EP0434685A1 - Endotoxine bacterienne gram negative bloquant des anticorps monoclonaux - Google Patents

Endotoxine bacterienne gram negative bloquant des anticorps monoclonaux

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Publication number
EP0434685A1
EP0434685A1 EP89904495A EP89904495A EP0434685A1 EP 0434685 A1 EP0434685 A1 EP 0434685A1 EP 89904495 A EP89904495 A EP 89904495A EP 89904495 A EP89904495 A EP 89904495A EP 0434685 A1 EP0434685 A1 EP 0434685A1
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EP
European Patent Office
Prior art keywords
antibody
gram
sepsis
cells
treating
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Withdrawn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
EP89904495A
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German (de)
English (en)
Inventor
James W. Larrick
Andrew A. Raubitschek
Kong Teck Chong
Jeffrey Flatgaard
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Cetus Oncology Corp
Original Assignee
Cetus Corp
Cetus Oncology Corp
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Publication date
Application filed by Cetus Corp, Cetus Oncology Corp filed Critical Cetus Corp
Publication of EP0434685A1 publication Critical patent/EP0434685A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1235—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of biotechnology and relates to somatic cell hybridization and immunology. More particularly, it concerns: monoclonal antibodies that bind to Gram-negative bacterial endotoxins and block the biological effects thereof; hybrid cell lines that produce the antibodies; and treatment of Gram-negative bacteremia and sepsis with the antibodies and antibiotics.
  • Bacteremia due to Gram-negative bacteria is a major public health problem that results in a substantial number of deaths per year. Symptoms of bacteremia include: fever, leudopenia and hypoglycemia, hypotension and shock, impaired perfusion of essential organs, activation of C5a and the complement cascade, intravascular coagulation and death.
  • LPS cell-wall lipopolysaccharide
  • LPSs are composed of three regions: Serotype-specific polysaccharide (O-antigen), core polysaccharide, and lipid A.
  • the O-antigen region is made up of repeating ol igosaccharide combinations that define type- specific haptenic determinants.
  • the core region is composed of an outer core of hexoses (N-acetylglucosamine, glucose, galactose), and an inner core of heptose, ethanolamine and 2-keto-3-deoxyoctonate (KDO).
  • Lipid A is composed of di glucosamine- 4- phosphate, long chain fatty acids, and ethanolamine.
  • KDO forms the link between lipid A and the core region.
  • Mutants of Gram-negative bacteria that lack the O antigen are called "rough” or “R” forms because they do not form smooth colonies on solid media.
  • chemotypes of mutant LPSs are known such as Ra, Rb, Re, Rd, and Re.
  • chemotype Re the enzyme UDP- galactose-4-epimerase is deficient so that glucose, but not galactose, is synthesized and incorporated into the core.
  • Re chemotypes lack the O and core regions up to KDO.
  • Ziegler, E. J., et al., N. Engl . J. Med. (1982) 307:1225-1230 describe the preparation of human antisera to the E. coli Re mutant J5 by vaccinating healthy patients with heat- killed J5. The antisera were used to treat bacteremia and were reported to block the biological effect of LPS.
  • Immun. Meth. (1984) 70:83-90 discloses human monoclonal antibody production from an EBV-trans formed B cell line by fusion to a human-mouse hybridoma.
  • Several fusion partners are described by D. Buck et al., Chapter 11 in Monoclonal Antibodies and Functional Cell Lines, ed. by R. Kennett et al., Plenum Publishing Corp., 1984 and by Larrick and Buck, Biotechniques (1984) 2:6-14.
  • Europ. Pat. Publications 107,528 and 105,804 describe cell lines capable of producing human monoclonal antibodies against a bacterial toxin.
  • GB 2,086,937; GB 2,113,715; EP 57,107; EP 62,409; EP 118,893; EP 124,301 and EP 131,878 all relate to manufacture of human monoclonal antibodies from hybrid cells.
  • Stable, permanent hybrid cell lines that produce the above- described antibodies and progeny of those lines, and a specific mouse x human B-lymphocyte fusion partner producing such cell lines and partially adapted to serum-free medium are another aspect of the invention.
  • compositions for treating one or more of the above-described antibodies comprise two or more of the antibodies each of which binds to a different determinant located as specified in (b) above.
  • Methods for treating bacteremia or sepsis in a human patient by administering an effective amount of such compositions to the patient are also part of the invention. Such methods for treating bacteremia include the administration of antibiotics and antibody to the subject in need of treatment.
  • Figure 1 shows the growth curve of D-234 cells in serum- free media HL-1 (Ventrex Labs, Portland, Me) spinner culture.
  • the circles represent IgM levels, the triangles represent cell yield, and the squares represent glucose levels.
  • Figure 2 shows the growth curve of D-267 cells in serum-free media HB104 (Hana Biologicals, Berkeley, CA) spinner culture, where the circles, triangles and squares are as in Figure 1.
  • FIG. 3 shows the purification scheme for T88.
  • Figure 4 shows the elution profile of T8810A from a Bakerbond ABx column.
  • Table X presents a comparison of u chain concentrations in peaks 1 and 2.
  • Table XI presents a comparison of peak 1 and peak 2 LPS binding.
  • cell line refers to individual cells, harvested cells, and cultures containing cells so long as they are derived from cells of the cell line referred to.
  • progeny is intended to include all derivatives, issue, and offspring of the cell lines regardless of generation or karyotypic identity.
  • the term "monoclonal antibody” refers to an antibody selected from antibodies whose population is substantially homogeneous, .i.e., the individuals of the antibody population are identical except for naturally occurring mutations. Thus, the antigen-binding fragment of the antibody population is the same, but the constant regions may vary.
  • the term "functional equivalent” means a monoclonal antibody that recognizes the same determinant as and crossblocks the monoclonal antibody referred to. It is intended to include antibodies of the same or different immunoglobul in class and antigen binding fragments (e.g., Fab, F(ab') 2 , Fv) of the monoclonal antibody.
  • treat and conjugates thereof refers to therapy and/or prophylaxis.
  • the terms "permanent” and “stable” mean that the lines remain viable over a prolonged time, typically at least about six months, and maintain the ability to produce the specified monoclonal antibody through at least about 25 passages.
  • the term "binds strongly” means that the antibody exhibits a relatively strong binding affinity to lipid A determinants of the E. coli Re mutant LPS or Salmonella Re mutant LPS.
  • the term “intact” means that the LPS has '0' antigen carbohydrates.
  • whole Gram-negative bacteria means any Gram-negative bacteria with all of its component parts, not just the intact or core LPS, lipid A or rough mutant portions thereof.
  • antibiotics refers to a group of organic chemicals that inhibit the growth and proliferation of microorganisms, especially prokaryotic microorganisms, or that kill such microorganisms. Many of said antibiotics are the products of microorganisms, especially the soil microorganisms of the Streptomycetes. A number of synthetic and/or semi-synthetic antibiotics are known, e.g., amoxicillin. Of particular importance to the invention are antibiotics that have a broad spectrum of activity against Gram-negative microorganisms. Such antibiotics as are known to be useful in the treatment of Gram-negative bacteremia and Gram- negative sepsis, are particularly important.
  • Gram-negative bacteremia and sepsis is commonly treated with aminoglycoside antibiotics and, usually, at least one of the following antibiotics: cephalosporins, penicillins, beta-lactams, chloramphenicol , erythromycin, vancomycin, trimethaprim sulpha, clindamycin, rifampicin, metronidizole and quinolone antibiotics.
  • Monoclonal antibodies that meet the functional criteria of the invention may be made using cells of diverse mammalian origin. Rat and human embodiments have been made.
  • the antibodies may be of any isotype, including IgG and IgM, with IgM types being specifically exemplified herein.
  • the human embodiments are the products of triomas synthesized by somatic cell hybridization using a mouse x human parent hybrid cell line and Epstein-Barr virus (EBV)-transformed human PBLs or splenoeytes from non- immunized volunteers or volunteers immunized with available Gram-negative bacterial vaccines or inactivated Gram- negative bacteria.
  • EBV Epstein-Barr virus
  • Fresh PBLs or splenoeytes may be used, if desired.
  • the rat embodiments are the products of hybridomas synthesized by somatic cell hybridization using a rat myeloma line and splenoeytes from rats immunized with an E. coli Re mutant.
  • a preferred strategy for preparing and identifying hybrids that produce antibodies of the invention follows. Cells (PBLs, splenoeytes, etc.) are panned on LPS coated tissue culture plates, then EBV transformed and fused to the tumor fusion partner (mouse myeloma x human B cell or rat myeloma).
  • Panning involves incubation of the population of immunocompetent cells on a plastic surface coated with the relevant antigen. Antigen-specific cells adhere. Following removal of non-adherent cells, a population of cells specifically enriched for the antigen used is obtained. These cells are transformed by EBV and cultured at 10"* cells per microtiter well using an irradiated lym phobias to id feeder cell layer. Supernatants from the resulting lymphoblastoid cells are screened by ELISA against an E. coli Re LPS and a Salmonella Re LPS. Cells that are positive for either Re or Re lipid A LPS are expanded and fused to a 6-thioguanine- resistant mouse x human B cell fusion partner.
  • hybrids are selected in ouabain and azaserine.
  • Supernatants from the Re or Re positive hybrids are assayed by ELISA against a spectrum of Gram-negative bacteria and purified Gram-negative bacterial LPSs. Cultures exhibiting a wide range of activity are chosen for in vivo LPS neutralizing activity.
  • Many but not all antibodies so produced are of the IgM class and most demonstrate binding to a wide range of purified lipid A's or rough LPS's. The antibodies demonstrate binding to various smooth LPS's and to a range of clinical bacterial isolates by ELISA.
  • neutralizing is used to denote the ability of an antibody to block the adverse biological effects of Gram-negative bacteria endotoxin in vitro or in mammals regardless of the particular mechanism involved. It is intended to include, without limitation, mechanisms in which the antibody affects the biological activity of the endotoxin by binding thereto, causes the endotoxin to be degraded, or affects the activity of the endotoxin by altering the kinetics and/or site of its clearance. Neutralizing activity may be assayed in vivo using a murine model. Balb/C mice caged at 37oC, for example, may be injected i.p. or i.v.
  • LPS lethal dose of LPS
  • Antibody may be injected i.p. or i.v. at 0.2 to 20 mg/kg before or after the LPS injection.
  • the neutralizing effect is determined by comparing the morbidity of test mice with that of control mice (e.g., mice given no antibody, mice given non- binding antibody, etc.).
  • the hybridomas that produce the invention antibodies may be grown in suitable culture media such as Iscove's media or RPMI-1640 medium (Gibco, Grand Island, NY) or in vivo in immunodeficient laboratory animals. If desired, the antibody may be separated from the culture medium or body fluid, as the case may be, by conventional techniques such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, electrophoresis, microfiltration, and ultracentrifugation.
  • suitable culture media such as Iscove's media or RPMI-1640 medium (Gibco, Grand Island, NY) or in vivo in immunodeficient laboratory animals.
  • the antibody may be separated from the culture medium or body fluid, as the case may be, by conventional techniques such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, electrophoresis, microfiltration, and ultracentrifugation.
  • the monoclonal antibodies of this invention may be used passively to immunize individuals who suffer from bacteremia or sepsis or who are at risk with respect to bacterial infection.
  • a plurality of different monoclonal antibodies each of which recognizes and binds to a distinct determinant of the cell wall LPS located interiorly of the core region are employed.
  • the antibody/antibodies will normally be administered parenterally (e.g., intravenously, intraarterially, intramuscularly, intraperitoneally), preferably intravenously.
  • the dose and dosage regimen will depend upon whether the antibody/antibodies is/are being administered for therapeutic or prophylactic purposes, the patient, and the patient's history.
  • the total amount of an antibody adminstered per dose will typically be in the range of about 0.1 to 20 mg/kg of patient body weight, preferably 0.1 to 10 mg/kg of patient body weight.
  • the antibody/antibodies will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers.
  • the vehicle may contain minor amounts of additives such as substances that maintain isotonicity and chemical stability, e.g., buffers and preservatives.
  • the antibody will typically be formulated in such vehicles at a concentration of about 1.0 mg/ml to 100 mg/ml .
  • All cell lines were maintained in Iscove's DME medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 5 x 10 -5 M 2-mercaptoethanol. The cell lines were checked routinely for the presence of mycoplasma. For large-scale production of human monoclonal antibodies, cell lines were adapted to serum-free growth in HL-1 medium obtained from Ventrex Labs, Portland, ME, and in HB104 medium obtained from Hana Biologicals, Berkeley, CA.
  • FBS fetal bovine serum
  • HB104 Hana Biologicals, Berkeley, CA.
  • Volunteers with naturally acquired high titer serum antibodies to E. coli J5 or S. minnesota R595 core glycolipids or vaccinated with a standard available typhoid injection to produce high LPS antibody titers were used as sources of peripheral blood lymphocytes. Fifty ml of venous blood was drawn from the volunteers on days 5 and 7. The blood was centrifuged, the buffy coat was harvested, and the harvested buffy coat was gradient centrifuged using Ficoll/Hypaque to separate mononuclear cells from lymphocytes. Alternatively, peripheral blood was gradient centrifuged using Ficoll/Hypaque for the same purpose.
  • Ficoll/Hypaque in a 1 liter quantity is prepared by dissolving 64 g of Ficoll (Sigma) in 600 ml of distilled water using a stir bar rotor at low speed and then adding 99 g of diatrizoate sodium (Sterling Drug, N.Y.). After both substances were dissolved, more water was added to 1 liter volume and 0.7 g NaCl was added.
  • monocytes were depleted by adherence to plastic.
  • T cells were depleted by aminoethylthiouronium-treated sheep red blood cells (AET-SRBC) rosetting, using the technique described by Madsen and Johnsen, J. Immunol. Meth. (1979) 27:61-74.
  • the remaining B cell-enriched lymphocyte population was transformed with Epstein- Barr virus using the technique described by Foung et al., J. Immunol. Meth. (1984) 70:83-90, except that cells were generally cultured at 10 3 -10 4 cells/well in 96-well culture plates.
  • the cultures were assayed after 20 days by ELISA using E. coli J5 as antigen (described below).
  • the cells in one well exhibiting a high antibody titer were subcultured at 1000 cells/well, then at 500 cells/well, and positive cultures containing core- specific, antibody-secreting cells were pooled.
  • the mononuclear cells were isolated from the peripheral blood and were incubated on plastic to dep.lete plastic-adherent monoeytes.
  • the remaining cells (T and B) were panned on plates coated with LPS of S. minnesota Re R595 obtained from Ribi ImmunoChemResearch, Inc.
  • Adherent cells were then transformed by EBV and maintained in culture or cultured in microtiter plates and screened for those positive for Re LPS. Those cells maintained in culture were panned again ten days later on LPS of E. coli J5 from Ribi. These cells also were then cultured in microtiter plates. Panning on a specific antigen can at least double the number of recoverable antigen-specific cells and can improve the secretion rate of antibody.
  • B. Rat Splenoeytes S. minnesota Re R595 obtained from Ribi ImmunoChemResearch, Inc.
  • Adherent cells were then transformed by EBV and maintained in culture or cultured in microt
  • E. coli Re mutant J5 in saline at 10* cells/ml was used as antigen.
  • Rats were injected i.p. and sq with 0.5 ml of bacterial suspension + 0.5 ml of complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • the rats were boosted on day 25 with an identical injection and on day 29 with 0.2 ml of bacterial suspension injection i.v. Splenectomies were carried out on day 32.
  • C. F3B6 Mae x Human Line
  • a mouse-human heterohybrid fusion partner designated F3B6 (adapted to 99% serum-free medium and deposited with the ATCC under ATCC Accession No. HB8785 on April 18, 1985) was constructed by fusing human peripheral blood lymphocyte (PBL) B cells obtained from a blood bank with the murine plasmacytoma cell line NS1 obtained from ATCC under ATCC No. TIB18(P3/NS1/1-AG4-1). The PBL cells from random buffy coat were transferred to a 50 ml centrifuge tube and diluted with 30 ml Hanks' balanced salt solution (Ca 2+ -free/Mg 2+ -free) (HBSS-/-).
  • PBL peripheral blood lymphocyte
  • the NS-1 cells were grown in 4 x T75 flasks and harvested, washed with HBSS-/- and resuspended in HBSS-/-. The cells were counted.
  • HBSS HBSS-/- and resuspended in Iscove's medium in several T150 flasks.
  • the cells were washed in HBSS-/- in 50 ml centrifuge tubes. A total of 10 ml of Ficoll-Hypaque was added to the tubes. The tubes were centrifuged at 1500 rpm for 15 minutes at room temperature and the live cells at the interface were removed. The pellet was washed twice with RPMI-1640 (Gibco) and resuspended in an enriched hypoxanthine/azaserine selection medium (EHA) consisting of 100 ⁇ M hypoxanthine (Sigma), 5 ⁇ g/ml azaserine (Sigma) and Iscove's medium (Gibco), 10% NCTC (M. A. Biologicals), 20% heat-inactivated- FBS. The density was adjusted to 2.5 x 10 4 cells/ml medium.
  • EHA hypoxanthine/azaserine selection medium
  • the suspensions were washed twice with HBSS-/- and resuspended in 10 ml Iscove's medium. Live cells were separated by Ficoll-Hypaque density gradient centrifugation as described above. Cells were washed twice with RPMI-1640 + 20% FBS, and then plated out in 96-well plates at 10 6 cells/ml. At days 7, 9 and 12 the EHA selection medium described above was added each time. At days 15 and 18 the plates were fed with EHMT medium containing hypoxanthine, methotrexate and thymidine. The supernatants were assayed for Ig secretion and Ig secreting hybrid cell lines were cloned by limiting dilution in U bottom 96 well plates.
  • Well F3B6 was selected for 6-thioguanine selection.
  • Several roller bottles of F3B6 were grown up. A total of 10 ⁇ g/ml of 6- thioguanine was added to the roller bottles. Dead cells were removed by Ficoll-Hypaque density gradient centrifugation on days 2, 5 and 7. A 6-thioguanine resistant clone was grown up. Test fusions were performed, and the cell line was tested for ouabain resistance.
  • the resultant cell line was adapted to growth and maintenance in 99% serum- free medium and 1% FBS for more reproducible manufacturing by the following multi-step process:
  • the cells were fed with a mixture of the Iscove's DME in which they were growing, 50% of the amount of FBS in the medium in which they were growing, and 50% by weight of serum-free medium HL-1 supplied by Ventrex, Inc.
  • the cells were subcultured and planted with 50% of Iscove's DME medium and 50% of the serum-free medium. The cells were removed from the latter medium by centrifugation at 200 x g for five minutes. The Iscove's DME medium was mixed with 50% of the serum-free medium to form a 50:50 mixture, in which the cell pellet was suspended and then counted. An appropriate amount of cell suspension was planted in the vessel with 50% Iscove's DME and 50% serum-free medium. The planted cell densities preferably do not fall below 5 x 10 ⁇ cells/ml and not exceed 1 x 10 5 cells/ml.
  • the cells were cultured on serum- free medium only.
  • the cell densities were between 1 x 10 5 to 8-9 x 10 5 cells/ml.
  • the final medium was HL1 with 1% FBS.
  • the fusion mixture contained polyethylene glycol (PEG) 4000, 40% (w/v); dimethyl sulfoxide (DMSO), 10% (v/v) in Hanks' balanced salt solution (HBSSJ-/+ (Ca 2+ -free, 2 mM MgSO 4 ). Forty g of PEG 4000 was combined with 10 ml of DMSO and 50 ml of HBSS-/+. The mix was autoclaved for 25 minutes. Before use, the pH of the fusion mixture was adjusted to between 7.5 and 8.5 with sterile 0.1 N NaOH.
  • Plates (6-well cluster, 35 mm well diameter) were prepared as follows: 2 ml of HBSS-/+ and 50 ⁇ l of a filter sterilized, 20- 100 ⁇ g/ml, peanut agglutinin (PNA, Sigma) were added to each well. Plates were incubated at 37oC for at least one hour prior to use. PNA stock was stored frozen, and a freshly thawed aliquot was used to coat fusion cells. Smaller sized wells were used if cell numbers were limited.
  • FDM fusion dilution mixture
  • Immulon microtiter plates (Dynatech) were used or plates were prepared as follows for use in ELISAs. Fifty-100 ⁇ l of 0.5-1.0% glutaraldehyde (Sigma) in deionized water was coated onto flat/bottom microtiter plates (Dynatech). After one to four hours of incubation at room temperature, the wells were aspirated or washed twice with distilled water.
  • Bacteria were grown overnight, spun down, washed in saline three times and reconstituted to .25% (v/v). One-hundred ⁇ l of this bacteria suspension was used to coat each well of a 96-well flat bottom Immulon microtiter plate or plates prepared as above. Plates were spun for 20 minutes at 2000 rpm. The suspensions were incubated overnight or for a minimum of two hours.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase conjugated goat anti-human IgG
  • HRP horseradish peroxidase conjugate goat anti-human IgM
  • ABTS substrate was then added to each well, the substrate consisting of 55 mg (ml of ABTS aqueous stock soltuion diluted 1:1000 with .1 M sodium citrate buffer pH 4.5 to which 1:1000 of 30% H 2 O 2 was added immediately at 37oC in the dark.
  • HRP horseradish peroxidase
  • Immulon II flat-bottom microtiter plates were coated at 100 ⁇ l/well with goat anti-human IgM (Tago) diluted 1:100 in 50 mM bicarbonate buffer (pH 9.6). After 90 minutes at 37 oC, plates were washed with PBS ++ , 0.05% Tween 20, and preferably 0.01% thimerosal up to five times by immersion or with automated plate washer. Then 100 ⁇ l of PBS ++ , 1% BSA, 0.05% Tween 20, 0.01% thimerosal was preferably added to each well. A total of 100 ⁇ l of test supernatant was added to first wells and preferably duplicae two-fold dilutions were made. One well was preferably left as control.
  • Protein A-coated sheep erythrocytes (1.0%) were added to the upper layer of soft agar according to the method of Gronowicz et al., Eur.
  • Some antibodies demonstrate broad cross-reactivity to not only core antigenic determinants, but to O-antigenie determinants found on whole bacteria.
  • + is off scale with the plate reader set at 2.0 at 2.0 absorbance full scale.
  • Antibody D250 binds only to J5 E. coli (Re) core determinants with minimal binding to other core lipopolysaccharides and rough mutant bacteria. It gives spotty binding to clinical isolates of E. coli.
  • Antibody D244 binds to E. coli J5 and Salmonella minnesota R595 LPS and bacteria, but not to any other bacteria or lipopolysaccharides.
  • Antibodies D234 and D267 show considerable binding to rough lipopolysaccharides and lipid A's with spotty binding on clinical isolates.
  • the 'L' series and 'W' series of antibodies show high binding on rough lipopolysaccharides with a few 'holes' in the binding patterns.
  • Antibodies within the 'L' series bind to more clinical isolates with a higher binding affinity. In general, those monoclonals showing the highest amount of core LPS or rough bacterial binding demonstrate the most cross-reactivity on clinical isolates.
  • Tables I -VI indicate that all of the monoclonal antibodies produced in accordance with this invention, except L116-2-4, which is not part of the invention in view of its properties, meet the binding criteria defined herein, i.e., they bind strongly to the purified lipid A determinants of E. coli Re and/or S. minn. Re LPS (Tables I, IV and V) and they bind to these determinants in intact LPS (Table III) and in whole bacteria (Table VI).
  • Tables IV and V show that the S261 comparative antibody does not bind or binds very weakly (one-seventh the strength of D234) to the purified lipid A determinants of E. coli Re and/or S. minn. Re.
  • mice were injected intraveneously with a dose of 0.2 ml each of a 1:1 dilution of a hybridoma culture supernatant of negative control antibody D-253 or invention antibody D250, described above.
  • mice were challenged i.p. with 7, 10-fold dilutions of E. coli J5 core LPS bacteria, five mice per dilution, where before infection the bacteria were suspended in 0.5 ml of hog gastric mucin, 5% in normal saline, to which 15 mg of D-galactosamine had been added.
  • Table VII indicates the results, where LD 50 is the lethal dose of LPS bacteria at which 50% of the mice die.
  • the cells were fed with a mixture of the Iscove's DME in which they were growing, 50% of the amount of FBS in the medium in which they were growing, and 50% by weight of serum-free medium HL-1 supplied by Ventrex, Inc. or HB104 supplied by Hana Biologicals.
  • the cells were subcultured and planted with 50% of Iscove's DME medium and 50% of the serum-free medium. The cells were removed from the latter medium by centrifugation at 200 x g for five minutes. The Iscove's DME medium was mixed with 50% of the serum-free medium to form a 50:50 mixture, in which the cell pellet was suspended and then counted. An appropriate amount of cell suspension was planted in the vessel with 50% Iscove's DME and 50% serum-free medium. The planted cell densities preferably do not fall below 5 x 10 4 cells/ml and not exceed 1 x 10 5 cells/ml.
  • Step 3 was ' repeated for another passage.
  • the cells were cultured on serum-free medium only. When the cells were planted in the serum- free medium for the first time the cell densities were between 1 x 10 5 to 8-9 x 10 5 cells/ml.
  • Figure 1 shows the growth curve of D-234 cells in serum-free medium HL-1 (Ventrex).
  • Figure 2 shows the growth curve of D-267 cells in serum- free medium HB104 (Hana Biologicals). Under these conditions, as much as 50-75 ⁇ g/ml of specific human monoclonal antibody was produced.
  • a monoclonal antibody producing hybridoma designated T88 was obtained as the fusion product of human splenoeytes with cell line F3B6.
  • a human spleen specimen from a lymphorna patient was mascerated in Hank's balanced salt solution to release lymphocytes from the parenchyma of the spleen and suspend the cells.
  • the suspended cell fraction was collected and was gradient centrifuged using Ficoll/Hypaque to separate lymphocytes.
  • T cells were removed by aminoethylthiouronium treated sheep red blood cells (AET-SRBC) rosetting, using the technique described by Madsen and Johnsen, J. Immunol. Meth. (1979) 27::1-74.
  • the remaining cells were panned on plates coated with LPS of S. minnesota Re R595 obtained from Ribi ImmunoChemResearch, Inc.
  • Adherent cells enriched in B-cell population were transformed with Epstein-Barr virus using the technique described by Foung et al., J. Immunol. Meth. (1984) 70:83-90, except that cells were generally cultured at 10 3 -10 4 cells/well in 96-well culture plates.
  • the cells were maintained in culture and assayed after 20 days by ELISA using E. coli J5 as antigen (described above).
  • the cells in one well exhibiting a high antibody titer were subcultured at 1000 cells/well, then at 500 cells/well, and positive cultures containing core-specific, antibody-secreting cells were retained for fusion.
  • Fusions were carried out with cell line F3B6 using 40% (w/v) PEG 4000; DMSO, 10% (v/v) and 5*g/ml poly I arginine in Hank's balanced salt solution (HBSS)-/+ (Ca 2+ -free, 2 mM MgSO 4 ) using the fusion protocol described hereinabove.
  • Hybrids produced by the fusion were selected using azaserine, hypoxanthiene and oubain resistance and assayed by ELISA on LPS on whole J5 bacteria (described hereinabove).
  • T88 was identified as a cell producing antibody reactive with LPS antigen and has been deposited under accession number CTCC 10235 in applicant's depository. It has been accepted for deposit in the American Type Culture Collection under the terms of the Budapest Treaty for the deposit of microorganisms for patent purposes and has accession number HB7431. Therapeutic Effects of Human Anti-J5 Monoclonal Antibodies
  • outbred CD-1 female mice weighing 23-25 grams were injected intraperitoneally (i.p.) with 6 x 10 7 colony forming units of E. coli strain SM18.
  • antibody in 0.5 ml PBS was injected intravenously (i.v.) in the tail vein of experimental mice.
  • One and three hours after administration of the bacteria 3.2 mg/kg gentamicin was injected intramuscularly (i.m.) in each mouse using 0.05 ml saline as a carrier.
  • Type-specific polyclonal rabbit antibody to SM18 and an irrelevant (IgM) antibody (Rockland) were run as controls in some experiments as indicated. In all experiments, a bacterial control was run in which the mice were sham injected with PBS i.v. and i.m.
  • D234 was aministered as indicated above in four separate experiments.
  • VIII-1 and VIII-2 polyclonal type-specific rabbit immune globulins to E. coli strain SM18 was also used as a positive control.
  • VIII-3 and VIII-4 myeloma human IGM was used as a negative non-specific antibody control.
  • the amount of D234 was measured in an IgM ELISA with D234 as a standard. However, 10 mg of D234 by ELISA equals 14 mg by standard
  • T88 and L118 were tested for efficacy in the therapeutic infection model in the same manner as D234. These antibodies were tested at 10 mg/mouse (363 mg/kg) (IgM ELISA) and for T88 also at the lower doses of 2.5, 1.0, and 0.5 mg/mouse. These results are shown in Tables IX-1 and IX-2. Results
  • both T88 and L118 treated mice showed 80% survival as compared to 35% for the mice treated with Gentamicin alone.
  • the level of survival enhancement is highly significant at P less than 0.001.
  • T88 did not show significant protective effect.
  • both T88 and L118 preparations as employed in the tests were not free of endotoxin, the protective effect observed was not due to endotoxin because these antibodies were administered to mice 30 minutes after infection when endotoxin administration no longer influenced the outcome of infection.
  • T88 was shown to be protective against endotoxic shock in baboons.
  • the antibody from one culture supernatant was purified as shown in Figure 3 into two preparations. Both preparations were shown by chromatography on Bakerbond ABx resin, using a salt gradient for elution, to contain two peaks.
  • the T88-10 preparation contained 70% peak 1 and 30% peak 2, while the T88-10A preparation contained 15% peak 1 and 85% peak 2.
  • the fractionation of the T88 into two peaks was not unique to the Bakerbond ABx resin, but can also be seen with gradient salt elution on other chromato graphic matrices such as S- Sepharose and Q-Sephrose.
  • the elution profile for the T88-10A preparation from the Bakerbond ABx column is shown in Figure 4.
  • the T88-10A preparation was used to test for protective activity in baboons.
  • a total of 5 mg/kg of T88-10A was administered to three baboons. Three mg/kg was administered in a single I.V. bolus 60 minutes before the animals were challenged with a lethal dose of E. coli, and 2 mg/kg simultaneously with the E. coli challenge. About 4 x 10 10 organisms were used. The E.coli dose was infused over a two hour period. This dose of E. coli was shown in control experiments to be lethal in 100% of the animals that received it. The animals generally expire within 16 to 32 hours.
  • T88-10A is a useful antibody to prevent or to treat septic shock.
  • Tables 10 and 11 summarize the T88 u chain assay and LPS binding studies, respectively, for the peak 1 and 2 antibody seen in the two separate purification runs of T88, that is T8810 and T8810A.
  • ⁇ pk(l+2) represents the total amount of antibody present in peaks 1 and 2.
  • "u" represents the amount of u chain to yield an O.D. of 1.
  • 1/ng is the reciprocal of the concentration of antibody required to yield an O.D. of 1.
  • ⁇ /(1/ng) is the ratio of the total amount of antibody, and the reciprocal of the concentration needed to yield an O.D. of 1. It is clear from the data that the amount of u chain is nearly identical for both peaks of either T8810 or T8810A.
  • Table XI compares the areas of peaks 1 and 2 to LPS binding activity measured by ELISA.
  • the areas of peak 1 and 2 are represented by pkl and pk2, respectively.
  • ng.LPS represents the amount of LPS required to produce and OD of 1.
  • 1/ng.LPS is the reciprocal of the LPS concentration required to give an OD of 1.
  • pk2/(1/ng) is the ratio of the amount of peak 2 antibody to the amount of the reciprocal of LPS required to give an OD of 1. It is apparent from the data that peak 2 exhibits enhanced LPS binding activity.
  • mouse x human fusion partner F3B6 adapted to 99% serum- free medium which partner was the source of these hybridomas was deposited with the ATCC, with the deposit date and accession number given below:
  • antibiotic may be administered i.m., i.v., i.p. or if required, intrathecally into the eerebrospinal fluid.
  • Antibody will be administered i.v. or i.p., but most preferred is i.v. Antibody and antibiotic may be coadministered if the pharmaceutically acceptable carriers are compatible.
  • human anti-LPS monoclonal antibodies affect endotoxic shock associated with sepsis caused by Gram-negative bacteremia by either (1) enhanced host clearance of whole bacteria infecting the host, thereby reducing the source of endotoxin, and (2) enhanced host clearance and subsequent detoxification of endotoxin by the reticuloendothelial system and liver.

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Abstract

Anticorps monoclonaux qui se lient à des déterminants définis par les parties des lipides A des lipopolysaccharides de membrane cellulaire de mutants Rc E. coli et de mutants Re Salmonella et neutralisent l'endotoxine bactérienne Gram négative, et qui sont séparables en deux fractions par chromatographie d'échange ionique. On prend comme exemple des IgM humains. Ces anticorps sont utiles pour traiter la bactérémie ou la septicémie par administration parentérale.
EP89904495A 1988-09-19 1988-09-19 Endotoxine bacterienne gram negative bloquant des anticorps monoclonaux Withdrawn EP0434685A1 (fr)

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GB9105292D0 (en) * 1991-03-13 1991-04-24 Sandoz Ltd Improvements in or relating to organic compounds
US6749831B1 (en) 1997-05-16 2004-06-15 Medical Defense Technology, Llc Vaccine against lipopolysaccharide core
US6790661B1 (en) 1999-07-16 2004-09-14 Verax Biomedical, Inc. System for detecting bacteria in blood, blood products, and fluids of tissues
WO2007102442A1 (fr) * 2006-03-05 2007-09-13 Biomedical Research Group Inc. Anticorps monoclonal, hybridome, et procédé de quantification de lipopolysaccharide
KR20180101341A (ko) * 2015-11-10 2018-09-12 비스테라, 인크. 리포폴리사카라이드에 특이적으로 결합하는 항체 분자-약물 접합체 및 그의 용도
EP3571223B1 (fr) 2017-01-18 2024-09-18 Visterra, Inc. Conjugués molécule d'anticorps-médicament et leurs utilisations
JP2023521319A (ja) 2020-04-03 2023-05-24 ビステラ， インコーポレイテッド 抗体分子－薬物コンジュゲートおよびその使用

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FI853395L (fi) * 1984-09-05 1986-03-06 Cetus Corp Monoklonala antikroppar som blockerar gram-negativ bakterieendotoxin.

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