • the invention encompasses antibodiotics with reactivity against gram- negative organisms as well as antibodiotics with reactivity to gram-positive organisms, a wider spectrum of protection is expected than any other known approach.
  • diagnostic applications include methods to detect LPS from particular organisms or the surface structures present on organisms which are recognized by antibiotics (i,e., the receptors expressed on cell surfaces which bind antibiotic).
  • Section III describes the use of antibodiotics for: (A) Prophylactic Use in Humans; (B) Acute Therapy in Humans; and (C) Veterinary Care.
  • antibodiotics all types of antibody (e.g., IgG, pentameric and monomeric
  • IgM secretory and monomeric IgA, IgE and IgD
  • Table 1 compares the characteristics of IgG and IgM. While IgM has the advantage of better opsonization and complement activation, IgG has a longer half-life in vivo and can be raised to higher titers because of the fact that it is the primary antibody raised during secondary responses to antigen. Consequently, the preferred antibody for conjugation according to the present invention is IgG.
  • antigen-specific IgG can be employed (e.g., bacteria-seeking antibodies), antigen-specificity may result in a shorter half-life of the compound (and/or greater cost). Consequently, the preferred antibody is non-specific.
  • Goers et al (U.S. Patent No. 4,867,973) describe the use of antibody conjugated to antimicrobials, but with antigen-specific antibody. In contrast, the conjugates of the present invention utilize non-specific antibody. Goers et al describe in particular the conjugation to antigen-specific monoclonal antibodies. Monoclonal antibodies have not been a step forward in the prevention and/or treatment of bacteremia and sepsis. While these preparations should possess greater potency and specificity than polyclonal sera, they are: a) prohibitively expensive; b) frequently immunogenic; and c) exhibit unusually short circulating half-lives (typically less than 24 hours).
  • Centoxin a commercially produced antigen-specific monoclonal antibody
  • the price was approximately $3,700.00 per 100 mg dose.
  • Pharmacoeconomic analysis indicated that - even if the product was used under strict guidelines for acute cases - "its use could add $2.3 billion to the nation's health care budget.” [K.A. Schulman et al., JAMA 266:3466-3471 (1991).]
  • the expense of Centoxin is such that it simply could not be used prophylactically.
  • the conjugates of the present invention are produced from materials costing a fraction of this figure (e.g., $2.00 per 100 mg dose) because of the readily available inexpensive source of pooled donor IgG.
  • Non-specific IgG is easily and cheaply obtained, requiring no immunization and eliciting no immune response in a syngeneic setting.
  • Non-specific IgG does not have the standardization problems of antigen-specific antibody. Simply put, there is no antigen-specific titer to be concerned about (let alone variability in the titer from unit to unit). Rather, standardization comes from the conjugated ligand; conjugation of non-specific IgG results in >1000-fold increase in LPS- binding titer and by standardization of the ligand that is attached, one standardizes the activity of the therapeutic.
  • non-specific IgG unlike monoclonals, has a long half-life needed for a prophylactic (compare the >21 day half-life of pooled polyclonal human IgG with the mean serum half-life of 16 hours for the human monoclonal antibodies discussed above).
  • IgG For purposes of expense, IgG from donors (i.e., human and animal) rather than cell lines is desirable. In this regard, typically large pools of plasma are used as starting material. Large scale fractionation techniques known in the art include ethanol precipitation and precipitation with high concentrations of salt. [See H.F. Deutsch in Methods in Immunology and Immunochemistry, (CA. Williams and M.W. Chase, eds.), Academic Press, New York, pp. 315-321 (1967).] There is also the somewhat complicated procedure where the immunoglobulin is isolated from Cohn Effluent III by diafiltration and ultrafiltration. [See E.J. Cohn et al., J. Am. Chem Soc. 68:459-475 (1946).]
  • Each milliliter (ml) contains approximately 50 mg of protein, of which not less than 98% has the electrophoretic mobility of gamma globulin. Not less than 90% of the gamma globulin is monomeric. There are traces of IgA and IgM. The distribution of IgG subclasses is similar to that found in normal serum.
  • the commercial product displays a broad spectrum of opsonic and neutralizing antibody activities.
  • IgG antibodies are immediately available in the recipient's circulation.
  • the in vivo half-life equals or exceeds the three week half-life reported for IgG in the literature. It is therefore quite acceptable for use in the preparation of antibody-antibiotic conjugates of the present invention.
  • an antibodiotic sensitivity test performed before administration of the antibody-antibiotic conjugates of the present invention to humans. This can be done by subcutaneously injecting a small amount of the conjugate in the arm of the patient. A salt solution is injected in the other arm as a control. Normally, a positive
  • hypersensitivity test is indicated by no more than formation of a welt on the skin surface with surrounding swelling.
  • the usual dosage of the commercial intravenous immunoglobulin product is 100-200 mg/kg (2-4 ml/kg) of body weight administered approximately once a month by intravenous infusion.
  • the dosage may be given more frequently or increased as high as 400 mg/kg (8 ml/kg) body weight, if the clinical response is inadequate, or the level of IgG achieved in the circulation is felt to be insufficient.
  • the present invention contemplates a typical dosage for antibodiotics that is much less than that given for the commercial immunoglobulin preparations. This is particularly true where the number of conjugated antibiotic molecules exceeds one (1) per immunoglobulin molecule.
  • the present invention contemplates a conjugate dosage range of 0.1-100 mg/kg, and a preferred range of 1-20 mg/kg.
  • the amount of PMB (assuming 3 molecules per IgG molecule) contained in a dose for this preferred range will be 0.025 - 0.5 mg/kg.
  • antibody-antibiotic conjugate In the design of antibody-antibiotic conjugate, a primary consideration is the mode of action of the antibiotic. Since the conjugates will be much larger molecules than the parent antibiotics, only antibiotics that bind to exposed or secreted components (e.g., toxins) of the bacteria, fungus, virus, or parasite are likely to target the antibody carrier to the pathogen or its products. For example, penicillin antibiotics disrupt bacterial cell wall synthesis and bind to surface-exposed components of certain bacteria whereas aminoglycoside antibiotics commonly bind to ribosome subunits in the cell cytoplasm. The former is a much better candidate for effective antibody-antibiotic conjugates than the latter.
  • exposed or secreted components e.g., toxins
  • Antibiotics vary greatly in the type and species of organisms upon which they are active. For example, certain antibiotics such as the polymyxins are far more effective against gram-negative bacteria, whereas other antibiotics such as vancomycin tend to be more effective against gram-positives. Some, like the cephalosporins, and broad-spectrum penicillins are comparably effective against both types. Other antibiotics, such as
  • amphotericin are primarily antifungal agents whereas amantadine exhibits activity against certain influenza viruses.
  • antibody-antibiotic conjugates for the prevention or treatment of disease one must consider the spectrum of antibiotic activity desired and select those antibiotic(s) that are active against the target pathogen(s) and, as described above, act primarily on exposed components of the pathogen(s).
  • pathogen refers to any organism which is associated with infection or disease, whether that organism is among those traditionally considered pathogens (e.g., S. aureus, S. pyogenes, S. dysenteriae, S. flexneri, etc.) or is an opportunistic pathogen (e.g., P. aeruginosa, S. marcesens, S. mitis, etc.).
  • antibiotics e.g., penicillins, cephalosporins, polymyxins
  • these structural differences within an antibiotic family are important from two perspectives. First, the activity spectrum may influence the choice of antibiotic; and, second, the chemical differences between antibiotics will influence the range of cross- linking chemistries available to conjugate the antibiotic.
  • variable side chain component of penicillin antibiotics is a methyl benzyl group in penicillin G but the variable side chain group is a phenolic group with a primary amine side chain in amoxicillin.
  • the latter antibiotic presents a wider array of potential modes for cross-linking than does penicillin G.
  • a preferred antibiotic of the present invention is polymyxin B (PMB). As noted above, this antibiotic binds to and neutralizes endotoxin. However, when used in vivo, PMB is short-lived, and furthermore, at the recommended therapeutic dose for systemic infections, there is a significant risk of nephrotoxicity.
  • PMB polymyxin B
  • Centoxin-HA-IA is capable of binding endotoxin and neutralizing its biological activity.
  • the monoclonal antibody is costly and suffers from low affinity and short half-life. The latter characteristics may explain why the human clinical studies have yet to yield clear benefits.
  • dextran has a half-life in humans of only about a day.
  • immunoglobulin according to the present invention, a much longer half-life is achieved (see Table 4 and Examples 24 and 25).
  • Dextran having no
  • Fc receptor also has no known capacity to promote opsonization or activate
  • the present invention contemplates the use of conjugates which are effective against the organisms of interest, yet are non-toxic to the host.
  • the non-toxic character of IgG-PMB is demonstrated in Example 27.
  • the present invention also contemplates antibodiotics having reactivity with gram-positive organisms and their toxins.
  • the present invention contemplates the use of bacitracin conjugated to immunoglobulin.
  • Bacitracin is a polypeptide produced by a strain of Bacillus subtil is (Tracy strain), which is primarily bactericidal for gram-positive organisms including Streptococcus pyogenes, other ⁇ -haemolytic streptococci, Pneumococcus pneumoniae, and certain strains of Clostridium species. Bacitracin exerts its effect by inhibiting early steps in the biosynthesis of Bacillus subtil is (Tracy strain), which is primarily bactericidal for gram-positive organisms including Streptococcus pyogenes, other ⁇ -haemolytic streptococci, Pneumococcus pneumoniae, and certain strains of Clostridium species. Bacitracin exerts its effect by inhibiting early steps in the biosynthesis of
  • bacitracin is stable and poorly absorbed from the intestinal tract or wounds. Because of the proteinuria, hematuria and nitrogen retention observed upon systemic administration, its use is usually restricted to topical application.
  • R. Berkow and A.J. Fletcher eds.
  • the Merck Manual 16th ed., 1992, p. 46; and G.F. Brooks et al., Jawetz, Melnick & Adelberg 's Medical Microbiology, 19th ed., 1991 , pp. 172-173.
  • bacitracin Despite the unacceptable occurrence of nephrotoxicity associated with systemic administration of free bacitracin, when it is conjugated to immunoglobulin according to the present invention, the advantages of bacitracin can be achieved without this side-effect. It is not intended that the present invention be limited by the mechanism of action of any particular antimicrobial.
  • Vancomycin is active, principally, against gram-positive organisms including
  • the method involves conjugating the vancomycin to non-specific immunoglobulin by first treating the vancomycin and the immunoglobulin with different heterobifunctional crosslinking agents, and second reacting the derivatized species with each other to form a conjugate.
  • the method involves conjugating the vancomycin to non-specific immunoglobulin by first reacting the same heterobifunctional crosslinking agent with both the vancomycin and the non-specific immunoglobulin, then second reacting both derivatized species with each other forming a conjugate.
  • crosslinker combinations For both synthetic schemes a variety of crosslinker combinations have been contemplated and tested. Below is a table which lists the crosslinking compounds which have been tested to date for reaction with vancomycin.
  • crosslinking agents upon reaction with vancomycin, were insoluble in aqueous solution and were not further pursued. It is recognized, however, that should steps be taken to render them soluble (e.g. addition of solvents, further side group modification of the base vancomycin structure, etc.) that such crosslinking agents could prove useful.
  • the table describes the crosslinking approach, the group on the modified vancomycin that is reactive ("reactive group") with either immunoglobulin or the corresponding linker on immunoglobulin (if any), the solubility, and the biological activity of the conjugate.
  • reactive group the group on the modified vancomycin that is reactive
  • solubility the group on the modified vancomycin that is reactive
  • biological activity of the conjugate the group on the modified vancomycin that is reactive
  • antibodies have many reactive groups that can be used in direct conjugation schemes (amino acids containing primary amine, carboxyl, hydroxyl, thiol [after reduction]) or modified groups (glycosylated amino acids that can be oxidized to aldehyde; or primary amines that can be made thiol- reactive) for conjugation schemes.
  • reactive groups that can be used in direct conjugation schemes (amino acids containing primary amine, carboxyl, hydroxyl, thiol [after reduction]) or modified groups (glycosylated amino acids that can be oxidized to aldehyde; or primary amines that can be made thiol- reactive) for conjugation schemes.
  • Individual antibiotics will not, in general, possess very many different reactive groups and offer fewer choices for conjugation to antibodies.
  • the selection of an antibiotic from a family of related compounds and the selection of a cross- linking scheme must take into consideration the reactive groups on an antibiotic.
  • a key concern in modifying an antibiotic is the preservation of its ability to bind to the surface or secreted products of a pathogen.
  • the modification of individual reactive groups or excessive modification of more than one reactive group with cross-linking agents, or the steric hindrance created by attachment to a large protein such as immunoglobulin may abolish antibiotic activity. Therefore, before conjugate activity is considered, conditions for preservation of antibiotic activity must be determined by examining the biological activity of the modified or cross-linked antibiotic in simple antimicrobial assays. Preferably, one chooses a cross-linker type and concentration that preserves antibiotic activity.
  • cross-linkers may influence the activity of individual antibiotics and the efficiency with which they are conjugated to antibodies.
  • the discovery of more optimal cross-linkers relies on the empirical analysis of conjugates prepared using varying concentrations of different cross-linkers.
  • the in vivo safety and efficacy of antibody-antibiotic conjugates will depend upon their activity, toxicity and stability.
  • the selection of the cross-linking agent may also affect these aspects of conjugate performance.
  • the cross-linker employed may affect the properties of the antibody. Effector functions dependent upon the Fc region of the antibody such as opsonization or complement fixation may be influenced by which reactive groups are utilized and their location on the antibody molecule.
  • some cross-linkers may cause adverse reactions by eliciting an immune response to the haptenic groups on the cross- linker.
  • the in vivo stability of the bonds created by the cross-linking scheme may vary in important ways. Disulfide bonds linking the antibiotic and antibody may not be as stable, for example, as amide bonds created by other cross-linkers. Dissociation between antibody and antibiotic may not be tolerable in cases where long-term prophylaxis is desired.
  • Antibody analogues are those compounds which act in an analogous manner to antibodies.
  • the present invention contemplates fragments of antibodies (e.g., Fc fractions) to make antibody-antibiotic conjugates.
  • Fc fractions fragments of antibodies
  • immunoglobulin are meant to include antibody analogues.
  • Antibiotic compounds have been isolated from many different microbial, plant, and animal sources and new promising compounds continue to be discovered.
  • synthetic derivatives of natural compounds as well as wholly synthetic compounds such as small peptides are also being screened for antibiotic activities in many laboratories.
  • antibiotic refers to any chemical compound which destroys, inhibits the growth of, or binds to microorganisms (i.e., "antimicrobials"). It is not intended that the term
  • Antibiotic therefore includes compounds which are produced synthetically, as indeed many of the antibiotics are now produced in the chemistry lab rather than by microorganisms. Polymyxin and other compounds discussed herein may be produced synthetically or obtained from
  • Figures 2-4 outline the methods by which new antibiotics can be screened for use as compounds for conjugation with immunoglobulins.
  • the "Screening Modes” consist of the following temporal steps: Mode 1: Conjugate the antibiotic to a cross-linker only and then assess for inhibition of organism growth in liquid culture and on a disc inhibition lawn assay (e.g., Kirby-Bauer).
  • Mode IIA Conjugate the antibiotic via the cross-linker to immunoglobulin and then assess for binding to bacteria and bacterial toxin by a solid phase assay.
  • Mode IIB Conjugate the antibiotic to immunoglobulin without the use of a cross- linker (e.g., periodate oxidation of the carbohydrate groups ["CHO"] of IgG) and then assess for binding to bacteria and bacterial toxin by a solid phase assay.
  • a cross- linker e.g., periodate oxidation of the carbohydrate groups ["CHO"] of IgG
  • Mode III Check specificity of the antibodiotic by inhibition of bacterial toxin binding with the antibiotic.
  • Mode IV Assess the antibodiotic for inhibition of organisms growth in liquid culture.
  • NCLS National Committee for Clinical Laboratory Standards
  • antibiotic X may initially be evaluated by Mode I.
  • Mode I In this Mode, X is only conjugated to a cross-linker "c" to create "X-c"; this compound is then added to a liquid or solid phase culture.
  • a cross-linker "c" to create "X-c”
  • This compound is then added to a liquid or solid phase culture.
  • Mode I only addresses compatibility of "X" with the conjugation chemistry.
  • the assay is performed and the results are compared to an identical assay of unconjugated antibiotic X.
  • an agar-filled petri dish is inoculated with the organism (Step 1, Figure 2).
  • a small filter-paper disc containing a known amount of antibiotic X or X-c is placed on the agar surface and allowed to diffuse into the medium over an 18- to 24-hr period (Step 2, Figure 2). After this incubation, a zone of growth inhibition is apparent with X and this is compared to the zone (if any) achieved with X-c (Step 3, Figure 2).
  • MIC bactericidal activity
  • an aliquot is taken from a tube showing bacteriostatic activity, and this aliquot is added to agar plates ( Figure 3B). If growth occurs, then the agent is bacteriostatic; if no growth occurs, the agent is bactericidal.
  • the minimal bactericidal (lethal) concentration is the lowest concentration of X-c or X that produces a 99.9% reduction in organisms from the original inoculum of approximately 100,000 organisms. In this manner the minimum bactericidal concentration (“MBC”) is established.
  • Mode IIA If the activity of X-c is good, it is further evaluated in Mode IIA. If the activity of X- c is poor, X is evaluated in Mode IIB. Both Modes IIA and IIB contemplate covalent attachment; Mode IIA uses a cross-linker to create "X-c-Ig", while Mode IIB does not use a cross-linker and generates "X-CHO-Ig.” In both cases, the antibody-antibiotic conjugate, or simply the "antibodiotic", is assayed on a solid phase assay such as shown schematically in Figure 4.
  • Toxin or organisms may be used in the solid phase assay to coat a microwell or other appropriate surface (Step 1, Figure 4A).
  • the antibodiotic is then added to test for binding (Step 2, Figure 4A).
  • Standard washing procedures are used to avoid non-specific binding.
  • the antibody portion of the conjugate may thereafter serve as a target for secondary reagents (e.g., goat anti-human IgG antibody having an enzyme reporter group such as horseradish peroxidase) (see Step 3, Figure 4A).
  • An appropriate substrate for the enzyme may then be added (not shown) to generate a colorimetric signal.
  • X-c-Ig binding may be compared with that of X-CHO-Ig. Where the organism is used, care must be taken that binding is not via the Fc receptor of lg. Unconjugated lg can be used as a control for this purpose.
  • Mode IIA or Mode IIB conjugates showing reactivity in Mode IIA or Mode IIB should be evaluated in Mode III. As shown in Figure 4B, this simply involves adding free antibiotic to show that it will compete specifically for binding. The next portion of the evaluation involves testing the antibody-antibiotic conjugate for growth inhibition and/or bactericidal activity (Mode IV). This is the same assay as shown in Figure 2, the difference being that now the complete conjugate X-c-lg (or X-CHO-Ig) is evaluated rather than just the antibiotic (X-c).
  • Both X-c-Ig and X-CHO-Ig may show good toxin binding in Mode II but poor antibacterial activity in Mode IV. If the specificity of the binding is nonetheless confirmed in Mode III, these compounds are candidates for diagnostic reagents. Alternatively, they may be used in vivo simply to bind free toxin and thereby reduce toxin load.
  • antibody and “immunoglobulin” are meant to include antibody analogues.
  • Antibiotic precursors as herein defined are reactants used in the synthesis of semisynthetic antibiotics that possess limited in vitro and in vivo anti-microbial activity.
  • the present invention contemplates, however, that the "latent" anti-microbial potential of such compounds can be activated upon conjugation, according to the methods herein described, with immunoglobulin.
  • the method involves, as a first step, the unlocking of the antimicrobial potential of antibiotic precursors by derivatizing them with a crosslinking agent. These derivatized antibiotic precursors by themselves possess increased anti-microbial effects compared to the free antibiotic precursors.
  • conjugates are made between the derivatized antibiotic precursors and human immunoglobulin. Through this latter step, a conjugate is created that possesses the benefits exhibited by the earlier described conjugates (increased half life, reduced nephrotoxicity, etc.).
  • immunoglobulin is not simply an inert carrier.
  • the Fc portion of the antibody can mediate pathogen elimination by two mechanisms that are distinct from the effects of the antibiotic. First, it is known that following binding of antibody to antigen, the Fc region can activate the classical pathway of complement, ultimately resulting in the lysis of organisms. Second, binding of the conjugate to bacteria can lead to the ingestion or opsonization of the organism by recognition of the Fc region by phagocytes (e.g.,
  • the present invention contemplates antibody-antibiotic conjugates with the capability of binding Fc receptors on phagocytes. It is preferred that in competition binding, the binding of the antibody-antibiotic conjugates of the present invention to such cells is substantially similar to that of normal IgG.
  • the present invention contemplates antibody-antibiotic conjugates which, while not activating complement systemically, are capable of binding complement to facilitate pathogen killing. Furthermore, conjugates are contemplated which bind phagocytes via the Fc region to facilitate pathogen elimination. Thus, it is contemplated that the antibody-antibiotic conjugates will mediate or enhance opsonization and removal (opsonophagocytosis) of the etiologic agent(s) of sepsis in the treated patient.
  • the conjugate will be present in a background of the entire repertoire of host immune mediators.
  • immune mediators include, of course, humoral immune mediators such as endogenous antibodies directed against bacteria and their toxins.
  • the present invention contemplates, in one embodiment, determining the immune status of the host prior to administration of the antibodiotic. This determination can be made by screening potential risk groups for total and endotoxin core antigen-specific IgG and IgM levels. [B.J. Stoll et al. , Serodiagnosis and Immunotherapy 1 :21-31 (1987).] Screening is believed to be particularly important with the elderly, full-term and pre-term neonates [W. Marget et al., Infection 1 1 :84-86 (1983)], patients with malignancies [C. Stoll et al., Infection 13:115-119 (1985)], abdominal surgery candidates, individuals under long-term catheterization or artificial ventilation, and burn and other trauma victims.
  • the efficacy of the antibody-antibiotic conjugate is expected to be most dramatic. Where the host's immune status is good, use of the conjugate will support the endogenous anti-bacterial defenses.
  • the conjugate itself must be effective against clinically relevant organisms, non-toxic and non-immunogenic.
  • the conjugates of the present invention will be effective against gram-positive and gram-negative organisms which are commonly associated with sepsis (e.g., E. coli, K. pneumoniae, P.
  • conjugates will be non-toxic to the host animal. As with any chemotherapeutic, the conjugate must be effective against the infecting organisms, but not harm the host. In addition, in order to enhance the host's response to the infecting organism and to prevent such complications as serum sickness upon subsequent administration of conjugate, the conjugates themselves must be non-immunogenic. This characteristic permits the immune system of the host to focus on battling the infecting organisms, rather than attack the conjugates intended as treatment. As it is contemplated that these conjugates may be administered to the same animal multiple times
  • Conjugates which are non-immunogenic or poorly immunogenic due to high concentrations of D-configuration amino acids are also contemplated.
  • Synthetic polypeptides entirely comprised of D-amino acids are generally unable to elicit an immune response.
  • conjugation of a synthetic antimicrobial comprised entirely of D-amino acids to the antibody would be beneficial in the present invention.
  • antibodiotics in humans prior to the onset of symptoms (e.g., prophylactically).
  • the present invention contemplates the use of antibodiotics as a prophylactic in patients at high risk for infection, as well as sepsis.
  • Trauma patients are particularly difficult to examine because of the multitude of invasive procedures that they have undergone. Trauma patients are also typically hooked up to a number of devices, including intravascular lines, mechanical ventilators and Foley catheters. While every attempt is made to change intravascular lines, this is frequently impossible because of the extent of trauma and the lack of venous accessibility. [E.S. Caplan and N. Hoyt, Am. J. Med. 70:638-640 (1981).]
  • the present invention contemplates treating all trauma patients
  • the present invention contemplates administering antibodiotics immediately to the trauma patient upon admission. Indeed, the antibodiotics may successfully be used at the first moment that clinical care is available (e.g., emergency mobile care).
  • the use of the antibiotic-antibody conjugates of the present invention should protect the trauma patient during the entire period of risk.
  • Burn patients have many of the same problems with respect to the diagnosis and therapy for infection. Since the magnitude of thermal injury is related to the level of trauma in a burn victim, this even becomes more of a problem with acute cases.
  • therapy with the conjugates of the present invention is particularly appropriate immediately after the burn injury as a means of preventing a septic reaction. Furthermore, in severe cases, consideration should be given to the topical administration of antibodiotics to prevent wound sepsis.
  • Burn victims are exposed equally to both gram negative and gram positive organisms. Burn victims are particularly good candidates for therapeutic preparations having bactericidal activity for both gram-positive and gram-negative organisms.
  • This includes conjugates using a single antibiotic with reactivity for both groups of organisms (e.g., antibiotics such as a cephalosporin or broad-spectrum penicillin) and well as therapeutic "cocktail" preparations comprising: (i) a first conjugate consisting of a first antibiotic covalently bound to non-specific immunoglobulin; and (ii) a second conjugate consisting of a second antibiotic covalently bound to non-specific immunoglobulin (e.g., where the first antibiotic is polymyxin and the second antibiotic is bacitracin).
  • two different antibiotics can be covalently bound to the same immunoglobulin molecule.
  • the longer serum half- life of the conjugates of the present invention provide extended protection against sepsis without the expense of multiple dosing. Furthermore, since the distribution of
  • conjugates of the present invention may reduce the risk of disruption of endogenous flora. Consequently, the conjugates of the present invention may be used liberally (e.g., in more categories of surgical procedures).
  • the present invention also contemplates the use of antibodiotics in a therapeutic preparation for acute treatment.
  • treatment involves administration of the antibody-antibiotic conjugates after infection is detected and/or sepsis is suspected.
  • Evidence suggestive of gram-negative infection includes the following: (1) core temperature higher than 38°C or lower than 35°C; (2) peripheral blood leukocyte count greater than 12 x 10 9 /L or less than 3 x 10 9 /L (not due to chemotherapy), or at least 20% immature forms; (3) growth of gram-negative organisms from a blood culture drawn within the preceding 48 hours; or (4) documented or suspected site of gram-negative infection.
  • a systemic septic reaction is characterized by at least one of the following: arterial hypotension (systolic blood pressure ⁇ 90 mm Hg or an acute drop of 30 mm Hg); metabolic acidosis (base deficit >5 mEq/L); decreased systemic vascular resistance (systemic vascular resistance ⁇ 800 dynes/s ⁇ cm 5 ); tachypnea (respiratory rate >20/min or ventilation >10 L/min if mechanically ventilated); or otherwise unexplained dysfunction of the kidney (urine output ⁇ 30 ml/h), or lungs.
  • the antibodiotics of the present invention should ideally be used prior to a systemic infection, if possible.
  • the conjugates can be
  • conjugate(s) can be administered where there is an obvious sign of infection at a particular site (e.g., wounds, sinusitis, meningitis, respiratory, gastrointestinal, or urinary tract infections, etc.).
  • Primary bacteremia is typically defined as two or more blood cultures with the same bacterial organism occurring in a patient with no other obvious site of infection.
  • Sinusitis is diagnosed in a patient who has at least two of the following: purulent nasal discharge, roentgenographic evidence of sinusitis or purulent material aspirated from the sinuses.
  • the lower respiratory tract is a common site of infection.
  • Pneumonia in the intubated patient is diagnosed in a patient when there is fever, leukocytosis and a Gram stain with many polymorphonuclear leukocytes. Pneumonia may also be diagnosed in the patient with a new infiltrate that has not cleared with intensive physical therapy (this last criterion helps rule out atelectasis).
  • Septicemia and sepsis are by no means limited to human beings. Infection by gram- negative bacteria accounts for significant morbidity and mortality in neonatal livestock, such as calves. [D.D. Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986).] Interestingly, humoral immune status is again related to susceptibility to sepsis and this is largely dependent on passive transfer from colostrum. For this reason, the present invention contemplates, in one embodiment, determining the immune status of the animal prior to administration of the antibodiotic. This determination can be made by screening neonatal calves for total circulating serum immunoglobulin (e.g., by ELISA).
  • the conjugate should be used prophylactically. Where the animal's immune status is good, use of the conjugate may be needed for acute therapy of gram-negative bacterial sepsis, which remains prevalent in neonatal calves even with high antibody levels.
  • the present invention contemplates the treatment of other animals as well.
  • foals less than 10 days of age in critical distress sepsis is the most serious problem.
  • Symptoms highly indicative of sepsis risk include weakness, metabolic disturbance and dehydration.
  • the invention contemplates using antibodiotics for prophylactic treatment of foals less than 10 days of age having these indicators, or those at risk of infection.
  • the present invention therefore contemplates using antibodiotics for acute treatment of any animal with evidence of septicemia, with or without culture-proven cases.
  • the present invention contemplates using therapeutic compositions of soluble antibodiotics. It is not intended that the present invention be limited by the particular nature of the therapeutic preparation.
  • such compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients.
  • antibodiotics may be used together with other therapeutic agents, including unconjugated immunoglobulin.
  • these therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts.
  • the antibodiotics may be employed for intravenous, intramuscular, intrathecal or topical (including topical ophthalmic) administration.
  • Formulations for such administrations may comprise an effective amount of antibodiotic in sterile water or physiological saline.
  • formulations may contain such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • binders such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • binders such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbon
  • compositions are preferably prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the antibodiotics of the present invention are often mixed with diluents or excipients which are compatible and physiologically tolerable. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
  • purification of products from reactants is performed using various types of chromatography. Standard terms understandable to those skilled in the art are employed to describe this purification.
  • "eluent” is a chemical solution capable of dissociating desired products bound to the column matrix (if any) that passes through the column matrix and comprises an "eluate”. Products that are dissociated (if any) are freed from the column matrix and pass by elution with the "eluent" into the "eluate”.
  • EDC 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride
  • the example involved: (a) EDC-mediated cross-linking of PMB and IgG; and (b) enzyme-linked immunoassay (ELISA) of conjugate binding to LPS.
  • samples of the conjugates prepared in (a) above were diluted in PBS containing 1 mg/ml BSA to an initial concentration of 10 ⁇ g/ml IgG followed by five-fold dilutions.
  • a positive control antiserum of commercially prepared rabbit anti-E. coli 0111 :B4 antiserum was initially diluted 1 :100.
  • the wells incubated with the human antibody conjugates were incubated with 100 ⁇ l of a 1 :500 dilution of goat anti-human IgG (whole molecule)-alkaline phosphatase conjugate (Sigma) and the wells incubated with the rabbit serum were incubated with 100 ⁇ l of a 1 :500 dilution of goat anti-rabbit IgG (whole molecule)-alkaline phosphatase conjugate (Sigma) for 2 hours at room temperature.
  • Pyrogen-free PBS was prepared in pyrogen-free water (Baxter), and stock solutions of human IgG (40 mg/ml) and PMB (40 mg/ml) were dissolved in pyrogen-free PBS.
  • a 60 mM stock solution of DSS was prepared in 100% dimethylsulfoxide (DMSO). This solution was diluted to 6.0 mM DSS in PBS where some precipitation was noted.
  • a stock solution of human IgG and PMB was prepared containing 20 mg/ml IgG and 20 mg/ml PMB in PBS.
  • the ELISA was performed essentially as in Example 1(b) using the DSS conjugates at starting concentrations of 10 ⁇ g/ml and the same control rabbit anti-E. coli 01 1 1 :B4 antiserum.
  • the results of the initial binding assay are shown in Table 10.
  • cross-linkers were present in molar excess over IgG and were mixed simultaneously with both antibody and antibiotic.
  • IgG was first modified with the cross-linker, the cross-linker removed, and then PMB added to the coupling reaction. In this way, the binding activity of PMB might be improved and the non-specific binding of the IgG reduced.
  • BS 3 Pierce
  • a water soluble analogue of DSS was employed.
  • a 0.75 ml of a 4 mg/ml IgG solution in MES buffer was prepared as described in Example 1, and mixed with 0.75 ml of a 0.4 M EDC solution in MES buffer at room temperature for 2 hours.
  • the unreacted cross-linker was removed by passing the 1.5 ml reaction mixture over a Sephadex G-10 (Pharmacia) column that was poured into a sterile 10 ml pipette and equilibrated with pyrogen-free MES buffer.
  • the void volume was collected and the IgG content was determined by measuring the OD 280 of a 1 :40 dilution of each fraction.
  • the peak fraction containing 2.37 mg IgG/ml was divided into two fractions: 1.5 mg of PMB was added and dissolved in one volume; nothing was added to the other
  • the 0.2 M EDC IgG-PMB conjugate exhibited a high level of binding but this was partly due to non-specific binding as evidenced by the binding to control wells containing no LPS. Further evidence of non-specific binding created by EDC cross-linking is shown by the results for the conjugate containing no PMB (which exhibited somewhat comparable levels of binding to the wells regardless of whether antigen was present or not).
  • the BS 3 conjugates exhibited no specific binding to LPS whatsoever at the
  • the second step of this procedure involved preparation of malemide-activated PMB.
  • the third step of the procedure involved incubation of 0.65 ml of the reduced IgG with 0.65 ml of the SMCC-activated PMB.
  • the concentrations of the two reactants were 0.0265 mM PMB and 0.013 mM IgG (a
  • the LPS binding assay procedure was the same as that described in Example 1(b) except that the LPS was coated at 2 ⁇ g/well, the BBS-Tween 20 washes were eliminated, and the Tween 20 concentration in the PBS-Tween 20 wash was lowered to 0.05%.
  • the blocking solution and sample diluent were prepared using pyrogen-free PBS and low-endotoxin BSA (Sigma). The results are shown in Table 16.
  • the SMCC IgG-PMB exhibited slightly higher binding to LPS than the control but the overall level of binding was far below that of the positive control rabbit anti-E. coli 01 1 1 :B4 antiserum (1.097 at a 1 :25,000 dilution). It is possible that reduced IgG possesses only a few thiol groups available for cross-linking and that higher concentrations of activated PMB might drive the reaction more effectively.
  • coli HB101 was plated on Trypticase Soy Agar (TSA; BBL) to create a confluent lawn of bacteria.
  • TSA Trypticase Soy Agar
  • BBL Trypticase Soy Agar
  • One-quarter inch blank paper discs (BBL) were then applied to the surface of the lawn and 20 ⁇ l of each test solution applied. After incubation at 37°C overnight, zones of inhibition surrounding the disc were observed.
  • TSA Trypticase Soy Agar
  • BBL One-quarter inch blank paper discs
  • zones of inhibition surrounding the disc were observed.
  • the results indicate that PMB derivatized at 2:1 or 3: 1 molar ratios of SPDP-PMB were still active whereas antibiotic derivatized at a 4:1 molar ratio was inactive. Therefore, derivatization of PMB with SPDP should be carried out at ratios of SPDP to PMB of less than or equal to 3:1.
  • conjugates were prepared between SPDP-PMB and IgG by reacting the derivatized antibiotic with IgG to which sulfhydryl (-SH) groups were introduced with Traut's reagent.
  • SPDP (2.1 mg) in 50 ⁇ l of dimethyl-sulfoxide to 10 mg of PMB in 1 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0 and incubating at room temperature for 30 minutes on a rotating shaker.
  • the unconjugated cross-linker was removed by applying the sample to 15 ml.
  • Swift desalting column (Pierce) equilibrated with 20 mM NaPO 4 , 150 mM NaCl, 1 mM EDTA, pH 7.2 (PBS-EDTA). Peak fractions were pooled and stored at 4°C.
  • a) Derivatization of PMB with SPDP was carried out as in Example 7.
  • b) Derivatization of IgG with SPDP was carried out by adding 20 ⁇ l of 20 mM SPDP to 10 mg of IgG in 1 ml of 50 mM sodium borate, 300 mM NaCl, pH 9.0 and incubating 30 minutes at room temperature with shaking.
  • SPDP-modified PMB has a four-fold lower antibiotic activity than free PMB, the actual degree of IgG conjugation with PMB is probably at least four-fold higher than that calculated above (i.e., there is probably at least one PMB conjugated to each IgG molecule).
  • the conjugated polymyxin was also active.
  • the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the SPDP-conjugated IgG-PMB (107-fold molar excess of PMB, Example 8) and the periodate mediated IgG-PMB conjugate (3:1 ratio of PMB, Example 11) were determined.
  • the example involved: (a) preparation of an E. coli bacterial inoculum; (b) determination of the MIC; and (c) determination of the MBC. a) Preparation of an E. coli bacterial inoculum was initiated by first culturing E.
  • the MIC was found to be 0.031 ⁇ g/ml while for the SPDP conjugate, the MIC was found to be 0.25 mg/ml.
  • the MIC was found to be 0.031 mg/ml, which is approximately 1000-fold higher than for native PMB and eight-fold lower than for the SPDP IgG-PMB conjugate.
  • the 1 ml reaction mixture was applied to a 15 ml Swift desalting column equilibrated in 50 mM NaPO 4 , pH 7.2 and the peak IgG fractions were pooled to an IgG concentration of 7.1 mg/ml.
  • the reaction mixture was adjusted to pH 6.5 with 1.0 N HCI, and 10 ⁇ l of a 10 mg/ml NaBH 3 CN solution was added and incubated at room temperature for 4 hours.
  • the new periodate conjugate is four times as potent, and the 3:1 SPDP-PMB conjugate is twice as potent.
  • the periodate conjugate exhibits lower LPS-binding activity by ELISA but stronger antibacterial activity than the 3:1 SPDP-PMB conjugate.
  • the modification of PMB and IgG with SPDP improves the conjugation efficacy but decreases the antibiotic activity compared to the conjugation of native PMB to periodate-treated IgG.
  • the samples were prepared by transferring each to a glass hydrolysis tube using three rinses of 100 ⁇ l of pure water and then concentrated to dryness in a vacuum centrifuge. To each of the sample tubes, 500 ⁇ l of distilled 6N HCI, 10 ⁇ l of 2-mercaptoethanol, and 10 ⁇ l of a 50% aqueous phenol solution were added. The tubes were then purged with nitrogen gas and capped. The samples were hydrolyzed by heating at 110°C for 22 hours and then concentrated again to dryness. The PMB sample was suspended in 500 ⁇ l of 0.2 N sodium citrate buffer, pH 2.2 while the other four samples were suspended in 250 ⁇ l of this buffer. After thorough mixing, the sample solutions were passed through a 0.2 ⁇ m pore nylon membrane syringe filter.
  • a Beckman instruments 6300 Amino Acid Analyzer was used to analyze 20 ⁇ l of each filtered hydrolysate solution.
  • the machine was equipped with a Beckman 10 cm cationic exchange HPLC column, a Beckman sodium buffer system, a 60 minute analysis
  • the detector sensitivity was set at 1.0 AUFS for the PMB sample and 0.5 AUFS for the other four samples.
  • the 3:1 SPDP conjugate contained, on average, twice the number of PMB molecules than the 2: 1 SPDP conjugate, which would explain the two-fold greater activity of the 3: 1 SPDP conjugate in the LPS-binding ELISA.
  • the periodate Ig-PMB is also well conjugated and it exhibited the highest degree of antibacterial activity. It appears that the SPDP linkage affords the highest degree of LPS-binding activity while the periodate linkage provides greater antibacterial activity. This may reflect steric differences in the way PMB is attached to the IgG and/or the different effects of the two conjugation chemistries on PMB activity.
  • IgG-PMB Conjugates As A Diagnostic: Cross- Reactivity Of Different Gram-Negative LPS Antigens with IgG-PMB
  • the IgG-PMB conjugates exhibited binding to E. coli 0111 :B4 LPS, and this species is only one of many potential gram-negative agents of endotoxemia and bacteremia, it was of interest to determine whether the IgG-PMB conjugate was capable of detecting other species of LPS in a diagnostic format using a competitive ELISA.
  • the example involved: (a) coating of E. coli 011 1:B4 LPS to microtiter wells; (b) incubation of IgG-PMB conjugates with different concentrations of several species of LPS; and (c) assay of conjugate binding to E. coli 0111 :B4 LPS in the presence of competitor. a) Coating of E.
  • coli 01 11 :B4 LPS to the wells of 96-well microtiter ELISA plates was performed as described in Example 1, (100 ⁇ l/well of pyrogen-free PBS containing 1 mM EDTA and 2 ⁇ g of LPS was coated onto the wells and allowed to incubate overnight at 4°C). The wells were washed with PBS-0.05% Tween 20 and blocked with PBS containing 10 mg/ml endotoxin-free BSA at 37°C for 90 minutes.
  • IgG-PMB conjugate is capable of neutralizing the lethal effects of endotoxin
  • a well- characterized and accepted murine model of endotoxic shock was utilized.
  • the example involved: (a) determination of a minimum lethal dose of endotoxin in galactosamine-sensitized mice; and (b)
  • mice Twenty (20) CS7BL/6 mice weighing twenty (20) grams each were administered 200 ⁇ g (5 mice) or 400 ⁇ g (8 mice) of IgG-PMB conjugate (periodate conjugate prepared as in Example 14) or 400 ⁇ g control human IgG (7 mice) in 100 ⁇ l of PBS through their tail vein.
  • mice received 10 ng E. coli 01 11 :B4 endotoxin and 20 mg D-galactosamine in 200 ⁇ l of PBS administered intraperitoneally. After 24 hrs, the number of mice surviving in each group was recorded. The results are shown in Table 24. TABLE 24
  • IgG-PMB Conjugates Fc Receptor Binding One bf the functions of IgG is to opsonize and facilitate clearance of organisms, toxins, antigens, etc. by phagocytic cells. In order to determine whether this property of IgG, which is facilitated by the Fc region of the native molecule, remains intact in IgG conjugates that have been prepared with SPDP or periodate, the binding of IgG-PMB to human monocyte/macrophage cells was examined in a competition assay. This assay is similar to that employed to examine the Fc receptor binding activity of hybrid recombinant antibody fragments fused to cell surface viral receptors. [D.J.
  • Conjugation of albumin with PMB was carried out in three steps similar to the scheme described in Example 7.
  • the first step involved derivatization of 10 mg of PMB in 50 mM sodium borate, 300 mM NaCl, pH 9.0 with 2:1 mg of SPDP dissolved in 50 ⁇ l of dimethylsulfoxide for 30 minutes at room temperature.
  • the free cross-linker was removed on a 15 ml Swift desalting column as described in
  • the reduced, derivatized albumin was conjugated with SPDP-PMB by mixing the two solutions prepared above and incubating overnight at room temperature.
  • the conjugate was separated from SPDP-PMB by gel filtration on a 50 ml P- 10 column.
  • monocyte/macrophage cell line was assayed in a manner similar to that described by Capon el al. [Nature 337:525-531 (1989).]
  • the cell suspensions were incubated for 60 minutes at 37°C, centrifuged for 3 minutes at 1500 x g and washed three times with incubation buffer. The cell pellets were then counted for radioactivity with a Bioscan "Quick Count" benchtop radioisotope counter (Bioscan, Inc., Washington D.C). The binding was found to saturate at 1 x 10 -8 M 125 I-Ig so this
  • the human albumin-PMB exhibited no competitive binding activity at concentrations up to 10 -6 (data not shown) and is therefore unable to facilitate opsonization.
  • Gram-positive organisms are responsible for approximately one-third of sepsis cases. It would be desirable to have IgG-antibiotic conjugates with activity against these organisms. To this end, conjugates were made between IgG and bacitracin and vancomycin, two surface- active gram-positive antibiotics. The example involved: (a) periodate activation of IgG; and (b) conjugation to bacitracin and vancomycin. a) Periodate activation of IgG was carried out as described in Example 5(b), using 30 mg of human IgG and 50 mM sodium periodate in 1 ml of 50 mM NaPO 4 , pH 7.2 for 30 minutes at room temperature.
  • the activated IgG was purified on a 15 ml Swift desalting column (Pierce) and the peak fractions pooled.
  • Conjugation to bacitracin and vancomycin was carried out by adding 18.6 mg of bacitracin to 7.1 mg of activated IgG and 19.7 mg of vancomycin to 7.1 mg of activated IgG and each solution was incubated overnight at 4°C The mixtures were then clarified by centrifugation to remove any precipitates formed during incubation. The reaction mixtures were adjusted to pH 6.5 with 1.0 N HCI, and 10 ⁇ l of a NaCNBH 3 solution (10 mg/ml) was added and incubated for 4 hours at room temperature. The conjugate was then purified on a 15 ml Swift desalting column equilibrated in PBS-EDTA, pH 7.2.
  • the strain is gram- positive, DNase negative, mannitol salt negative, coagulase negative and novobiocin sensitive.
  • the MIC was defined as the minimum concentration of the compounds that inhibited visible growth and the MBC defined as the concentration that killed 99.9% or more of the initial organisms present in the inoculum (measured by plating those solutions that do not exhibit visible growth; see Example 10). The results are shown in Table 25. TABLE 25
  • the present invention contemplates screening for patients having a poor immune status for determining a subpopulation having the greatest need for antibodiotics.
  • the example involves: (a) assay of patient total IgG and IgM levels; (b) assay of patient endotoxin core antigen-specific IgG and IgM levels; (c) comparison of patient immunoglobulin levels to healthy normal controls; (d) administration of immunoglobulin and/or immunoglobulin-antibiotic conjugate to patients with significant deficiencies in total or core antigen-specific immunoglobulin levels.
  • Patients with ⁇ 80% of the normal control level of total IgG and/or ⁇ 60% of the normal control level of endotoxin core antigen-specific IgG and IgM are defined as at risk for gram-negative infection and endotoxemia.
  • Administration of immunoglobulin and/or immunoglobulin antibiotic conjugate to patients with significant deficiencies in total or core antigen-specific immunoglobulin levels is carried out to restore normal or near normal total and antigen-specific humoral defenses.
  • the IgG-PMB conjugates of the present invention comprise a population of antibody molecules all of which are capable of binding to endotoxin, much less IgG-PMB conjugate is required than total IgG to restore or increase levels antigen-specific antibody.
  • a single intravenous dose consisting of 1-20 mg of IgG-PMB conjugate per kg of body weight is administered to restore endotoxin-specific antibody levels to > 100% of normal levels.
  • b) Administration of a cocktail of antibody-antibiotic conjugates and, if necessary, total pooled human immunoglobulin to restore antigen-specific and total immunoglobulin levels is carried out by injecting a single intravenous dose of IgG-PMB (1-20 mg/kg) and a single intravenous dose of IgG-bacitracin conjugate (1-20 mg/kg) to increase the levels of gram-negative and gram-positive-reactive antibodies, respectively.
  • mice Twenty-four hours after the administration of PMB-conjugated or control IgG, both groups of mice were challenged intraperitoneally with a lethal dose of E. coli 01 1 1 :B4 endotoxin (# 201 ; List Biological Laboratories, Campbell, CA), prepared as described below. A 1 mg/ml stock solution of endotoxin was sonicated for 2 minutes in a Branson 2000 water bath sonicator and diluted 100-fold in PBS to make a 10 ng/ ⁇ l working solution. Two hundred and forty mg of D-galactosamine hydrochloride (# G-1639; Sigma Chemical Co., St.
  • This example describes experiments to determine if there was any effect on the half-life of HIgG in rabbits when conjugated to PMB.
  • the pharmacokinetic study was conducted using male New Zealand White rabbits (10 lb, 12 months old). Two rabbits each received a single dose of 3 mg of PMB-HlgG conjugate in 10 mM phosphate buffer (pH 7.1) containing 150 mM sodium chloride intravenously on day 0. At the same time, two control rabbits received intravenous injections of 3 mg of HIgG in the same buffer. Both the test samples and control samples were tested and found to be pyrogen-free. Rabbits were bled at one hour and 5 hours after the initial injection and then at days 1, 2, 3, 4, 7, 10, and 14 after the initial injection. Serum samples were collected and stored at -70°C until tested for the presence of HIgG.
  • a sandwich ELISA was developed in order to detect the presence of HIgG in rabbit serum samples.
  • Each well of a microtiter plate (Coming) was coated with 100 ⁇ l of a solution containing 25 ⁇ g/ml of goat-anti human IgG (Sigma) in 50 mM carbonate buffer, pH 9.5. After an overnight incubation at 4°C, the coating solutions were removed and all wells were washed three times with PBS-Tween-20 (.05% Tween-20 in PBS).
  • the remaining antigen binding sites were blocked by the addition of PBS containing 10 mg/ml BSA (Sigma) for one hour at 37°C
  • the test serum samples which were stored at -70°C, were thawed just prior to assay and diluted 1 :10 in PBS-Tween-20 containing 0.1% BSA. All samples were added (200 ⁇ l/well) as duplicate aliquots to wells of the microtiter plate. Negative control wells were prepared by adding 200 ⁇ l/well of 1 :10 diluted normal rabbit serum in the same diluent as used with the test serum samples.
  • normal HIgG normal HIgG was diluted in the same diluent at 20 ⁇ g/ml and subsequently underwent eight serial 1 :4 dilutions up to 0.00031 ⁇ g/ml. The corresponding O.D. values were used to generate a standard curve from which corresponding HIgG levels from test serum samples were determined. Plates were incubated at 37°C for 1 hour and washed three times in PBS-Tween-20. Alkaline phosphatase conjugated goat anti-human IgG (Sigma), diluted 1 :500 in 0.1 % BSA in PBS-Tween-20 was added to the wells and incubated at 37°C for one hour.
  • This example describes an experiment to determine if PMB-HlgG LPS-binding activity is still present after two weeks of circulation in rabbits.
  • the study was conducted using male New Zealand White rabbits (10 lb, 12 months old). Two rabbits each received a single dose of 3 mg of PMB-HlgG conjugate in 50 mM phosphate buffer containing 150 mM sodium chloride intravenously on day 0. At the same time, two control rabbits received intravenous injections of 3 mg of HIgG in the same buffer. Both the test samples and control samples were tested and found to be pyrogen-free. Rabbits were bled at one hour and 5 hours after the initial injection and then at days 1, 2, 3, 4, 7, 10, and 14 after the initial injection. Serum samples were collected and stored at -70°C until tested for the activity of PMB-HlgG conjugate.
  • the remaining antigen binding sites were blocked by the addition of PBS containing 10 mg/ml BSA (Sigma, tissue culture grade) for 1 hour at 37°C
  • the blocking solution was removed and test rabbit serum samples diluted 1 :10 in PBS-Tween-20 were added.
  • PMB-HlgG conjugate was also diluted in 10% normal rabbit serum and added to the wells. Samples were incubated in duplicate at 37°C for 1 hour and the plates were washed three times with PBS-Tween-20.
  • the wells were incubated with 100 ⁇ l of a 1 :500 dilution of goat anti -human IgG-alkaline phosphatase labeled antibody (Sigma) and incubated for 1 hour at 37°C After removing the secondary antibody solutions, the wells were washed 4 times with PBS-Tween-20. Substrate [p-nitrophenylphophate (Sigma)] at 1 mg/ml in 50 mM Na 2 CO 3 , and 10 mM MgCl 2 was added to each well. The color developed after 15-20 minutes of incubation at room temperature was measured at 410 nm using a Dynatech MR700 microplate reader. The results of the LPS binding assay are as shown in Table 27. The conjugate from rabbit sera collected on day 14 bound to the LPS coated wells indicating that the conjugate was still active after circulating for two weeks in rabbits.
  • This example describes an experiment to determine if anti-PMB antibodies are elicited in rabbits by conjugate administration.
  • Two rabbits were each given 3 mg of PMB-HlgG conjugate intravenously on day 0. These rabbits received additional injections (boosts) at 2 weeks, 4 weeks and 7 weeks.
  • boosts additional injections
  • 2 rabbits each received 3 mg of HIgG alone at the same scheduled day and time as with the experimental group. All rabbits were bled every two weeks after receiving either conjugate or IgG alone. Sera were collected and stored at -70°C until tested for anti-PMB antibodies.
  • a positive control antiserum (chicken anti-PMB immunoglobulin, Ophidian Pharmaceuticals Inc., Madison, WI) was also diluted as for the test rabbit serum samples. Samples were incubated in duplicate at 37°C for 1 hour. Following this incubation, the plates were washed three times with PBS-Tween-20.
  • the wells incubated with rabbit serum were incubated with 100 ⁇ l of a 1 :500 dilution of goat anti-rabbit IgG-alkaline phosphatase labeled antibody (Sigma) and the wells incubated with chicken antibody were incubated with 100 ⁇ l of 1 :500 dilution of goat anti-chicken IgG (whole molecule)-alkaline phosphatase conjugate (Sigma) for 1 hour at 37°C
  • the wells were washed 4 times with PBS-Tween-20 and p-nitrophenylphosphate (Sigma) at 1 mg/ml in 50 mM Na 2 CO 3 , 10 mM MgCl 2 was added to each well.
  • the color developed after 15-20 minutes of incubation at room temperature was measured at 410 nm using a Dynatech MR700 microplate reader.
  • results in Table 28 show that the positive control antibody, as expected, bound to PMB. This validates that the design of ELISA is capable of detecting PMB-binding antibodies.
  • results in Table 29 show that none of the rabbit semm samples bound to PMB, indicating the absence of anti-PMB antibodies. These results demonstrate that PMB is not immunogenic, even on an heterologous protein carrier with repeated injections when given intravenously.
  • the lack of immunogenicity of this peptide may be related to its D-amino acid content, as these residues may not be recognized by the immune system.
  • mice served as a normal control (no injection of either conjugate nor normal rat IgG). The rats were bled by cardiac puncture and sacrificed for organ pathology as indicated in Table 30.
  • All rat serum samples (18 total: 3 from groups 1, 2, 4, and 5; and 6 from group 3) were analyzed on the DuPont Dimension AR (DuPont Co., Wilmington, DE) for the following 12 tests (SMAC 12): glucose, blood urea nitrogen (BUN), creatinine, uric acid, calcium, albumin, total protein, cholesterol, total bilirubin, alkaline phosphatase, aspartate transferase (SGOT/AST), and lactate dehydrogenase (LDH).
  • the values for each group were averaged (Table 31) and the experimental groups (1, 2, and 3) were compared with the control groups (4 and 5) to detect any significant differences.
  • the laboratory results were also compared to the normal ranges for each assay, determined by analyzing laboratory data for 20 female Sprague-Dawley rats (data provided by Harlan Sprague-Dawley).
  • the standard laboratory tests for liver disease include measurement of serum levels of bilimbin, AST, alkaline phosphatase, LDH, albumin, and, to a lesser extent, glucose. Kidney function can be assessed by measuring plasma levels of urea, creatinine, and calcium. [J. F. Zilva, P.R. Pannall, Clinical Chemistry in Diagnosis and Treatment, Yearbook Medical Publishers, Chicago, IL (1984).] With the exception of the lactate dehydrogenase value (LDH), which will be discussed below, Table 31 shows no significant differences when the values for the experimental groups 1, 2, and 3 are compared with the control groups 4 and 5. Furthermore, all the values are within or close to the normal ranges for each assay for this strain of rat.
  • LDH lactate dehydrogenase value
  • Lactate dehydrogenase is found in high concentrations in the liver, heart, skeletal muscle, brain, kidney and in erythrocyt ⁇ s. Elevated values of particular isozymes can indicate liver or cardiac muscle damage, however in this study the control rats also show elevated LDH values, suggesting the elevated LDH values are not associated with the IgG-PMB conjugate.
  • Hemolysis which may have occurred in vitro (as the blood samples were being drawn, or if the serum was not separated from the blood cells soon enough), also increases serum LDH values (J. F. Zilva and P. R. Pannall, Clinical Chemistry in Diagnosis and Treatment, supra) . and may explain the elevated values in this study.
  • day 5 3 days after the second injection
  • day 7 5 days after the second injection
  • day 14 (12 days after the second injection)
  • the day 5, 7 and 14 values for all of the serum components measured are within or close to their respective normal ranges (except LDH) and show little or no significant change over time, as would be expected if the conjugate brought about any acute changes in the condition of the test rats.
  • Table 32 shows no significant differences in the percentages of each cell type from group to group.
  • the differentials do show some variation from the normal reference values for rats obtained from Harlan Sprague-Dawley (about 5-10% more lymphocytes and 5-10 % fewer neutrophils than expected), however this is found in both the normal control and experimental groups, suggesting this finding is not related to the administration of the IgG-PMB conjugate.
  • the red blood cell morphology appeared normal, and platelets were abundant on all slides examined.
  • the organs of interest were removed and fixed in phosphate buffered formaldehyde. Sections were made as described below and stained with hematoxylin and eosin.
  • Kidney Full length mid-longitudinal section through center
  • Immunoglobulin and immunoglobulin complexes have the potential to activate the complement system. Complement activation of this type, mediated by IgG-PMB conjugates, would exacerbate the inflammatory response to endotoxemia or bacteremia. In addition, inhibition of normal complement function would impair complement-mediated host defense mechanisms. In this example, the in vivo effect of IgG-PMB conjugate on serum complement activity was investigated.
  • Rat serum samples were analyzed for total hemolytic complement activity (CH 50 ) using the EZ Complement CH 50 Assay (Diamedix Corp., Miami, FL).
  • CH 50 total hemolytic complement activity
  • EZ Complement CH 50 Assay Diamedix Corp., Miami, FL
  • the resulting CH 50 values obtained from untreated control rats were compared to the CH 50 values obtained from the IgG and IgG-PMB conjugate-treated rats.
  • PMB was conjugated to rat IgG using periodate oxidation of IgG. This involved a) periodate oxidation of IgG in phosphate buffer followed by conjugation of PMB to the periodate-oxidized IgG. a) Periodate oxidation of IgG in phosphate buffer was achieved by dissolving 25 mg rat IgG (Sigma) in 1 ml of 50 mM NaPO 4 , pH 7.2 buffer and adding 10.7 mg of sodium metaperiodate (final concentration 50 mM ).
  • Opsonic IgG class antibodies mediate an important immune effector function by enhancing the phagocytic clearance of extracellular bacteria [Raff, et al. , J. Infect. Dis.
  • the pinpose of this example was to investigate whether the IgG component of IgG-PMB conjugates retains this important effector function. This was done by assessing whether the pre-treatment of E. coli organisms with IgG-PMB conjugate potentiates phagocytic uptake (opsonophagocytosis) by the human monocytic cell line U937.
  • Opsonophagocytosis assays provide a useful means by which the potential therapeutic efficacy of immunoglobulin preparations, used for the treatment of bacterial infection, can be assessed. [Hill, et al. Am. J. Med. 61-66 (1984).] This example involved (a) Assay for
  • E. coli strain HB101 was grown for approximately 20 hours at 37°C on TSA (BBL). The organisms were then suspended in PBS, pH 7.2, at a concentration of 1 x 10 8 CFU./ml. Aliquots of 1.0 ml volumes of this suspension were placed into separate microfuge tubes and the tubes centrifuged at approximately 14,000 x g for 5 min. at 4°C Each of the resulting pellets was then resuspended in a 1.0 ml volume of one of the following opsonin or control solutions:
  • IgG-PMB Conjugate prepared by periodate oxidation, as described in Example 14(a) at the MIC for E. coli HB101 (0.062mg/ml) (The MIC was determined as described in Example 12).
  • IgG-PMB Conjugate (same as above) at 2x the MBC for E. coli HB101 (0.25 mg/ml) (The MBC was determined as described in Example 12).
  • IgG Control (unconjugated) at 0.062mg/ml (control for #1 above;
  • PBS Control (no IgG or conjugate). PBS, pH 7.2 only.
  • the five suspensions were opsonized by incubation at 37°C for 60 min. with periodic mixing. Following opsonization, the suspensions were centrifuged as above, and the resulting pellets were each resuspended in 0.5ml of RPMI 1640 medium which was supplemented with 10% FCS (this will be referred to as "medium” for the remainder of this example).
  • medium 0.5ml of RPMI 1640 medium which was supplemented with 10% FCS (this will be referred to as "medium” for the remainder of this example).
  • RPMI 1640 medium which was supplemented with 10% FCS (this will be referred to as "medium” for the remainder of this example).
  • RPMI 1640 medium which was supplemented with 10% FCS (this will be referred to as "medium” for the remainder of this example).
  • S/P polypropylene culture tubes
  • 1.0 ml of a U937 cell suspension which was prepared in medium, and contained 1 x 10 6 U937 cells/
  • a sixth control group was also prepared which contained 1.0 ml of the U937 cell suspension and 0.1 ml of PBS (PBS control). At this point, each tube contained 1 x 10 6 U937 cells, and 2 x 10 7 E. coli organisms, thus providing an E. coli to U937 cell ratio of 20:1.
  • the 6 tubes were then incubated at 37°C for 60 min. with constant shaking, in order to allow phagocytosis to occur. Following incubation, the tubes were placed on ice for several minutes to prevent further phagocytosis. The 6 tubes were then centrifuged for 10 min.
  • the resulting pellets were washed three times (centrifuging as in the previous step) with chilled PBS, to remove extracellular E. coli organisms.
  • the final pellets were each resuspended in 0.2 ml of chilled PBS, and smears were prepared by applying 40 ⁇ l volumes of the suspensions to glass microscope slides. The smears were allowed to air-dry, and were then fixed by immersion in 100% methanol for 5 sec. and again allowed to air-dry.
  • the smears were stained using a modified version of the Sowter-McGee staining procedure [Sowter and McGee. J. Clin. Pathol. 29:433-437 (1976)], which chromatically differentiates between intracellular bacteria and the surrounding cytoplasm of the host cells.
  • the slides were hydrated by immersion in water for approximately 60 sec, and were then placed in a methyl green-pyronin (MGP) solution (Sigma) for 5 min.
  • MGP methyl green-pyronin
  • the slides were washed in water for 15-20 sec. and then immersed in light green counterstain (0.25% Sigma Light Green SF Yellowish in distilled H 2 O) for 3-5 sec. Following a 15-20 sec.
  • the minimum concentration of IgG-PMB required to mediate opsonophagocytosis was determined by testing the conjugate at the MIC and at fractional concentrations of the MIC (sub-MIC).
  • a parallel series of albumin-PMB (Alb-PMB) conjugate solutions were also tested at concentrations comparable to the IgG-PMB conjugate.
  • the following conjugate and control solutions were assayed for opsonophagocytic activity by the procedure described in part (a) of this example:
  • IgG-PMB Conjugate (same as that used in part (a) of this Example) at the MIC for E. coli HB101 (0.062 mg/ml).
  • IgG-PMB Conjugate (same as above) at 1/2 the MIC for E. coli HB101 (0.031 mg/ml).
  • IgG-PMB Conjugate (same as above) at 1/4 the MIC for E. coli HB101 (0.0155 mg/ml).
  • Alb-PMB Conjugate prepared as described in Example 18(a) at 0.062 mg/ml (this group served as a control for #1 above).
  • Alb-PMB Conjugate (same as above) at 0.031 mg/ml (this group served as a control for #2 above).
  • Alb-PMB Conjugate (same as above) at 0.0155 mg/ml (this group served as a control for #3 above).
  • Alb-PMB Conjugate (same as above) at 7.75 ⁇ g/ml (this group served as a control for #4 above).
  • IgG Control (unconjugated) at 0.062 mg/ml.
  • MIC and MBC values were determined for IgG-PMB conjugate and native PMB control against bacterial strains which are known to be human pathogens (see Example 29 below). This example involved (a) Preparation of the Conjugate, (b) Preparation of the Bacterial Inocula, and (c) Determination of the MIC and MBC.
  • the IgG-PMB conjugate was prepared by periodate oxidation as described in Example 14(a) with the following modification, which was performed in order to more effectively remove free (unconjugated) PMB from the final conjugate preparation.
  • the final conjugate solution was adjusted to contain 1.0% Tween-20, and then was chromatographed on a P- 10 column using PBS containing 0.1 % Tween-20 as the eluent. The material in the void volume was concentrated and then further purified by column chromatography as described in the previous sentence.
  • Organisms were grown, and separate inocula were prepared for the following test organisms, as described in Example 12 (a): E. coli strain EC 5; Pseudomonas aeruginosa strain ATCC 27312; Pseudomonas aeruginosa strain Strong; and Pseudomonas aeruginosa strain 3.
  • the MIC and MBC of IgG-PMB conjugate and native PMB control were determined for each of the test organisms, as described in Examples 12(b) and 12(c).
  • IgG-PMB conjugates against pathogenic bacterial strains demonstrate that these compounds may be effective for the prophylaxis and/or treatment of bacteremia.
  • E. coli sepsis continues to be associated with an unacceptably high mortality rate, despite the availability of potent antibiotics.
  • E. coli strains with the K1 capsular type have been identified as the etiologic agent in up to 24% of blood culture isolates [G. W. Count and M. Turck, J. Clin. Microbiol. 5:490 (1977)], 80% of the cases of neonatal meningitis. [L.D. Sarff et al., Lancet 1 :1099 (1975).] It is the most frequent cause of nosocomial gram negative bacteremia in adults [M.P. Weinstein et al., Rev. Infect. Dis. 5:35-53 (1983)] and
  • E. coli serotype O18:Kl is a very virulent human pathogen, as defined by either D. Rowley, Br. J. ⁇ xp. Pathol. 35:528-538 (1954) or H. Smith, J. Gen. Microbiol. 136:377-383 (1990), they can grow in vivo from a small inoculum, evade host defenses and cause extraintestinal infections.
  • the established animal infection model described by D. ⁇ . Schiff et al., Infect. Immun. 61 :975-980 (1993) was utilized.
  • This model fulfills many criteria important in evaluating the toxicity, efficacy and safety of immunotherapeutics, some of which have been outlined by A.S. Cross et al., Infect. Immun. 61:2741-2747 (1993).
  • the model is an infection rather than an intoxication model in which rats are challenged with low doses of a virulent bacteria instead of using large doses of an avirulent strain.
  • E. coli O18:K1 E. coli O18:K1.
  • the E. coli strain designation C5 obtained from K.S. Kim, Children's Hospital (Los
  • IgG-PMB conjugate To determine if the administration of IgG-PMB conjugate can protect in vivo, rat pups were pretreated with IgG-PMB or control IgG followed by an administration of a lethal dose of E. coli C5.
  • the myeloma IgG was used as the carrier to produce the PMB-conjugate, in order to ensure the reactivity between the E. coli and IgG-PMB conjugate was exclusively due to binding between PMB and lipopolysaccharide.
  • the eluent was 50 mM phosphate buffer (pH 6.65) flowing at 0.5 ml/minute.
  • the eluate i.e. the liquid collected at the bottom of the column
  • the eluate was monitored for absorbance at 280 nm.
  • 0.5 ml fractions were collected.
  • Two major absorbance peaks were evident - one centered at 42 minutes and the other at 47.5 minutes (aminocephalosporanic acid and reaction products, respectively).
  • Fractions corresponding to the leading edge of the first peak were pooled (2.0 ml, 3.42 A 280).
  • 1.4 ⁇ l of 100% beta-mercaptoethanol was added to the mixture which was then filtered using a sterile Whatman 0.45 micron Puradisc
  • hydroxyethylthio-maleimidobenzoyl-N-aminocephalosporanic acid ester was determined to be 0.86 A 280 with Staph. aureus compared to 3.7 A 280 for 7-aminocephalosporanic acid.
  • This example describes the attachment of the heterobifunctional crosslinking agent of Example 31 to a different antibiotic precursor.
  • This example outlines the derivatization of 6- aminopenicillanic acid, an antibiotic precursor exhibiting no significant anti-microbial properties, with sulfo-MBS.
  • 6-aminopenicillanic acid 12.1 mg of 6-aminopenicillanic acid (ICN) was dissolved in 2.5 ml of 50 mM phosphate buffer (pH 6.65). The solution was continuously mixed with a stir bar and magnetic stirrer and the pH was monitored. Sulfo- MBS (24.1 mg, Prochem, Inc.) was added and the pH was adjusted to 6.85 with a 1.0 N sodium hydroxide solution. The mixture was incubated at ambient temperature for 2.5 hours.
  • ICN 6-aminopenicillanic acid
  • the reaction mixture was applied to a 1.5 x 20 cm column of Whatman LRP-2 resin (C18 reverse phase), equilibrated with 10% medianol in water.
  • the column was developed at 1.0 ml/min. with 10% methanol for 5 min., followed by a linear, 30 min. gradient of 10 to 90%) methanol in water.
  • the eluate containing the last peak of material absorbing at 280 nm (eluted at 26 min.) was collected and concentrated to dryness under reduced pressure using a Labconco Centravap concentrator.
  • the derivatized aminopenicillanic acid was dissolved in 1.0 ml of 50 mM phosphate buffer plus 1.0 mM EDTA, pH 6.65.
  • the MIC of the derivatized aminopenicillanic acid was determined to be 8 ⁇ g/ml against S. aureus, compared to 250 ⁇ g/ml for the native aminopenicillanic acid.
  • Purified human IgG (40 mg, Sigma) was dissolved in 2.5 ml of 50 mM
  • the derivatized aminopenicillanic acid (0.5 ml, 9.0 mg/ml) was mixed with 1.75 ml of iminothiolated IgG and incubated at ambient temperature for 10 min with mixing and then at 2-8°C overnight. The mixture was transferred to ambient temperature and incubated with agitation. After 20 minutes, 146 ⁇ l of 10 mM N-ethylmaleimide (Pierce) was added and the incubation was continued for 4 hours.
  • the reaction mixture was passed through a Unifio-Plus filter (S&S) and applied to a 2.5 X 20 cm column of Spectra/Gel ACA 202 (Spectrum).
  • the column was eluted at 2.0 ml/min with 50 mM sodium phosphate buffer, pH 6.5. The absorbance at 280 nm was monitored.
  • the material in the void volume, containing MBS aminopenicillanic acid:IgG was collected, pooled, concentrated using a Centricon concentrator (Amicon) and passed through a Unifio-Plus filter (S&S).
  • the MBS aminopenicillanic acid:IgG was found to be inactive at 0.65 mg/ml against S. aureus.
  • This example outlines the derivatization of amoxicillin, an antibiotic exhibiting significant anti-microbial properties, with sulfo-SMCC.
  • amoxicillin trihydrate ICN
  • 50 mM phosphate buffer pH 6.65
  • Sulfo SMCC 23 mg, Prochem, Inc.
  • the mixture was incubated at ambient temperature for 4 hours. (The reaction mixture was initially turbid due to suspended amoxicillin, but the mixture became clear with time.)
  • reaction mixture was applied to a 1.5 X 20 cm column of Whatman LRP-2 resin
  • cefadroxil an antibiotic active against gram-positive bacteria, was reacted with the crosslinking agent sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-c-rboxylate ("sulfo-SMCC").
  • Cefadroxil (Sigma) was dissolved at 3.0 mg/ml in 50 mM phosphate (pH 6.65). sulfo- SMCC was added and dissolved at 2.6 mg/ml. After a 1 hour and 55 minute incubation at ambient temperature with agitation, ethanolamine was added at 3.4 mg/ml and the incubation was continued for an additional 42 minutes.
  • reaction mixture (0.5 ml) was applied to a 1.5 X 13 cm column of Sephadex G10 resin (Pharmacia).
  • the eluent was 50 mM phosphate buffer, pH 6.65, flowing at 0.5 ml/minute.
  • the eluate was monitored for absorbance at 280 nm.
  • 1.0 ml fractions were collected. Two major absorbance peaks were evident - one centered at 22 minutes and the other at 36 minutes (reaction products and cefadroxil, respectively). Fractions corresponding to the leading edge of the first peak were pooled (3.0 ml, 3.5 A 280). 300 ⁇ l of 100 mM beta-mercaptoethanol was added to the mixture which was then filtered using a sterile
  • maleimidomethyl cyclohexane carboxyl-N-cefadroxil ester was determined to be 1.6 A 280 against Staph. aureus compared to a MIC of 0.028 A 280 for native cefadroxil. Thus, the derivatized cefadroxil was relatively inactive.
  • vancomycin an antibiotic active against gram-positive bacteria, was reacted with four different heterobifunctional crosslinking agents. Vancomycin in phosphate buffer was reacted with each of the following compounds: sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamide] hexanoate (“sulfo- LC- SPDP”), m- maleimidobenzoyl-N-hydroxysulfosuccinimide ester (“sulfo-MBS”), sulfosuccinimidyl (4- iodoacetyl) aminobenzoate (“sulfo-SIAB”), and sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (“sulfo-SMPB”).
  • sulfo-LC- SPDP m- maleimidobenzoyl-N-hydroxysulfosuccinimide ester
  • Vancomycin derivatized by sulfo-LC-SPDP possesses a sulfhydryl group which can be exposed under the proper conditions and can be reacted with a maleimide on derivatized IgG.
  • Vancomycin derivatized by sulfo-MBS possesses a maleimide group which can react with a sulfhydryl group on either reduced IgG or derivatized IgG, by addition to the maleimide's carbon-carbon double bond.
  • vancomycin derivatized by sulfo-SIAB posesses an iodo group, which can also react with a sulfhydryl group on either reduced IgG or derivatized IgG, by nucleophilic substitution for the iodo group.
  • Sulfo MBS, sulfo SIAB, sulfo SMPB or sulfo SMCC (Pierce) were dissolved with mixing at a concentration of 20 mM in a solution of 10 mM vancomycin (ICN) in 50 mM sodium phosphate buffer, pH 7.15. The mixtures were incubated with agitation at ambient temperature. Precipitates formed in all four mixtures and the products of the reaction were not further pursued.
  • the mixture (0.5 ml) was applied to a 1.5 x 13 cm column of Sephadex G10 (Pharmacia).
  • the eluent was freshly degassed 50 mM phosphate, 1.0 mM EDTA, pH 6.65 buffer flowing at 0.5 ml/min.
  • 1.0 ml fractions were collected and the fractions in the first peak (void volume of column) were pooled (3.0 ml).
  • the pool from the G10 column was applied to a 4 ml, 1.0 cm diameter column of
  • Bio-Rad Affi-Gel 501 an organomercury resin.
  • the 501 resin had been previously washed with 25 ml 50 mM sodium acetate, pH 5.0 (acetate buffer), 25 ml of 4.0 mM mercuric acetate in acetate buffer, 25 ml of acetate buffer and the equilibrated to 50 mM phosphate, 1.0 mM EDTA, 0.5% Tween 20, pH 6.65.
  • the pool was applied at 0.2 ml/min using 50 mM phosphate, 1.0 mM EDTA, 0.5% Tween 20, pH 6.65 to wash the column. The flow was increased to 1.0 ml/min after 13 minutes.
  • the MIC of vancomycin is 1-2 ⁇ g/ml.
  • the sample was loaded onto a 2.5 x 20 cm column of Spectra Gel ACA 202 resin (Spectmm) and eluted, at 1.0 ml/min, with PBS plus 0.1 % Tween 20. 1.5 ml fractions were collected. The fractions in the void volume were pooled and sterile filtered. The activity of the conjugate was determined by standard MIC testing against S. aureus. The conjugate against S. aureus was found to be inactive at 1.1 mg/ml.
  • vancomycin-IgG conjugate with antibacterial activity an alternative crosslinking method was investigated using S-acetyl mercapto succinic anhydride (“SAMSA”) to derivatize the vancomycin and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“sulfo- SMCC”) to derivatize the IgG.
  • SAMSA S-acetyl mercapto succinic anhydride
  • sulfo- SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • nonspecific immunoglobulin is derivatized with sulfo-SMCC.
  • the purified derivatized vancomycin and the derivatized IgG are reacted with each other forming a conjugate. a) Reaction Of Vancomycin With SAMSA
  • vancomycin (Sigma, Cat # V2002, Lot # 43H1090) [14 ⁇ moles] was dissolved in 200 ⁇ l of water. 1.2 ml of saturated sodium succinate was added slowly with stirring and this mixture was cooled to 4°C by placing the reaction mixture on ice. The mixture appeared slightly cloudy. To this mixture was added 121.8 mg of SAMSA (Sigma, Cat # A1251, Lot 3 # 120H5017) dissolved in 200 ⁇ l of dimethyl sulfoxide (DMSO,
  • vancomycin was collected and stored at 4°C c) Purification Of Modified Vancomycin From Unmodified Vancomycin By Affinity Chromatography On Organomercurical Column
  • a 5ml Affi Gel 501 Organomercurial agarose column (Bio-Rad) was prepared according to the manufacturer's instmctions. The column was equilibrated with 50 mM phosphate, pH 7.1. SAMSA modified vancomycin contains protected sulfhydryl groups which were deprotected with hydroxylamine hydrochloride before applying to the column. Hydroxylamine hydrochloride was added to the modified vancomycin solution to a final concentration of 0.2 M and the mixture was incubated at room temperature for five minutes.
  • the sample was applied to the Affi Gel 501 column at a flow rate of 0.5 ml/min and the column was then washed with 10 mM 2-(N-Morpholino) ethane sulfonic acid, 1 mM EDTA, 0.5%) Tween-20, pH 6.5 buffer until the baseline A 280 was obtained.
  • the bound modified vancomycin was eluted with the same wash buffer containing 20 mM 2-mercaptoethanol. The activity of the modified vancomycin was determined to be 2.6 ⁇ g/ml by MIC testing against S. aureus. d) Reaction Of IgG With Sulfo-SMCC
  • Modified vancomycin was in a buffer containing 20 mM 2-mercaptoethanol which was removed by dialysis, using benzoylated dialysis tubing (Sigma Cat # D7884, Lot # 43H7085). 750 ⁇ g vancomycin (-0.5 ⁇ mole) and 7.5 mg (0.05 ⁇ mole) maleimide activated IgG was used. The mixture was incubated at room temperature for one hour. Unreacted maleimide sites were blocked by adding 30 moles of 2-mercapto ethyl amine per mole of IgG and incubating the mixture at room temperature for 20 min. The conjugate was purified from excess 2- mercapto ethyl amine and unreacted vancomycin by gel filtration chromatography.
  • the sample was applied to a AcA 202 gel filtration column (2.5 x 20 cm, Spectrum) equilibrated with 0.01 M phosphate buffered saline, pH 7.2 with 0.1 % Tween-20. Absorbance at 280 nm was monitored. The first peak containing vancomycin-IgG conjugate was collected. The activity of the conjugate was determined by MIC testing against S. aureus. MIC of this conjugate against S. aureus was found to be 0.438 mg/ml.
  • This example demonstrates that conjugation of vancomycin derivatized by SAMSA with IgG derivatized by sulfo-SMCC results in an active conjugate. This conjugate was found to be an effective anti-microbial agent when tested against S. aureus.
  • LALF Limulus antilipopolysaccharide factor
  • amoebocytes from L. polyphemus are collected under endotoxin-free conditions, lysed by the addition of distilled water, and centrifuged at 5,000 x g for 30 min. The pellet is extracted with 3 M urea. The extract is filtered through a membrane with a 30,000-Da cutoff and concentrated by a membrane with a
  • CM Sepharose cation exchange column
  • LALF was dissolved at 15 mg/ml in 50 mM sodium acetate, pH 5.0.
  • LALF against E. coli HB101 was found to be 16 ug/ml.
  • E. coli 0111 :B4 lipopolysaccaride was obtained from Sigma and was dissolved at 0.02 mg/ml in PBS plus 0.005% thimerosol.
  • E. coli HB101 was diluted to 10,000,000 CFU/ml in PBS. 100 ⁇ l aliquots of LPS solution, E. coli HB101 suspension or PBS were added to wells of Falcon Pro-Bind 96 well microtiter plates. The plates were incubated for 18 hours at 2-8°C . The wells were washed 3 times with PBS. 100 ⁇ l of PBS plus 5 mg/ml BSA (Sigma Chemical Co.) was added to each well of the plates and the plates were incubated for 2.0 hours at room temperature.
  • the plates were decanted and 100 ⁇ l of sample (e.g. conjugate, antibody, etc.) was added per well and the plates were incubated at ambient temperature for 2.0 hours.
  • the wells were washed 6 times with BBS (0.125 sodium borate, 1.0 M NaCl, pH 8.3) plus 0.5% Tween 20, 3 times with 50 mM sodium carbonate, pH 9.5.
  • Three Sigma 104 phosphatase substrate tablets were dissolved in 15 ml of 50 mM sodium carbonate buffer plus 10 mM MgCl 2 and added at 100 ml per well. After approximately 20 minutes at ambient temperature, the absorbance at 410 nm of each well was determined. The results are shown in Table 40.
  • LALF-IgG conjugate is capable of neutralizing the lethal effects of endotoxin
  • murine model of endotoxic shock discussed in Example 16 (see above) was utilized. Neutralization of endotoxin lethality was assessed by LALF-IgG.
  • the minimal effective lethal dose of endotoxin was 15 ng, and the injection volume was 200 ⁇ l per mouse.
  • E. coli 0111 :B4 endotoxin (Sigma) was prepared as described in Example 23 (see above), except that the diluent used was PBS and .0.1% Tween-20 (without BSA).
  • the endotoxin and variable amounts of LALF-IgG were incubated in varying amounts as in Example 16(b) (see above). The results are shown in Table 42. As shown in Table 42, the LALF-IgG conjugate was 100% effective at 5 ⁇ g per mouse in neutralizing the lethal dose of 15 ng endotoxin per mouse.
  • An antibiotic-antibody conjugate comprising antibiotic covalently bound to non-specific immunoglobulin, wherein said conjugate is capable of binding to bacteria via said antibiotic.
  • said conjugate of Claim 1 wherein said immunoglobulin is IgG having an Fc region.
  • a method of treatment comprising:
  • a method of treatment comprising:
  • a therapeutic preparation comprising an antibiotic capable of binding to a microorganism, covalently bound to a non-specific immunoglobulin; and c) administering said preparation to said mammal, prior to the onset of any symptoms of sepsis.
  • said therapeutic preparation comprises a mixture of a first conjugate comprising an antibiotic capable of reacting with a surface component present on a gram-positive bacteria covalently bound to a non-specific
  • immunoglobulin and a second conjugate comprising an antibiotic capable of reacting with a surface component present on a gram-negative bacteria covalently bound to a non-specific immunoglobulin.
  • a method of diagnosis comprising:
  • said gram negative bacteria is selected from the group consisting of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Vibrio cholerae, Shigella flexneri, Klebsiella pneumoniae, Salmonella enteritiditis, Serratia marcescens and Rhodobacter sphaeroides.
  • a method of synthesizing a conjugate comprising the steps of:
  • microorganisms 59.
  • polymyxin is polymyxin B.
  • a method of synthesizing a conjugate comprising the steps of: a) reacting a non-specific immunoglobulin with a first modifying reagent to form an oxidized immunoglobulin preparation;and
  • a method of synthesizing a conjugate comprising the steps of:
  • said antibiotic precursor is selected from the group consisting of 7-aminocephalosporanic acid and 6-aminopenicillanic acid.
  • said first crosslinking agent is bifunctional.
  • said first bifunctional crosslinking agent is m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester.

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Abstract

L'invention concerne des compositions et des procédés pour la prévention et le traitement de la septicémie chez les êtres humains et les animaux. Des patients ayant subi une opération chirurgicale, des nouveau-nés de faible poids, des victimes ayant subi des brûlures et des traumatismes ainsi que d'autres individus à risque peuvent être traités de manière prophylactique. Des procédés de traitement d'infections aiguës présentant des avantages par rapport aux thérapies actuelles sont décrits.
EP94909422A 1992-12-21 1993-12-20 Prevention et traitement de la septicemie. Withdrawn EP0679083A4 (fr)

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US99538892A 1992-12-21 1992-12-21
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US16406793A 1993-12-08 1993-12-08
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US08/169,701 US5545721A (en) 1992-12-21 1993-12-17 Conjugates for the prevention and treatment of sepsis
US169701 1993-12-17
PCT/US1993/012381 WO1994014437A1 (fr) 1992-12-21 1993-12-20 Prevention et traitement de la septicemie

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US6579696B1 (en) * 1992-12-21 2003-06-17 Promega Corporation Polymyxin B conjugates
US6485726B1 (en) 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6030613A (en) * 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
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AU693433B2 (en) 1998-07-02
AU6226994A (en) 1994-07-19
WO1994014437A1 (fr) 1994-07-07
CA2151386A1 (fr) 1994-07-07
JPH08504824A (ja) 1996-05-28

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