NL8701554A - MONOCLONAL ANTIBODIES FOR PSEUDOMONAS AERUGINOSA FLAGELLA. - Google Patents

Description

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Monoclonal antibodies to Pseudomonas aeruginosa flagella.

The invention relates to the use of immunological techniques to provide new materials useful for the diagnosis and treatment of bacterial infections, in particular to the preparation and use of monoclonal antibodies capable of reaction with Pseudomonas aeruginosa flagella.

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

In recent decades, antibiotics have been the preferred therapy for fighting gram-negative diseases. However, the continuing serious illness and high mortality associated with gram-negative bacterial disease indicates the limitations of antibiotic therapy, particularly with respect to P. aeruginosa.

{See, e.g., V.G. Andriole, J. Lab. Clin. Med. 94 (1978) 196-199).

This has led to the search for alternative methods for prevention and treatment.

One method that has been investigated is the enhancement of the host's immune system by active or passive immunization.

For example, it has been found that active immunization of humans or experimental animals with whole cell bacterial vaccines or purified bacterial endotoxins from P. aeruginosa leads to the development of specific opsonic antibodies directed primarily against determinants on the repeating oligosaccharide units of the lipopolysaccharide (EPS) molecules, located on the outer cell membrane of P. aeruginosa (see M. Pollack, Immunoglobulins: Characteristics and 30 Uses of Intravenous Preparations, biz. 73-79, US Department of Health and Human Services, 1979). Such antibodies, whether actively raised or passively transferred, have been shown to be protective against the lethal effects of P. aeruginosa infection in a variety of animal models (Pollack, supra) and in some preliminary studies with 701554 * *? -2- people (see L.S. Young and M. Pollack, Pseudomonas aeruginosa, biz. 119-132, Hans Huber, 1980).

The said reports suggest that immunotherapeutic approaches could be used to prevent and treat bacterial disease caused by P. aeruginosa, such as by administering combined human immunoglobulins containing antibodies against the infecting strain or strains. Human immunoglobulins are defined herein as that portion of fragmented human plasma enriched for antibodies, including specific antibodies against P. aeruginosa strains. Due to certain inherent limitations in the use of human immunoglobulin components, this approach to treating disease due to P. aeruginosa remains under investigation (see, for example, MS Collins and re Roby, Am. J. Med. 76 (3A) (1984) 15 168-174, and hitherto no commercial products are available where these components have been used.

One of the limitations associated with immunoglobulin compositions is that they consist of combinations of samples from a thousand or more donors, which samples are selected for the presence of particular anti-Pseudomonas antibodies. The combination results in an averaging of individual antibody titers, which at best results in modest increases in the resulting titer of the desired antibodies.

Another limitation is that the selection process itself requires a costly continuous screening of the donor combination to ensure product consistency. Despite these efforts, the immunoglobulin products may 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 co-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 AIDS), with the potential to cause adverse biological effects. The combination of low titers of desired antibodies and a high content of external substances can often limit sub-optimal levels of the amount of specific and therefore beneficial immune globulin (s) to the patient. can be administered.

In 1975, Kohier and Milstein reported their discovery that certain mouse cell lines could be fused to murine mouse cells 5 to form hybridomas, each of which can secrete antibodies of a single specificity / notably monoclonal antibodies (G. Kohier and C. Milstein, Nature, 256 (1975)). 495-497). The advent of this technology made it possible in some cases to prepare large amounts of excellently specific murine antibodies for a particular IQ determinant or determinants on antigens. Subsequently, using later developed technologies, it became possible to produce human monoclonal antibodies (see, for example, U.S. Patent 4,464,465).

It is recognized that in some situations, mouse monoclonal antibodies or compositions containing such antibodies may cause problems for use in humans. For example, it has been reported that murine monoclonal antibodies used in studies for the treatment of certain human diseases can elicit an immune response, rendering them ineffective (RL Levy 20 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. Also, methods for the production of human monoclonal antibodies are now available (see Human Hybridomas and Monoclonal Antibodies, Plenum Publishing Corp., 1985).

A number of groups have reported on the production by hybridoma and / or cell transformation technology of monoclonal antibodies, protective against P. aeruginosa infections. Monoclonal antibodies have been produced which are reactive with various epitopes of P. aeruginosa, including single and multi-serotype specific surface epitopes, such as found in LPS molecules of the bacteria (see, for example, U.S. Patent Nos. 734,624 and 807,394). Protective monoclonal antibodies specific for P. aeruginosa exotoxin A were also prepared (see, for example, U.S. Patent Application 742,170).

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Although the use of monoclonal antibodies specific for the LPS region of P. aeruginosa or the exotoxins of the bacterium may provide sufficient protection in certain situations, it is generally preferred to have broader protection options. For example, for prophylactic treatment for potential infections in humans, it would be preferable to administer one or more antibodies protecting against a number of P. aeruginosa strains. Likewise, 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 effective against most, if not all, clinically important P. aeruginosa serotypes, ideally by providing antibodies that are reactive across traditional serotype schemes.

One aspect of P. aeruginosa physiology, which has been shown to contribute to the virulence of the organism, is motility, an ability primarily resulting from 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-like structure. Model studies in mice with burns 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.

Burns 6 (1980) 235-239 and T. Montie et al. Infect, and Immun. 38 (1982) 1296-1298). Other studies regarding the pathogenesis of P. aeruginosa 25 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. 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. Sci. 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, supra) or flagella type b (R. Ansorg, supra) was serologically uniform, i.e., no subgroups were identified. This serologically uniform S701554 "* -5" flagellar type will be referred to as type b. The other main 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 aQ, a., , aa ^ and a ^. The five subgroups of type a are expressed in varying combinations on various strains of type a carrying P. aeruginosa with the exception of antigen a. The antigen was found on each type of a flagella, although the extent varied between strains.

A serotyping scheme based on the heat-stable major somatic antigens of P. aeruginosa is referred to as the Habs scheme, which has recently been included in the International Antigenic Typing System scheme (see Int. J. Syst. Bacteriol. S3 (1983) 256 ). The flagella types of P. aeruginsa Habs reference strains have been 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 strains and Habs strains 1, 6, 8 and 9 carry type a flagella. Thus, a large number of P. aeruginosa strains could potentially be recognized by a small number of monoclonal antibodies specific for flagellar proteins.

There is therefore a significant need for monoclonal antibodies, which can react with epitopes on flagellar proteins and in some cases also protect against multiple sero-types of P. aeruginosa. Furthermore, some of these antibodies would be suitable for use as prophylactic and therapeutic treatments for P. aeruginosa infections, as well as for the diagnosis of such infections. The invention meets these needs.

New cell lines are provided which can produce monoclonal antibodies capable of binding flagella present on most strains of P. aeruginosa bacteria. The monoclonal antibodies specifically react with epitopes on P. aeruginosa flagella proteins and can distinguish between type a and type b flagella of the bacterium. Furthermore, a method is provided for the treatment of people who are susceptible to or already infected with P. aeruginosa by administering a prophylactic or therapeutic amount of a composition containing at least one monoclonal antibody or binding fragment thereof capable of reacting with the flagella of P. aeruginosa strains, which composition preferably also contains a physiologically acceptable carrier. The composition 5 may also contain one or more of the following ingredients: further monoclonal antibodies capable of reaction with P. aeruginosa exotoxin A; monoclonal antibodies capable of reacting with serotype determinants on the LPS of P. aeruginosa; a gamma globulin fraction of human blood plasma; a gamma globulin fraction of human blood plasma, which plasma may have been obtained from a human, which 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 capable of producing monoclonal antibodies and compositions containing such antibodies, which compositions are capable of selectively recognizing the flagella present on a number of P. aeruginosa strains, where individual antibodies typically recognize one type of P. aeruginosa 20 flagella. The subject cells have identifiable chromosomes in which their germline DNA or a precursor cell has been rearranged to encode an antibody with an epitope binding site on a flagellar protein common to certain P. aeruginosa strains. Pan-reactive monoclonal antibodies can be produced for type a flagellar proteins; for type b flagellar proteins, antibodies are included which are pan-reactive or react with at least about 70% of the flagellar-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 likely inhibiting the motility of the organism and / or other effects beneficial to the infected host.

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The preparation of monoclonal antibodies can be accomplished by immortalizing a cell line capable of expressing nucleic acid sequences encoding antibodies specific for an epitope on the flagellar proteins of P. aeruginosa strains.

The immortalized cell line can be a mammalian cell line transformed by oncogenesis, by transfection, mutation or the like. Such cells include myeloma lines, lymphoma lines, or other cell lines 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 murmur or from human sources, produced by transformation of a lymphocyte, in particular a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, eg a myeloma, to form a hybrid cell line. The splenocyte will typically be obtained from an animal immunized against flagellar 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 20 and Practice, Academic Press (1983).

The hybrid cell lines can be cloned and screened according to conventional techniques and antibodies discovered in the supernatant cell fluid capable of binding to P. aeruginosa flagellar 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 ascites fluid production.

In one embodiment of the invention, the cells are transformed human lymphocytes, which form human monoclonal antibodies, preferably protective in vivo, into accessible epitopes specific for at least one flagellar protein. The lymphocytes can be obtained from human donors who have been or have been exposed to the appropriate flagella-bearing strains of P. aeruginosa. A preferred cell-driven transformation process is described in detail in U.S. Patent 4,464,465.

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By having the antibodies of the invention, which are known to be specific for the flagellar proteins, in some cases the supernatants from subsequent experiments can be screened in a comparative study with the subject monoclonal antibodies as a means of identifying further examples of anti-flagellar monoclonal antibodies.

Thus, hybrid cell lines can be easily produced from a variety of sources based on the availability of the subject antibodies specific for the particular flagellar antigens.

When hybrid cell lines are available which produce antibodies specific for the subject epitopic sites, these hybrid cell lines can also be fused to other neoplastic B cells, such other B cells serving as genome DNA encoding receptacles for the receptors. While neoplastic B cells from rodents, especially mice, are most commonly used, other mammalian species may be used, such as bovine, ovine, equine, porcine, monkey, and monkey lagomorphs.

The monoclonal antibodies can be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgE, or subclasses of IgG, known for any animal species. In general, the monoclonal antibodies can be used intact or as binding fragments, such as Fv, Fab, F (ab ') 2, but usually intact.

The cell lines of the invention may find uses other than for the direct production of the monoclonal antibodies.

The cell lines can be fused with other cells (such as appropriate drug-labeled human myeloma, mouse myeloma or human lymphoblastic cells) to form hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies. the cell lines are used as a source of the chromosomes encoding 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 the 3701554 * · * -9-

In particular, by forming cDNA libraries from messenger RNA, a single cDNA clone encoding the immunoglobulin and free of introns can be isolated and placed into suitable prokaryotic or eukaryotic expression vectors and then transformed into a host for final mass production (see generally, U.S. Pat. Nos. 4,172,124, 4,350,683, 4,363,799, 4,381,292, and 4,423,147; see further Kennett et al., Monoclonal Antibodies, Plenum, New York (1980) and 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 encoding the light and heavy chains of the monoclonal antibodies produced by a cell line of 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. This process can be repeated as necessary to further increase the desired mRNA levels. The subtracted mRNA composition can then be transferred in opposite directions to provide a cDNA mixture enriched for the desired sequences. The RNA can be hydrolyzed with an appropriate RNase and the ssDNA can be double-stranded with DNA polymerase I and random primers, eg statistically fragmented sweetbread DNA. The resulting dsDNA can then be cloned by insertion into a suitable vector, for example virus vectors, such as lambda 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 encoding for the desired light and heavy chains can be identified by hybridization.

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

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By appropriate means, mammalian hosts (e.g., mouse cells) can be used to treat the chain (e.g., linking the heavy and light chains) to produce an intact immunoglobulin and further, optionally, secrete the immunoglobulin free of the leader sequence. One can also 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, coding for the heavy chain.

10 On this. Alternatively, the immunoglobulins can be prepared and treated to be assembled and glycosylated in cells other than mammalian cells. If desired, any of the chains can be truncated to retain at least the variable region, which regions can then be manipulated to provide other immunoglobulins or fragments specific for the flagellate epitopes.

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

Monoclonal antibodies of the invention also find a wide variety of applications in vitro. For example, the monoclonal antibodies can be used to characterize microorganisms, isolate specific P. aeruginosa strains, 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 complex formation by the binding of the monoclonal antibody to the flagellum of the P. aeruginosa organism. If not labeled, the antibodies find use in agglutination studies. Furthermore, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies), which are reactive with the monoclonal antibody, such as antibodies, which are specific for S 7 0 1 5 5 4 * i -11- immunoglobulin. The monoclonal antibodies can also be directly labeled. A wide variety of labels can be used, such as radionuclides, fluorescent substances, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (especially haptens), etc. Numerous immunoassays are available and for example example include some 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 containing P. aeruginosa of a given serotype, such as human blood or lysate thereof, is combined with the subject antibodies, binding takes place between the antibodies and the molecules displaying the desired epitope. Such cells can then be separated from the unbound reagents and a second antibody (labeled with an enzyme label) can be added. Then, the presence of the antibody-enzyme conjugate, which is specifically bound to the cells, is determined. Other conventional techniques which are known to the person skilled in the art can also be used.

Packages may also be provided for use with the subject antibodies for detection of B. aeruginosa in solutions or the presence of P. aeruginosa flagellar antigens. Thus, the present monoclonal antibody composition of the invention can be provided, usually in a lyophilized form, either alone or together with further antibodies specific for other gram negative bacteria. The antibodies, which can be conjugated with a label or unconjugated, are included in the package with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, eg bovine serum albumin, or the like.

Generally, these materials will be present in less than about 5 wt% based on the amount of active antibody and usually in a total amount of at least about 0.001 wt% based on the antibody concentration. Often it will be desirable to include an inert excipient or excipient to dilute the active ingredients, the excipient being present in 870? 534 i -12- an amount of 1-99 wt% of the total composition. When a second antibody capable of binding the monoclonal antibody is used, it will usually be in a separate one. ampoule. The second antibody is typically conjugated to a label and assembled in an analogous manner to the above-described composition with the antibody.

The monoclonal antibodies, especially human monoclonal antibodies of the invention, may also be included as components of pharmaceutical compositions containing a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of 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, dispersants) can also be included in the pharmaceutical composition. Such compositions may contain a single monoclonal antibody to be specific for one P. aeruginosa flagellar type strains. Also, a pharmaceutical composition can contain two or more monoclonal antibodies to form a "cocktail". For example, a cocktail containing monoclonal antibodies to both types of flagella or to groups of the various P. aeruginosa strains (eg different serotypes) would be a universal product with activity against the vast majority of clinical isolates of the bacterium concerned.

The molar ratio of the different monoclonal antibody components will usually differ by no more than a factor of 10, usually no 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 globulins and immunoglobulin products, used for the prophylactic or therapeutic treatment of P. aeruginosa disease in humans. Preferably, the plasma for 570 1 55 4% immunoglobulins is obtained from human donors showing elevated levels of immunoglobulins reactive with P. aeruginosa (see generally the Compendium "Intravenous Immune Globulin and the Compromised Host", Smer, J. Med, 76 (3a) (March 30, 1984), 1-231).

The subject 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 (eg carbenicillin) together with an aminoglycoside (eg 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 of the invention are particularly suitable for oral or parenteral administration. Preferably, the pharmaceutical compositions are 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, 0.3% glycine and the like. These solutions are sterile and generally free from particulate matter. These compositions can be sterilized by conventional and known sterilization techniques.

The compositions may contain pharmaceutically acceptable excipients, as necessary to approximate physiological conditions, such as pH regulators and buffers, toxicity regulators and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the antibody in these compositions can vary within wide limits, typically from less than about 0.5%, usually at least about 1% to as much as 15. or 20% by weight, and is selected primarily on a liquid basis. preferred volumes, viscosities and the like for the chosen dosage form.

For example, a typical pharmaceutical composition for intramuscular injection may contain 1 ml of sterile buffered water and 50 mg of monoclonal antibody. For example, a typical composition for intravenous 8701554-14 injection may 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 understood to those skilled in the art and are described in more detail in, for example, Remington's 5 Pharmaceutical Science, 15d ed., Mack Publishing Company (1980).

The monoclonal antibodies of the invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has proven effective with conventional immunoglobulins, using known lyophilization and IQ reconstitution techniques. It would be apparent to those skilled in the art that lyophilization and reconstitution may result in varying degrees of loss of activity of the antibody (eg, with conventional immunoglobulins, igM antibodies tend to have greater loss of activity than igG antibodies) and 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 use, 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 arrest the infection and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose." Amounts effective for this use depend on the severity of the infection and the general state 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 containing dose of 5-25 mg per kilogram are used. It is to be remembered that the materials of the invention will generally be used in severe disease states, ie, life-threatening or potentially life-threatening situations, in particular bateremia and endotoxemia caused by P. aeruginosa.

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

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

EXAMPLE I

Example I shows the methodology used to prepare 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 of 6 7 were 8 x 10 and 1 x 10 organisms per mouse for, respectively

Fisher immunotype 1 and Fisher immunotype 2, and the dose was increased to 30- to 60-fold over the course of the immunizations.

Three days after the last injection, the spleen from 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 slides. Mononuclear spleen cells were combined in a 4: 1 ratio with log phase mouse myeloma 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 ml in RPMI hybrid HAT (RPMI 1640, containing 15% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 100 µg / ml penicillin and streptomycin , 35 -4 -7 -5

1.0 x 10 M hypoxanthine, 4.0 x 10 M aminopterin and 1.6 x 10 M

g thymidine) containing 2.0 x 10 per ml freshly prepared BALB / c thymocytes as 8701554-16 feed cells.

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

The supernatants of the hybrid cells were assayed 10 '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 seeded in trypticase soy culture fluid (TSB) and grown 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 Na2HPC> 4.7 HO, 1.5 mM KH2PC> 4, PH 7.2) containing 150 trypsin inhibitory units (TIU) aprotinin per ml.

The pellet from the last centrifugation was resuspended in 0.17 M triethanolamine, 20 mM disodium ethylenediamine tetraacetic acid (EDTA) and then homogenized on ice for 10 minutes. The solid was granulated by centrifugation of the homogenate at 14,900 x g and decanted, and the supernatant centrifuged again as described. The solid was sagged again and the membranes were granulated by centrifuging the supernatant at 141,000 x g for 1 hour. The supernatant was collected 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 vortexing, 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 placed in each well of 96-well plates, sealed and incubated overnight at 37 ° C. Unbound antigen was removed from the plates and 100 µl 5% (weight / volume) bovine serum albumin (BSA) in PBS was added to each well and the plates incubated at 37 ° C for 1 hour.

After removing the unadsorbed BSA, the supernatant (50 µl) culture fluid was copied from each well of the fusion plates into corresponding wells of antigen plates and incubated at 37 ° C for 30 minutes. The unbound antibody was removed from the wells and the plates were washed three times with 10 µl 1% (weight / volume) BSA-PBS 10 per well. Subsequently, 50 µl of appropriately diluted biotinylated goat anti-mouse IgG was added to each well per well and incubated at 37 ° C for 30 minutes. The plates were washed three times as described above, after which 50 μΐ of a pre-formed avidin: biotinylated horseradish peroxidase complex (Vectastain ABC Kit, Vector 15 Laboratories, Inc., Burlingame, CA), prepared according to the manufacturer's specifications, wells added. After 30 minutes at room temperature, the Vectastain reagent was removed from the wells, after which the wells were washed as described and per well 100 μΐ substrate o-phenylenediamine (0.8 mg / ml in 0.1 M citrate buffer, pH 5.0 , mixed with an equal volume of 0.03% (volume / volume) was added The substrate was incubated for 30 minutes at room temperature in the dark, after which the reactions were terminated by adding 50 µl 3N H 2 SO ^ per well.

Hybridoma cells secreting monoclonal antibodies that bound to one of the two antigen preparations were detected by measuring the absorbance at 490 nm of the colorimetric reactions 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 Pisher 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 miniaturized and cloned by limiting dilution techniques, as described in Infect. Immun. 36 (1982) 1042-1053.

Ascites fluid containing the high titer monoclonal antibody was prepared in CB6 mice (BALB / c (female) x C57BL / 6 5791554-18 (male) F ^) according to procedures described in Infect. Ixnmun. 315 (1982) 1042-1053. Two- to three-month-old male CB6 F ^ mice were injected intraperitoneally with 0.5 ml Pristane (2, 5, 10, 14-tetramethylpentadecane, Aldrich Chemical Co.) 10-21 days before intra-5 peritoneal log phase injection Pa3 IVC2 cells in RPMI. Each mouse 7 was injected with 0.5-1 x 10 cells in 0.5 ml. After about two weeks, the liquid 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 10: ascites containing 5 mg / ml or more of the antibody were pooled, sampled and frozen at -70 ° C.

Culture supernatant from the cloned Pa3 IVC2 cell line was examined by ELISA, as described above, on outer membrane preparations of all seven P. aeruginosa Fisher immunotype strains (ATCC 27313-27318), P. aureofaciens (ATCC 13985) and Klebsiella pneumoniae (ATCC 8047). ), all prepared as described above. Antibody PA3 IVC2, 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 radioimmunoprecipitation. Briefly, this includes incubating radially-labeled antigens 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 µg) of soluble P. aeruginosa Fisher immunotypes 2, 3, 4 and 5 outer membrane preparations were radiolabelled in 125 solid phase with I using Iodo gene (Pierce Chemical Co) (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.

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To reduce nonspecific binding of the outer membrane antigens to antibody Pa3 IVC2, the radiolabeled 6 preparations (5 x 10 counts per minute per analysis) were first incubated with BALB / c normal mouse serum for 1 hour at 4 ° C (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 incubation of 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 an additional 10 minutes at 4 ° C was incubated (J. Immunol. 115 (1975) 1617-1622).

IgGSORB was prepared according to the manufacturer's specifications and just before use, nonspecific reactions were prevented by blocking potentially reactive sites with culture media by washing the IgGSORB twice with RPMI hybrid (RPMI hybrid HAT media exclusive • 15 HAT) .

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 Tris-HCl, pH 8.0, 0.5 M LiCl, 1% (volume / volume) 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., µl. SA 76 25 (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 / volume) beta mercaptoethanol and 20% (volume / volume) glycerol) and collected in the supernatant after centrifugation at 1500 xg for 10 min.

The supernatant samples were applied to 14% polyacrylamide gels containing SDS prepared by 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 anti-genes were separated in 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) 870 1 55 4 -20-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 from this experiment showed that Pa3 IVC2 bound to only one antigen in the outer membrane preparation of P. aeruginosa Fisher immunotype 2 and not any other antigen present in the other outer membrane preparations. The molecular weight (MW) of the antigen in the gel was about 53,000 daltons, as determined by equation 14 of. its mobility with that of 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. aeruginsoa (Infect. Immun. 35 (1982) 281-288).

Furthermore, Pa3 IVC2 was tested by ELISA using P. aeruginosa Habs strains 1-12 (A.T.C.C. 33348-33359) fixed with ethanol on 96-well microtiter plates. The antigen plates were prepared as follows.

Overnight fluid cultures from each organism were centrifuged, washed twice with PBS and then resuspended in PBS to one of 0.2 O.D. units. The diluted bacteria were placed -660 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 to type b flagella of 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 monoclonal 35 Pa3 IVC2, the P. aeruginosa carry reference strains, Fisher immunotype 2, Fisher immunotype 6 and Fisher immunotype 7 type b flagella. From the preliminary experimental data, it was concluded that Pa3 1VC2 binds specifically to P. aeruginosa flagellin type b.

In vivo protective activity of Pa3 XVC2

Animal studies were performed to determine whether monoclonal antibody Pa3 IVC2 would protect a mouse threatening multiple LDg0 from live P. aeruginosa bacteria. 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 LD ^ s of Fisher immunotype 7. 10 Before the burn and the threat, monoclonal antibody was administered intraperitoneally as high titer ascites (10). 0.2 ml intraperitoneally). In the animals treated with Pa3 IVC2, no increase in the number of survivors was observed compared to the animals, which received no antibody.

EXAMPLE II

Example II shows the methodology for the preparation of a mouse hybridoma cell line, which produces a mouse monoclonal 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 60 (ATCC 27317) (8 x 10 organisms) followed two weeks later by an injection of viable P. aeruginosa Fisher immunotype 50 (ATCC 27316) (4 x 10 organisms). During the subsequent 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-fold the starting dose. Four days after the last viable Bacteria injection, a final injection of P. aeruginosa Fisher immunotype 6 outer membrane preparations (50 µg 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 as described in Example I.

The culture supernatants of the hybridoma cells were examined for the presence of anti-P. aeruginosa antibodies by ELISA on the day 10 after the fusion according to the 6701554 -22 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 µg / ml in PBS) was added to each well of the 96-well plates and incubated at room temperature for 30 minutes. Unadsorbed 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 left to bind for 1 hour at 37 ° C. Unbound bacteria were removed and the wells washed three times with brine-Tween (0.9% (weight / volume) NaCl, 0.05% (volume / volume) Tween-20).

Nonspecific binding of the antibodies was blocked by adding 200 μl / well of blocking buffer (PBS containing 5% (weight / volume) non-fat dry milk, 0.01% (volume / volume) Anti-foam A (Sigma ) and 0.01% (weight / volume) thimerosal) at the wells and incubation for 1 hour at room temperature. Excess blocking buffer was driven off and the wells were washed three times with brine-Tween as previously described.

Culture supernatants (50 µl) were copied into the corresponding wells of the assay plates and incubated at room temperature for 30 minutes. The supernatant broths were removed and the wells were washed five times with brine-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 / volume) BSA according to titrations previously performed, after which 50 µl 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 brine-Tween; 100 µl o-phenylene-diamine substrate was added per well, incubated for 30 minutes as described in Example 1. Reactions were terminated as stated in Example I and then read at A ^ ^ on a Bio-Tek 35 EL-310 Automated EIA Plate Reader.

8701554 -23-

According to the methods described, the supernatants of the fusion culture were examined for the presence of antibodies which bound to P. aeruginosa Fisher immunotypes 1, 2, 3 or 4, but not to control plates prepared by the same PLL and blocking-5. procedure, but without bacteria. Supernatants containing antibody that bound to one of these four Fisher immunotypes were tested a second time with each of the seven Fisher immunotype bacteria separately. Antibody present in the single well supernatant, PaF ^ IVE8, bound only to 10. Aeruginosa Fisher immunotypes 2, 6, and 7 cells from well PaF4 IVE8 were cloned by limiting dilution methods as described in Example 1. The monoclonal antibody and clonal cell line from this well are both identified in the following text by the designation PaF4 IVE8 . High-titer monoclonal antibody-containing ascites fluid was generated as described in Example I, except that BALB / c mice were used instead of CB6F1.

Specificity of PaF4 IVE8

An investigation conducted to identify the antigen bound by monoclonal antibody PaF4 IVE8 was indirect immuno-fluorescence on bacterial organisms. Each of the seven reference

Fisher immunotypes of P. aeruginosa plus a non-flagellated strain of P. aeruginosa (PA103, A.T.C.C. 29260, J. Bacteriol. 62 (1951) 377-389) and Escherichia coli (G.S.C. A25) were grown overnight in TSB at 37QC. The bacteria were centrifuged and then washed twice in PBS. Each strain was resuspended in PBS to an O.D.-__ = 2.2.

660 nm

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

After the slides were dried, fluorescein isothio-cyanate (FITC) -conjugated goat anti-mouse IgG + IgM (25 μΐ per well of a 1:40 dilution in PBS) for 30 min at room temperature in 70701554 -24- a humidified room incubated on the slides in the dark. The slides were again washed in distilled water, dried and then covered with a cover slip, mounted with glycerol in PBS (9: 1). The slides were viewed with a fluorescent microscope.

Fluorescent staining was only observed in P. aeru ginosa Fisher immunotypes 2, 6 and 7 and showed a sinusoidal pattern (line) 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 immunofluidity analysis. Outer membrane antigens of 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, MW 25,700; ovalbumin, MW 43,000; bovine serum albumin, MW 68,000; phosphorylase B, MW 97,400; and myosin, MW 200,000). the same polyacrylamide gel included.

Antigens were transferred from the polyacrylamide gel overnight at 4 ° C at a constant amperage of 200 mA to the nitrocellulose membrane (NCM) (0.45 µl) in a Tris-glycine methanol buffer (Proc. Natl. Acad. Sci.,). USA 76 (1979) 4350-4354), containing 0.05% (w / v) SDS. After the transfer, the NCM was incubated in 0.05% (volume / volume) Tween-20 in PBS (PBS-Tween) (J. Immunol. Meth. 55 (1982) 297-307) for 1 hour at room temperature. . In this treatment and all subsequent treatments, the tray containing the NCM was placed on a rocking platform to ensure distribution of the solution throughout the 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, 5 minutes each, with PBS-Tween to remove unbound antibody-Alkaline phosphatase-conjugated goat anti-mouse IgG + IgM (Tago, Ine.), Diluted according to manufacturer's specifications and NCM incubated for 1 hour at room temperature 8701554 ii -25-. The NCM was washed five times as described, after which substrate containing bromochloroindolyl phosphate and nitra blue tetrazolium (Sigma) was 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 reaction was stopped by washing the substrate away 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 immunoflowering show that PaF4 IVE8 binds to the flagella of P. aeruginosa.

The flagella type, which recognized PaF4 IVE8, was determined by ELISA. Habs strains 1-12 (A.T.C.C. 33348-33359) were each bound to wells of 96-well microtiter plates (Linbro) with PLL, after which the ELISA was performed as described earlier in this example. The source of

PaF4 IVE8 antibody was supernatant culture fluid. Positive reactions 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 are presented in Example IV.

EXAMPLE III

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

The lymphoid cell source for the fusion was a spleen from an immunized BALB / c mouse injected intraperitoneally four times with purified type a flagella (10-20 µg protein) from Habs strains 6 and 8 (ATCC) over a six week period. 33353 and 33355). Flagella 30 were purified according to the method described in Infect. Immun. 35 (1982) 281-288, except that the final centrifugation of flagella was at 100,000 x g for 1 hour instead of 40,000 x g for 3 hours. A second modification, which was 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).

£ 721554 -26-

Protein concentrations of each preparation were determined by 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 fla-5 gellable proteins were determined by comparing their migration in an SDS polyacrylamide gel with the migration of standard protein markers (BRL) (see Example II). The molecular weight of Habs 6 flagellin was 51,700 daltons and that of Habs 8 flagellin was 47,200 daltons. These values correspond to those of Infect. Immun. 49 (1985) 770-774.

10. 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 grew to a confluence of about 40% (day 7), the culture supernatants were copied into corresponding wells of three different antigen plates, PLL-bound P. aeruginosa Fisher immunotype 1 (see Example II for the preparation) and formalin - fix the Habs 6 and Habs 9.

The bacteria for the formalin-fixed antigen plates were grown, washed and diluted as described for PLL-linked anti-gene plates. Diluted bacteria (0.2 O.D. units at Agg ^) were added to individual wells (50 µl per well) of Linbro 96-well microtiter plates and the plates centrifuged at 1200 x g for 20 minutes at room temperature. The supernatants were removed from the wells and 75 µl 0.2% (volume / volume) formalin in 25 PBS was added to each well and incubated at room temperature for 15 minutes *. 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 agglutinate 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-flagellated, as was shown by staining (Manual of Clin. Microbiol. (1985) ed. Amer. Soc. Microbiol., P. 1099). Wellbore hybrid cells, designated 35 FA6 11 (35 produced an antibody, which bound to Habs 6 and Habs 9 (both flagella type a carrying strains), but not to Fisher immunotype 1.

8701554 -27-

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

Specificity of FA6 IIGS

The specificity of the FA6 IIG5 antibody was determined by indirect immunofluorescence and immunofluidity. The indirect immuno-fluorescence was performed essentially 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 Aggg of 0.2 OD units. Formalin (0.37% (volume / volume) in PBS final concentration) was added to the suspension under vortexing. After incubation at room temperature for 15 minutes, the bacteria were diluted 1:12 in PBS and 20 μΐ 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 supernatant culture fluid from the FA6 IIG5 cell line.

Fluorescent staining by the FA6 IIG5 antibody was observed only with P. aeruginosa strains carrying type a flagella and none. who wear type b. 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 treatment was not necessary to visualize flagellar staining with the antibody.

Immunofluid was performed as described in Example II.

The sources of flagella type a antigens were the purified flagellar preparations (see this example). Antigens were separated into 10% polyacrylamide gels containing SDS (Nature 227 (1970) 680-685) and transferred to an NCM. Formulations of FA6 IIG5, either 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.

570 1 55 4.

-28-

Immunofluid showed that FA6 IIG5 specifically bound to Habs 6's 51,700 MW flagellin and Habs 8's 47,200 MW flagellin.

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

EXAMPLE IV

Example IV shows the protection of mice passively immunized with antibodies PaF4 IVE8 and FA6 IIG5 against threats with P. aeruginosa 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 carrier strain used for the animal experiments was P. aeruginosa PA220 (from Dr. James Pennington, Boston) and the flagella type b strain was the reference P. aeruginosa Fisher immunotype 2 (A.T.C.C. 27313).

Per mouse, 40 µg of purified monoclonal antibody was administered intravenously 1-2 hours before burning and the threat. Immediately after the branding, the animals received 0.5 ml cold PBS subschar containing the threatening bacteria. The threat dose was approximately 10 LD ^ Js for each organism. The results of the animal experiments are shown in Tables A and B.

8701554 -29-

TABLE · A

Protection study of an anti-flagella type alpha monoclonal antibody in the burned mouse model.

% survival per day 2

Treatment 1 2 3 4 5 6 7 8 9 MFA6fXIG5lla 100 100 100 100 100 90 90 80 80 b "100 20 10 10 10 10 10 10 10

PaF4 IVES

Non-specific anti-LPS monoclonal 100 40 30 20 10 10 10 10 10 antibody PBS, without 7 7 .- 80 80 80 80 80 80 80 80 80 bacteria 1 Mice were endangered with approximately 10 LDPs of P. aeruginosa PA 220.

2 ...

The percentage rs based on the survival of muxzen m from 10 animal groups, excluding the PBS control group only, which consisted of 5 mice. The days are counted after the threat has burned.

.3 7 C * 55 4 -30-

TABLE B

Protection study of an anti-flagella type b monoclonal antibody in the burned mouse model.

% consultation per day 1

Treatment 123456789

AnpaF41I ^ 8la b '100 100 90 90 90 90 90 90 90

AnFA6fIIG5lla ^ 100 20 10 10 10 10 10 10 10

Non-specific anti-LPS monoclonal 100 20 20 20 20 20 20 20 20 antibody poe zoTifipy z '^ · - · 100 100 100 100 100 100 100 100 100 bacteria

Mice were threatened subeschar with approximately 10 LD ^ s of P. aeruginosa Fisher immunotype 2.

8701554

See footnote 2 to table A.

-31-

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 threatened mice or animals treated with an inappropriate anti-flagellar monoclonal antibody or nonspecific anti-LPS antibody died. 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 against a fatal threat from type a-bearing P. aeruginosa PA220, confirmed in vivo the specificity of the antibodies, which had been assumed in vitro. Survival from burned, but uninfected mice showed that the burn itself was not fatal.

EXAMPLE Y

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 came from a variety of isolation sites, including blood, wounds, respiratory system, urine, and ears. A total of 157 isolates were examined.

PaF4 IVE8 specifically bound to 34 clinical isolates (22%), while the flagella type a antibody, FA6 IIG5, bound to 102 clinical isolates (65%), making a total of 136 of 157 isolates (87%).

Of the 21 strains, which were not recognized by either antibody, 19 were non-flagellated, as shown by stain staining- Both antibodies in combination therefore bound 136 of 138 (98%) flagellated clinical isolates, confirming previous reports 30 (Zbl. Bakt. Hyg., I Abt. Orig. A 242 (1978) 228-238).

EXAMPLE VI

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

370 1 554 * * -32-

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 in T cells using a modified E-rosette procedure. The cells first turned 7

resuspended to a concentration of 1 x 10 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 g 10 1 x 10 with 2-aminoisothiouronium bromide (AET) -treated red sheep blood cells was added from a 10% (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 at 4 ° C for 5-10 minutes, 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 blood mononuclear cells (E PBMC), which united at the interface, were collected and washed once in Iscove's medium, then resuspended in the same medium containing 15% (volume / volume) FCS, L-glutamine (2 mxnol / 1) penicillin -4 20 (100 lü / ml), streptomycin (100 µg / ml), hypoxanthine (1 x 10 M), amino- -7 -5 pterine (4 x 10 M) and thymidine (1 , 6 x 10 M). This medium is further referred to as HAT medium.

Cell-driven transformation of the E PBMC was accomplished by co-cultivating these cells with a transforming cell line.

25 'The transforming cell line was an Epstein-Barr nuclear antigen (EBNA) 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 µg / ml 6-thioguanine to the cells hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient and thus render 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 were suspended in HAT medium and then combined with the E PBMC1s at 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 an 870 1 554 -33 volume of 200 µl per well, and incubated at 37 ° C in a humidified atmosphere, containing 6% 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 to remove and test supernatants for anti-P. aeruginosa antibodies.

Supernatants were screened for the presence of anti-P. aeruginosa antibodies using the ELISA-10 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 DOÜ flat bottom 96-well microtiter plates (Immulon II, Dynatech) pretreated with poly-L-lysine, incubated and washed as described in Example II. After blocking non-specific binding sites and washing the plates, 50 µl PBS containing 0.1% (volume / volume) Tween-20 and 0.2% (weight / volume) BSA was added per well. The culture supernatants 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 μΐ per well), horseradish peroxidase-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, was added to the wells and the assay was completed as described in Example II.

Supernatants containing antibody that bound to the combination of Fisher immunotypes, but not the control plate, were tested a second time using each of the seven Fisher immunotype bacteria separately. Antibody present in the single well supernatant, 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 with growth antibody separated. The cell line and the monoclonal antibody (IgM isotype) are both referred to below as 20H11.

8701554 -34-

A second transformation was performed in which the source of B cells came from the peripheral blood of a bladder fibrosis patient known to have a chronic P. aeruginosa infection. E PBMCs were prepared as described above and co-cultured with the transforming cell line, 1A2, at a ratio of 72 1A2 cells per E PBMC. The cell mixture was plated in fifteen round-bottom 4 96-well microtiter plates at a concentration of 7.4 x 10 cells per well and then cultured as above.

Supernatants were examined 16 days after plating the transformation according to ELISA for the presence of anti-P. aeruginosa antibodies. The assay was performed as described for the previous transformation, except that the combination of P. aeruginosa strains used for the initial screening was composed of Fisher immunotype reference strains F2, F4, F6 and F7 (ATCC 27313, 27315, 15 27316 and 27317 ) and three clinical isolates from the Genetic Systems

Corporation Organism Bank (GSCOB), which had different LPS immunotypes and flagella types. The clinical isolate PSA 1277 (GSCOB) carries type a flagella and Fisher immunotype 1 LPS; the second isolate PSA G98 (GSCOB) carries type a flagella and Fisher immunotype 3 LPS? and the third, PSA F625 (GSCOB) carries type b flagella and Fisher immunotype 5 LPS. This mixture of reference strains and clinical isolates will be referred to as the P. aeruginosa flagellated combination. Supernatants containing antibody which bound to plates containing the P. aeruginosa flagellated combination, but not to the PLL-coated control plates, were tested a second time according to ELISA for the individual strains of the combination. One well, 3C1, bound to reference strains F2, F6 and F7 and to the clinical isolate F625.

Cloning of the 3C1 cell line was done by first subculturing the cells in two rounds of low density subculture, first at 20 cells per well of 96 well plates, followed by culture at 2 cells per well. Formal cloning of the specific antibody-producing cells was accomplished by plating the cells at a density of approximately 1 cell / well in 72-well Terasaki plates in a volume of 10 µl HAT medium without the aminopterin component (HT medium) per pit.

The plates were placed in an incubator for 2-3 hours to allow the cells to settle 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 the outgrowth wells were examined by ELISA 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 below as 3C1.

The antigen identified by 20H11 and 3C1 was flagellar, as shown by indirect immunofluorescence and immunofluidity.

The techniques were basically used as described in Examples II and III. For the indirect immunofluorescence study, P. aeruginosa strains with type b flagella, reference Fisher immuno types F2, F6 and F7 (ATCC 27313, 27317 and 27318) and a strain with type 15 a flagella, reference Fisher immunotype 4 (ATCC 27315) ) 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. Slides were prepared for inspection as described in Example II. The sources of both antibodies were supernatant culture fluids and the FITC conjugated reagent was FITC conjugated goat anti-human Ig (polyvalent) diluted 1: 100 in PBS containing 0.5% (weight / volume) bovine gamma globulins (Miles Scientific, Cat No. 82-041-2) and 0.1% (weight / volume) sodium azide as a preservative.

Fluorescent staining by the 20H11 and 3C1 antibodies was observed only with P. aeruginosa strains with type b flagella and not with the flagella type a carrying strain, reference Fisher immunotype 4.

The observed fluorescence pattern was a sinus oxalin line pattern, starting from one end of the bacterium, showing that the antibodies bound to the bacterial flagella.

Immunofluid 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.

57 ö155 4 -36-

Culture supernatants containing 20H11 or 3C1 antibodies, culture supernatant containing non-specific human antibody and culture media were incubated with the NCM and the reaction was discovered with an alkaline phosphatase-conjugated goat anti-human Ig 5 (polyvalent) diluted in PBS containing 0.05% (volume / volume) Tween-20. Enzyme substrate was prepared as described in Example II. The immunofluidity 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 nonspecific human antibody or with the culture media.

Further confirmation that antibodies 20H11 and 3C1 bound only 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 microtiter plates. The antibodies bound only to Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, which are type b carrying strains (J. Clin. Microbiol. 20 (1984) 84).

EXAMPLE VII

Example VII shows methods for the production of a human monoclonal antibody that 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 separated from the blood and then depleted in T cell slate 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-cultivating the E PBMCs with 1A2 cells at a ratio of 30 1A2 cells per E PBMC. The cell mixture was plated into 96 well tissue culture plates at a concentration of 62,000 cells per well. On the seventh day 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.

8701554 -37-

The supernatants were tested for the presence of anti-P by ELISA. aeruginosa antibodies using the flagellated P. aeruginosa combination and PLL-treated plates as a control, as described in Example VI. Supernatants containing antibodies that bound to the flagellated combination but not to the PLL control plates were re-examined for the individual bacterial strains of the flagellated combination.

One well, 21B8, contained antibody that bound to PSA 1277, PSA G98 and reference Fisher immunotype 4, i.e., the three strains of 10: the flagellated combination bearing type a flagella.

Cloning of the 21B8 cell line was done as described in Example VI for the 3C1 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 15 Terasaki plate was transferred to one individual well of a 96-well round-bottom culture plate in a volume of 100 μΐ HAT medium without the aminopterin component ( HT medium). In all wells, non-transforming HAT-sensitive lymphoblast oxide cells were included at a density of 500 cells / well as feeder cells. Five days after plating, 100 µl HAT medium was added to the wells to selectively kill the nutrient cells. On 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 the outgrowth wells produced antibody which bound to flagella type a carrying 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 observed only with P. aeruginosa reference strain Fisher immunotype 4 (A.T.C.C. 27315) carrying type a flagella, and not with P. aeruginosa reference strain immunotype 2 (A.T.C.C. 35 27313) carrying type a flagella. The fluorescence pattern observed was a sunusol line pattern, starting from one end of the 8701354-38 bacterium, showing that the antibody was bound to the bacterial flagella.

Immunofluid was performed as described in Example II. Purified type a flagella from the P. aeruginosa reference strain Habs 65 (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. Culture supernatants 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. Immunofluid 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 passively immunized with human anti-flagella antibodies, 20H11, 3C1 and 21B8, against threats with P. aeruginosa in the roasted mouse model.

The human anti-flagella monoclonal antibodies were tested in the roasted mouse model (see Example IV). 21B8 and 20H11 antibodies were prepared by precipitation of the above culture liquids 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 administration to animals. The source of anti-body 3C1 and the nonspecific anti-LPS antibody used as negative control in the study was supernatant culture fluid. As a positive control for each study, the appropriate purified mouse monoclonal antibody PaF4 IVE8 or FA6 IIG5 was included.

The flagella type a carrying strain used in the animal studies was the clinical isolate PSA A522 (GSC0B), capable of expressing Fisher immunotype 1 LPS, and the flagella type b strain was clinical isolate PSA A447 (GSCOB), capable of expressing Fisher immunotype 6 LPS. The human antibodies (0.45 ml) were pre-mixed with the bacteria (more than 5 DD ^ g's in 0.05 ml) and seeded sub-scar immediately after the burn was caused. The results of the animal experiments are shown in Tables C, D and E.

3701554 J * -40-

TABLE · C

Protection study of the human anti-flagella type a monoclonal antibody 21B8 in the burned mouse model.

% survival per day 3

Treatment 123456789

Shellazella a '100 100 100 100 88 88 88 88 88

Human anti-a. 100 100 100 100 100 100 100 100 100 flagella a, 21B8

Human anti-100 00000000 flagella b, 21B8 PBS 100 25 12 12 12 12 12 12 12

Mice were endangered subeschar with more than 5 LDs of 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 µg in 0.45 ml PBS) was premixed with the bacteria and sub-shed after burning and threatening.

8701554 -41-

TABLE D

Protection study of the human anti-flagella type ^ b monoclonal antibody 20H11 in the burned mouse model.

% survival day 2

Treatment 1 2 3 4 5 6 7 8 9

Mouse anti-flagella b, 100 1Q0 1Q0 1 (J0 100 100 100 1Q0 1QQ

PaF4 IVE81

Human anti-100 100 100 100 100 100 100 100 100 flagella b, 20H11

Human anti-100 0 0 0 0 0 0 0 0 flagella a, 21B8

Media 80 00000000 ^ Mice were endangered subeschar with more than 5's of 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.

8701554

See footnote 3 to table C.

3 * -42-

TABLE E

Protection study of the human anti-flagella type ^ b monoclonal antibody 3C1 in the burned mouse model.

2% survival per day

Treatment 123456789

Mouse anti-flagella b, 1Q0 100 100 100 100 100 100 100 i00

PaF4 IVE83

Human anti 100 100 80 80 80 80 80 80 80 flagella b, 3C1

Non-Specific Anti-LPS Monoclonal 100 00000000 Antibody 1 Mice were endangered with more than 5 LDVs of PSA A447.

2% consultation was based on the number of surviving mice in groups of 5 animals. The days are after burning and threatening.

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

8701554 -43-

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, then threatened with the corresponding antigen. Conversely, 88-100% of the untreated, but endangered, mice, or mice treated with an inappropriate anti-flagellar monoclonal antibody or non-specific anti-LPS antibody died. As observed with the mouse monoclonal antibodies (see Example IV), the human anti-flagella antibodies specifically protect against lethal threat only with the organisms containing it. wear corresponding flagella type, i.e., the human anti-flagella type a antibody conferred protection against lethal threat to the flagella type a carrier organism and not the type b, and that the anti-flagella type b antibodies protected mice threatened with flagella type b bearing strains, but not the type a carrying organism EXAMPLE IX

Example IX shows the cross-reactivity of the 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 flagella type a or type b by typing with the mouse monoclonal antibodies FA6 IIG5 or PaF4 IVE8 (see examples II, III and V). Of the clinical isolates, five-fifty-five were identified as carrying type a flagella by reaction with the murine monoclonal antibody FA6 IIG5, and 59 were identified as carrying type b by their reaction with the murine monoclonal antibody PaF4 IVE8.

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 reactivity of 20H11 with the isolates carrying type b flagella was also extensive: 20H11 recognized all 59 isolates (100%). In contrast, the other anti-flagella type b monoclonal antibody, 3C1, 35 bound only 43 of the 59 isolates (73%). These results show that 20H11 binds to a pan-reactive epitope (ie an epitope, present 8701554 * * -44- on at least 95% of the flagellated P. aeruginosa strains), while 3C1 binds to an epitope that is not present on all type b flagellin molecules are present. 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, which can be identified by monoclonal antibodies.

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

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 roasted mouse model.

A transformed cell line was prepared and cloned as described in Example VII, except that the transformed cell mixture was plated on 20 96-well tissue culture plates at a concentration of about 2250 U PBMC per well. The supernatants were first tested by ELISA on the flagellated 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 flagellated 25 'combination, including Fisher immunotype 2 and 4. The ultimately isolated cell line and monoclonal antibody (IgG isotype) are referred to below as 12D7.

The 12D7 human monoclonal antibody showed extensive cross-reactivity with anti-flagella type a isolates, recognizing 54 'of 56 tested flagella type a carrying clinical isolates (96%).

The protective activity of the 12D7 monoclonal antibody is shown in Table F. The protective study was performed as described in Example IV except that the threatening dose was 1 LD-q0 and that the threatening strain was 16.24, a clinical isolate, expressing Fisher immunotype 2 LPS and type a flagella.

8701554 -45-

TABLE · F

Protection study of the human anti-flagella type ^ a monoclonal antibody 12D7 in the burned mouse model.

% survival per day 1

Treatment ^ 123456789

Human antx- 100 3.00 100 100 100 100 100 100 100 flagella ar 12D7

Human aati 90 90 90 90 90 90 90 90 90 90

Fisher 2 LPS, 2H9

Mmze anti-flagelia 100 10q 100 100 100 100 100 100 100 a, 11G5

Human anti-flagella b, 15F4 1 Mice were endangered with 1 LD ^ qQ (950 CPU) of 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 µg in 0.5 ml PBS) was injected i.p. administered 8701554 hours before burning and threatening.

> «-46-

FORE IMAGE XI

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

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 the transformed cell mixture was plated on 15 plates at a concentration of about 1.0 spring 1930 U PBMC per well. Cells were also tested in a combination of P. aeruginosa strains, including G98 (Fisher 3 immunotype, flagella type a) and 1739 (a clinical isolate of Fisher 5 immunotype, flagella type b), and confirmed on another combination of P. ' aeruginosa strains; 1277, G98, 1739, and Fisher immunotypes F2, F4, F6, and F7.

The ultimately isolated cell line and the secreted monoclonal antibody (IgG ^ isotype) are referred to as 2B8 in the following.

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

The protective activity of 2B8 is shown in Table G.

The protective study was performed as described in Example X except that clinical isolate F164 (Fisher immunotype 4, flagella type b) was used for the threat.

370 f554 -47-

TABLE G

Screening study of the human anti-flagella typ | b monoclonal antibody 2B8 in the burned mouse model% consultation per day2

Treatment 12345 6 789

Human anti-g 100 µg 89 µg gg gg gg gg 8g flagella b, 2b8

Mouse anti-flagella 1οσ χ00 100 100 100 100 90 90 90 b, PaF4 IVE8

Mouse anti-Fisher 2 LPsV VH34 90 20 20 20 20 20 20 20 20

Mice were subeschar threatened with 1 (1Q5 cfü) of P. aeruginosa F164.

2% survival was based on the number of surviving mice per group of 10 animals, except group 2B8, which contained 9 animals - The days are from burning and threatening.

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

4

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

d / 01554

EXAMPLE XII

-48-

Example XII shows the production of another human monoclonal antibody, which is reactive with flagella b type P. aeruginosa, and the protection of mice passively immunized with this antibody against threats with P. aeruginosa in the roasted 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 about 2000 cells per well. Screening was done for combined Fisher 1-7 immunotypes. The ultimately 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 flagellated type b isolates were positive with 14C1. The protection study was performed as described in Example XI and the results are reported in Table H.

870 1 554 4-J; -49-TABLE Η

Protection study of the human anti-flagella type ^ b monoclonal antibody 14C1 in the burned mouse model.

-% of survivors per day

Treatment 123456789

Human anti-100io 100 100 100 90 90 90 90 flagella b, 14C1 MU ^ ZS, 100 100 100 100 100 100 100 100 100 b, PaF4 IVE8, 100 40 20 20 20 20 20 20 20

Fisher 2 LPS, VH3 1 Mice were endangered with 1 LD ^ Q0 (90 CFü) of 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.

3

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

From the foregoing, it will be apparent that the cell lines of the invention provide agents for the production of monoclonal antibodies and fragments thereof, which are reactive with P. aeruginosa flagella and cross-protective against various P. aeruginosa strains. Surprisingly, a number of monoclonal antibodies, which were reactive with different epitopes on each type of flagella, were isolated. The antibodies of the invention allow prophylactic and therapeutic compositions to be produced more economically and more readily for use against infections caused by most P. aeruginosa strains. Furthermore, the cell lines provide antibodies, which find use in immunoassays and other known procedures.

17 91554

Claims (32)

A composition comprising a monoclonal antibody or binding fragment thereof capable of specific reaction with flagella of Pseudomonas aeruginosa bacteria. A composition comprising a monoclonal antibody or binding fragment thereof capable of specific reaction with flagella of Pseudomonas aeruginosa bacteria. The composition of claim 1, wherein the monoclonal antibody or binding fragment thereof inhibits the motility of the bacteria. The composition of claim 1, wherein the monoclonal antibody or binding fragment thereof inhibits the motility of the bacteria. The composition of claim 1, wherein the monoclonal antibody or binding fragment thereof is protective in vivo. The composition of claim 1, wherein the monoclonal antibody or binding fragment thereof is protective in vivo. A composition comprising a monoclonal antibody capable of specific reaction with a flagellar protein epitope of Pseudomonas aeruginosa. 4. A composition comprising a monoclonal antibody capable of specific reaction with a flagellar protein epitope of Pseudomonas aeruginosa. The composition of claim 4, wherein the monoclonal antibody is capable of blocking binding to the epitope of monoclonal antibodies produced by cell lines designated A.T.C.C. Accession Numbers HB9129, HB9130, CEL 9300 and CEL 9301. The composition of claim 4, wherein the monoclonal antibody is capable of blocking binding to the epitope of monoclonal antibodies produced by cell lines designated as 6. Composition comprising one or more monoclonal antibodies, wherein a first monoclonal antibody is capable of reacting with an epitope of type a or type b flagella of Pseudomonas aeruginosa. c, u 1 ï o 4 545 r - - 6. Cell line producing a monoclonal antibody capable of specific reaction with type a or type b Pseudomonas aeruginosa flagella. The composition of claim 6, wherein a second monoclonal antibody is capable of reacting with a type of flagella that is not reactive to the first monoclonal antibody. The cell line of claim 6, wherein the monoclonal antibody 20 is protective against Pseudomonas aeruginosa in vivo. The composition of claim 6, wherein the first antibody is a human monoclonal antibody. The cell line of claim 7, which is a hybrid cell line. The composition of claim 6, wherein at least one of the monoclonal antibodies is protective in vivo. The cell line of claim 6, which produces human monoclonal antibodies. The composition of any of claims 1, 4, 6 or 8, 10 further comprising a gamma globulin fraction from human blood plasma and / or an antibiotic. The cell line of claim 6, which is one of A.T.C.C. Accession Pharmaceutical composition, comprising a composition according to any one of claims 1, 4, 6 or 8, as well as a physiologically acceptable carrier. 11. A method of producing monoclonal antibodies specific for flagellar proteins of Pseudomonas aeruginosa and capable of treating or preventing Pseudomonas aeruginosa infections, characterized in that at least one of the cell lines of claim 10 is cultured and the antibodies are recovered. Pharmaceutical composition for use in the treatment or prevention of a Pseudomonas aeruginosa infection, which composition is a monoclonal antibody reactive with a flagellar protein of Pseudomonas aeruginosa and in vivo protective, an antimicrobial agent, a gamma -20 globulin fraction of human blood plasma and comprises a physiologically acceptable carrier. 12. A method of treatment of a human susceptible to bacteremia and / or septicemia, characterized in that a prophylactic or therapeutic amount of monomonoclonal antibodies produced according to claim 11 is administered to the human. 8701554 -52- The pharmaceutical composition according to claim 12, wherein the antibody is a human monoclonal antibody and the gamma globulin fraction from human blood plasma is obtained from humans showing elevated concentrations of 8 7 0 1 S 5 4 "* 7 immunoglobulins that are reactive to relative to Pseudomonas aeruginosa bacteria and / or products thereof. A composition comprising a monoclonal antibody or fragment thereof that is reactive with an epitope capable of binding to a monoclonal antibody produced according to claim 11. 14. Pharmaceutical composition comprising at least two monoclonal antibodies, each specifically reactive with a different type of Pseudomonas aeruginosa flagellar protein and suitable for treatment or prevention of Pseudomonas aeruginosa infections. Pharmaceutical composition comprising at least two monoclonal antibodies, each specifically reactive with a different type of Pseudomonas aeruginosa flagellar protein and suitable for treatment or prevention of Pseudomonas aeruginosa infections. The composition of claim 14, wherein at least one of the monoclonal antibodies is a human monoclonal antibody. The composition of claim 14, wherein at least one of the monoclonal antibodies is a human monoclonal antibody. 15 A.T.C.C. Accession Numbers HB9129, HB9130, CRL 9300, CEL 9301, CEL 9422, CEL 9423 and CEL 9424. The composition of claim 14, further comprising 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 composition of claim 14, further comprising 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. 17. Method for the treatment of a human susceptible to bacteremia and / or septicemia, characterized in that the human is administered a prophylactic or therapeutic amount of a composition according to any one of claims 1, 6, 8, 12, 14 or 15. Method for the treatment of a human susceptible to bacteremia and / or septicemia, characterized in that the human is administered a prophylactic or therapeutic amount of a composition according to any one of claims 1, 6, 8, 12, 14 or 15. 18. Cell line producing a monoclonal antibody capable of specific reaction with type a or type b Pseudomonas aeruginosa flagella. 18. Cell line producing a monoclonal antibody capable of specific reaction with type or type b Pseudomonas aeruginosa flagella. The cell line of claim 18, wherein the monoclonal? Λ antibody protects against Pseudomonas aeruginosa in vivo. The cell line of claim 18, wherein the monoclonal antibody is protective against Psudomonas aeruginosa in vivo. The cell line of claim 19, which is a hybrid cell line. The cell line of claim 19, which is a hybrid cell line. The cell line of claim 18, which produces human monoclonal antibodies. The cell line of claim 18, which produces human monoclonal antibodies. The cell line of claim 18, which is one of A.T.C.C. Accession Numbers is HB9129, HB9130 or CRL 9300 and CRL 9301. The cell line of claim 18, which is one of A.T.C.C. Accession Numbers is HB9129, HB9130 or CRL 9300 and CRL 9301. 23. A method of producing monoclonal antibodies specific for flagellar proteins of Pseudomonas aeruginosa and suitable for treatment or prevention of Pseudomonas aeruginosa infections, characterized in that at least one of the cell lines of claim 22 is cultured and the antibodies be won. 23. A method of producing monoclonal antibodies specific for flagellar proteins of Pseudomonas aeruginosa and suitable for treatment or prevention of Pseudomonas aeruginosa infections, characterized in that at least one of the cell lines of claim 22 is cultured and the antibodies are recovered . A method of treatment of a human susceptible to bacteremia and / or septicemia, characterized in that a prophylactic or therapeutic amount of monoclonal antibodies produced according to claim 23 is administered to the human. 2Q 25. Monoclonal antibody or fragment thereof, which is reactive with an epitope capable of binding to a monoclonal antibody produced according to claim 23. 24. A method of treatment of a human susceptible to bacteremia and / or septicemia, characterized in that a prophylactic or therapeutic amount of monoclonal antibodies produced according to claim 23 is administered to the human. 8701554 -53- A monoclonal antibody or fragment thereof, which is reactive with an epitope capable of binding a monoclonal antibody produced according to claim 23. 25 Numbers is HB9129, HB9130, CEL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. 26. Composition comprising a monoclonal antibody or fragment thereof, which is reactive with an epitope capable of binding to a monoclonal antibody in 77 ft -1% “P * ö ƒ V i -J 4 _s-5_, tried. dipped according to claim 23. 26. Composition comprising a human monoclonal antibody, which is specifically reactive with an epitope, present on Pseudomonas aeruginosa flagella. 27. A composition comprising a human monoclonal antibody, which is specifically reactive with an epitope, present on Pseudomonas aeruginosa flagella. The composition of claim 26, wherein the epitope is present on type a or type b flagella, but not both. The composition of claim 26, wherein the epitope is present on type a or type b flagella, but not both. The composition of claim 26, wherein the epitope is exhibited by at least about 70% of type b flagella. The composition of claim 26, wherein the epitope is exhibited by at least about 70% of type b 10 flagella. The composition of claim 26, wherein the epitope is displayed only by type b flagella. The composition of claim 26, wherein the epitope is displayed only by type b flagella. The composition of claim 29, wherein the antibody is pan-reactive. The composition of claim 29, wherein the antibody is pan-reactive. A method of treatment for a human susceptible to bacterial infections, characterized in that a prophylactic or therapeutic amount of a monoclonal antibody capable of binding to the flagellum of Pseudomonas aeruginosa is administered to the human in combination with one or more of: a propylactic or therapeutic amount of a monoclonal antibody capable of reaction with pseudomonas aeruginosa exotoxin A; a monoclonal antibody capable of reacting with at least one serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa; a gamma globulin fraction of human blood-25. plasma; a gamma globulin fraction of a human blood plasma, which exhibits elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa and / or products thereof; or an antimicrobial agent. 32. Monoclonal antibody composition having the same binding specificity as one of the monoclonal antibodies, designated FA6 IIG5, Pa3 IVC2, PaF4 IVE8, 20H11 and 21B8. The monoclonal antibody of claim 32, conjugated with a label capable of providing an observable signal. The monoclonal antibody of claim 33, wherein the label is 35 fluorescent or an enzyme. 870 1 554 -54- 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. 36. Package for use for detecting the presence of Pseudomonas aeruginosa bacteria, containing a monoclonal antibody composition, containing at least one monoclonal antibody, which reacts with a type-specific flagellar protein of the bacterium, and labels to provide a detectable signal, covalently bound to the antibody or bound to second antibodies, which are reactive with the monoclonal antibody. 8701554 - COMPANY PATENT OFFICES O.A. No. 8701554 * s ^ srav £ nhace (holland) Beh. when writing dd. August 25, 1987 Ln / 657 32. A method of treatment of a human susceptible to bacterial infection, characterized in that a prophylactic or therapeutic amount of a monoclonal antibody capable of binding to the flagellum of Pseudomonas aeruginosa is administered to the human. in combination with one or more of: a prophylactic or therapeutic amount of a monoclonal antibody capable of reacting with at least one serotype determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa; a gamma globulin fraction from human blood plasma; Showing elevated levels of immunoglobulins, which