AU5991786A - Protective human monoclonal antibodies to pseudomonas aeruginosa exotoxin a - Google Patents

Description

PROTECTIVE HUMAN MONOCLONAL ANTIBODIES TO PSEUDOMONAS AERUGINOSA EXOTOXIN A

Field of the Invention 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.

BACKGROUND OF THE INVENTION Over the past three decades, the pattern of potentially fatal bacterial infections in humans has changed dramatically, apparently concomitant with the discovery and widespread use of new antimicrobial agents. The incidence of potentially fatal infections caused by Pseudomonas aeruginosa is particularly a problem for immunocompromised human patients, i.e., those having hematologic malignancies, solid tumors, extensive burns, etc. Indeed, any hospital patient receiving broad-spectrum antibiotics, or undergoing treatment with corticosteroids, cytotoxic drugs, or other agents that deleteriously affect human defense mechanisms, is susceptible to Pseudomonas bacteremia. 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. Also, this bacteria produces a number of extra-cellular products that may contribute to its pathogenicity, including hemolysins, proteases and other extra-cellular enzymes.

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. Specifically, 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

NAD - nicotinamide adenine dinucleotide EF-2 - elongation factor 2 of protein synthesis ADPR - adenosine diphosphate ribosyl

The resulting ADPR-EF-2 complex can no longer function properly (i.e. , as native EF-2) in eucaryotic protein synthesis. As 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).

Research has shown that as little as 60 ng of purified exotoxin A is lethal for mice and correspondingly small quantities .are lethal for other mammals as well (Pollack, M. et al_^., "Neutralizing Antibody to Pseudomonas aeruginosa Exotoxin in Human Sera: Evidence for In Vivo Toxin Production During Infections," Infect. Immun. (1976) 14:942-947). Also, exotoxin A production has been demonstrated in 85-90% of clinical Pseudomonas aeruginosa strains isolated from human infections (Bjorn, M. et al. , "Incidence of Exotoxin Production by Pseudomonas Species," Infect. Immun. (1977) 16:362-366). Moreover, anti-exotoxin A activity has been observed in the sera of humans and other animals recovering from P. aeruginosa infections (Pollack, M. et al. (1976)). And, high acute serum antitoxin titers have been associated with survival of humans with P. aeruginosa infections. (Pollack, M. and Young, L., "Protective Activity of Antibodies to Exotoxin A and Lipopolysaccharide at the Onset of Pseudomonas aeruginosa Septicemia in Man," J. Clin. Invest. (1979) 63:276-286).

For these and additional reasons, exotoxin A is presumed to play a central role in human Pseudomonas aeruginosa infections. Thus, methods of neutralizing the effects of exotoxin A in vivo have been studied as possible means to control such infections.

One method of 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. The data suggested that exotoxin A contributed to the mortality of burned, infected mice and that survival was increased by passive administration of rabbit antitoxin serum (Pavlovskis, O. et al., "Passive Protection by Anti-exotoxin A in Experimental Pseudomonas aeruginosa Burn Infection," Infect. Immun. (1977) 18:596-607).

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. At the very least, this recognition can lead to accelerated clearance of the mouse antibody, mitigating its therapeutic potential (see Levy, R. and Miller, R. , "Tumor Therapy with Monoclonal Antibodies," Fed. Proc. (1983) 42_^:2650-2656). More seriously, the immune response to the murine protein could result in shock and even death caused by, inter alia, an allergic reaction analogous to "serum sickness." Clinical experience has shown that such human immune system responses have limited the effectiveness of mouse monoclonals in approximately one-half of the patients receiving mouse monoclonals for treatment of various tumors (Sears, H. et al., "Phase I Clinical Trial of Monoclonal Antibody in Treatment of Gastrointestinal Tumor," Lancet (1982) 1:762-764; and Miller R.A. et al. , "Monoclonal Antibody Therapeutic Trials in Seven Patients with T-cell Lymphoma," Blood (1983) 6_2:988-995). Murine monoclonals would therefore be anticipated to be minimally useful for passive immunization in humans. Thus, there exists a need for 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.

More specifically, 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. Further, by way of example and not limitation, 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.

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.

Other features and advantages of the invention will become apparent from the following detailed description, which describes, by way of example, the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is an immunoblot analysis of two

_* different human monoclonal antibodies of the present invention and shows that these antibodies recognize exotoxin A as evidenced by their binding of a 71,000 dalton molecule also recognized by specific rabbit anti-exotoxin A antibody.

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.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the present invention, methods are provided for the production and use of human monoclonal antibodies specific for and capable of neutralizing toxic effects of Pseudomonas aeruginosa exotoxin A. As used hereinafter, the word "neutralize" shall refer to the result of any reaction (antibody, clinical, physical or the like) resulting in the diminution and, desirably abrogation, of the toxic effects of exotoxin A as may be evidenced by, for example, cytotoxicity inhibition assays, protection of a mammal against a lethal dose of exotoxin A, etc. The human monoclonal antibodies may be administered for passive immunization to a human host exhibiting symptoms of susceptible to bacteremia and/or septicemia. For such treatments, 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. Alternatively, 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. Typically, 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.

Preferably, the monoclonal antibodies of the present invention are produced by cell-driven Epstein Barr virus (EBV) transformation of the B-lymphocyte cells. 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.

Alternatively, the lymphocytes can be transformed with EBV to generate immortalized lymphoblastoid cells, which can be used in a subsequent fusion with a fusion partner. (See e.g., Brown and Miller, J. Immunol. (1982) 128:24-29.)

A wide variety of fusion partners may be employed which provide for the secretion of human immunoglobulins of the various isotypes described above. 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. Preferably, however, 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. As necessary, 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. In some instances, it may be desirable to conjugate the antibodies to other compounds (e.g., cytotoxic agents, labels, etc.) for achieving particular results. More specifically, 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. Alternatively, 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. In addition, 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. Particularly, by preparing cDNA libraries from messenger RNA, 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. (See generally, "Immunological Methods", Vols. I & II, Eds. Lefkovits, I. and Pernis, B., Academic Press, New York, N.Y. [1979 & 1981]; and "Handbook of Experimental Immunology", Ed. Weir, D., Blackwell Scientific Publications, St. Louis, MO [1978] . For example, 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.

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. Alternatively, pharmaceutical compositions may contain two or more monoclonal antibodies to form a "cocktail." By way of example, for Pseudomonas aeruginosa infections, 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. Preferably, for immune globulins 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. Med., §.(^3a)' March 30, 1984, pgs 1-231, which is incorporated herein by reference. The monoclonal antibodies of the present invention can be used as separately administered compositions given in conjunction with antibiotics or antimicrobial agents. Typically, the antimicrobial agents may include an anti-pseudόmonal 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

Anti-exotoxin A + Gamma Globulin + Antibiotics Anti-exotoxin A + Immune Globulin Anti-exotoxin A + Immune Globulin + Antibiotics Anti-exotoxin A + Gamma Globulin

The human monoclonal antibodies of this invention are particularly suitable for oral, topical or parenteral administration. Preferably, the pharmaceutical compositions may be administered parenterally, i.e. , subcutaneous, intramuscular or intravenous. Thus, in a preferred embodiment, 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. A variety of 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. The 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. The concentration of the antibody(ies) of this invention in these formulations can vary widely, i.e., from less than 1% to as much as Ϊ5% to 20% by weight, and will be selected primarily based on fluid volumes and viscosities etc., preferably for the particular mode of administration selected. Thus, 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). Actual methods for preparing 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.

The 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. In therapeutic applications, 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. It must be kept in mind that 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. In such cases, in view of the 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.

In prophylactic applications, 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. Such an amount is defined to be a "prophylactically effective dose." In this use, 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.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by a treating physician. In any event, the 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 (lymphocytes) were isolated from peripheral blood samples of cystic fibrosis patients known to have had chronic infection with Pseudomonas aeruginosa. The mononuclear cell fraction, containing the B cells, was separated from the blood by standard centrifugation techniques through Ficoll-Paque (Boyum, A., "Isolation of Mononuclear Cells and Granulocytes from Human Blood," Scand. J. Clin. Lab. Invest. (1968) 21 Suppl. 97, 77-89). 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 μg/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.

B. Cell-Driven Transformation of B Cells

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

(hypoxanthxne 1x10 -4 M, amx.nopterxn 4x10-7 M, and thymx .dx.ne 1.6x10-5 M) supplemented growth medxum. Transformation #1

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 μl 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. On days 10, 12 and 13, one-half, three-fourths and three-fourths, respectively, of the culture fluid volumes per well were removed and replaced with the corresponding volume of fresh Iscove's medium supplemented with HT (no aminopterin) and cyclosporin A. Growth of EBV-transformed B cells was microscopically evident in virtually all wells of the ten 96-well plates in this experiment.

Transformation #2

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 μl 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. C. Detection of Specific Antibody Secreting Cells

Assay of culture supernatants for the presence of anti-exotoxin A antibodies was performed on day 15 for Transformation #1 and day 17 for Transformation #2. The assay was a cytotoxicity inhibition assay which was performed as follows:

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). For assays, the indicator cells were plated at lxl0⁴/ 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% C0₂.

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. That dilution was the lowest exotoxin concentration which reproducibly generated a 100% cytotoxic effect on the L cells. Assay of human antibody-containing supernatants from Transformations #1 and #2 were performed as follows. Approximately 150 μl of RPMI growth medium was removed from each well of the indicator cell cultures and discarded. Supernatant fluids (40 μl) from each of the wells of the transformed B cell cultures (from B) were mixed with one 100% cytotoxic dose (in 20 μl) of exotoxin A as described above. Independent mixtures were prepared for each of the 96 wells per plate of transformed B cell cultures. The resulting mixtures were transferred to separate wells of indicator cells and then incubated at 37°C, 6% CO,. After 1 hour, 100 microliters of Iscove's medium was added to each well. Plates were returned to the same incubation conditions and observed after an additional 48-72 hours. If exotoxin A-specific, neutralizing antibody was present in the culture fluid of the transformed B cells then the indicator L cells were unaffected by the toxin dose and grew over the 48-72 hour period. However, if the test supernatant did not contain toxin-neutralizing antibody, the indicator cells were killed. These effects were apparent by microscopic evaluation of each well for evidence of cell growth or cell death by change in number and/or morphology of the indicator cells compared to wells containing controls, including anti-exotoxin A antiserum or Iscove's alone. Using this assay, the supernatant from one well, designated 8B9, was identified as containing toxin-neutralizing activity from transformation #1, and a second well, designated 4A4, with similar toxin-neutralizing activity was identified from transformation #2.

D. Cloning of Specific Antibody-Producing Cells from 8B9 and 4A4

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.

Seven days later, an additional 1x10 irradiated PBLs were added to each well. The resulting clonal cell lines, producing anti-exotoxin A human monoclonal antibody were designated 8B9 and 4A4.

Prior to filing of this patent application, 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

8833 and 4A4 - A.T.C.C. CRL 8834.

E. Characterization of Monoclonal Antibodies 4A4 and 8B9

Isotype of Monoclonal Antibodies 4A4 and 8B9.

The isotype of monoclonal antibodies 4A4 and 8B9 was determined with the use- of an enzyme linked immunosorbent assay (ELISA). Partially purified exo- toxin A was diluted 1:50 in PBS and 60 μl 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 μl 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 μl of horseradish peroxidase (HRP) conjugated goat anti-human immunoglobulin G (IgG) (American ualex International #A1114) or anti-human immunoglobulin M (IgM) (A I

#A1124) was added to the wells. 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. Following a 30-minute incubation at 25°C, 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% H₂0₂ in deionized H_0, mixed in equal volumes just before plating) added to each well. The plate was incubated 30 minutes in the dark, at which time the reactions were terminated by the addition of 50 μl of 3N H_S0₄ to each well. In these experiments, positive color development was observed with monoclonal antibodies 4A4 and 8B9 only when the anti-human IgG reagent was used as the second step reagent, thereby demonstrating an IgG isotype for both monoclonal antibodies.

Effect of Monoclonal Antibodies on ADP-Ribosylation.

The effect of monoclonal anti-exotoxin A con- taining cell supernatants on blocking the

ADP-ribosylation activity of exotoxin A was assessed by the capacity of the supernatants to reduce or inhibit incorporation of radxoactxvxty from [adenxne- 14C]NAD into trichloroacetic acid-precipitable material in the presence of crude EF-2 according to the following reaction scheme: exotoxin A ₊

NAD + EF-2 -*• ADP-ribose-EF-2 + nicotinamide + H

Detailed methods for the preparation of EF-2 and exotoxin have been previously cited herein. However, it will be appreciated that substantial variations can occur among individual preparations of these components. In prxncxple, the [adenxne- 14C]NAD and EF-2 are present in excess amounts to encourage the above reaction to proceed from left to right in the presence of exotoxin A. The amounts of 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. For antibody-mediated inhibition assays, 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.

In the present example, 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 μl was preincubated with 2 μl 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. (1977) 16_:832-841) was diluted 2:3 in 100 mM Tris-HCl, 0.2 mM EDTA, 100 mM dithiothreitol, pH 8.2, and 15 μl added to the reaction tube. This was immediately followed by the addxtxon of 1 μl (0.01 μCx) of [adenxne- 14C]NAD and the tube incubated for 1 hour at 25°C. Following the second incubation step, the reaction was stopped by placing 20 μl of each reaction mixture on 1-inch squares of Whatman 3MM paper that had been previously impregnated with 10% trichloroacetic acid (TCA) (Bollum, F. , "Filter Paper Disk Techniques for Assaying Radioactive Macromolecules," Methods Enzy . (1968) 12B:169-173). 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.

TABLE 1

Treatment Antibody Toxin EF-2 14 C-NAD CPM^a

l^b Buffer 1 + + + 9202

2^C Buffer 1 - + + 116

3 Rabbit , anti-toxin + + + 328

4 MAb 6Fll^e + + . + 9120

5 MAb 4A4 + + + 7770

6 MAb 8B9 + + + 104 a TCA-insoluble radioactive counts per minute. b Represents maximal incorporation - 2 μl of Buffer

1 instead of antibody.

Represents minimal incorporation - 22 μl of Buffer

1 instead of antibody and toxin.

Rabbit anti-exotoxin A was diluted 1:10 in Buffer

1.

Human monoclonal antibody (MAb) 6F11 (A.T.C.C. CRL

8562) is directed against the lipopolysaccharide of the Fisher immunotype 2 of P. aeruginosa.

From the results presented in Table 1, it is evident that monoclonal antibody 8B9 very effectively inhibited the ADP-ribosylation activity of exotoxin A in vitro, whereas monoclonal antibody 4A4 demonstrated a relatively negligible effect. The difference in in vitro inhibition between the two antibodies indicates that each is directed to a different epitope on the exotoxin A molecule. Because the ADP-ribosylation activity of exotoxin A was completely inhibited by monoclonal antibody 8B9, it is possible that this antibody is directed at an epitope within the enzymatic site of the exotoxin A molecule or an epitope in close proximity to the enzymatic site. In the latter case, binding between the antibody and the toxin could still sterically hinder the enzymatic function of the molecule. In comparison, 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).

Immunoblot Analysis of Human Monoclonal Anti-Exotoxin A Antibodies.

Confirmation of the specificity of monoclonal antibodies 4A4 and 8B9 as anti-exotoxin A antibodies was accomplished by immunoblot analysis. For this pur¬ pose, partially purified exotoxin A from strain PA-103 of P. aeruginosa (prepared as described above) was first diluted 1:15 in PBS. Four identical samples were prepared as follows: 30 μl of diluted exotoxin A was combined with 5 μl of dissociation buffer (0.3125 M Tris-hydrochloride, pH 6.8, 25% SDS, and 0.72 M dithiothreitol), heated at 100°C for 5 minutes, cooled to room temperature, and combined with 20 μl of 75% glycerol/25% stock 0.25% bromphenol blue. Each sample was then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel according to Laemmli, U., "Cleavage of Structural Protein During the Assembly of the Head of Bacteriophage T4," Nature (1970) 227:680-685. Separated molecular species were transferred from the gel to a nitrocellulose membrane (NCM) as described by Towbin, H., et al., "Electrophoretic Transfer of Proteins From Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications," Proc. Natl. Acad. Sci. (1979) 76:4350-4354; and the NCM blot blocked for 2 hours in PBS-Tween (Batteiger, B. et al. , "The Use of Tween 20 as a Blocking Agent in the Immunological Detection of Proteins Transferred to Nitrocellulose Membranes," J. Immunol. Meth. (1982) 55_:297-307). Individual tracks were cut from the NCM blot and separately incubated for 1 hour at 25°C in 40 mis of rabbit anti-exotoxin A (prepared as described in Bjorn, supra) previously diluted 1:1000 in PBS-Tween or human monoclonal antibodies diluted 1:10 in PBS-Tween. Following five 5 minute washes in PBS-Tween, each of the NCM blots was incubated for 1 hour at 25°C in a

1:1000 dilution (in PBS-Tween) of alkaline pliosphatase conjugated goat anti-human IgG + IgA + IgM (note: this reagent from Zymed apparently has extensive cross-reactivity to rabbit immunoglobulins). These blots were each subjected to five 5 minute washes in PBS-Tween, after which time antigen-antibody interactions were visualized by incubating the blots for 20-30 minutes at 25°C in 30 ml of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) substrate as described by Leary, J., et al. , "Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots," Proc. Natl. Acad. Sci. (1983) 80:4045-4059. Color de- velopment was stopped by rinsing the blot several times in deionized water.

As shown in Figure 1, the reaction patterns of monoclonal antibodies 4A4 and 8B9 were nearly identical to that of a specific ϊrabbit anti-exotoxin A antibody. The reaction pattern in all three cases was dominated by the recognition of a 71,000 dalton molecule representing the native exotoxin A polypeptide (Vasil, supra), demonstrating that monoclonal antibodies 4A4 and 8B9 were directed against exotoxin A. Additional evidence for this specificity was provided by the non-reaction of a control human monoclonal antibody (C5B7) with the exotoxin A preparation.

Also notable in the blots was the reaction of the 8B9 monoclonal antibody with several lower molecular weight exotoxin A degradation products which were apparently not recognized by the 4A4 monoclonal antibody. This further supports the proposition that the two monoclonal antibodies recognize different epitopes on the exotoxin A molecule.

Immunoblot analysis was also utilized to dem- onstrate that monoclonal antibodies 4A4 and 8B9 reacted with the exotoxin A produced by each of the seven Fisher immunotypes of P. aeruginosa (Fisher, M.W. , et al. , "New Immunotype Scheme for Pseudomonas aeruginosa Based on Protective Antigens," J. Bacteriol. (1969) 9_8_:835-836). For these experiments, crude preparations of exotoxin A were prepared from each of the seven Fisher immunotypes and strain PA-103 of P. aeruginosa as follows. Bacteria-free culture supernatant from broth cultures of each immunotype and strain PA-103 of P. 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 μl of each crude exotoxin preparation were combined with 20 μl dissociation buffer, and after heat treatment, 50 μl 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.

F. Protection Studies with Monoclonal Antibodies Experiments to assess the capability of monoclonal antibodies 4A4 and 8B9 to neutralize the toxic effects of exotoxin A in vivo were performed as follows. Both antibodies were first concentrated from spent culture supernatant by precipitation with saturated ammonium sulfate (50% final concentration) (Good, A. et al., "Purification of Immunoglobulins and Their Fragments" in Selected Methods in Cellular Immunology, Mishell, B. and Shiigi, S., eds., W.H. Freeman and Company, San Francisco, California, (1980) 279-286). Precipitated material was reconstituted in PBS, extensively dialyzed against PBS, and sterile filtered. 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 H₂0. 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. This material was frozen at -70°C and was used freshly thawed in protection studies. 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. In the experiment, all animals that had received no antibody or C5B7 antibody were dead within 36 hours. Of the animals that had received 8B9 antibody, all but one were alive at 36 hours and went on to full recovery by the end of the observation period. Similarly, in the 4A4 experiment all animals that had received 4A4 antibody were alive at 36 hours and fully recovered by day 5. These results demonstrated that both 8B9 and 4A4 anti-exotoxin A antibodies are capable of effectively neutralizing an otherwise lethal challenge of exotoxin A.

From the foregoing, it will be appreciated that the human monoclonal antibodies of the present invention provide practical means for neutralizing the toxic effects of exotoxin A. Importantly, these antibodies are minimally immunogenic to humans, and are suitable for therapeutic or prophylactic use, either alone or with other agents. Moreover, these antibodies can be readily and economically produced from cell lines, for example, in tissue culture. Although the foregoing invention has been described in some detail by way of illustration and example, it will also be apparent that various changes and modifications can be made without departing from the scope and spirit of the appended claims.

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.

Claims (22)

WE CLAIM:

1. A composition comprising a human monoclonal antibody capable of binding with and neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A.

2. A composition according to Claim 1, wherein the monoclonal antibody blocks exotoxin A enzymatic activity.

3. A composition according to Claim 1, wherein the monoclonal antibody exhibits IgG or IgM isotype.

4. A composition comprising a human monoclonal antibody of the IgG isotype capable of specifically reacting with and neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A.

5. A composition comprising at least two human monoclonal antibodies, wherein at least one of said monoclonal antibodies is capable of reacting with and neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A.

6. A composition according to Claim 5, wherein another of said monoclonal antibodies is capable of reacting with a serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa.

7. A composition according to any of

Claims 1, 4 or 5, further comprising a gamma globulin fraction from human blood plasma.

8. A composition according to Claim 7, wherein the plasma is obtained from a human exhibiting elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa or products thereof.

9. A composition according to any of Claims 1, 4 or 5, further comprising an antimicrobial agent.

10. A pharmaceutical composition comprising a composition according to any of Claims 1, 4 or 5 and a physiologically acceptable carrier.

11. A pharmaceutical composition comprising at least one human monoclonal antibody capable of specifically reacting with and neutralizing the toxic effects of Pseudomonas aeruginosa exotoxin A, an antimicrobial agent, a gamma globulin fraction from human blood plasma and a physiologically acceptable carrier.

12. A pharmaceutical composition according to Claim 11, wherein the gamma globulin fraction from human blood plasma is obtained from humans exhibiting elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa bacteria and/or products thereof.

13. A pharmaceutical composition comprising at least one human monoclonal antibody capable of specifically reacting with and neutralizing the toxic effects of Pseudomonas aeruginosa bacteria exotoxin A, at least one human monoclonal antibody capable of reacting with a serotype determinant on a lipopolysaccharide molecule of the bacteria, an antimicrobial agent, and a physiologically acceptable carrier.

14. A method for treating a human susceptible to bacteremia and/or septicemia, which comprises: administering to said human a prophylactic or therapeutic amount of a composition according to any of Claims 1, 4, 5, 11, 12 or 13.

15. A cell line which produces a human monoclonal antibody capable of specifically reacting with and neutralizing Pseudomonas aeruginosa exotoxin A.

16. A cell line according to Claim 15, which is an immortal human lymphocyte cell line.

17. A cell line according to Claim 16, which is a hybrid cell line.

18. A cell line according to Claim 16, which is an Epstein Barr virus transformed B lymphocyte.

19. A cell line according to Claim 18, which is one of A.T.C.C. Accession Nos. CRL 8833 or CRL 8834.

20. A method of producing human monoclonal antibodies specific for and capable of blocking the toxic effects of Pseudomonas aeruginosa exotoxin A, said method comprising: cultivating at least one of the cell lines of Claim 19 and recovering said antibodies.

21. A method for treating a human susceptible to bacteremia and/or septicemia which comprises: administering to said human a prophylactic or therapeutic amount of monoclonal antibodies produced according to Claim 20.

22. A method of treating a human susceptible to bacterial infections, which comprises: administering to said human a prophylactic or therapeutic amount of a human monoclonal antibody capable of neutralizing the toxic effects of

Pseudomonas aeruginosa exotoxin A in combination with one or more of: a prophylactic or therapeutic amount of a human monoclonal antibody capable of reaction with a serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from a human blood plasma exhibiting elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa and/or products thereof; or an antimicrobial agent.