NL194961C - Composition comprising a component capable of reacting with Pseudomonas aeruginosa flagella, pharmaceutical preparation, cell line, monoclonal antibody, method for determining Pseudomonas aeruginosa, and package for detecting thereof. - Google Patents

Description

1 194961

Composition comprising a component capable of reacting with Pseudomonas aeruginosa flagella, pharmaceutical preparation, cell glue, monoclonal antibody, method for determining Pseudomonas aeruginosa, and the package for detecting it. The present invention relates to a composition comprising a component which is able to react with Pseudomonas aeruginosa bacteria.

Such a composition is known from Pollack, Immunoglobulins: Characteristics and Uses of Interventions, biz. 73-79, US Department of Health and Human Services, 1979.

Gram-negative diseases and their most serious complications, for example bacteremia and endotoxemia, are the cause of serious cases of disease and death in human patients. This is particularly the case for the gram-negative organism Pseudomonas aeruginosa, which is increasingly associated with bacterial infections, especially noscomic infections, during the past 50 years.

In recent decades, antibiotics have been the preferred therapy for combating gram-negative diseases. However, the ongoing severe disease cases and high mortality associated with 15 gram-negative bacterial disease points to the limitations of antibiotic therapy, particularly with regard to P. aeroginosa (see, for example, VG Andriole, J. Lab Clin Med. Med 94 (1978) 196-199).

A method for the prevention and treatment of Pseudomonas in the article mentioned in the preamble. It concerns the amplification of the host's immune system by active or passive immunization. For example, it has been found that active immunization of humans or laboratory animals with whole-cell bacterial vaccines or purified bacterial endotoxins from P. aeruginosa leads to the development of specific opsonic antibodies, primarily directed against determinants on the repeat oligosaccharide units of the lipopolysaccharide (LPS) ) molecules located on the outer cell membrane of P. aeruginosa. Such antibodies, whether actively raised or passively transmitted, have been found to be protective against the lethal effects of P. aeruginosa infection in a variety of animal models (Pollack, see above) and in some preliminary human studies (see LS Young and M. Pollack, Pseudomonas aeruginosa, biz. 119-132, Hans Huber, 1980).

The reports mentioned suggest that immunotherapeutic approaches could be used to prevent and treat bacterial disease caused by P. aerugi-nosa, such as by administration of combined human immunoglobulins containing antibodies against the infecting strain or strains. Human immunoglobulins are defined herein as that portion of fractionated human plasma that is enriched in antibodies, including specific antibodies against strains of P. aeruginosa. In connection with certain inherent limitations in the use of human immunoglobulin components, this approach to treatment of P. aeruginosa disease remains under investigation (see, for example, MS Collins and RE Roby, Am. J. Med. 76 (3A) (1984) 168-174, and so far no commercial products are available in which these components have been applied One of the limitations associated with immunoglobulin compositions is that they consist of combinations of samples from a thousand or more donors, which samples have been selected for the presence of special anti-Pseudomonas antibodies The combination leads to an averaging of individual antibody titers, which at best results in modest increase in the resulting titer of the desired antibodies.

Another limitation is that the selection process itself requires costly continuous screening of the donor combination to ensure product consistency. Despite these attempts, the immunoglobu-45 line products can show significant variations from batch to batch and among products from different geographical areas.

Yet another limitation inherent in immunoglobulin compositions is that their use results in the simultaneous administration of large amounts of external proteinaceous substances, which may contain viruses, such as those recently found to be associated with Acquired Immune Deficiency Syndrome, or 50 AIDS) with the possibility of causing adverse biological effects. The combination of low titers of desired antibodies and a high content of external substances can often mean a limitation to suboptimal levels of the amount of specific and therefore beneficial immunoglobulin (s) that can be administered to the patient.

In 1975, Kohier and Milstein reported their discovery that certain mouse cell lines could be fused with mouse spleen cells to form hybridomas, each of which can secrete antibodies of a single specificity, particularly monoclonal antibodies (G. Kohier and C. Milstein, Nature, 256 (1975) ) 495-497). With the advent of this technology it became possible in some cases to prepare large amounts of excellently specific murine antibodies for a particular determinant or determinants for antigens. Subsequently, the use of later developed technologies made it possible to produce human monoclonal antibodies (see, for example, U.S. Pat. No. 4,464,465).

It is recognized that in some situations monoclonal antibodies of mice or compositions containing such antibodies may cause problems for use in humans. For example, it has been reported that mouse monoclonal antibodies used in research for the treatment of certain human diseases can elicit an immune response that makes them ineffective (RL Levy and RA Miller, Ann. Rev. Med. 34 (1983) 107 -116). However, it is possible that with recent advances in recombinant DNA technology, such as the production of mouse / human monoclonal antibodies, these problems can be overcome. Methods for the production of human monoclonal antibodies are now also available (see Human Hybridomas and Monoclonal Antibodies, Plenum Publishing Corp., 1985).

A number of groups have reported on the production using hybridoma and / or cell transformation technology of monoclonal antibodies, protecting against P. aeruginosa infections. Monoclonal antibodies have been produced that are reactive with various P. aeruginosa epitopes, including single and multi-serotype specific surface epitopes, as found in bacteria LPS molecules (see, for example, U.S. Patent Applications 734,624 and 807,394). Protective monoclonal antibodies were also prepared specifically for P. aeruginosa exotoxin A (see, e.g., U.S. Patent Application No. 742,170).

Although the use of monoclonal antibodies specific to the LPS region of P. aeruginosa or the exotoxins of the bacterium can provide adequate protection in certain situations, it is generally preferred to have wider protection options. For prophylactic treatment for potential infections in humans, it would be preferable to administer one or more antibodies that protect against a number of P. aeruginosa strains. Similarly, in therapeutic applications where the serotypes of the infecting strains are not known, it would be preferable to administer an antibody or combination of antibodies that are effective against most, if not all, clinically important P. aeruginosa serotypes in ideally by providing antibodies that are reactive across traditional serotype schemes.

One aspect of P, aeruginosa physiology, which appears to contribute to the virulence of the organism, is motility, a capacity that is primarily the result of the presence of a flagellum (see T. Montie et al., Infect, and Immun. 38 (1982) 1296-1298).

P. aeruginosa is characterized by a single flagellum at one end of its rod-shaped structure. Model studies with mice with burn wounds have shown that a greater percentage of mice survived when non-motile P. aeruginosa strains were inoculated in experimental burns than when motile strains were used (A. McManus et al., Bums 6 (1980) 235-239 and T. Montie et al. Infect, and Immun. 38 (1982) 1296-1298). Other studies on the pathogenesis of P. aeruginosa suggest that animals immunized with flagella antigen preparations were protected from burns and infected with motile strains of the bacterium (see I. Holder et al., Infect, and Immun. 40 35 (1982) 276- 280).

Importantly, P. aeruginosa flagella have been studied by serological methods and appear to fall into two main antigenic groups, designated H1 and H2 by B. Lanyi (Acta Microbiol. Acad. Sd. Hung. 17 (1970) 35-48) and as type a and type b by R. Ansorg (Zbl. Bakt. Hyg. I. Abt. Orig. A, 242 (1978) 228-238). Serological typing of flagella by both laboratories showed that H1 flagella (B. Lanyi, see above) or 45 flagella type b (R. Ansorg, see above) was serologically uniform, i.e. no subgroups were identified. This serologically uniform flagellar type will be referred to as type b. The other major antigen, H2 (B. Lanyi, see above) or type a flagella (R. Ansorg, see above) contained five subgroups. This antigen will be referred to as flagella type a and the five subgroups as ag, a1t a2, a3 and a4. The five subgroups of type a are expressed in varying combinations on various strains of type a, which are P.

Wear 50 aeruginosa with the exception of antigen ag. The 3g antigen was found on each type of a flagella, although the degree of variation between the strains.

A serotyping scheme based on the heat-stable principal somatic antigens of P. aeruginosa is referred to as the Habs scheme, recently included in the International Antigenic Typing System scheme (see Int. J. Syst. Bacteriol. 33 (1983) 256). The flagella types of P. aeruginsa Habs 55 reference strains are characterized by immunofluorescence with polyclonal sera by R. Ansorg (Zbl. Bakt. Hyg., I. Abt. Orig. A, 242 (1978) 228-238) or by coagglutination (R Ansorg et al., J. Clin. Microbiol. 20 (1984) 84-88). Habs strains 2,3,4,5,7,10,11 and 12 are flagella type b bearing 3 194961 strains and Habs strains 1, 6, 8 and 9 bear type a flagella. Thus, a large number of strains of P. aeruginosa could possibly be recognized by a small number of monoclonal antibodies, specifically for flagella proteins.

There is therefore a substantial need for monoclonal antibodies that can respond with epitopes to flagella proteins and in some cases also offer protection against multiple serotypes of P. aeruginosa. Furthermore, some of these antibodies would be suitable for use as prophylactic and therapeutic treatments of P, aeruginosa infections, as well as for the diagnosis of such infections.

To this end, the composition described in the opening paragraph is characterized according to the invention in that this component is a monoclonal antibody or binding fragment thereof that is capable of specific reaction with flagella of Pseudomonas aeruginosa bacteria.

A composition according to the invention is useful for the diagnosis and / or treatment of bacterial infections and may offer protection against single or multiple serotypes of P. aeruginosa.

A preferred embodiment of the invention is characterized in that the monoclonal antibody or binding fragment thereof is protective in vivo. This embodiment is preferred because such an antibody or fragment thereof can be used both for diagnostic purposes and for therapeutic purposes.

A further preferred embodiment of the invention is characterized in that said component is a monoclonal antibody or binding fragment thereof that is capable of specific reaction with flagella protein epitope from Pseudomonas aeruginosa. This embodiment is preferred because antibodies that bind to a flagella protein epitope are useful for diagnosis or treatment of infections by multiple serotypes of Pseudomonas aeruginosa.

A further preferred embodiment of the invention is characterized in that the monoclonal antibody is capable of blocking the binding to the epitope of monoclonal antibodies produced by cell lines, designated ATCC Accession Numbers HB9129, HB9130, CRL9300 and CRL9301. This embodiment is preferred because such antibodies are useful for selecting antibodies that recognize Pseudomonas aeruginosa epitopes on flagella. The selected antibodies are useful for diagnosis and / or treatment of a Pseudomonas aeruginosa infection.

A further preferred embodiment of the invention is characterized in that said component is a monoclonal antibody or binding fragment thereof that is capable of reacting with type A or type B flagella of flagella protein epitope from Pseudomonas aeruginosa. This embodiment is preferred because it allows a reaction with both type a and type b flagella protein to be achieved. A preferred variant of this embodiment is characterized in that a second monoclonal antibody is capable of reacting with a type of flagella that is not reactive with respect to the first monoclonal antibody. This embodiment is preferred because when one antibody reacts with type b flagella and the other with type a flagella, multiple serotypes of Pseudomonas aeruginosa are covered. A preferred embodiment of these embodiments is characterized in that the first antibody is a human monoclonal antibody. The reason for this preference is that when a human monoclonal antibody is used, no immune response to the antibody occurs, as is the case when a heterologous antibody is used. It is further preferred in these embodiments that at least one of the monoclonal antibodies is protective in vivo. The reason for this preference is that the composition is then useful both for diagnosis and for treatment of a Pseudomonas aeruginosa infection.

A further preferred embodiment of the invention is characterized in that the composition further comprises a gamma globulin fraction of human blood plasma and / or an antibiotic. This embodiment is preferred because a better antibacterial response can thus be obtained than with the antibody, gamma globulin or antibiotic alone.

The invention further provides a pharmaceutical composition characterized in that it comprises a composition as defined above as well as a physiologically acceptable carrier. Such a composition has the advantage that it is suitable for administration to patients.

The invention also provides a pharmaceutical composition characterized in that it is for use in the treatment or prevention of a Pseudomonas aeruginosa infection, which composition is a monoclonal antibody reactive with a flagella protein of Pseudomonas aeruginosa and in vivo protective, an antimicrobial agent, a gamma globulin fraction of human blood plasma 55 and a physiologically acceptable carrier. Such a composition has the advantage of a special effectiveness for killing Pseudomonas aeruginosa bacteria. A preferred variant of this composition is characterized in that the antibody is a human monoclonal antibody and the 194961 4 gamma-globulin fraction of human blood plasma is obtained from people exhibiting increased concentrations of immunoglobulins reactive to Pseudomonas aeruginosa bacteria and / or products thereof. This embodiment is preferred because of greater protection against infection.

The invention further provides a pharmaceutical composition characterized in that it comprises at least two monoclonal antibodies, each specifically reactive with a different type of Pseudomonas aeruginosa flagellair protein and suitable for treatment or prevention of Pseudomonas aeruginosa infections. Such a composition is preferred because it is suitable for treatment of a wider selection of multiple serotypes of Pseudomonas aeruginosa bacteria. Preferably, at least one of the monoclonal antibodies herein is a human monoclonal antibody. The advantage of this is that it does not elicit such a strong immune response as a heterologous antibody. It is also preferred that the composition further contain at least one human monoclonal antibody capable of reacting with at least one serotypic determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa and / or a monoclonal antibody reactive with exotoxin A. The benefit of such a combination of antibodies is a greater effectiveness against Pseudomonas aeruginosa.

The invention further provides a cell line characterized in that it produces a monoclonal antibody capable of specific reaction with type a or type b Pseudomonas aeruginosa flagella.

A preferred embodiment of this is characterized in that the monoclonal antibody is protective in vivo against Pseudomonas aeruginosa. This embodiment is preferred because the antibody produced by the cell line is widely applicable both for diagnosis and for treatment of a Pseudomonas aeruginosa infection.

A preferred embodiment of the cell line is characterized in that it is a hybrid cell line. Advantageously, such a hybrid cell line produces an antibody that recognizes only a single epitope of Pseudomo-25 nas aeruginosa flagella.

A further preferred embodiment of the cell line is characterized in that it produces human monoclonal antibodies. An advantage thereof is that they do not elicit an immune response to the antibody when administered to a human.

A further preferred embodiment of the cell line is characterized in that it is one of ATCC Accession Numbers HB9129, HB9130 or CRL9300 and CRL9301. These are accessible to the public by depositing the numbers mentioned at the ATCC.

The invention further provides a method for producing monoclonal antibodies, specifically for Pseudomonas aeruginosa flammable proteins and suitable for treatment or prevention of Pseudomonas aeruginosa infections, which method is characterized in that at least one of the cell lines specified above is cultured and the antibodies be won.

The invention also provides a monoclonal antibody characterized by the same binding specificity as one of the monoclonal antibodies, designated FA6 IIG5, Pa3 IVC2, PaF4 IVE8,20H11 and 21B8. The advantage of such antibodies is that they are useful for diagnosis and treatment of infections caused by multiple Pseudomonas aeruginosa serotypes.

A preferred embodiment of this monoclonal antibody is characterized in that it is conjugated to a label capable of providing a detectable signal. Such labeled antibodies are particularly suitable for use in diagnostic tests for Pseudomonas aeruginosa infections. Preferred is that the label is fluorescent or an enzyme. The reason for this preference is that fluorescent and enzyme labels can be easily configured in a standard immunoassay.

The invention further relates to a method for determining the presence of Pseudomonas aeruginosa in a sample, which method is characterized in that the sample is combined with a monoclonal antibody that is reactive with Pseudomonas aeruginosa flagella and complex formation is observed.

The invention also provides a package for the detection of the presence of 50 Pseudomonas aeruginosa bacteria, characterized in that this package comprises a monoclonal antibody composition, which contains at least one monoclonal antibody, which reacts with a type-specific flagellain protein of the bacterium and labels for providing a detectable signal, covalently bound to the antibody or bound to second antibodies, which are reactive with the monoclonal antibody.

New cell lines are provided that can produce monoclonal antibodies capable of binding flagella present on most strains of P. aeruginosa bacterium. The monoclonal antibodies specifically react with epitopes on P. aeruginosa flagella proteins and can, between 194961, separate between type a and type b flagella of the bacterium. Furthermore, a method is provided for the treatment of people who are susceptible to infection or have already been infected with P. aeruginosa by administering a prophylactic or therapeutic amount of a composition containing at least one monoclonal antibody or binding fragment thereof, contained in is capable of reacting with the flagella of 5 P. aeruginosa strains, which composition preferably also contains a physiologically acceptable carrier, the composition may also contain one or more of the following components: further monoclonal antibodies capable of reacting with P aeruginosa exotoxin A; monoclonal antibodies capable of reacting with serotype determinants on the P. aeruginosa LPS; a human blood plasma gamma globulin fraction; a human blood plasma gamma globulin fraction, which plasma may be obtained from a human who exhibits elevated levels of P. aeruginosa reactive immunoglobulins and one or more antimicrobial agents. Furthermore, clinical applications of the monoclonal antibodies are provided, including the manufacture of diagnostic packages.

The invention relates to new cells which are capable of producing monoclonal antibodies and compositions containing such antibodies, which compositions are capable of selective recognition of the flagella present on a number of P. aeruginosa strains, where individual antibodies typically recognize one type of P. aeruginosa flagella. The subject cells have identifiable chromosomes in which the germ-line DNA thereof or a precursor cell is rearranged for encoding an antibody with a binding site for an epitope on a flagella protein that is common among certain P. aeruginosa strains. For type a flagellain proteins, pan-reactive monoclonal antibodies can be produced; for type b flagella proteins, antibodies are included that are pan-reactive or react with at least about 70% of the flagella-bearing strains. These monoclonal antibodies can be used in a wide variety of ways, including! diagnosis and therapy.

The monoclonal antibodies thus provided are particularly suitable for the treatment or prophylaxis of serious diseases caused by P, aeruginosa. The surface proteins on the P. aeruginosa flagella would be available for direct contact by the antibody molecules, thereby probably inhibiting the motility of the organism and / or facilitating other effects beneficial to the infected host.

The preparation of monoclonal antibodies can be effected by immortalizing a cell line capable of expressing nucleic acid sequences encoding antibodies specific for an epitope on the flagella proteins of P. aeruginosa strains. The immortalized cell line can be a mammalian cell line transformed by oncogenesis, transfection, mutation or the like.

Such cells include myeloma lines, lymphoma lines, or other cell lines that are capable of supporting the expression and secretion of the immunoglobulin or binding fragment thereof in vitro. The Immunoglobulin or fragment thereof may be a naturally occurring immunoglobulin from a mammal, other than the preferred mouse or from human sources, produced by transformation of a lymphocyte, in particular a splenocyte, by virus or by fusion of the lymphocyte with a neoplastic cell, for example a myeloma, to form a hybrid cell line. The splenocyte will typically be obtained from an animal immunized against flagella antigens or fragments thereof containing an epitopic site. Immunization protocols are well known and can vary considerably and still remain effective. (See Golding, Monoclonal Antibodies: Principles and Practice,

Academic Press (1983).

The hybrid cell lines can be cloned and screened by conventional techniques and antibodies can be detected in the above cell fluid, which are capable of binding with 45 P. aeruginosa flagella determinants. Subsequently, the appropriate hybrid cell lines can be grown in large-scale culture or injected into the peritoneal cavity of a suitable host for production of ascites fluid.

In one embodiment of the invention, the cells are human lymphocytes transformed, forming human monoclonal antibodies, preferably protecting in vivo, into accessible epitopes, specific for at least one flagellain protein. The lymphocytes can be obtained from human donors who have been or have been exposed to the appropriate strain-bearing strains of P. aeruginosa. A preferred cell-driven transformation process is described in detail in U.S. Patent No. 4,464,465.

By having the antibodies of the invention that are known to be specific to the flagella proteins, in some cases the supernatants of subsequent experiments can be screened in a comparative study with the present monoclonal antibodies as a means of identifying further examples of anti-flammable monoclonal antibodies. Thus, 194961 6, hybrid cell lines can be easily produced from a variety of sources based on the availability of the subject antibodies, which are specific to the particular flagella antigens.

When hybrid cell lines are available that produce antibodies specific to the present epitope sites, these hybrid cell lines can also be fused with other neoplastic B cells, such other B cells serving as recipients for genomic DNA coding for the receptors. While neoplastic B cells from rodents, in particular from mice, are most commonly used, other species of mammals can be used, such as lagomorpha from cattle, sheep, horses, pigs, monkeys and the like.

The monoclonal antibodies may be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgE, or subclasses of IgG, known for each animal species. In general, the monoclonal antibodies can be used intact or as binding fragments, such as Fv, Fab, F ^ b ^, but usually intact.

The cell lines according to the invention can find other uses than for the direct production of the monoclonal antibodies. The cell lines can be fused with other cells (such as suitable drug-labeled human myeloma, mouse myeloma or human lymphoblastoid cells), to form hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies. The cell lines can also be used as the source of the chromosomes, which encode the immunoglobulins, which can be isolated and transferred to cells by techniques other than fusion. Furthermore, the genes encoding the monoclonal antibodies can be isolated and used according to recombinant DNA techniques for the production of the specific immunoglobulin in a variety of hosts. In particular, by forming cDNA libraries from messenger RNA, a single cDNA clone encoding the immunoglobulin and free of introns, it can be isolated and placed in suitable prokaryotic or eukaryotic expression vectors and then transformed into a host for final mass production (see in U.S. Pat. Nos. 4,172,124, 4,350,683, 4,363,799, 4,381,292 and 4,423,147, see also Kennett et al., Monoclonal Antibodies,

Plenum, New York (1980) and the references cited therein).

More specifically, in accordance with hybrid DNA technology, the immunoglobulins or fragments of the invention can be produced in bacteria or yeast (see Boss et al., Nucl. Acid.

Res. 12, 3791 and Wood et al., Nature 314, 446). For example, the messenger RNA transferred from the genes coding for the light and heavy chains of the monoclonal antibodies produced by a cell line according to the invention can be isolated by differential cDNA hybridization using cDNA from BALB / c lymphocytes other than the present clone. The mRNA, which does not hybridize, will be rich in the messages encoding the desired immunoglobulin chains. If necessary, this process can be repeated to further increase the desired mRNA levels.

The subtracted mRNA composition can then be transferred in the opposite direction to provide a cDNA mixture that is enriched for the desired sequences. The RNA can be hydrolyzed with a suitable RNase and the ssDNA can be double-stranded with DNA polymerase I and statistical primers, for example statistically fragmented calf thymus DNA. The resulting dsDNA can then be cloned by insertion into a suitable vector, for example virus vectors, such as lambda 40 vectors or plasmid vectors, (such as pBR322, pACYC184, etc.). By developing probes based on known sequences for the constant regions of the light and heavy chains, the cDNA clones with the gene coding for the desired light and heavy chains can be identified by hybridization.

The genes can then be excised from the plasmids, manipulated to remove excess DNA upstream from the initiation code or constant region DNA, and then introduced into a suitable vector for transformation of a host and eventual expression of the gene.

Suitably, mammalian hosts (e.g., mouse cells) can be used to treat the chain (e.g., joining the heavy and light chains) to produce an intact immunoglobulin and further optionally secrete the immunoglobulin free from the leader sequence. It is also possible to use unicellular microorganisms for the production of the two chains, where further manipulation may be required to remove the DNA sequences, encoding the secretory leader and process signals, while providing an initiation code at the 5 'terminus of the sequence encoding the heavy chain. In this way, the immunoglobulins can be prepared and treated in order to be assembled and glycosylated in cells other than mammalian cells. If desired, each of the chains can be truncated in order to at least retain the variable region, which regions can then be manipulated to provide other immunoglobulins or fragments specific to the flagelate epitopes.

The monoclonal antibodies according to the invention are particularly useful because of their specificity for 7 194961 antigens over almost all P. aeruginosa variants currently known. Also, some of the monoclonal antibodies are protective in vivo, allowing them to be incorporated into pharmaceutical products, such as antibody combinations for bacterial infections.

Monoclonal antibodies according to the invention also find a wide variety of applications in vitro.

For example, the monoclonal antibodies can be used to characterize microorganisms, to isolate specific P. aeruginosa strains, to selectively remove P. aeruginosa cells in a heterogeneous mixture of cells, and the like.

For diagnostic purposes, the monoclonal antibodies may or may not be labeled. Diagnostic studies typically include the detection of the formation of a complex by the binding of the monoclonal antibody to the flagellum of the P. aeruginosa organism. If not labeled, the antibodies find application in agglutination studies. Furthermore, non-labeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for immunoglobulin. The monoclonal antibodies can also be labeled directly.

A wide variety of labels can be used, such as radionuclides, fluorescent substances, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (in particular haptens), etc. Numerous immunoassays are available and, for example, some include such described in U.S. Pat. Nos. 3,817,827, 3,850,752, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074 and 4,098,876.

Usually, the monoclonal antibodies of the invention are used in enzyme immunoassays, where the subject antibodies or second antibodies from another species are conjugated to an enzyme. When a sample comprising P. aeruginosa of a particular serotype, such as human blood or lysate thereof, is combined with the subject antibodies, binding takes place between the antibodies and the molecules that exhibit the desired epitope. Such cells can then be separated from the unbound reagents and a second antibody (provided with an enzyme label) can be added. The presence of the antibody-enzyme conjugate, which is specifically bound to the cells, is then determined. Other conventional techniques may also be used which are known to the skilled person.

Packages may also be provided for use with the subject antibodies for detection of P. aeruginosa in solutions or the presence of P. aeruginosa flagella antigens. Thus, the present monoclonal antibody composition of the invention can be provided, usually in a lyophilized form, either by itself or together with further antibodies specific to other gram-negative bacteria. The antibodies, which may be conjugated labeled or unconjugated, are included in the package with buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, for example, bovine serum albumin, or the like. Generally, these materials will be present in less than about 5% by weight based on the amount of active antibody and usually in a total amount of at least about 0.001% by weight based on antibody concentration. It will often be desirable to include an inert excipient or excipients to dilute the active ingredients, wherein the excipients may be present in an amount of 40 to 99% by weight of the total composition. When a second antibody capable of binding the monoclonal antibody is used, it will usually be present in a separate ampoule. The second antibody is typically conjugated to a label and assembled in an analogous manner to the composition described above with the antibody.

The monoclonal antibodies, in particular human monoclonal antibodies according to the invention, can also be included as components of pharmaceutical compositions which contain a therapeutic or prophylactic amount of at least one of the monoclonal antibodies according to the invention with a pharmaceutically effective carrier. A pharmaceutical carrier should be a compatible non-toxic substance suitable for delivering the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes, and inert solids can be used as a carrier.

Pharmaceutically acceptable excipients (buffering agents, dispersing agents) can also be included in the pharmaceutical composition. Such compositions may contain a single monoclonal antibody to be specific for strains of one flagella type from P. aeruginosa.

A pharmaceutical composition may also contain two or more monoclonal antibodies to form a "cocktail." For example, a cocktail containing monoclonal antibodies against both types of 55 flagella or against groups of the various P. aeruginosa strains (eg different serotypes) would be a universal product with activity against the vast majority of the clinical isolates of the bacterium concerned.

194961 8

The molar ratio of the different monoclonal antibody components will usually not differ by more than a factor of 10, usually not more than a factor of 5, and will usually be in a molar ratio of about 1: 1-2 to each of the other antibody components.

The monoclonal antibodies of the invention can also be used in combination with existing blood plasma products, such as commercially available gamma globulin and immunoglobulin products, used for the prophylactic or therapeutic treatment of P. aeruginosa disease in humans. Preferably, the immunoglobulin plasma is obtained from human donors showing elevated levels of immunoglobulins reactive with P. aeruginosa (see generally the Intravenous Immune Globulin and the Compromised Host compendium, American J. Med. 76 ( 3a) (March 30, 1984), 10 pp. 1-231).

The present monoclonal antibodies can be used as separately administered compositions given in conjunction with antibiotics or antimicrobial agents. The antimicrobial agents may typically contain an anti-pseudomonal penicillin (e.g., carbenicillin) together with an aminoglycoside (e.g., gentamicin, tobramycin, and the like.), But numerous further agents (cephalosporins known to those skilled in the art) may also be used .

The monoclonal antibodies and pharmaceutical compositions thereof according to the invention are particularly suitable for oral or parenteral administration. The pharmaceutical compositions are preferably administered parenterally, in particular subcutaneously, intramuscularly or intravenously. The invention thus provides compositions for parenteral administration containing a solution of the monoclonal antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.4% saline, 0.1% glycine and the like. These solutions are sterile and generally free of particulate material. These compositions can be sterilized according to conventional and known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients, as needed to approximate physiological conditions, such as pH adjusters and buffers, toxicity regulators, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium contactate and the like. The concentration of the antibody in these compositions can vary within wide limits, in particular from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight, and is primarily selected based on liquid volumes, viscosities and the like, which are preferred for the chosen dosage form.

A typical pharmaceutical composition for intramuscular injection may contain, for example, 1 ml of sterile buffered water and 50 mg of monoclonal antibody. A typical composition for intravenous injection may, for example, contain 250 ml of sterile Ringer's solution and 150 mg of monoclonal antibody. Actual methods for the preparation of parenterally administrable compositions are known or clear to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15d ed., Mack Publishing Company (1980).

The monoclonal antibodies of the invention can be lyophilized for storage and can be reconstructed in a suitable carrier prior to use. This technique has been found to be effective with conventional immunoglobulins, using known lyophilization and reconstitution techniques. It would be apparent to those skilled in the art that lyophilization and reconstitution may lead to varying degrees of antibody loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies) and that the levels used may need to be adjusted for compensation.

The compositions containing the present monoclonal antibodies or a cocktail thereof can be administered for the prophylactic and / or therapeutic treatment of P. aeruginosa infections. In therapeutic application, compositions are administered to one patient, already infected with one or more P. aeruginosa serotypes, in an amount sufficient to cure or at least partially stop the infection and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose." Amounts effective for this use are dependent on the severity of the infection and the general condition of the patient's own immune system. but are generally from about 1 to about 200 mg of antibody per kilogram of body weight, usually using a dose of 5-25 mg per kilogram, It should be borne in mind that the materials of the invention will generally be used in severe disease states that that is, life-threatening or potentially life-threatening situations, in particular bacteremia and endotoxemia, caused by P. aeruginosa.

In prophylactic applications, compositions containing the subject antibody or a cocktail thereof are administered to a patient who has not yet been infected by P. aeruginosa to improve the patient's resistance to such a potential infection. Such an amount is defined as a "prophylactically effective dose." In this application, the precise amounts also depend on the health condition of the patient and his general immunity level, but are generally 0.1-25 mg per kilogram, in particular 0.5-2.5 mg per 5 kilograms.

Single or multiple administrations of the compositions can be performed with doses and according to one pattern selected by the treating physician. In any case, the pharmaceutical compositions should provide an amount of the one or more antibodies of the invention that is sufficient to effectively treat or protect the patient.

10

EXAMPLE I

Example I shows the methodology used for the preparation of a mouse monoclonal antibody that is specifically bound to P. aeruginosa flagella.

Three-month-old BALB / c mice were immunized intraperitoneally eight times with viable P. aeruginosa Fisher immunotype 1 and Fisher immunotype 2 (A.T.C.C. 27312 and 27313) bacteria every one to two weeks for a total of nine weeks. The initial bacterial doses were 8 x 10 8 and 1 x 10 7 organisms per mouse for Fisher immunotype 1 and Fisher immunotype 2, respectively, and the dose was increased to 30 to 60-fold during the course of the immunizations.

Three days after the final injection, the spleen of one mouse was aseptically removed and a single cell suspension was prepared by gently rotating the organ between the frozen ends of two sterile glass plates. Mononuclear spleen cells were combined in a 4: 1 ratio with log phase mouse hemoma cells (NSI-1, obtained from Dr. C. Milstein, Molecular Research Council, Cambridge, England) and fused to form hybridomas according to the procedure described in Infect. Immun. 36 (1982) 1042-1053. The final hybrid cell suspension was diluted to a concentration of 1.5 x 10® cells per 25 ml in RPMI hybrid HAT (RPM11640, containing 15% heat-inactivated fetal calf serum, 1 mM

sodium pyruvate, 100 pg / ml penicillin and streptomycin, 1.0 x 10 "4 M hypoxantine, 4.0 x 10'7 M aminopterine and 1.6 x 10'5 M thymidine), which is 2.0 x 10" 6 per ml of freshly prepared BALB / c thymocytes as feed cells.

The mixture was applied to 96-well plates (200 µl per well). The cultures were fed by removal and replacement of 50% of the volume of each well with fresh RPMI hybrid HAT every two to three days. The above culture fluid was tested for the presence of anti-P. aeruginosa antibodies by enzyme-linked immunosorbent assay (ELISA) when cell growth reached approximately 40% confluence in the wells, usually within 7-10 days.

The above culture fluids from the hybrid cells were tested simultaneously for outer membrane preparations of each of the two immunizing bacteria. Outer membrane preparations were isolated by a modification of the method described in Infect. Immun. 36 (1982) 1042-1053. Bacteria (P. aeruginosa Fisher immunotype 1 and Fisher immunotype 2) were inoculated into trypticase soybean culture fluid (TSB) and cultured at 34 ° C for 16-18 hours with aeration in a revolving shaking bath. The bacteria were harvested by centrifugation and washed twice with phosphate buffered saline (PBS, 0.14 M NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 .7 H 2 O, 1.5 mM KH 2 PO 4, PH 7.2) containing 150 trypsin -inhibiting units (TIU) aprotinin per ml.

The pellet from the final centrifugation was resuspended is 0.17 M triethanolamine, 20 mM disodium ethylenediamine tetraacetic acid (EDTA) and then homogenized on ice for 10 minutes. The solid was pelleted by centrifuging the homogenate at 14,900 x g and decartagated, 45 and the supernatant was recentrifuged as described. The solid was again cartoned and the membranes were granulated by centrifuging the supernatant at 141,000 x g for 1 hour. The supernatant was taken and the membrane solid was stored overnight at 4 ° C in 10 ml PBS containing 75 T.I.U. aprotinin per ml. The solid was resuspended the next day by vortication and then sampled and stored at -70 ° C. The protein content of each sample was determined by the method of Lowry et al. (J. Biol. Chem. 193 (1951) 265-275).

The antigen plates for the ELISA were prepared as follows. The outer membrane preparations were diluted to 5 µg per ml of protein in PBS and 50 µl of the solutions were introduced into each well of 96-well plates, sealed and incubated overnight at 37 ° C. Unbound antigen was removed from the plates 55 and 100 μΙ 5% (weight / volume) bovine serum albumin (BSA) in PBS was added to each well, after which the plates were incubated for 1 hour at 37 ° C.

After removal of the non-adsorbed BSA, the supernatant culture fluid (50 μΙ) from each well 194961 of the fusion plates was copied into corresponding wells of antigen plates and incubated for 30 minutes at 37 ° C. The unbound antibody was removed from the wells and the plates were washed three times with 10 µl of 1% (weight / volume) BSA-PBS per well. Then 50 μ of diluted biotinylated goat anti-mouse IgG per well was added to each well and incubated for 30 minutes at 37 ° C. The plates were washed three times as described above, after which 50 μΙ of a preformed avidin: biotinylated horseradish peroxydase complex (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA), prepared according to the manufacturer's specifications, was added to the wells. After 30 minutes at room temperature, the Vectastain reagent was removed from the wells, after which the wells were washed and 100 μ en substrate o-phenylenediamine 10 (0.8 mg / ml in 0.1 M citrate buffer, pH 5) per well. 0, mixed with an equal volume of 0.03% (volume / volume) H 2 O 2) was added. The substrate was incubated for 30 minutes at room temperature in the dark, after which the reactions were terminated by adding 50 μ 3N H 2 SO 4 per well.

Hybridoma cells secreting monoclonal antibodies bound to one of the two antigen preparations were detected by measuring the absorbance at 490 nm of the colorimetric responses in each well on a Dynatech Model 580 MicroELISA reader. The cells in a well, designated Pa3 IVC2, produced antibody that bound only to the Fisher Immunotype 2 antigen plate. This well was further studied as described below. The monoclonal antibody and the clonal cell line are both identified in the following text by the Pa3 IVC2 designation. Pa3 IVC2 cells from the master well were mincloned and cloned by limiting dilution techniques, such as described in Infect. Immun. 36 (1982) 1042-1053.

Ascites fluid containing the high titre monoclonal antibody was prepared in CB6 F, mice (BALB / c (female) x C57BL / 6 (male) Fn) according to procedures described in Infect. Immun. 36 (1982) 1042-1053. Two to three month old male CB6 F mice were injected intraperitoneally with 0.5 ml Pristane (2, 6, 10, 14-tetramethylpentadecane, Aldrich Chemical Co.) 10-21 days before intraperitoneal injection with log phase Pa3 IVC2 cells in RPMI. Each mouse was injected with 0.5-1 x 10 7 cells in 0.5 ml. After about two weeks, the fluid that had collected was removed from the mice every two to three days. The concentration of antibody in the ascites fluid was determined by agarose gel electrophoresis, and all ascites containing 5 mg / ml or more antibody were pooled, sampled, and frozen at -70 ° C.

The above culture fluid from the cloned Pa3 IVC2 cell line was examined by ELISA, as described above, for outer membrane preparations of all seven P. aeruginosa Fisher immunotypes (ATCC 27313-27318), P. aureofaciens (ATCC 13985) and Klebsiella pneumoniae (ATCC 8047 ), all prepared as described above. PA3 IVC2 antibody, bound to the outer membrane preparations of P. aeruginosa Fisher immunotypes 2, 6 and 7 and not to other Fisher immunotypes, P. aureofaciens or K. pneumoniae.

The specific antigen identified by the Pa3 IVC2 antibody was identified by radioimmune precipitation. This briefly includes incubating antigens that are radially labeled with Pa3 IVC2 antibody and a particulate source of protein A, resulting in the formation of insoluble antibody: antigen complexes. These complexes are washed to remove non-specifically bound antigen, after which the complexes are dissociated and separated in a polyacrylamide gel. The predominant radioactive species found in the gel are thereby identified as the corresponding antigens to which the Pa3 IVC2 antibody binds.

Samples (25 pg) of soluble P. aeruginosa Fisher immunotypes 2, 3, 4 and 5 outer membrane preparations were radiolabeled in solid phase with 12SI using lodogen (Pierce Chemical Co) 45 (Biochem. Biophys. Res. Commun. 80 (1978) 849-857; Biochemistry 17 (1978) 4807-4817). This procedure resulted in the iodination of exposed tyrosine residues from most, if not all, proteins in the outer membrane preparations.

To reduce non-specific binding of the outer membrane antigens to antibody Pa3 IVC2, the radiolabeled preparations (5 x 10® counts per minute per analysis) were first incubated for 1 hour at 50 4 ° C with BALB / c normal mouse serum (1:40 final dilution). Then supernatant (0.5 ml) of the Pa3 IVC2 culture containing the Pa3 IVC2 antibody was added to each outer membrane sample. After incubating the antigen and antibody for 1 hour at 4 ° C, the protein A source, IgGSORB (0.095 ml per sample) (The Enzyme Center, Ine., Boston, MA) was added, followed by a further 30 minutes at 4 ° C incubated (J. Immunol. 115 (1975) 1617-1622).

IgGSORB was prepared according to the manufacturer's specifications and just before use, non-specific reactions were prevented by blocking potentially reactive sites with culture media by washing the IgGSORB twice with RPMI hybrid (RPMI-hyride-HAT media excluding HAT).

11, 194961

The antigen-antibody IgGSORB complexes were centrifuged at 1500 xg for 10 minutes at 4 ° C, washed twice with phosphate-RIPA buffer (10 mM phosphate, pH 7.2, 0.15 M NaCl, 1.0% (volume / volume)

Triton X-100, 1.0% (weight / volume) sodium deoxycholate, 0.1% (weight / volume) sodium dodecyl sulfate (SDS), and 1.0% (volume / volume) aprotinin); twice with a high salt buffer (0.1 M 5 Tris-HCl, pH 8.0, 0.5 M LiCl, 1% (volume / volume) of beta-mercaptoethanol); and once with lysis buffer (0.02 M Tris-HCL, pH 7.5, 0.05 M NaCl, 0.05% (volume / volume) Nonidet P-40) (Proc. Natl. Acad. Sci., USA 76 (1979) 4479-4483). Antigen bound to the complex was released by incubation for 10 minutes at 95 ° C with sample buffer (0.125 M Tris-HCl, pH 6.8, 2% (weight / volume) SDS, 2% (volume / 10 volume) betamercaptoethanol and 20% (volume / volume) glycerol) and collected in the supernatant after centrifugation at 1500 xg for 10 minutes.

The supernatant fluid samples were applied to 14% polyacrylamide gels containing SDS prepared according to the method of B. Lugtenberg et al. (FEBS Lett. 58 (1978) 254-258), as modified by Hancock and Carey (J. Bacteriol. 140 (1979) 902 -910) and the antigens were separated into the gel by electrophoresis overnight at a constant voltage of 50 V. After fixation of the gel in 40% (volume / volume) methanol, 10% (volume / volume) acetic acid and 5% ( volume / volume) of glycerol overnight, it was dried on Whatman 3 MM paper via a Biorad money dryer (Richmond, CA). The dried gel was covered with plastic wrap and exposed to Kodak X-AR film for 18 hours at room temperature.

Results of this experiment showed that Pa3 IVC2 bound to only one antigen in the outer membrane preparation of P. aeruginosa Fisher immunotype 2 and not to any other antigen present in the other outer membrane preparations. The molecular weight (MW) of the antigen in the gel was approximately 53,000 daltons, determined by comparing its mobility with that of 14 C-labeled protein standards (phosphorylase B, MW 92,500; BSA, MW 69,000; ovalbumin, MW 46,000; carbonic anhydrase, Mw 30,000 cytochrome C, MW 12,000), which were separated in the same gel. The molecular weight of this antigen correlated with the molecular weight of flagellin, the protein of the flagella of P. aeruginosa (Infect. Immun. 35 (1982) 281-288).

Furthermore, Pa3 IVC2 was tested according to ELISA using P. aeruginosa Habs strains 1-12 (A.T.C.C. 33343-33359), fixed with ethanol on 96-well microtiter plates. The antigen plates were prepared as follows.

Supernatant fluid cultures from each organism were centrifuged, washed twice with PBS and then resuspended in PBS to an A 2 of 0.2 O.D. units. The diluted bacteria were put into wells (50 µl per well) and then centrifuged at 1500 x g for 15 minutes at room temperature. The PBS was aspirated and then ethanol (95%) was added to the wells for 15 minutes at room temperature. After the ethanol was removed from the wells, the plates were air dried and then covered and stored at 4 ° C until use.

The results of the ELISA tests performed as described above showed that Pa3 IVC2 bound to ethanol-fixed Habs strains 2, 3, 4, 5, 7, 10, 11 and 12. This specificity pattern showed that Pa3 IVC2, bound by type b flagella from P. aeruginosa (Zbl. Bakt. Hyg., I. Abt. Orig. A 242 (1978) 228-238; J. Clin. Microbiol. 20 (1984) 84-88). Based on this assignment of specificity to monoclo-40 sew Pa3 IVC2, the P. aeruginosa reference strains, Fisher immunotype 2, Fisher immunotype 6 and Fisher immunotype 7 carry type b flagella. From the foregoing experimental data, it was concluded that Pa3 IVC2 specifically binds to P. aeruginosa flagelline type b.

In vivo protective activity of Pa3 IVC2

Animal tests were performed to determine if Pa3 IVC2 monoclonal antibody would protect a mouse, threatened with multiple LDM from live P. aeruginosa bacterium. The model chosen was the burned mouse model (J. Trauma 23 (1983) 530-534). Groups of mice were given a severe burn according to the described protocol and then immediately threatened with 5-10 LD50s of Fisher immunotype 7. Before the burn and the threat, monoclonal antibody was administered intraperitoneally as high titer ascites (0.2 ml) intraperitoneal). In the 50 animals treated with Pa3 IVC2, no increase in the number of survivors was observed compared to the animals that received no antibody.

EXAMPLE II

Example II shows the methodology for the preparation of a mouse hybridoma cell line, which produces a mouse mono-55 clonal antibody against P. aeruginosa flagellin type b, which is protective in vivo.

Adult female BALB / c mice were first injected intraperitoneally with viable P, aeruginosa Fisher immunotype 6 (ATCC 27317) (8 x 10 6 organisms) followed two weeks later by 194961 12 an injection of viable P. aeruginosa Fisher immunotype 5 (ATCC 27316) (4x10® organisms). During the following two-week period, viable P, aeruginosa Fisher immunotype 5 and Fisher immunotype 6 were co-administered in two injections per week. The dose of each organism was increased such that the final dose was ten times the initial dose. Four days after the last viable bacterial injection, a final injection of P. aeruginosa Fisher immunotype 6 outer membrane preparations (50 pg protein), prepared according to J. Bacteriol, was given. 136 (1978) 381-390). Three days after the last immunization, the spleen of one mouse was removed and the spleen cells were prepared for hybridization in the manner described in Example I.

The above culture fluids from the hybridoma cells were examined for the presence of anti-P. aeruginosa antibodies by ELISA on the day 10 post-fusion according to the procedures described in Example I, except that the antigen for the ELISA plates consisted of viable bacteria immobilized in the wells of the 96 well microtiter plates. The plates were prepared as follows.

Fifty microliters of poly-L-lysine (PLL) (1 pg / ml in PBS) was added to each well of the 96-well plates and incubated for 30 minutes at room temperature. Un adsorbed PLL was removed and the wells were washed three times with PBS. The bacterial cultures grown overnight in TSB were washed once with PBS and then resuspended in PBS to O-D. = 0.2. Fifty microliters of the bacterial suspensions were added to each well of the plate and allowed to bind for 1 hour at 37 ° C. Unbound bacteria were removed, after which the wells were washed three times with saline Tween (0.9% (weight / volume) NaCl, 0.05% (volume / Volume) Tween-20).

Non-specific binding of the antibodies was blocked by the addition of 200 µl / well blocking buffer (PBS, containing 5% (weight / volume) non-fat dry milk, 0.01% (volume / volume) Antifoam A (Sigma) and 0.01% (weight / volume) of thimerosal) at the wells and incubation for 1 hour at room temperature. Excess bio-buffer was discarded and the wells were washed three times with saline Tween as previously described.

The above culture fluids (50 µl) were copied in the corresponding wells of the assay plates and incubated for 30 minutes at room temperature. The above culture fluids were removed and the wells were washed five times with saline Tween.

An enzyme-conjugated second-stage antibody (horseradish peroxidase-conjugated goat anti-mouse IgG + IgM) was diluted in PBS containing 0.1% (volume / volume) Tween-20 and 0.2% (weight / 30 volume) BSA according to previously performed titrations, after which 50 μΙ of the reagent was added to each well and incubated for 30 minutes at room temperature. The excess reagent was removed; the wells were washed five times in saline Tween; 100 μΙ o-phenylenediamine substrate was added per well, after which 30 minutes were incubated as described in example I. The reactions were terminated as described in example I and then read at Α4Θ0 m on a Bio-Tek EL-310 35 Automated EIA Plate Reader.

According to the methods described, the above culture fluids of the fusion were examined for the presence of antibodies that bound to P. aeruginosa Fisher immunotypes 1, 2.3 or 4, but not to control plates prepared by the same PLL and blocking procedure, but without bacteria . The above liquids, containing antibody, which bound to one of these four Fisher 40 immunotypes, were examined a second time with each of the seven Fisher immunotype bacteria separately. Antibody present in the supernatant fluid of one well, PaF4 IVE8, bound only to P, aeruginosa Fisher immunotypes 2, 6 and 7. Cells from well PaF4 IVE8 were cloned by limiting dilution methods as described in Example I. The monoclonal antibody and the clonal cell line from this well are both identified in the following text by the designation PaF4 45 IVE8. High titre monoclonal antibody-containing ascites fluid was generated as described in Example 1 except that BALB / c mice were used instead of CB6F.

Specificity of PaF4 IVE8

A study carried out to identify the antigen bound by PaF4 IVE8 monoclonal antibody was indirect immunofluorescence on bacterial organisms. Each of the seven reference Fisher 50 immunotypes from P. aeruginosa plus a non-fluted P. aeruginosa strain (PA 103, ATCC 29260, J. Bacteriol. 62 (1951) 377-389) and Escherichia coli (GSC A25) were overnight grown in TSB at 37 ° C. The bacteria were centrifuged and then washed twice in PBS. Each strain was resuspended in PBS to an O-D. = 2.2.

The bacterial suspensions were then further diluted 1: 150 and 20 μ samples were placed in individual wells of Carlson slides and dried on the slide at 40 ° C. The above culture fluids (25 μΙ) of PaF4 IVE8 were incubated on the slides in a humidity chamber at room temperature for 30 minutes on the dried bacterial samples. Unbound antibody 13 194961 was washed off the slides by immersing the slides in distilled water.

After the slides were dried, fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse IgG + IgM (25 µl per well of a 1:40 dilution in PBS) was incubated on the slides for 30 minutes at room temperature in the dark room in the dark . The slides were washed again in distilled water, dried and then covered with a cover glass mounted with glycerol in PBS (9: 1). The slides were examined with a fluorescent microscope.

Fluorescent staining was only observed with P. aeruginosa Fisher immunotypes 2, 6 and 7 and showed a sinusoidal pattern (line) starting from only one end of the organisms. This is consistent with the morpholophy and location of the single polar flagellum of these bacteria.

The reaction of PaF4 IVE8 with flagella was confirmed by immuno-fluid analysis. Outer membrane antigens from P. aeruginosa Fisher immunotype 6 (see example I) were separated by electrophoresis in a 14% polyacrylamide gel containing SDS as described in example I, except that the electrophoresis was performed for 5 hours at a constant amperage of 80 mA. Pre-stained molecular weight markers (lysozyme, Mw 14,300; beta-lactoglobulin, MW 18,400; alpha-chymotrypsinogen, 15 Mw 25,700; ovalbumin, MW 43,000; bovine serum albumin, Mw 68,000; phosphorylase B, MW 97,400; and myosin in 200,000) polyacrylamide gel.

Antigens were transferred from the polyacrylamide gel overnight at 4 ° C at a constant amperage of 200 mA to a nitrocellulose membrane (NCM) (0.45 µm) in a Tris-glycine-methanol buffer (Proc.

Natl. Acad. Sci., U.S.A. 76 (1979) 4350-4354), containing 0.05% (weight / volume) SDS. After the transfer, the NCM was incubated for 1 hour at room temperature in 0.05% (volume / volume) Tween-20 in PBS (PBS-Tween) (J. Immunol. Meth. 55 (1982) 297-307). In this treatment and all subsequent treatments, the tray containing the NCM was placed on a rocking platform to ensure distribution of the solution over the entire NCM.

After 1 hour, the PBS-Tween solution was decanted and PaF4 IVE8 ascites (diluted 1: 1000 in PBS-Tween) was added and incubated with the NCM for 1 hour at room temperature. The NCM was then washed five times, each time 5 minutes, with PBS-Tween to remove unbound antibody. Alkaline phosphatase-conjugated goat anti-mouse IgG + IgM (Tago, Inc.) was diluted according to the manufacturer's specifications and incubated with the NCM for 1 hour at room temperature. The NCM was washed five times in the manner described, then substrate-containing bromochlorindolyl phosphate and nitro blue tetrazolium (Sigma) prepared as described in Proc. Natl. Acad. Sci., U.S.A. 80 (1983) 4045-4049) was added and incubated for 10-20 minutes at room temperature. The response was! terminated by washing away the substrate with distilled water. !

The results of this experiment showed that PaF4 IVE8 specifically bound to a single antigen with a molecular weight of 53.00 daltons in the outer membrane preparation. The results of the indirect immunofluorescence study and immunoflowing show that PaF4 IVE8 binds to the flagella of P, aeruginosa. j

The flagella type, which recognized PaF4 IVE8, was determined by ELISA. Habs strains 1-12 (A.T.C.C. j

33348-33359) were each bound to wells of 96-well microtiter plates (Linbro) with PLL, after which the I

ELISA was performed as previously described in this example. The source of PaF4 IVE8 antibody was! 40 above culture fluid. Positive responses were observed in wells containing Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, showing that PaF4 IVE8 binds to type b flagella. The in vivo protection data is presented in Example IV. i

EXAMPLE III

Example III shows the methodology for the preparation of a mouse hybridoma cell line which produces a monoclonal antibody reactive with anti-P. aeruginosa flagella type a, which is protective in vivo.

The lymph cell source for the fusion was a spleen from an immunized BALB / c mouse, which was intraperitoneally injected four times over six weeks with purified type a flagella (10-20 pg protein) from Habs strains 6 and 8 (ATCC 33353 and 33355). Flagella were purified according to the 50 method described in Infect. Immun. 35 (1982) 281-288, with the difference that the last centrifugation was flagella at 100,000 x g for 1 hour instead of 40,000 x g for 3 hours. A second modification, used for some procedures, was to separate the flagella from the bacterium for 30 seconds in a mixer instead of 3 minutes (Infect. Immun. 49 (1985) 770-774).

Protein concentrations of each preparation were determined with the Bio-Rad Protein Assay (Bio-Rad) and the presence of contaminating lipopolysaccharide (LPS) was determined by measuring the KDO content (Anal. Biochem. 85 (1978) 595-601). The molecular weights of the flagella proteins were determined by comparing their migration in an SDS polyacrylamide gel with the migration of standard protein markers 194961 14 (BRL) (see Example II). The molecular weight of Habs 6 flagelline was 51,700 dalton and that of Habs 8 flagelline 47,200 dalton. These values correspond to those of Infect. Immun. 49 (1985) 770-774.

Fusion of splenocytes from flagellin immunized mice and NS-1 myeloma cells was performed three days after the last immunization, as described in Examples I and II. When the hybridoma cells 5 grew to a confluence of about 40% (day 7), the above culture fluids were copied into corresponding wells of three different antigen plates, PLL-bound P. aeruginosa Fisher immunotype 1 (see Example II for preparation) and formalin-fixed Habs 6 and Habs 9.

The bacteria for the formalin-fixed antigen plates were grown, washed and diluted as described for PLL-bound antigen plates. Diluted bacteria (0.2 O.D. units at A 2) were added to individual wells (50 µl per well) of Linbro 96-well microtiter plates, after which the plates were centrifuged at 1200 x g for 20 minutes at room temperature. The supernatants were removed from the wells and 75 µl of 0.2% (volume / volume) formalin in PBS was added to each well and incubated for 15 minutes at room temperature. After the formalin was removed from the wells, the plates were air dried and stored at 4 ° C until use.

Formalin did not alter the antigenicity of the flagella, as evidenced by the ability of anti-flagella antisera to agglutination of formalin-treated organisms (Acta Microbiol. Acad. Sci., Hung. 17 (1970) 3548). P. aeruginosa Fisher immunotype 1 strain was used as a control because this strain was non-flared, as shown by stain staining (Manual of Clin. Microbiol. (1985) ed. Amer. Soc. Microbiol., P. 1099). Hybrid cells in the well, designated FA6 IIG5, produced an antibody that bound to Habs 6 and Habs 9 (both strains of flagella type A), but not to Fisher immunotype 1.

Cells from the well FA6 IIG5 were grown in a subculture and cloned as described in the previous examples. The monoclonal antibody and the clonal cell line from this well are hereinafter referred to as FA6 IIG5. Ascites was produced in BALB / c mice, as described in Example II.

Specificity of FA6 IIG5

The specificity of the FA6 IIG5 antibody was determined by indirect immunofluorescence and immunoflux. The indirect immunofluorescence was essentially performed as described in Example II with the following modifications.

Bacterial cultures grown overnight on trypticase soy agar at 30 ° C were removed from the plates with cotton swabs and resuspended in PBS to an Aeeo of 0.2 O.D. units.

Formalin (0.37% (volume / volume) in PBS final concentration) was added to the suspension with swirling. After incubation at room temperature for 15 minutes, the bacteria were diluted 1:12 in PBS, after which 20 μΙ of the suspension was placed in individual wells of Carlson slides. After drying, the slides were prepared for viewing as described in Example II. The source of antibody was the culture culture supernatant from the FA6 IIG5 cell line.

Fluorescent staining by the FA6 IIG5 antibody was only observed with P. aeruginosa strains that carry type a flagella and do not carry type b in any of them. The observed fluorescence pattern was a sinusoidal line pattern, showing that the FA6 IIG5 bound to the flagella. The fluorescence signal was enhanced by treatment of the bacteria with formalin, but the treatment was not necessary to visualize the flagella staining with the antibody.

Immunoflowing was performed as described in Example II. The sources of flagella type a antigens were the purified flagella preparations (see this example). Antigens were separated into 10% polyacrylamide gels containing SDS (Nature 227 (1970) 680-685) and transferred to an NCM.

Preparations of FA6 IIG5, either culture supernatant or ascites diluted 1: 1000, were reacted with the NCM and the reaction was discovered with a suitable enzyme-conjugated reagent and enzyme substrate as described in Example II.

The immuno-flow showed that FA6 IIG5 specifically bound to the 51,700 MW flagellin of Habs 6 and the 47,200 MW flagellin of Habs 8.

Confirmation that FA6 IIG5 only reacted with flagella type a and was not obtained with type b by ELISA, in which Habs strains 1-12 were individually bound with PLL to the wells of Linbro 96-well microtiter plates. The antibody only bound to Habs strains 1, 6, 8 and 9, which are the only flagella type a strains of the 12 (J. Clin. Microbiol. 20 (1984) 84-88). Research on protection In vivo is presented in Example IV.

15, 194961

EXAMPLE IV

Example IV shows the protection of mice that are passively immunized with antibodies PaF4 IVE8 and FA6 IIG5 against P. aeruginosa threat in the burned mouse model.

The anti-flagella monoclonal antibodies were tested in the burned mouse model according to the method described in J. Trauma 23 (1983) 530-534. For the protection study, all antibodies were purified by protein A-Sepharose chromatography (Immunochemistry 15 (1978) 429-436) and dialyzed in PBS buffer. The flagella type a strain used for the animal experiments was P. aeruginosa PA220 (from Dr. James Pennington, Boston) and the flagella type b strain was the reference K aeruginosa Fisher immunotype 2 (A.T.C.C. 27313).

Per mouse, 40 µg of purified monoclonal antibody was administered intravenously 1-2 hours prior to burning and the threat. Immediately after the surf, the animals received 0.5 ml of cold PBS subeschar, which contained the threatening bacteria. The threat dose was approximately 10 LDs for each organism. The results of the animal tests are given in Tables A and B.

TABLE A

Protection study of an anti-flagella type a monoclonal antibody in the burned mouse model.1 20% survival per day2

Treatment 123456789

Anti-flagella a, 100 100 100 100 100 90 90 80 80 FA6 IIG5 25 Anti-flagella b, 100 20 10 10 10 10 10 10 10

PaF4 IVE8

Non-specific 100 40 30 20 10 10 10 10 10 anti-LPS monoclonal antibody PBS, without 80 80 80 80 80 80 80 80 80 bacteria 1 Mice were sub-scarcely threatened with approximately 10 LDs of P. aeruginosa PA 220.

2 The percentage is based on the survival of mice in 10 animal groups, with the exception of the PBS only control group, which consisted of 5 mice. The days are counted after the threat is burned.

TABLE B

Protection study of an anti-flagella type b 40 monoclonal antibody in the burned mouse model.1% survival per day2

Treatment 123456789 45 Antiflagella b, 100 100 90 90 90 90 90 90 90

PaF4 IVE8

Anti-flagella a, 100 20 10 10 10 10 10 10 10 FA6 IIG5

Non-specific 100 20 20 20 20 20 20 20 20 50 anti-LPS monoclonal antibody PBS, without 100 100 100 100 100 100 100 100 100 bacteria 55___

Mice were sub-threatened with approximately 10 LDs of P. aeruginosa Fisher immunotype 2.

194961 16 * See footnote 2 to Table A.

Very significant survival was observed in mice treated with the anti-a antibody or anti-b antibody and then threatened with the corresponding antigen. Conversely, 80-90% of the untreated but endangered mice or animals died, treated with an inappropriate anti-flagella monoclonal antibody or non-specific anti-LPS antibody. The inability of the anti-flagella type a antibody to protect mice from a lethal threat of flagella type b bearing P. aeruginosa Fisher immunotype 2, and the inability of the anti-flagella type b antibody to protect mice from a lethal threat of type A bearing P, aeruginosa PA220, confirmed in vivo the specificity of the antibodies, which was assumed in vitro. Survival of burned but not infected mice showed that the burn itself was not fatal.

EXAMPLE V

Example V shows the extensive cross-reactivity of PaF4 IVE8 and Fa6 IIG5 with P. aeruginosa clinical isolates, demonstrating the clinical utility of these antibodies for the immunotherapy of P. aeruginosa infections.

Clinical isolates were obtained from hospitals and clinics. The isolates were from a variety of isolation sites, including blood, wounds, respiratory tract, urine, and ears. A total of 157 isolates were investigated.

PaF4 IVE8 specifically bound to 34 clinical isolates (22%), while the flagella type a antibody, FA6 IIG5, bound to 102 clinical isolates (65%), thus a total of 136 of 157 isolates (87%). Of the 21 strains that were not recognized by either antibody, 19 were non-flared, as shown by staining. Both antibodies in combination therefore bound to 136 of 138 (98%) fluted clinical isolates, confirming previous reports (Zbi. Bakt. Hyg., I Abt. Orig. A 242 25 (1978) 228-238).

EXAMPLE VI

Example VI shows methods for the production of human monoclonal antibodies that bind P. aeruginosa type b flagella.

A peripheral blood sample from an individual immunized with a high molecular weight polysaccharide preparation (Infect. Immun. 45 (1984) 309) served as a source of B cells.

Mononuclear cells were separated from the blood by standard centrifugation techniques on Ficoll-Paque (Scand. J. Clin. Lab. Invest. 21 (1968) 77) and washed twice in calcium / magnesium-free phosphate buffered saline (PBS).

The mononuclear cells were depleted on T cells using a modified E-rosette procedure. The cells were first resuspended to a concentration of 1 x 10 7 cells / ml in PBS of 4 ° C containing 20% fetal calf serum (FCS). 1 ml of this suspension was placed in a 17 x 100 ml polystyrene round bottom tube, to which 1 x 10 s with 2-amino-isothiouronium bromide (AET)-treated red sheep blood cells was added from a 10% 40 (volume / volume) solution in Iscove's modified Dulbecco's medium (Iscove's medium) (see J. Immun. Methods 27 (1979) 61). The suspension was mixed very gently for 5-10 minutes at 4 ° C, after which the E-rosette cells were removed by centrifugation on Ficoll-Paque for 8 minutes at 2500 x g at 4 ° C. E-rosette negative peripheral mononuclear blood cells (E'PBMC), which were joined at the interface, were collected and washed once in Iscove's medium, and then resuspended in the same medium containing 15% (volume / volume) FCS, L-glutamine (2 mmol / l) penicillin (100 lU / ml), streptomycin (100 pg / ml), hypoxanthine (1 x 10 "4 M), aminopterin (4 x 10'7 M) and thymidine (1.6 x 10 ') 5 M. This medium is further referred to as HAT medium.

Cell-driven transformation of the E'PBMC was accomplished by co-cultivation of these cells with a transforming cell line. The transforming cell line was an Epstein-Barr nuclear antigen (EBNA) 50 positive human lymphoblastoid cell line, derived by ethyl methane sulfonate (EMS) mutagenesis of the GM 1500 lymphoblastoid cell line, followed by selection in the presence of 30 pg / ml 6-thioguanine to the hypoxanthine-guanine phosphoribosyl transferase cells (HGPRT) deficient and thus make HAT sensitive. This cell line is referred to as the 1A2 cell line and was deposited with the American Type Culture Collection (A.T.C.C.) on March 29, 1982 under A.T.C.C. No. CRL 8119. 1A2 cells in logarithmic growth phase 55 were suspended in HAT medium and then combined with the E'PBMCs in a ratio of fifteen 1A2 cells per PBMC. The cell mixture was plated in thirty round-bottom 96-well microtiter plates (Costar 3799) at a concentration of 32,000 cells / well in a volume of 200 µl per well, and incubated at 19 ° C in a humidified atmosphere at 37 ° C for 6% Contained CO2. The cultures were fed on days 5 and 8 after plating by replacing half of the supernatant with fresh HAT medium. Sixteen days after plating, 100% of the wells showed proliferating cells and in most of the wells the cells were of sufficient density for removal and testing of supernatants for anti-P. aeruginosa antibodies.

The above liquids were screened for the presence of anti-P. aeruginosa antibodies using the ELISA technique, as described in Example II with the following modifications. A combination of the seven Fisher immunotype reference strains (ATCC 27312-27318) (A ^ = 0.2 OD units) was bound to flat-bottom 96-well microtiter plates (Immulon II, Dynatech) pre-treated with poly-L-lysine, incubated and washed as described in Example II. After blocking non-specific binding sites and washing the plates, 50 µl of PBS containing 0.1% (volume / volume) Tween-20 and 0.2% (weight / volume) BSA was added per well. The above culture fluids were then copied by plating into the corresponding wells of assay plates and in control plates, which were treated with PLL and blocked but did not contain bacteria. After incubation and washing, enzyme-conjugated second-stage antibodies (50 μΙ per well), horseradish peroxy-dase-conjugated goat anti-human IgG and goat anti-human IgM were appropriately diluted in PBS containing 0.1% (volume) / volume) Tween-20 and 0.2% (weight / volume) BSA added to the wells and the assay was completed as described in Example II.

Supernatants containing antibody bound to the combination of Fisher immunotypes but not to the control plate were tested a second time using each of the seven Fisher immunotype bacteria individually. Antibody present in the supernatant of one well, 20H11, bound only to P. aeruginosa Fisher immunotypes 2, 6 and 7. The cells were repeatedly subcultured at decreasing low cell densities until all wells separated with growth antibody. The cell line and the monoclonal antibody (IgM isotype) are both referred to as 20H11 in the following.

A second transformation was performed in which the source of B cells came from the peripheral blood of a bladder fibrosis patient who was known to have a chronic P. aeruginosa infection. E'PBMCs were prepared as described above and co-cultured with the transforming cell line, 1A2, in a ratio of 72 1A2 cells per E'PBMC. The cell mixture was plated in vipen 30-well 96-well microtiter plates at a concentration of 7.4 x 104 cells per well and then cultured as above.

The supernatants were tested for the presence of anti-P 16 days after the plating transformation by ELISA. aeruginosa antibodies. The assay was performed as described for the previous transformation, except that the combination of P. aeruginosa strains used for the first screening was composed of Fisher immunotype reference strains F2, F4, F6 and F7 (ATCC 27313, 27315, 27316 and 27317 ) and three clinical isolates from the Genetic Systems Corporation Organism Bank (GSCOB), which had different LPS immunotypes and flagellate types. The clinical isolate PSA I277 (GSCOB) bears type a flagella and Fisher immunotype 1 LPS; the second isolate PSA G98 (GSCOB) bears type a flagella and Fisher immunotype 3 LPS; and the third, PSA F625 (GSCOB) carries type b flagella 40 and Fisher immunotype 5 LPS. This mixture of reference strains and clinical isolates will be referred to as the P. aeruginosa fluted combination. Supernatants containing antibody bound to plates containing the P. aeruginosa fluted combination, but not to the PLL-coated control plates, were tested a second time for individual strains of the combination according to ELISA. One well, 3C1, bound to reference strains F2, F6 and F7 and to the clinical isolate F625.

Cloning of the 3C1 cell line was accomplished by first subculturing the cells in two rounds of low density subculture, first at 20 cells per well of 96-well plates, followed by culturing at 2 cells per well. Formal cloning of the specific antibody-producing cells was accomplished by plating the cells at a density of about 1 cell / well in 72-well Terasaki plates in a 50 volume of 10 μΙ HAT medium without the aminopterin component (HT medium) per well . The plates were placed in an incubator for 2-3 hours to settle the cells to the bottom of the wells and were then examined microscopically by two individuals for wells containing a single cell. The wells were fed daily with HT medium and when the outgrowth was sufficient, the cells were transferred to a 96-well round-bottom plate. All wells with outgrowth were examined according to ELISA 55 for P. aeruginosa strains, carrying type b flagella, and all were found to produce the appropriate antibody. The cell line and the monoclonal antibody (IgM isotype) are referred to as 3C1 in the following.

194961 18

The antigen identified by 20H11 and 3C1 was flagella, as shown by indirect immunofluorescence and immunoflux. The techniques were used in principle as described in Examples II and III. For the indirect immunofluorescence study, P. aeruginosa strains with type b flagella, reference Fisher immunotypes F2, F6 and F7 (ATCC 27313, 27317 and 27318) and a strain with type a flagella, reference Fisher immunotype 4 (ATCC 27315) were prepared as described in Example III. The flagella type of the reference strains was determined by typing with the mouse monoclonal antibodies PaF4 IVE8 and FA6 IIG5. The slides were prepared for inspection as described in Example II. The sources of both antibodies were culture fluids above and the FITC-conjugated reagent was FITC-conjugated goat anti-human Ig (polyvalent) diluted 1: 100 in PBS containing 0.5% (weight / volume) of bovine gamgoglobulins (Miles Scientific, Cat No. 82-041-2) and 0.1% (weight / volume) of sodium azide as a preservative.

Fluorescent staining by the 20HI1 and 3CI antibodies was only observed with P. aeruginosa strains with type b flagella and not with the flagella type a strain, reference Fisher immunotype 4. The observed fluorescence pattern was a sinusoidal line pattern, starting from one end of the 15 bacterium, which showed that the antibodies bound to the bacterium's flagella.

Immunoflowing was performed as described in Example II.

Purified type b flagella from the P. aeruginosa reference strains Fisher immunotype 2 (A.T.C.C. 27313) and purified flagella type a from reference strains Habs 6 and Habs 8 (A.T.C.C. 33353 and 33355) were prepared as described in Example III. Antigens were separated in a 10% polyacrylamide gel (see Example III) and transferred to an NCM.

The above culture fluids, containing 20H11 or 3C1 antibodies, the above culture fluid containing a non-specific human antibody and culture media were incubated with the NCM and the reaction was detected with an alkaline phosphatase-conjugated goat anti-human Ig (polyvalent), diluted in PBS containing 0 , 05% (volume / vofume) Tween-20. Enzyme substrate was prepared as described in Example II. The immunoinfluence showed that both antibodies bound to the 53,000 MW flagellin protein of Fisher immunotype 2 and not to the 51,700 MW flagellin protein of Habs 6 nor to the 47,200 MW flagellin protein of Habs 8. No reaction was observed with the non-specific human antibody or with the culture media.

Further confirmation that antibodies 20H11 and 3C1 only bound to type b flagella and not to type a was obtained by ELISA, in which Habs strains 1-12 were individually bound with PLL to the wells of Immulon 96-well mlciter plates. The antibodies only bound to Habs strains 2, 3, 4,5,7,10,11 and 12, which are type b bearing strains (J. Clin. Microbiol. 20 (1984) 84).

EXAMPLE VII

Example VII shows methods for the production of a human monoclonal antibody, which binds to P. aeruginosa type a flagella.

A peripheral blood sample from an individual immunized with a high molecular weight polysaccharide preparation (Infect. Immun. 34 (1981) 461) served as a source of B cells. The mononuclear cells were secreted from the blood and then depleted in T cells as described in Example VI. The cells were then frozen in FCS containing 10% dimethyl sulfoxide in a liquid nitrogen vapor tank. At a later date, the cells were rapidly thawed to 37 ° C, washed once in Iscove’s medium and resuspended in HAT medium. Cell-driven transformation was accomplished by co-cultivation of the E'PBMCs with 1A2 cells in a ratio of 30 1A2 cells per E'PBMC. The cell mixture was plated in 96-well tissue culture plates at a concentration of 62,000 cells per well. The seventh 45 days after plating the cultures were fed by replacing half the volume with HAT medium. On the fourteenth day after plating, cell proliferation was observed in 100% of the wells and supernatants were removed from the wells and examined

The supernatants were tested for the presence of anti-P by ELISA. aeruginosa antibodies by using the fluted P. aeruginosa combination and PLL-50 treated plates as a control, as described in Example VI. Supernatants containing antibodies that bound to the fluted combination, but not to the PLL control plates, were re-examined for the individual bacterial strains of the flared combination. One well, 21B8, contained antibody that bound to PSA I277, PSA G98 and reference Fisher immunotype 4, i.e., the three strains of the fluted combination carrying type a flagella.

55 Cloning of the 21B8 cell line was done as described in Example VI for the 3CI cell line with the following modifications in the formal cloning step. After the wells of the Terasaki plates were examined for the presence of only a single cell, each cell of the Terasaki plate was transferred to one individual well of a 96-well round-bottom culture plate in a volume of 100 μl! HAT medium without the aminopterin component (HT medium). In all wells, non-transforming HAT-sensitive lymphoblastoid cells were taken up at a density of 500 cells / well as feed cells. Five days after plating, 100 µl of HAT medium was added to the wells to selectively kill the feed cells. On 5 days 7 and 9 after plating, the wells were refed by replacing half of the supernatant with HAT medium. The cells were then fed with HT medium until the cells were of sufficient density to detect the presence of ELISA antibody. All wells with outgrowth produced antibody, which bound to flagella type A bearing P. aeruginosa strains. The cell line and the monoclonal antibody (IgG, isotype) are both referred to as 21B8 in the following.

The antigen identified by 21B8 was flagella, as shown by indirect immunofluorescence and immunoflowing (see Example VI for descriptions of the techniques). Fluorescent staining by the 21B8 antibody was only observed with P. aeruginosa reference strain Fisher immunotype 4 (A.T.C.C. 27315), which carries type a flagella, and not with P. aeruginosa reference strain, immunotype 2 (A.T.C.C. 27313), which carries type a flagella. The observed fluorescence pattern was a sinusoidal line pattern, starting from one end of the bacterium, showing that the antibody was bound to the bacterium's flagella.

Immunoflowing was performed as described in Example II. Purified type a flagella from the P. aeruginosa reference strain Habs 6 (A.T.C.C. 33353) and purified flagella type b from the P. aeruginosa reference strain Fisher immunotype 2 (A.T.C.C. 27313) were prepared as described in Example III. Antigens were separated in a 10% polyacrylamide gel (see Example III) and transferred to an NCM. The above culture fluids, containing 21B8 or a non-specific human antibody and culture media, were reacted with the NCM and the reaction was discovered with an alkaline phosphatase-conjugated goat anti-human Ig (polyvalent) and enzyme substrate, as described in Examples II and VI. Immunoflowing showed that the 21B8 antibody bound only to the 51,700 Mw flagellin protein of Habs 6 and not to the 53,000 Mw flagellin protein of Fisher immunotype 2. No reaction was observed with the non-specific human antibody or culture media.

EXAMPLE VIII

Example VIII shows protection of mice that are passively immunized with human anti-flagella antibodies, 20H11, 3C1 and 21B8, against threat with P. aeruginosa in the burned mouse model.

The human anti-flagella monoclonal antibodies were tested in the burned mouse model (see Example IV). 21B8 and 20H11 antibodies were prepared by precipitation of the above culture fluids, generated from the respective cell lines with ammonium sulfate (50% final concentration) (Selected Methods in Cellular Immunology, San Francisco, 1980, 279-286). The precipitate was solubilized in PBS, dialyzed against PBS overnight at 4 ° C and then sterile filtered before being administered to animals. The source of antibody 3C1 and the non-specific antl-LPS antibody used as the negative control in the study was culture supernatant. As a positive control for each study, the appropriate purified monoclonal mouse antibody PaF4 IVE8 or 40 FA6 IIG5 was included.

The flagella type a strain used in the animal studies was the clinical isolate PSA A522 (GSCOB), which is capable of expressing Fisher immunotype 1 LPS, and the flagella type b strain was clinical isolate PSA A447 (GSCOB) , which is capable of expressing Fisher immunotype 6 LPS. The human antibodies (0.45 ml) were pre-mixed with the bacteria (more than 5 LD100s in 0.05 ml) and inoculated sube * 45 dab immediately after the burn was caused. The results of the animal tests are given in Tables C, D and E.

194961 20

TABLE C

Protection study of the human anti-flagella type a monoclonal antibody 21B8 in the burned mouse model.1 5% survival per day2

Treatment 1 234567 69 10 Anti-100 100 100 100 88 88 88 88 88 flagella a, FA6 IIG53

Human 100 100 100 100 100 100 100 100 100 anti-flammable, 15 21B8

Human 100 00000000 anti-flagella b, 21B8 PBS 100 25 12 12 12 12 12 12 12 20 - 1 Mice were sub-threatened with more than 5 LD100s from P. aeruginosa PSA A522.

2% survival was based on the number of surviving mice per group of 8 animals. The days are from burning and threatening.

3 Purified antibody (10 pg in 0.45 ml PBS) was pre-mixed with the bacteria and administered subeschar after burning and threatening.

TABLE D

Protection study of the human anti-flagella type b monoclonal antibody 20H11 in the burned mouse model.1 30% survival day2

Treatment 1 2 3 4 5 6 7 8 9

Muize anti-100 100 100 100 100 100 100 100 100 35 flagella b, PaF4 IVE83

Human 100 100 100 100 100 100 100 100 100 anti-flagella b, 20H11 40 Human 100 00000000 anti-flagella a, 21B8

Media 100 0 0 0 0 0 0 0 0 1 "1 Mice were sub-threatened with more than 5 LD100s from P. aeruginosa clinical isolate, PSA A447.

2% survival was based on the number of surviving mice per group of 5 animals. The days are from burning and threatening.

3 See footnote 3 to table C.

«21, 194961

TABLE E

Protection study of the human anti-flagella type b monoclonal antibody 3CI in the burned mouse model.1 5 -% survival per day2

Treatment 123456789

Mouse anti- 100 100 100 100 100 100 100 100 100 10 flagella b, PaF4 IVE83

Human 100 100 80 80 80 80 80 80 80 anti-flagella b,

3CI

Non-specific 100 0 0 0 0 0 0 0 0 anti-LPS monoclonal antibody 2Q 1 Mice were endangered with more than 5 LD100s from PSA A447.

3% survival was based on the number of surviving mice in groups of 5 animals. The days are after burning and threatening.

3 Purified antibody (40 pg) was administered intravenously 2 hours before burning and threatening.

Very significant survival was observed in mice treated with the anti-flagella type a antibody or with one of the two anti-flagella type b antibodies, and then threatened with the corresponding antigen. Conversely, 88-100% of the untreated but endangered mice, or mice treated with an inappropriate anti-flagella monoclonal antibody or non-specific anti-LPS antibody, died. As was observed with the monoclonal mouse antibodies (see Example IV), the human anti-flagella antibodies specifically protect against lethal threat only with the organisms bearing the corresponding flagella type, that is, the human anti-flagella type a antibody protection against lethal threat with the flagella type a bearing organism and not the type b, and that the anti-flagella type b antibodies protected mice threatened with strains bearing flagella type b, but not the type a bearing organism.

EXAMPLE IX

Example IX shows the cross-reactivity of human anti-flagella antibodies 20H11, 3C1 and 21B8 with P. aeruginosa clinical isolates.

P. aeruginosa clinical isolates (115), obtained from hospitals and clinics and isolated primarily from burns and blood, were identified as carrying type A or type B flagella by:

typing with the FA6 IIG5 or PaF4 IVE8 monoclonal mouse antibodies (see Examples II, III and V). From I

40 the clinical isolates were identified as fifty-five bearing type A flagella by reaction with the FA6 IIG5 monoclonal mouse antibody, and 59 identified as type B bearing by their reaction; with the monoclonal mouse antibody PaF4 IVE8. j

The cross-reactivity of the anti-flagella type a human monoclonal antibody 21B8 was extensive.

The antibody recognized 54 of the 56 flagella type a-bearing clinical isolates (96%). The cross-45 reactivity of 20H11 with the isolates bearing type b flagella was also extensive: 20H11 recognized all 59 isolates (100%). In contrast, the other anti-flagella type b monoclonal antibody, 3C1, bound only 43 of the 59 isolates (73%). These results show that 20H11 binds to a pan-reactive epitope (i.e., an epitope present on at least 95% of the flagelated P. aeruginosa strains), while 3C1 binds to an epitope that is not present on all type b flagellin molecules. Even though the flagella type b antigen is serologically uniform when analyzed with polyclonal antisera, the cross-reactivity patterns of 20H11 and 3C1 surprisingly show that the type b flagella has at least two separate epitopes that can be identified by monoclonal antibodies.

The extensive cross-reactivity of antibodies 21B8 and 20H11 with P. aeruginosa clinical isolates demonstrates the particular clinical utility of these antibodies in the immunotherapy of P. aeruginosa 55 infections.

194961 22

EXAMPLE X

Example 10 shows methods for the production of another human monoclonal antibody that binds to P. aeruginosa type b flagella, as well as the protective activity of that antibody against P. aeruginosa threat in the burned mouse model.

A transformed cell line was prepared and cloned in the manner described in Example VII, except that the transformed cell mixture was plated on 96-well tissue culture plates at a concentration of approximately 2250 E'PBMC per well. The supernatants were first tested by ELISA on the fluted combination, as described in Example VI, except that Fisher immunotype 2 and 4 reference strains were missing in the combination. Positive wells were then examined for each of the strains in the fluted combination, including Fisher immunotypes 2 and 4. The finally isolated cell line and the monoclonal antibody (IgG isotype) are referred to as 12D7 below.

The 12D7 human monoclonal antibody showed extensive cross-reactivity with anti-flagella type a isolates, recognizing 54 of the 56 tested flagella type a bearing clinical isolates (96%). The protective activity of the 12D7 monoclonal antibody is shown in Table F. The protection study was conducted in the manner described in Example IV except that the threatening dose was 1 LD100 and that the threatening strain was I624, a clinical isolate, expressing Fisher immunotype 2 LPS and type a flagella.

TABLE F

Protection study of the human anti-flagella type a monoclonal antibody 12D7 in the burned mouse model.1 25% survival per day2

Treatment3 1 2 3 4 5 6 7 8 9

Human 100 100 100 100 100 100 100 100 100 anti-flammable, 12D7

Human 90 90 90 90 90 90 90 90 90 anti-Fisher 2 LPS, 2H9

Muizeanti- 100 100 100 100 100 100 100 100 100 35 flagella a, 11G5

Human 100 37.5 25 25 25 25 25 25 25 anti-flagella b, 15F4 40 1 Mice were sub-threatened with 1 L010O (950 CFU) from P. aeruginosa 1624.

2% survival was based on the number of surviving mice per group of 10 animals, except for the 15F4 group, which contained 8 animals.

The days are from burning and threatening.

3 Purified antibody (50 pg in 0.5 ml PBS) was injected i.p. administered 2 hours before burning and threatening. 1 2 3 4 5 6 7 8 9 10 11

EXAMPLE XI

2

Example XI shows the production of a human monoclonal antibody reactive with flagella b 3 type P. aeruginosa and the protection of mice passively immunized with this antibody 4 against threat with P. aeruginosa in the burned mouse model.

5

A transformed cell line was prepared and cloned in the manner described in Example X, except that the transformation ratio of the 1A2 to B cells was approximately 60: 1 and that the transformed 7 cell mixture was plated on 15 plates at a concentration of approximately 1930E ' PBMC per well. Also, 8 cells were examined in a combination of P. aeruginosa strains, including G98 (Fisher 3 immunotype, flagella type a) and I739 (a clinical isolate of Fisher 5, flagella type b), and 10 confirmed for another combination from P. aeruginosa strains; I277, G98,1739 and Fisher immunotypes 11 F2, F4, F6 and F7. The ultimately isolated cell line and the secreted monoclonal antibody (IgG1 isotype) are referred to as 2B8 in the following.

An immunofluorescence study performed with 2B8 was positive on a flagella type b, Fisher 2 f 194961 immunotype strain, but negative in a flagella type a, Fisher immunotype 4 reference strain. The result was positive for clinical isolate test 59/59 (100%) of fluted type B isolates.

The protective activity of 2B8 is shown in Table G. The protective study was performed as described in Example X, except that clinical isolate FI64 (Fisher immunotype 4, flagella type b) was used for the threat.

TABLE G

Protection study of the human anti-flagella 10 type b monoclonal antibody 2B8 in the burned mouse model.1% survival per day2

Treatment 1234567 8 9 15 Human 100 89 89 89 89 89 89 89 89 anti-flagella b, 2b83

Mouse anti- 100 100 100 100 100 100 90 90 90 flagella b, PaF4 20 IVE8

Mouse anti-Fisher 90 20 20 20 20 20 20 20 2 LPS, VH34 'Mice were sub-threatened with 1 LD100 (105 CFU) from P. aeruginosa F164.

2% survival was based on the number of overturing mice per group of 10 animals, except group 2B8, which contained 9 animals.

The days are from burning and threatening.

3 Purified antibody (50 pg in 0.5 ml PBS) was injected i.p. administered 2 hours before burning and threatening.

4 Purified antibody (5 pg in 0.5 ml PBS) was injected i.p. administered 2 hours before burning and threatening.

EXAMPLE XII

Example XII shows the production of another human monoclonal antibody reactive with flagella b type P. aeruginosa, and the protection of mice passively immunized with this antibody against P. aeruginosa threat in the burned mouse model.

A transformed cell line was prepared and cloned in the manner described in Example VII with the following exceptions. Another individual was immunized (Infect. Immun. 45 (1984) 309) and served as a source of B cells, and the plating level of E-PBMC was approximately 2000 cells per well. The screening was performed on combined Fisher 1-7 immunotypes. The finally isolated cell line and the secreted monoclonal antibody (IgG1 isotype) are both referred to as 14C1 in the following.

In clinical trials, 59/59 (100%) of fluted type B isolates were found to be positive with 14C1. The protection test was conducted as described in Example XI and the results are shown in Table H.

TABLE H

45 Protection study of the human anti-flagella type b monoclonal antibody 14C1 in the burned mouse model.1% survival per day2

Treatment 123456789 50 ---

Human 100 100 100 100 100 90 90 90 90 anti-flagella b, 14C1

Mouse anti- 100 100 100 100 100 100 100 100 100 55 flagella b, PaF4 IVE8

Claims (26)

A composition comprising a component capable of reacting with Pseudomonas aeruginosa bacteria, characterized in that said component is a monoclonal antibody or binding fragment thereof capable of specific reaction with flagella of Pseudomonas aeruginosa bacteria. 1 mice were sub-threatened with 1 LD100 (90 CFU) from P. aeruginosa F164. 2. survival was based on the number of surviving mice per group of 10 animals, except for the PaF4 IVE8 group, which contained 9 animals. The days are from burning and threatening. 2. Composition according to claim 1, characterized in that the monoclonal antibody or binding fragment thereof is protective in vivo. A composition comprising a component capable of reacting with Pseudomonas aeruginosa, characterized in that this component is a monoclonal antibody or binding fragment thereof that is capable of specific reaction with flagella protein epitope of Pseudomonas aeruginosa. 3 Purified antibody (50 pg in 0.5 ml PBS) was injected i.p. administered 2 hours before burning and threatening. It will be apparent from the foregoing that the cell lines of the invention provide means for the production of monoclonal antibodies and fragments thereof, which are reactive with P. aeruginosa flagella and cross-protecting against various P. aeruginosa strains. Surprisingly, a number of monoclonal antibodies were isolated, which were reactive with different epitopes on each type of flagella. The antibodies according to the invention allow prophylactic and therapeutic compositions to be produced more economically and more easily for use against infections caused by most P. aeruginosa strains. Furthermore, the cell lines provide antibodies that find use in immunoassay and other known procedures. 25 4. A composition according to claim 3, characterized in that the monoclonal antibody is capable of blocking the binding to the epitope of monoclonal antibodies produced by cell lines, designated as T.T.C.C. Accession Numbers HB9129, HB9130, CRL 9300 and CRL 9301. A composition comprising a component capable of reacting with Pseudomonas aeruginosa, characterized in that this component is a monoclonal antibody or binding fragment thereof capable of reacting with type a or type b flagella of flagella protein epitope of Pseudomonas aeruginosa. A composition according to claim 5, characterized in that a second monoclonal antibody is capable of reacting with a type of flagella that is not reactive with respect to the first monoclonal antibody. Composition according to claim 5, characterized in that the first antibody is a human monoclonal antibody. The composition of claim 5, characterized in that at least one of the monoclonal antibodies 45 is protective in vivo. Composition according to any of claims 1, 3, 5 or 7, characterized in that it further comprises a gamma-globulin fraction of human blood plasma and / or an antibiotic Pharmaceutical composition, characterized in that it comprises a composition according to any of claims 1, 3, 5 or 7, and a physiologically acceptable carrier A pharmaceutical composition, characterized in that it is used for the treatment or prevention of a Pseudomonas aeruginosa infection, which composition is a monoclonal antibody reactive with a flagella protein of Pseudomonas aeruginosa and an anti-microbial agent in vivo, a gamma globulin fraction of human blood plasma and a physiologically acceptable carrier. 12. Pharmaceutical composition according to claim 11, characterized in that the antibody is a human monoclonal antibody and the human blood plasma gamma globulin fraction is obtained from people showing elevated concentrations of immunoglobulins reactive to 194961 Pseudomonas aeruginosa bacteria and / or products thereof. Pharmaceutical composition, characterized in that it comprises at least two monoclonal antibodies, each specifically reactive with a different type of Pseudomonas aeruginosa flagellair protein and suitable for treatment or prevention of Pseudomonas aeruginosa infections. The composition according to claim 13, characterized in that it is at least one of the monoclonal antibodies a human monoclonal antibody. 15. A composition according to claim 13, characterized in that it further comprises at least one human monoclonal antibody capable of reacting with at least one serotypic determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa and / or a monoclonal antibody, which is reactive is with exotoxin A. A cell line, characterized in that it produces a monoclonal antibody capable of specific reaction with type a or type b Pseudomonas aeruginosa flagella. The cell line of claim 16, wherein the monoclonal antibody is invivo protective against Pseudomonas aeruginosa. Cell line according to claim 17, characterized in that it is a hybrid cell line. A cell line according to claim 16, characterized in that it produces human monoclonal antibodies. Cell line according to claim 16, characterized in that it is one of A.T.C.C. Accession Numbers is HB9129, HB9130 or CRL 9300 and CRL 9301. 21. A method for producing monoclonal antibodies, specifically for Pseudomonas aeruginosa flammable proteins and suitable for treatment or prevention of Pseudomonas aeruginosa Infections, characterized in that at least one of the cell lines of claim 20 is cultured and the antibodies be won. A monoclonal antibody composition, characterized in that it has the same binding specificity as one of the monoclonal antibodies, designated as FA6 IIG5, Pa3 IVC2, PaF4 IVE8, 20H11 and 21 BB. A monoclonal antibody according to claim 22, characterized in that it is conjugated to a label capable of delivering a detectable signal. A monoclonal antibody according to claim 23, characterized in that it is labeled fluorescent or an enzyme. 25. A method for determining the presence of Pseudomonas aeruginosa in a sample, characterized in that the sample is combined with a monoclonal antibody reactive with Pseudomonas aeruginosa flagella and complex formation is observed. A package for detecting the presence of Pseudomonas aeruginosa bacteria, characterized in that this package comprises a monoclonal antibody composition, which contains at least one monoclonal antibody, which reacts with a type-specific flagellain protein of the bacterium, and labels for the Providing a detectable signal, covalently bound to the antibody or bound to second antibodies, which are reactive with the monoclonal antibody.