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
  • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
• • 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 relates generally to the application of monoclonal antibody technology to the problems associated with bacterial infections in humans; and, more particularly, to the isolation of cell lines suitable for the production of human monoclonal antibodies specific for and capable of neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A.
• the continued prevalence and seriousness of Pseudomonas infections are indicative of the limited effectiveness of available treatment regimens (see, Andriole, V., J. Lab. Clin. Med. (1979) 94:196-199).
• the virulence associated with Pseudomonas aeruginosa appears to be the result of a number of bacterial products.
• the outer cell membrane contains lipopolysaccharide (endotoxin) which displays a series of toxic properties.
• this bacteria produces a number of extra-cellular products that may contribute to its pathogenicity, including hemolysins, proteases and other extra-cellular enzymes.
• exotoxin A and exoenzyme S Two of these extra-cellular enzymes, exotoxin A and exoenzyme S, have been shown to demonstrate eucaryotic protein synthesis inhibition by exhibiting adenosine diphosphate ribosyltransferase activity, similar to that previously found for diptherial toxin fragment A.
• Pseudomonas aeruginosa exotoxin A has been shown to catalyze the transfer of the adenosine 5'-diphosphate ribosyl moiety of nicotinamide adenine dinucleotide onto elongation factor 2 (EF-2) according to the following reaction: exotoxin A NAD + EF-2 -> ADPR-EF-2 + nicotinamide
• ADPR-EF-2 complex can no longer function properly (i.e. , as native EF-2) in eucaryotic protein synthesis.
• EF-2 serves a crucial role in protein synthesis by acting at the elongation step of polypeptide assembly, eucaryotic protein synthesis is effectively inhibited (see Iglewski, B. and Kabat, D., "NAD-Dependent Inhibition of Protein Synthesis by Pseudomonas aeruginosa Toxin," Proc. Nat. Acad. Sci. U.S.A. (1975) 72:2284-2288).
• exotoxin A production has been demonstrated in 85-90% of clinical Pseudomonas aeruginosa strains isolated from human infections (Bjorn, M. et al.
• exotoxin A is presumed to play a central role in human Pseudomonas aeruginosa infections.
• methods of neutralizing the effects of exotoxin A in vivo have been studied as possible means to control such infections.
• exotoxin A neutralization may be through the use of specific antibody.
• the passive protective effect of rabbit anti-exotoxin A has been studied in a mouse model entailing serious burn and subsequent lethal P. aeruginosa infection.
• Murine monoclonal antibodies reactive with exotoxin A have also been prepared and tested in animals by Galloway, D. et al. , "Production and Characterization of Monoclonal Antibodies to Exotoxin A from Pseudomonas aeruginosa,” Infect. Immun. (1984) 44:262-267. Some of the mouse monoclonals in this report were reported to be capable of neutralizing exotoxin A in vitro and one allegedly mediated increased survival in the burned, P. aeruginosa- infected mouse model. Of course, a mouse monoclonal antibody, while possibly useful in treating mice, presents major problems for human applications. The human immune system can be expected to generally recognize any mouse monoclonal antibodies as foreign substances.
• Murine monoclonals would therefore be anticipated to be minimally useful for passive immunization in humans.
• monoclonal antibodies capable of binding to and neutralizing exotoxin A, as well as antibodies that are only minimally immunogenic to humans. Also needed are methods for production of such monoclonal antibodies and compositions useful in treatment of P. aeruginosa infections.
• the present invention fulfills these needs. SUMMARY OF THE INVENTION
• the present invention provides human monoclonal antibodies capable of neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A, and methods of making such antibodies. These human monoclonal antibodies are useful in passive immunization against Pseudomonas aeruginosa infections.
• the invention provides a method for treating a human susceptible to bacteremia and/or septicemia by administration of a prophylatic or therapeutic amount of a composition comprising at least one human monoclonal antibody capable of specifically reacting with and neutralizing exotoxin A, the composition preferably also including a physiologically acceptable carrier.
• the composition may contain any one or more of the following: a second human monoclonal antibody capable or reacting with a serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from human blood plasma, where the plasma is obtained from a human exhibiting elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa and/or products thereof; and one or more antimicrobial agents.
• a second human monoclonal antibody capable or reacting with a serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa
• a gamma globulin fraction from human blood plasma a gamma globulin fraction from human blood plasma, where the plasma is obtained from a human exhibiting elevated levels of immunoglobulins reactive with Pse
• the human monoclonal antibodies of the present invention may be produced by a cell line, such as a human lymphocyte cell line immortalized by Epstein Barr virus transformation. Such cells can then be cultivated and the human monoclonal antibodies recovered, all by well-known procedures.
• FIGURES Figure 1 is an immunoblot analysis of two
• Figure 2 is an immunoblot demonstrating that two human monoclonal antibodies of the present invention react with exotoxin A produced by each of seven Fisher immunotypes of Pseudomonas aeruginosa.
• the human monoclonal antibodies may be administered for passive immunization to a human host exhibiting symptoms of susceptible to bacteremia and/or septicemia.
• the monoclonal antibodies have particular utility when administered as an adjunct therapy, i.e., in conjunction with human monoclonal antibodies specific for a serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa or other gram-negative bacteria; with a gamma globulin fraction fr,om human blood plasma, particularly when such plasma is obtained for a human exhibiting elevated level of immunoglobulins reactive with Pseudomonas aeruginosa; and with various antimicrobial agents.
• the human monoclonal antibodies are preferably produced by B-lymphocyte cells obtained from human donors who have been exposed to a Pseudomonas aeruginosa infection and who have developed a strong immune response to the infection.
• a human host may be sensitized with exotoxin A or other suitable antigens according to ' well-known techniques (see, e.g., Jones, R. et a ., "Control Trial of Pseudomonas Immunoglobulin and Vaccine in Burn Patients," The Lancet (1980) 1263-1265) and their blood collected.
• the hosts are vaccinated subcutaneously at one week intervals and bled three weeks after the last injection. If desired, the host may be revaccinated with a booster subcutaneous injection and again bled three weeks later.
• Mononuclear cells may be obtained from peripheral blood, spleen, bone marrow or lymph nodes and separated from the other components therein by standard techniques, such as through Ficoll-Paque.
• the T cells can be separated by any convenient technique, e.g. , mass E-rosetting.
• Variations in the mononuclear cell preparation for subsequent immortalization include the use of unseparated versus T cell depleted spleen cells, mitogen-stimulated versus unstimulated cells, and the like.
• the particular technique will, of course, vary depending upon the success of the particular immortalization procedure utilized.
• the monoclonal antibodies of the present invention are produced by cell-driven Epstein Barr virus (EBV) transformation of the B-lymphocyte cells.
• EBV Epstein Barr virus
• the transformed cells so-produced are characterized as continuously growing lymphoblastoid cells that possess a diploid karyotype, are Epstein Barr nuclear antigen positive, and secrete monoclonal antibody of either IgG, IgM, IgA, or IgD isotype, including subtypes IgGl, IgG2, IgG3 and IgG4.
• the cell-driven transformation process itself is an invention of M. E. Lostrom, a co-inventor of the present invention, and is described in detail in U.S. Patent No. 4,464,465, which is incorporated herein by reference.
• the lymphocytes can be transformed with EBV to generate immortalized lymphoblastoid cells, which can be used in a subsequent fusion with a fusion partner.
• EBV epigallocate virus
• the fusion partner may be a mouse myeloma line, a heteromyeloma line or a human myeloma or other immortalized line, such as described in PCT Application No. 81/00957; Schlom et al ⁇ ., Proc. Nat. Acad. Sci. U.S.A. (1980) 72:6841-6845; and Croce et al., Nature (1980) 288:488-489.
• Desirable characteristics of a fusion partner are high efficiency of fusion to provide for a high proportion of immunoglobulin-producing hybridomas, absence of the production of individual chains or immunoglobulins unassociated with the immunoglobulin of interest, and the maintenance of the capability of continuously secreting the desired immunoglobulin over long periods of time.
• the fusion is carried out generally in the presence of a non-ionic detergent, normally polyethylene glycol, for a short period of time, the detergent removed, and the cells subjected to selective conditions which are cytotoxic to the parent cells, but not to the fused cell (e.g., HAT, HAT and ouabain, etc.).
• Hybrid cells which grow out from the selective media are seeded into individual wells and are screened by any convenient technique for the monoclonal antibodies of interest.
• the screening is accomplished utilizing functional assays, such as cytotoxicity inhibition assays, to increase the likelihood that isolated clones will be capable of producing neutralizing antibodies.
• Cells secreting suitable monoclonal antibodies are then cloned by limiting dilution procedures, and the clones producing higher levels of specific antibody are then expanded.
• the antibodies may be further characterized as to class and subclass.
• the antibodies may be purified by any convenient technique, such as chromatography, electrophoresis, precipitation and extraction, or the like.
• the antibodies may then be employed without further change after purification, or may be modified by reduction to various size fragments, such as F(ab')_, Fab, Fv, or the like.
• the antibodies may be conjugated to various moieties capable of controlling the conjugate's half-life in serum after injection into a human host.
• the cell lines of the present invention may find use other than for the direct production of the human monoclonal antibodies.
• the cell lines may be fused with other cells, transferring the genes providing for expression of the monoclonal antibodies, and thus providing new hybridomas.
• the cell lines may be used as a source of the chromosomes encoding for the immunoglobulins,, which may be isolated and transferred to cells by techniques other than fusion.
• the genes encoding for the monoclonal antibodies may be isolated and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin in a variety of hosts.
• a single cDNA clone, coding for the immunoglobulin and free of introns may be isolated and placed into suitable prokaryotic or eucaryotic expression vectors and subsequently transformed into a host for ultimate bulk production.
• Monoclonal antibodies specific for exotoxin A also have utility in numerous research, production and diagnostic applications, in accordance with well-known techniques.
• these monoclonal antibodies can be purified from the cell line supernatants and bound to a solid support (e.g., cyanogen bromide-activated Sepharase 4B; Pharmacia Fine Chemicals, Piscataway, N.J.) for use in affinity chromatography purification of exotoxin A, particularly as modified or fragmented for vaccines.
• a solid support e.g., cyanogen bromide-activated Sepharase 4B; Pharmacia Fine Chemicals, Piscataway, N.J.
• the monoclonal antibodies of this invention can be incorporated as components of pharmaceutical compositions containing a therapeutic or prophylactic amount of at least one of such antibodies in conjunction with a pharmaceutically effective carrier.
• a pharmaceutical carrier can be any compatible, non- toxic substance suitable to deliver the monoclonal antibody(ies) to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically accepted adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition.
• Such compositions may contain a single monoclonal antibody so as to be specific against exotoxin A alone.
• compositions may contain two or more monoclonal antibodies to form a "cocktail.”
• a cocktail containing one or more human monoclonal antibodies against a lipopolysaccharide of immunotypes or serotypes most prevalent in human disease would be combined with one or more human monoclonal antibodies capable of neutralizing exotoxin A.
• the preparation of exemplary cell lines producing such monoclonal antibodies (Anti-LPS Monoclonals) useful in this regard is disclosed in commonly assigned U.S. Serial No. 614,184 and U.S. Serial No. 734,624, both of which are incorporated herein by reference. Such a combination would have enhanced activity against most strains of the bacterium.
• the human monoclonal antibodies of the present invention may also be used in combination with existing blood plasma products, such as commercially available gamma globulins and immune globulins products widely used in prophylactic or therapeutic treatment of P. aeruginosa or other gram-negative bacterial diseases in humans.
• existing blood plasma products such as commercially available gamma globulins and immune globulins products widely used in prophylactic or therapeutic treatment of P. aeruginosa or other gram-negative bacterial diseases in humans.
• the plasma will be obtained from human donors exhibiting elevated levels of immunoglobulins reactive with P. aeruginosa (e.g. , endotoxins, exotoxins, etc.). See generally, the compendium "Intravenous Immune Globulin and the Compromised Host," Amer. J.
• the monoclonal antibodies of the present invention can be used as separately administered compositions given in conjunction with antibiotics or antimicrobial agents.
• the antimicrobial agents may include an anti-pseud ⁇ monal penicillin (e.g. , carbenicillin) in conjunction with an aminoglycoside (e.g. , gentamicin, tobramycin, etc.), but numerous additional agents (e.g. , cephalasporins) well-known to those skilled in the art may also be utilized. Possible effective combination treatments or cocktails would include the following:
• Anti-exotoxin A Anti-LPS Monoclonals + Antibiotics Anti-exotoxin A + Anti-LPS Monoclonals Anti-exotoxin A + Antibiotics
• the human monoclonal antibodies of this invention are particularly suitable for oral, topical or parenteral administration.
• the pharmaceutical compositions may be administered parenterally, i.e. , subcutaneous, intramuscular or intravenous.
• this invention provides compositions for parenteral adminis ⁇ tration, the compositions comprising a solution of one or more of the human monoclonal antibodies dissolved in an acceptable carrier, typically an aqueous carrier.
• an acceptable carrier typically an aqueous carrier.
• aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
• These compositions may be sterilized by conventional, well-known techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances as required, to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
• concentration of the antibody(ies) of this invention in these formulations can vary widely, i.e., from less than 1% to as much as ⁇ 5% to 20% by weight, and will be selected primarily based on fluid volumes and viscosities etc., preferably for the particular mode of administration selected.
• a typical pharmaceutical composition for intravenous infusion could be made up to contain up to 250 ml of sterile Ringer's solution and 50 mg of monoclonal antibody(ies).
• parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference.
• the monoclonal antibodies may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be ef- fective with conventional immune globulins, and various well-known lyophilizations and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss and that use levels may be adjusted to compensate.
• compositions containing the present human monoclonal antibody(ies) or cocktail thereof can be administered for the prophylactic and/or therapeutic treatment of Pseudomonas aeruginosa bacterial disease.
• compositions are administered to a patient already infected with Pseudomonas aeruginosa, in an amount sufficient to cure or at least partially arrest the infection and its complications.
• An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own immune system, but generally range from about 1 to 200 milligrams of antibody per kilogram of body weight, with dosages from 5 to 25 milligrams per kilogram being more commonly used.
• the materials of this invention may generally be employed in serious disease states, including life- threatening or potentially life-threatening situations, especially bacteremia and toxemia.
• life- threatening or potentially life-threatening situations especially bacteremia and toxemia.
• substantial absence of "foreign substance" rejections which are achieved by the present human monoclonal antibodies of this invention, it is possible, and may be felt desirable by the treating physician, to administer substantial excess of these antibodies.
• compositions containing the present monoclonal antibody(ies) or cocktails thereof are administered to a person judged to be at risk to acquire a serious infection with P. aeruginosa, i.e., not already infected by this bacterium.
• P. aeruginosa i.e., not already infected by this bacterium.
• Such an amount is defined to be a "prophylactically effective dose.”
• the precise amount again depends upon the patient's state of health and general level of immunity, but generally ranges from 0.1 to 2.5 milligrams per kilogram, especially 0.5 to 2.5 milligrams per kilogram.
• compositions can be carried out with dose levels and pattern being selected by a treating physician.
• pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the disease and thereby the patient.
• EXPERIMENTAL The following demonstrates methods for the production of cell lines secreting human monoclonal antibodies against exotoxin A of Pseudomonas aeruginosa, and further demonstrates the protective activity of said antibodies in vivo against a lethal challenge of exotoxin A.
• A. Obtaining Suitable Human Cells
• Human B cells were isolated from peripheral blood samples of cystic fibrosis patients known to have had chronic infection with Pseudomonas aeruginosa.
• Mononuclear cells harvested from the interface, were washed twice in growth medium, Iscove's Modified Dulbecco's Medium (Gibco #430-2200), supplemented to 15% (v/v) with heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 ⁇ g/ml streptomycin and 100 I.U./ml penicillin. (This formulation is hereinafter referred to as Iscove's medium.) Washed cells were counted and assessed for viability via trypan blue vital stain on a hemacytometer by standard techniques. Cell samples from two different hviman donors were prepared in this manner.
• the transforming cell line designated 1A2 (A.T.C.C. CRL 8119), was an Epstein-Barr nuclear antigen (EBNA) positive human lymphoblastoid cell line derived by ethyl methanesulfonate (EMS) mutagenesis of the lymphoblastoid cell line GM1500, followed by subculture in the presence of 30 micrograms/ml of 6-thioguanine to render the surviving cells deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase and thus sensitive to HAT
• EBNA Epstein-Barr nuclear antigen
• EMS ethyl methanesulfonate
• 1A2 cells in logarithmic growth phase were mixed with the washed peripheral blood mononuclear cells from (A) in a ratio of 8:1. Approximately 10,000 mononuclear cells and 80,000 1A2 cells were plated per round-bottom well of 96 well plates (Costar 3799) in 200 ⁇ l of Iscove's medium supplemented with HAT and 0.5 micrograms/ml cyclosporin A (Sandoz). The cyclosporin A was used to inhibit T lymphocyte function. Cultures were fed once at day 6 by removal and replacement of one-half of the culture fluid volume with fresh Iscove's medium containing HAT and cyclosporin A supplements.
• 1A2 cells in logarithmic growth phase were mixed with the washed peripheral blood mononuclear cells from a second and different donor in a ratio of about 3:1.
• Approximately 73,000 1A2 cells and 24,000 PBLs were plated per round-bottom well of ten 96 well plates in 200 ⁇ l per well of Iscove's medium supplemented with HAT and cyclosporin A, as previously described in Transformation #1. Cultures were fed on days 6, 8, 10 and 13 post initiation by removal and replacement of 50% of the culture fluid volume per well with Iscove's medium supplemented with HT (no aminopterin) and cyclosporin A. .Growth of EBV-transformed B cells was microscopically evident in virtually all wells plated.
• Indicator cells mouse connective tissue, clone of L strain, A.T.C.C. CCL1 were grown as attached cell cultures in RPMI 1640 (Gibco) supplemented to 15% (v/v) with heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 micrograms/ml streptomycin and 100 I.U./ml penicillin (this formulation is hereinafter called RPMI).
• the indicator cells were plated at lxl0 4 / ell of 96 well flat-bottom plates (Costar 3596) in RPMI and incubated approximately 48 hours at 37°C in a humid chamber in ambient air supplemented to 6% C0 2 .
• Exotoxin A was partially purified from spent culture medium of P. aeruginosa strain PA103 (A.T.C.C. 29260) by the methods described by Iglewski, B. et al. , "Toxin Inhibitors of Protein Synthesis: Production, Purification, and Assay of Pseudomonas aeruginosa Toxin A," in Methods in Enzymology (1979) 60:780-793, Academic Press, Inc., New York. Dilutions of the partially purified exotoxin A in RPMI were incubated in wells with the L cells prepared as described. From this titration, a dilution of the exotoxin preparation was selected for subsequent assays.
• the cells from wells 8B9 and 4A4 were independently subjected to several (usually three) rounds of limiting dilution cloning until all clonal supernatants assayed gave a positive reaction. Clonings were performed in 96 well round bottom plates with 1x10 irradiated (2400 Rads) human peripheral blood lymphocytes (PBLs) per well as feeder cells.
• PBLs peripheral blood lymphocytes
• cell line 8B9 and cell line 4A4 described herein were deposited in the American Type Culture Collection and given the following designations: 8B9 - A.T.C.C. CRL
• the isotype of monoclonal antibodies 4A4 and 8B9 was determined with the use- of an enzyme linked immunosorbent assay (ELISA).
• ELISA enzyme linked immunosorbent assay
• Partially purified exo- toxin A was diluted 1:50 in PBS and 60 ⁇ l of this mate ⁇ rial was placed into the wells of a 96-well flat-bottom microtiter plate. Following an overnight incubation, unabsorbed antigen was aspirated and the wells were rinsed once with washing buffer (0.9% NaCl plus 0.05% (v/v) Tween 20).
• Spent culture supernatant containing monoclonal antibody 4A4 or 8B9 was diluted 1:1 with PBS, pH 7.2, containing 0.1% Tween 20 and 0.2% (w/v) bovine serum albumin (BSA), and 50 ⁇ l of diluted anti ⁇ bodies were added to separate wells.
• the plate was incubated at 25°C for 30 minutes, after which the supernatants were removed, the wells rinsed three times with washing buffer, and 50 ⁇ l of horseradish peroxidase (HRP) conjugated goat anti-human immunoglobulin G (IgG) (American ualex International #A1114) or anti-human immunoglobulin M (IgM) (A IRP) conjugated goat anti-human immunoglobulin G (IgG) (American ualex International #A1114) or anti-human immunoglobulin M (IgM) (A IRP) conjugated goat anti-human immunoglobulin G (IgG) (American ualex International #A1114) or anti-human immunoglobul
• HRP-goat anti-IgG and HRP-goat anti-IgM were diluted 1:5000 and 1:3000 respectively in PBS, pH 7.2, containing 0.05% Tween 20 and 0.1% BSA.
• the enzyme-conjugated goat antibodies were removed, the wells rinsed three times in washing buffer, and 100 icroliters of substrate (0.8 mg/ml ortho-phenylenediamine dihydrochloride in 100 mM citrate buffer, pH 5.0, plus 0.03% H 2 0 2 in deionized H_0, mixed in equal volumes just before plating) added to each well.
• exotoxin A, EF-2, and [adenxne- 14C]NAD were txtrated to provxde sufficient transfer (incorporatxon) of [adenine- 14C]NAD into the conjugated form [adenxne- 14C]ADP-rxbose-EF-2, total incorporation being 40-60% of the input counts per mxnute (cpm) of radxoactive 14C.
• it was important to adjust the exotoxin A concentration in the above reaction such that the toxin did not exist in such an excess as to prohibit detection of inhibition by the positive control anti-exotoxin A antibodies.
• a partially purified exotoxin A preparation (prepared as described in section C above, but stored frozen at -20°C) was thawed to potentiate the ADPR-transferase activity of the exotoxin A (Vasil, M. , et al., "Structure-Activity Relationships of an Exotoxin of Pseudomonas aeruginosa,” Infect. Immun. (1977) 16_:353-361).
• This preparation was then diluted 1:133 in Buffer 1 (50 mM Tris-HCl, pH 7.5) and 20 ⁇ l was preincubated with 2 ⁇ l of control rabbit anti-exotoxin A serum (Bjorn, supra) or monoclonal antibody containing supernatant in a 1 ml polypropylene tube for 1 hour at 25°C. After incubation, partially purified EF-2 from wheat germ (Chung, D. and Collier, R., "Enzymatically Active Peptide from the Adenosine Diphosphate-Ribosylating Toxin of Pseudomonas aeruginosa," Infect. Immun.
• TCA-soluble material was removed from the squares by three successive 30 minute washes in 10% TCA. The squares were then briefly washed once in acetone, air dried, and placed in individual vials containing 3 ml of scintillation fluid. TCA-precipitable radioactivity was measured on a Beckman LS7000 Liquid Scintillation System and the results shown in Table 1.
• MAb Human monoclonal antibody 6F11 (A.T.C.C. CRL
• monoclonal antibody 4A4 is possibly directed against an epitope on the molecule somewhat removed from the enzymatic site and may exercise its anti-toxic activity by reacting with that portion of the exotoxin A molecule which binds to the toxin receptor on susceptible cell surfaces (Vasil, supra).
• aeruginosa were prepared as described by Bjorn, M. et al., "Effect of Iron on Yields of Exotoxin A in Culture of Pseudomonas aeruginosa PA-103," Infect. Immun. (1978) 19_:785-791. Small volumes of each of the exotoxin A-containing supernatants (total of 8) were then concentrated approximately eight-fold with the use of Amicon Centricon-30 microconcentrators (Amicon 4208) according to manufacturer's instructions.
• Sample preparation for SDS-PAGE was performed as described above with the following modifications: 190 ⁇ l of each crude exotoxin preparation were combined with 20 ⁇ l dissociation buffer, and after heat treatment, 50 ⁇ l of glycerol/dye buffer added. Fifty microliter samples from each preparation were electrophoresed. The remainder of the immunoblot procedure was performed as described above, except that all eight concentrated crude exotoxin supernatants were exposed to monoclonal antibody 4A4 or 8B9 at neat concentration on one NCM blot rather than as individual tracks. As shown in Figure 2, monoclonal antibodies 4A4 and 8B9 reacted with the 71,000 dalton exotoxin A molecule produced by all seven Fisher immunotype strains and PA-103. These data suggest that the 8B9 and 4A4 monoclonal antibodies would react with the exotoxin A of substantially all P. aeruginosa strains that produce the toxin.
• a human monoclonal antibody (C5B7) directed against the lipopolysaccharide of Fisher immunotype 1 of P. aeruginosa was treated in a similar manner.
• a crude exotoxin A preparation was prepared as described earlier for immunoblot analysis, except that after removal of bacteria from the broth culture, the exotoxin A-containing supernatant was first diluted 1:4 with 4°C deionized H 2 0. It was then concentrated approximately 120-fold by: i) precipitation with saturated ammonium sulfate (75% final concentration); ii) solubilization of the precipitate in 0.01 M Tris-hydrochloride, pH 9.0, and 2 mM beta mercaptoethanol; and iii) extensive dialysis against the same buffer.
• mice Female BALB/c mice, between 20 and 22 gm body weight, were divided into three groups of ten mice each. One group was inoculated intraperitoneally (ip) with 0.5 ml of concentrated 8B9 or 4A4 antibody, while another group received 0.5 ml ip of concentrated C5B7 antibody as a negative control. The last group received nothing. Four hours later all animals were challenged ip with 0.3 ml of diluted (in saline) crude exotoxin A preparation representing a 2-3 LD 5Q dose. Animals were observed for a period of five days.
• the human monoclonal antibodies of the present invention provide practical means for neutralizing the toxic effects of exotoxin A.
• these antibodies are minimally immunogenic to humans, and are suitable for therapeutic or prophylactic use, either alone or with other agents.
• these antibodies can be readily and economically produced from cell lines, for example, in tissue culture.
• Two immortal human lymphocyte cell lines, designated 8B9 and 4A4, of the present invention have been deposited at the ATCC on June 4, 1985 and given accession numbers CRL 8833 and CRL 8834, respectively.

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