CH677796A5 - - Google Patents

Description

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

description

The present invention relates to monoclonal antibodies which are capable of recognizing Pseudomonas aeruginosa flagella and which are useful for the diagnosis and treatment of bacterial infections. The invention also relates to the use of such monoconal antibodies.

Gram-negative diseases and their most serious complications, e.g. Bacteremia and endotoxemia are the causes of significant morbidity and mortality in humans. This is particularly true for the gram-negative organism Pseudomonas aeruginosa, which has been increasingly associated with bacterial infections, especially nosocomial infections, in the past 50 years.

Over the past few decades, antibiotics have been the treatment of choice to fight gram-negative diseases. The still high morbidity and high mortality observed in Gram-negative bacterial diseases, however, show the limits of antibiotic therapy, especially with regard to P. aeruginosa (see e.g. Andriole, VG, «Pseudomonas Bacteremia: Can Antibiotic Therapy Improve Survival? », J. Lab. Clin. Med. (1978) 94: 196-199). This prompted the search for alternative methods of prevention and treatment.

One method that has been considered has been to boost the host's immune system through active or passive immunization. For example, it has been observed that active immunization of humans or experimental animals with whole-cell bacterial vaccines or with purified bacterial endotoxins from P. aeruginosa leads to the development of specific opsonized antibodies which are primarily against the determinants on the repeating oligosaccharide units of the lipopoly-saccharide LPS) molecules which are located on the outer cell membrane of P. aeruginosa (see Pollack, M., Immunoglobulins: Characteristics and Uses of Intravenous Preparations, Aiving, BM, and Finlayson, JS, editors, pages 73-79, US Department of Health and Human Services, 1979). It has been shown that such antibodies, whether actively generated or passively transmitted, offer protection against the lethal effects of a P. aeruoinosa infection in various animal models (Pollack, supra). Some preliminary experiments with humans point in the same direction (see Young, LS. And Poltack, M., Pseudomonas aeruginosa. Sabath, L, editor, pages 119-132, Hans Huber, 1980).

These reports suggest that an immunotherapeutic approach could be used to prevent and treat bacterial diseases due to P. aeruginosa. One such approach would be to administer collected human immunogiobulins that contain antibodies to the infecting strain. Human immunoglobulins are defined here as the portion of the fractionated human plasma that is enriched in antibodies. Specific antibodies against strains of P. aeruginosa are also represented. Because of certain inherent limitations in the use of human immunoglobulin components, this approach to treating diseases caused by R aeruginosa is still under investigation (see, e.g., Collins, MS and Roby, RE, Am. J. Med., 76: (3A) : 168-174), (1984)) and for the time being there are no commercial products available »which use these components.

One of the limits of using immunoglobulin compositions is that such compositions consist of the collected samples from thousands or more donors, the samples being selected for the presence of a particular anti-Pseudomonas antibody. The individual antibody titers are averaged by this collection; at best, this leads to a modest increase in the resulting titer on the desired antibody.

Another limit is that the selection process itself requires expensive and constant monitoring of the donor pool in order to guarantee a uniform product. Despite these efforts, immunoglobulin products can vary significantly from series to series and products of different geographic origins.

Yet another limit inherent in immunoglobulin compositions is that their use results in the simultaneous administration of large amounts of foreign proteinaceous substances (including viruses such as those associated with AIDS) that can cause deleterious biological effects. The combination of low titer of desired antibodies and high content of foreign substances, often at suboptimal levels, limits the amount of specific and therefore useful immunoglobulins that can be administered to a patient.

In 1975, Köhler and Milstein reported their fundamental discovery that certain mouse cell lines can be fused with spleen cells from mice to produce hybridomas. These hybridomas are able to secrete antibodies of a single specificity, i.e. monoclonal antibodies (Koehler, G., and Milstein, C., Nature, 256: 495-497 (1975)). With the advent of this technology, it has become possible in some cases to produce large amounts of highly specific murine antibodies against a certain determinant or against determinants on antigens. As a result, using technologies developed later, it became possible to produce human monoclonal antibodies (see e.g. U.S. Patent No. 4464465).

The use of mouse monoclonal antibodies or compositions containing such antibodies is known to be problematic in humans in certain cases. For example, it has been reported that mouse monoclonal antibodies used in experimental studies to treat certain human diseases

2nd

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796 A5

have elicited an immune response that rendered them useless (Levy, R.L. and Miller, R.A., Ann, Rev. Med., 34: 107-116 (1983)), thanks to recent advances in recombinant DNA technology, e.g. the production of chimeric mouse human monoclonal antibodies, these difficulties could be reduced. Furthermore, methods for the production of monoclonal human antibodies are available today (see Human Hybridomas and Monoclonal Antibodies, Engleman, E.G., et al., Editor, Plenum Publishing Corp. (1985)).

Several groups reported the production of monoclonal antibodies that protect against P. aeru-ainosa infections and that were obtained using hybridomas and / or cell transformation technologies. Monoclonal antibodies that react with various P. aeruginosa epitopes have also been prepared. Included are single and multi-serotype specific surface epitopes, such as those found in bacterial LPS molecules (see, e.g., pending U.S. Patent Application Nos. 734,624 and 807,394). Protective antibodies specific for P. aeruginosa exotoxin A have also been made (see, e.g., pending U.S. Patent Application No. 742,170),

Although the use of monoclonal antibodies specific for the LPS region or for the P. aeruginosa exotoxins may provide adequate protection in some cases, it is desirable to have broader protection options. For example, in the case of prophylactic treatment against potential human infections, the administration of one or more antibodies that protect against a plurality of P. aeruoinosa strains would be desirable. Analogously, in the case of therapy in which the serotype or the serotypes of the infecting strain are unknown, the administration of an antibody or a combination of antibodies would be desirable which against most, if not all, of the clinically important P aeruginosa-Serotvoen are effective. Ideally, this would require the provision of antibodies that are reactive across the traditional serotype schemes.

One aspect of P. aeruoinosa-Phvsioiooie. which has been shown to contribute to the virulence of the microorganism is its motility. This results primarily from the presence of a flagella thread (see Montie, T., et al., (1982), Infect, and Immun., 38: 1296-1298). P. aeruginosa is characterized by a single flagella thread at one end of its rod-like structure. Studies in burned mice have shown that a higher percentage of mice survived when molar strains of P. aeruoinosa were not vaccinated in the experimental burns than when motile strains were used (McManus, A., et al., (1980) , Bums, 6: 235-239 and Montie, T., et al., (1982), Infect, and Immun., 38: 1296-1298). Further studies on the pathogenesis of P. aeruginosa have claimed that animals immunized with flagella antigen preparations were protected when burned and infected with motile strains of the bacteria (see Holder, I., et ai ., 1982, Infect, and Immun., 35: 276-280).

It is important that P. aeruginosa can be divided into two larger antigenic groups according to studies based on serological methods. These groups were designated H1 and H2 by B. Lanyi (1970, Acta Microlbiol. Acad. Sei. Hung., 17: 35-48) and by Ansorg, R. with type a and type b (1978, Zbl, Bakt. Hyg., I. Dept. Orig. A, 242: 228-238). Serological typing of the flagella by both laboratories showed that H1 flagella (Lanyi, B., supra) or flagella type b (Ansorg, R., supra) were serologically uniform, i.e. no subgroups were identified. This serologically uniform flagellar type is referred to below as type b. The other major antigen, H2 (Lanyi, B., supra) or type a-flagella (Ansorg, R., supra) contained five subgroups. This antigen will be referred to as flagella type a and the five subgroups as ao, ai, az, a3 and a4. The five subgroups of type a are expressed in different combinations on the different strains of the type a-containing P. aeruoinosa strains, with the exception of the antigen ao. The ao antigen has been found on all types of flagella, although the extent to which it is expressed varies between strains.

A serotyping scheme based on the heat stable, more important P. aeruginosa somatic antigens is called the Habs scheme. It has recently been included in the scheme of the International Antigenic Typing System (see, Liu, Int. J. Syst. Bacteriol., 33: 256 (1983)). The R. aeruoinosa-flaoella types of the Habs reference strains were all immunofluorescent using polyclonal sera (Ansorg, R., 1978, Zbl. Bakt. Hyg., I. Dept. Orig. A, 242: 228-238) or characterized by coagulation smear (Ansorg, R., et al., 1984, J. Clin. Microbiol., 20: 84-88). The Habs strains 2, 3, 4, 5, 7, 10, 11 and 12 are flagella type B strains, while the Habs strains 1, 6, 8 and 9 are flagella type A strains. A large number of P. aeruoinosa strains with a small number of monoclonal antibodies that are specific for flagellar proteins can thus be detected.

Accordingly, there is an important need for monoclonal antibodies that are capable of reacting with epitopes of flagellar proteins and that in some cases can provide protection against a plurality of P. aeruoinosa serotypes. Furthermore, some of these antibodies should be useful in the prophylaxis and therapy of P. aeruoinosa infections and for the diagnosis of such infections. The present invention fulfills this need.

The present invention relates to the monoclonal antibodies, cell lines which secrete the antibodies, pharmaceutical compositions, a detection method which the anti3

5

10th

15

20th

25th

30th

35

40

45

50

55

60

CH677 796 A5

Body used and a set of reagents as defined in claims 1,10,14,20 and 21 respectively.

New cell lines are provided which can produce monoclonal antibodies capable of binding to the goat threads found on most strains of P. aeruainosa bacteria. The monoclonal antibodies react specifically with the epitopes on the flagella proteins of P. aeruginosa and can differentiate between type a and type b flagella threads of the bacteria. In addition, a method for treating people who are susceptible to, or already infected with, P. aeruginosa infection is provided. The method is based on the administration of a prophylactic or therapeutic amount of a composition containing at least one monoclonal antibody or binding fragment thereof capable of reacting with the goat strings of the P. aeruginosa strains. The composition preferably contains a physiologically acceptable carrier. The composition may also contain one or more of the following: additional monoclonal antibodies capable of reacting with P. aeruoinos exotoxin A; monoclonal antibodies capable of reacting with serotype determinants to P. aeruginosa LPS; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from human blood plasma, the plasma being from humans who have elevated levels of immunoglobulins that react with P. aeruginosa; and one or more antimicrobial agents. Clinical applications of the monoclonal antibodies are also provided, including the production of diagnostic «kits».

As previously mentioned, the present invention provides new cells that are capable of producing monoclonal antibodies and compositions that contain these antibodies. These compositions can selectively recognize the flagella present on a plurality of P. aeruoinosa strains, with individual antibodies typically recognizing a type of P. aeruginosa flagella. The above cells have identifiable chromosomes in which their germ lines, DNA or that of a progenitor cell, are rearranged to code for an antibody that has a binding site for an epitope to one of the flagellar proteins found on some P. aeruoinosa strains occurrence. Pan-reactive monoclonal antibodies can be produced for type a flagellar proteins; and for type b flagellar proteins, antibodies are included that are either pan-reactive or react with at least about 70% of the flagella-bearing strains. These monoclonal antibodies can be used in a variety of ways, including diagnosis and therapy.

The monoclonal antibodies thus provided are particularly useful in the treatment or prophylaxis of serious diseases caused by P. aeruginosa. The surface proteins on the flagella of P. aeruginosa should be available for direct contact with the antibody molecules. This is likely to inhibit the organism's motility and / or enable other beneficial effects for the host.

Monoclonal antibody production can be accomplished by immortalizing a cell line capable of expressing nucleic acid sequences encoding antibodies specific for an epitope on the P. aeruginosa strains of the flagellar proteins. Immoralized cell lines can be mammalian cell lines transformed by oncogenesis, transfection mutation, or the like. Such cells include myeloma lines, lymphoma lines or other cell lines which can withstand the in vitro expression and secretion of immunoglobulin or a binding fragment thereof. The immunoglobulin or its fragment can be a naturally occurring immunoglobulin that comes from a source other than the preferred mouse or human source. It can be made by transforming a lymphocyte, especially a splenocyte, by means of a virus or by fusing the lymphocyte with a neoplastic cell, e.g. a myeloma cell to produce a hybrid cell line. The splenocyte is usually obtained from an animal which has been immunized against flagellar antigens or their fragments, such as those containing an epitope. Immunization protocols are well known and can vary widely, but remain effective (see Golding, Monoclonal Antibodies: Principles and Practice, Academic Press, N.Y. (1983)).

The hybrid cell lines can be cloned and screened in accordance with conventional techniques, whereby antibodies that can bind to P. aeruginosa flagellar determinants are detected in the supernatant cell fluid. The appropriate hybrid cell line can then be grown on a large scale or injected into the peritoneum of a suitable host to produce Ascltes fluid.

In one embodiment of the present invention, the cells are transformed human lymphocytes, which preferably produce in vivo protective, human monoclonal antibodies for accessible epitope that are specific for at least one flagellar protein. The lymphocytes can be obtained from humans who have been or are exposed to appropriate flagella strains of P. aeruginosa. A preferred cell-powered transformation process is described in detail in U.S. Patent No. 4,464,465.

With the antibodies according to the present invention, which are known to be specific for flagellar proteins, the supernatant from late experiments can be screened in some cases. In a competitive experiment, the monoclonal antibodies according to the invention are used as a means of identifying additional representatives of anti-flagellar mono-

4th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

clonal antibodies used In this way, hybrid line lines can be easily prepared from different sources. The method is based only on the presence of the antibodies according to the invention which are specific for certain flagellar proteins.

Alternatively, if there are hybrid cell lines that produce antibodies specific for the corresponding epitopes, these hybrid cell lines can be fused with other neoplastic B cells. These B cells serve as receivers for genomic DNA that code for the receptors. Mostly neoplastic B cells are used, in particular of mice, but those of other mammals can also be used, such as lagomorphic, bovine, ovine, equine, porcine, aviane or the like.

The monoclonal antibodies can be from any of the classes or subclasses of the immunoglobulins. For example IgM, IgD, IgA, IgE or subclasses of IgG, which are known for each animal species. The monoclonal antibodies are preferably used intact, but binding fragments such as Fv, Fab, F (ab ') 2 can also be used.

The line lines of the present invention can also find use other than the direct production of monoclonal antibodies. They can be fused with other cells (for example, appropriately drug-labeled human myeloma, mouse myeloma or Hurnan lymphoblastold cells) to produce hybridomas, thus transferring the genes encoding monoclonal antibodies. Furthermore, the cell lines can serve as a source for chromosomes which code for immunoglobulins. These can be isolated and transferred to cells by techniques other than fusion. In addition, the genes encoding monoclonal antibodies can be isolated and used in accordance with recombinant DNA techniques for the production of specific immunoglobulins in a variety of hosts. In particular, by preparing cDNA libraries starting from messenger RNA, a single cDNA clone can be isolated, which can then be used in suitable prokaryotic or eukaryotic expression vectors and subsequently transformed into a host for mass production (see generally, US patent No. 4,172,124; 4,350,683; 4,363,799; 4,381,292; and 4,423,147. See also Kennett, et al., Monoclonal Antibodies, Plenum, New York (1980) and the references cited therein) .

According to the present description, in accordance with hybrid DNA technologies, specific immunoglobulins or fragments thereof 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 which has been transcribed from genes which code for the light and heavy chains of the monoclonal antibodies produced with a cell line according to the invention can be isolated by means of differential cDNA hybridization using cDNA from BALB / c lymphocytes other than the clone according to the invention. The mRNA that does not hybridize will be enriched for messages that code for the desired immunoglobulin chains. If necessary, this process can be repeated to further increase the desired mRNA levels. The stripped mRNA composition can then be reverse transcribed to provide a cDNA mixture enriched for desired sequences. The RNA can be hybridized with a suitable RNAase and the ssDNA can be made double-stranded with DNA polymerase 1 and with random sensitizers (random primers), e.g. indiscriminately fragmented calf thymus DNA. The resulting dsDNA can be cloned into a suitable vector by infection, for example virus vectors such as lambda vectors or plasmid vectors (e.g. pBR322, pACYC184 etc.). By developing samples based on known sequences for the constant portions of the light and heavy chains, those cDNA clones with genes coding for the desired light and heavy chains can be identified by hybridization. The genes can then be cut out of the plasmids, treated to remove superfluous DNA upstream of the starter codon or of the constant DNA section and then introduced into a suitable vector for transforming a host and finally expressing the genes.

Mammalian cells (e.g., mouse cells) are advantageously used as hosts for building the chain (i.e. linking the light and heavy chains), to produce an intact immunoglobulin and, furthermore, if desired, to excrete the immunoglobulin free of the leader sequence. Alternatively, unicellular microorganisms can be used to make the two legs. Thereafter, further manipulations may be necessary to remove the DNA sequences encoding the secretion leader and processing signals while introducing a starter codon at the 5 'end of the heavy chain coding sequence. In this way, the immunoglobulins can be produced and processed so that they can be assembled and glycosylated in cells other than mammalian cells. If desired, each of the chains can be trimmed to leave at least the variable section. These sections can then be treated to provide other immunoglobulins or fragments thereof that are specific for flagellate epitopes.

The monoclonal antibodies of the present invention are particularly useful because of their specificity for antigens, almost all currently known P. aeruainosa variants. In addition, some of the monoclonal antibodies are protective in vivo. This allows them to be included in pharmaceutical products, such as antibody combinations against bacterial infections.

The monoclonal antibodies of the present invention also find a variety of in vitro applications. For example, the monoclonal antibodies for microorganism typing,

5

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796 A5

for the isolation of specific P. aeruqinosa strains, for the selective removal of P. aeruainosa strains from a heterogeneous cell mixture, etc.

For diagnostic purposes, monoclonal antibodies can be labeled or unlabeled. Typically, diagnostic tests require detection of the formation of a complex by binding a monoclonal antibody to the flagellum of a P. aeruainosa oranism.

If unmarked, the antibodies are used in agglomeration tests. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that react with the monoclonal antibodies, e.g. immunogiobulin-specific antibodies. Alternatively, the monoclonal antibodies can be labeled directly. A variety of labels can be used, for example radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors, ligands (especially haptens) etc. Many types of immunoassays are available, e.g. those in the U.S. Patent 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.

In general, the monoclonal antibodies of the present invention are used in enzyme immuno-assays, in which the antibodies according to the invention or second antibodies of another species are conjugated with an enzyme. If a sample containing P. aeruginosa of a particular serotype, e.g. human ßlut or a lysate thereof, combined with the antibodies according to the invention, the antibodies are bound to those molecules which have the desired epitope. Such cells are then separated from unbound reagents and a second antibody labeled with an enzyme is added. The presence of antibody-enzyme conjugates which are specifically bound to the cells is then determined. Other common techniques well known to those skilled in the art can also be used.

Kits can also be provided to detect P. aeruginosa in solution or the presence of FL. to detect aeruainosa-Flaaellar antibodies. The monoclonal antibodies according to the invention are usually provided in lyophilized form, either alone or together with additional antibodies which are specific for other Gram-negative bacteria. The antibodies, which can be unconjugated or conjugated with a label, are in the kits together with buffers such as Tris, phosphates, carbonates etc., stabilizers, biocides, inert proteins, e.g. Bovine serum albumin or the like. In general, the proportion of these substances is less than about 5% by weight, based on the amount of active antibodies, while their total proportion is usually at least about 0.001% by weight, again based on the antibody concentration . It will often be desirable to add an inert extender or carrier to dilute the active ingredients. The proportion of the carrier can be between 1 and 99% by weight of the total composition. When a second antibody is used that is capable of binding to the monoclonal antibody, it is generally present in a second ampoule. The second antibody is usually conjugated to a label and formulated analogously to the antibody formulation described above.

The monoclonal antibodies according to the invention, in particular monoclonal human antibodies, can likewise be components of pharmaceutical compositions which contain a therapeutic or prophylactic amount of at least one monoclonal antibody according to the invention and a pharmaceutically active carrier. The pharmaceutical carrier can be any compatible, non-toxic substance which is suitable for administering the monoclonal antibody to a patient. Sterile water, alcohol, fats, waxes and inert solids can also be used. Pharmaceutically acceptable adjuvants (buffers, dispersants) can also be added to the pharmaceutical composition. Such compositions can contain a single monoclonal antibody so that they are specific for strains of a flagellar type of P. aeruginosa. Alternatively, a pharmaceutical composition can contain two or more monoclonal antibodies as a "cocktail". For example, a cocktail containing monoclonal antibodies to groups of different P. aeruoinosa strains (i.e. different serotypes) would be a universal product, active against the vast majority of the clinical isolates of these particular bacteria.

The mole ratio of the various monoclonal antibodies will generally differ by no more than a factor of 10, usually no more than a factor of 5. Most of the time, the mole ratio will be about 1: 1 to 2 for each of the other antibodies.

The monoclonal antibodies of the present invention can also be used in combination with plasma products, such as commercial gamma globulin or immunoglobulin preparations as used for the prophylactic or therapeutic treatment of P. aeruainosa disease in humans. For immunoglobulins, the plasma will preferably be obtained from donors who show elevated levels of immunoglobulin that react with P. aeruginosa (see generally, the manual "Intravenous Immune Globulin and the Compromised Host", Amer, J, Med., 76 ( 3a), March 30, 1984, pages 1-231).

The monoclonal antibodies according to the invention can be used as preparations to be administered separately, which are given together with antibiotics or antimicrobial agents. The antimicrobial agents typically include anti-pseudomonal penicillin (e.g. carbenicillin) along with an aminoglycoside (e.g. gentamicin, tobramycin, etc.); however, many other additional substances (e.g. cephalosporins) that are well known to those skilled in the art can be used.

6

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

The monoclonal antibodies and the pharmaceutical compositions obtained therefrom according to this invention are particularly suitable for oral or parenteral administrations. Preferably the pharmaceutical compositions are administered parenterally, i.e. subcutaneously, intramuscularly or intravenously. The invention thus provides compositions for parenteral administration which contain a solution of a monoclonal antibody or a cocktail of such antibodies, dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g. Water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and usually free of solid particles. These compositions can be sterilized in a known manner. To achieve near physiological conditions, the compositions may contain pharmaceutically acceptable adjuvants such as pH or toxicity adjusters, buffers, etc., e.g. Sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations can vary widely, i.e. less than about 0.5%, mostly around or at least 1% up to 15 or 20% by weight. It is essentially determined on the basis of fluid volumes, viscosities, etc., preferably according to the chosen method of administration.

A typical pharmaceutical composition for intramuscular injections could contain, for example, 1 ml of sterile, buffered water and 50 mg of monoclonal antibodies. A typical composition for intravenous infusions could contain 250 ml of sterile Ringer's solution and 150 mg of monoclonal antibodies. Common methods for producing parenterally administrable compositions are known to the person skilled in the art or at least obvious. Incidentally, such methods are described in detail in, for example, Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pennsylvania (1980).

The monoclonal antibodies according to the invention can be lyophilized and stored for storage. reconstituted for use with a suitable carrier. This technique has been found to be effective with conventional immunoglobulins, so prior art lyophylization and reconstitution techniques can be used. It will be known to the person skilled in the art that lyophilization and reconstitution can lead to varying degrees of loss of antibody activity (for example conventional IgM antibodies tend to lose more activity than IgG antibodies), so that the application level is compensated for may need to be adjusted.

The compositions containing the antibodies according to the invention or a cocktail thereof can be administered for the prophylactic and / or therapeutic treatment of P. aeruainosa infections. In therapeutic use, the compositions are administered to a patient who is already infected with one or more P. aeruainosa serotonou in an amount sufficient to cure him or to at least partially stop the infection and its complications. An amount that is capable of achieving this is defined as a "therapeutically effective dose". Amounts effective for this purpose depend on the severity of the infection and the general condition of the patient's immune system. However, they generally range from about 1 to about 200 mg of antibody per kg of body weight, with dosages of 5 to 25 mg per kg of body weight being the most common. It must be remembered that substances according to the invention can generally be used in serious disease conditions, i.e. in life-threatening or potentially life-threatening situations, particularly bacteremia and endotoxemia caused by P. aeruginosa.

In the case of prophylactic use, the compositions containing the antibody according to the invention or a cocktail thereof are administered to a patient who has not yet been infected with P. aeruginosa in order to increase his resistance to a potential infection. A corresponding amount is defined as a "prophylactically effective dose". In this application, the exact amounts in turn depend on the patient's state of health and the general level of immunity. The doses are normally 0.1 to 25 mg per kg body weight, in particular 0.5 to 2.5 mg per kg.

The compositions can be administered one or more times, with the dose level and the administration pattern being determined by the attending physician. In any case, the pharmaceutical formulations according to the invention should provide a sufficient amount of antibodies according to the invention for prophylaxis or therapy.

example 1

This example illustrates the methodology used to produce monoclonal murine antibodies that specifically bind to P. aeruginosa.

3 month old BALB / c mice were immunized intraperitoneally eight times with viable P. aeru-ginosa bacteria of Fisher immunotype 1 and Fisher immunotype 2 (ATCC 27 312 and 27 313), every week to every two weeks for a total nine weeks. The initial doses of the bacteria were 8 x 106 and 1 x 107 organisms per mouse for P. aeruginosa Fisher immunotype 1 and Fisher immunotype 2, respectively, and were increased 30- to 60-fold during the course of the immunization.

7

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

3 days after the last infection, the mouse spleen was removed under aseptic conditions and a single cell suspension was prepared by gently rotating the organ between the cooled ends of two sterile slides. Monoclonal 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 together to produce hybridomas that of Tarn et al. (1982, Infect. Immun., 36: 1042-1053). The resulting hybrid cell suspension was diluted to a concentration of 1.5 x 10 6 cells per ml in RPMl hybrid HAT (RPMI 1640 (Gibco, Grand Island, NY) containing 15% heat-inactivated fetal calf serum, 1 mM sodium pyruvate). 100 ng / ml penicillin and streptomycin, 1.0 x 10- * M hypoxanthine, 4.0 x 10 ~ 7 M aminopterine and 1.6 x 10-5 M thymidine), which is fresh per ml 2.0 x 10B BALB / c-thymocytes produced as "feeder" cells.

The mixture was transferred to a 96 well plate (# 3596, Costar, Cambridge, MA) (200 ni per well). The culture was fed by removing and replacing 50% of the volume of each well with RPM1 hybrid HAT every other day or three. The culture supernatant was checked for the presence of Anti-P. aerualnosa antioxidants were examined by ELISA (enzyme-linked im-munosorbant assay) when cell growth in the holes had reached a confluence of about 40%, which was generally the case in 7 to 10 days.

The culture supernatants of the hybrid cells were examined simultaneously on preparations from outer membranes of each of the two immunizing bacteria. Preparations from outer membranes were isolated according to a modification of the method by Tarn et al. (1982, Infect. Immun., 36: 1042-1053). Bacteria (P. aeruginosa Fisher immunotype 1 and Fisher immunotype 2) were inoculated into trypticase soy broth (TSB) and grown for 16-18 hours at 34 ° C with aeration in a rotary shaker bath. The bacteria were harvested by centrifugation and twice with phosphate-buffered saline (PBS, 0.14 M NaCl, 3 mM KCl, 8 mM Na2HPÛ4-7 HaO, 1.5 mM KH2P04, pH 7.2), which inhibited 150 trypsin Unit (TLU) of aprotinin per ml (Sigma, St. Louis, MO).

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 min. The debris of the homogenate was pelleted at 14,900 x g and discarded and the supernatant centrifuged again as above. The pellet was again discarded and the membranes from the supernatant were pelleted by centrifugation at 141,000 x g for 1 hour. The supernatant was discarded and the membrane pellets overnight at 4 ° C in 10 ml PBS containing 75 T.L.U. Aprotinin saved per ml. The next day the pellets were resuspended by vortexing, separated into aliquots and stored at -70 ° C. The protein content of each pellet was determined by the method of Lowry et al., (1951, J. Biol. Chem., 193: 265-275).

The antigen plates were prepared for the ELISA as follows. Outer membrane preparations were diluted to 5 μg per ml protein in PBS and 50 μl of the solution transferred to each well of the 96-hole perforated plates (Linbro No. 76-031-05, Flow Laboratories, Inc., McLean, VA). sealed and incubated overnight at 37 ° C. Unbound antigen was removed from the plates, 100 p.1 5% (w / v) bovine serum albumin (BSA) in PBS was added to each well and the plates were incubated at 37 ° C for 1 hour.

After removing the non-adsorbed BSA, the culture supernatants (50 μl) from each well of the fusion plates were replica-replicated in the corresponding wells of the antigen plates and incubated for 30 min at 37 ° C. The unbound protein was removed from the wells and the plates were washed three times with 100 ml of 1% (w / v) BSA-PBS per well. Thereafter, 50 ml of a suitably diluted biotinylated goat anti-mouse IgG (Tago, Inc., Buriinga-me, CA) was added to each well and incubated at 37 ° C. for 30 min. The plates were washed three times as described above and then 50 ni of a preformed avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA), prepared according to the manufacturer's specifications, were added to the wells. After 30 min at room temperature, the Vectastain reagent was removed from the wells, the wells washed as above and then 100 ml of substrate, o-phenylenediamine (0.8 mg / ml in 0.1 M citrate buffer, pH 5.0 , mixed with the same volume of a 0.03% (v / v) HzOa solution). The substrate was incubated in the dark for 30 min at room temperature and the reactions were then ended by adding 50 μl of 3N H2SO4 per well.

Hybridoma cells that secreted monoclonal antibodies that could bind to either of the two antigen preparations were located by measuring the absorbance at 490 nm of the calorimetric response in each well. Measurements were made with a Dynatech Model 580 MicroELISA reader (Alexandria, VA). The cells in a well, which were designated Pa3 IVC2, produced antibodies which bound only to Fisher immunotype 2 antigen. These wells were further examined as described below. In the following text, monoclonal antibodies and the clonal cell line from this well, both designated Pa3 IVC2. Pa3 IVC2 cells from the original well were mini-cloned and cloned by limiting dilution techniques as described in Tarn et al., (1982, Infect Immun. 36 : 1042-1053).

Ascites fluid with a high content of monoclonal antibody was produced in CB6 Fi mice (BALB / c (female) x C57BL / 6 (male) Fi) according to the instructions of Tarn et al-,

8th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796A5

(1982, Infect. Immun. 36: 1042-1053). 2 to 3 month old male CB6 Fi mice were intraperitoneally injected with 0.5 ml pristane (2, 6, 10, 14-tetramethylpentadecane, Aldrich Chemical Co., Milwaukee, WI). An intraperitoneal injection with log-phase Pa3 IVC2 cells in RPMI was carried out 10 to 21 days later. Each mouse was injected with 0.5 to 1 x 107 cells in 0.5 ml. After about 2 weeks, the accumulated fluid was removed from the mice every 2-3 days. The concentration of the antibodies in the ascites fluid was determined by agarose gel electrophoresis (Paragon, Beckman Instruments, Inc., Brea, CA) and all ascites with an antibody concentration of 5 mg per ml or more were pooled, aliquoted and added -70 ° C frozen.

Characterization of the molecular target to which the monoclonal antibody is bound

The culture supernatant of the cloned Pa3 IVC2 cell line was, as indicated above, examined by ELISA for preparations of outer cell membranes from all seven P. aeruainosa-Fisher-Immunotvp strains (ATCC No. 27312-27318V P. aureofaciens (ATCC No. 13985) and Klebsiella pneumoniae fATCC No. 8047), all prepared as indicated above. The antibody Pa3 1VC2 was bound to the outer membrane preparations of P. aeruqinosa-Fisher immunotopes 2, 6 and 7, but not to the other Fisher immunotypes, P. aureofaciens or K. pneumoniae.

The specific antigen identified by the antibody Pa3 IVC2 was identified by radio immunoprecipitation. Briefly, the analysis involves incubating radio-labeled antigens with the Pa3 IVC2 antibody and a source of protein A, thereby forming insoluble antibody: antigeri complexes. These complexes are washed to remove non-specifically bound antigen, then the complexes are dissociated and separated on polyacrylamide gel. The predominantly radioactive species found in the gel are identified as the corresponding antigens to which the antibody Pa3 IVC2 has been bound.

Aliquots (25 jig) of soluble preparations of the outer membrane of P. aeruoinosa-Fisher immunotypes 2, 3, 4 and 5 were solid-phase with 12SI using iodo gene (Pierce Chemical Co., Rockford, IL) (Fraker and Speck, 1978, Biochem. Biophys. Res, Commun., 80: 849-857; Markwell and Fox, 1978, Biochemistry, 17: 4807-4817). This resulted in the exposure of the exposed tyrosine residues to most, if not all, of the proteins in the outer membrane preparations.

In order to reduce non-specific binding of the outer membrane antigens to the antibody Pa3 IVC2, the radiolabeled preparations (5 x 106 counts per minute and examination) were first run for 1 h at 4 ° C with BALB / c normal mouse serum (1:40 final dilution ) incubated. The supernatant from a Pa3 IVC2 culture (0.5 ml) containing Pa3 IVC2 antibody was then added to each sample of outer membrane preparations. After incubation of the antigen and antibody for 1 h at 4 ° C, the protein A source, IgGSORB (0.095 ml per sample) (The Enzyme Center, Inc., Boston, MA) was added and for an additional 30 min Incubated at 4 ° C (Kessler, SW, 1975, J. Immunol. 115: 1617-1622). IgGSORB was manufactured according to the manufacturer's specifications. Non-specific reactions were prevented by blocking potentially reactive sites with culture medium immediately before use by washing the IgGSORB twice with RPMI hybrid (HAT-free RPM1 hybrid HAT medium).

The antigen-antibody-IgGSORB complexes were pelleted at 1500 xg for 10 min at 4 ° C., twice with phosphate-RIPA buffer (10 mM phosphate, pH 7.2, 0.15 M NaCl, 1.0% ( v / v) Triton X-100, 1.0% (w / v) sodium deoxycholate, 0.1% (w / v) sodium dodecyl sulfate (SDS) and 1.0% (v / v) aprotinin); twice with high saline buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M LiCl, 1% (v / v) beta-mercaptoethanol); and once with lysis buffer (0.02 M Tris-HCl, pH 7.5, 0.05 M NaCl, 0.05% (v / v) Nonidet P-40) (Rohrschneider et al., 1979, Proc, Nati. Acad, Sci., USA, 76: 4479-4483). Antigen bound to the complex was freed by incubation with sample buffer (0.125 M Tris-HCl, pH 6.8, 2% (w / v) SDS, 2% beta-mercaptoethanol and 20% (v / v) glycerol ) at 95 ° C for 10 minutes and in the supernatant after centrifugation at 15,000 xg for 10 minutes.

The supernatant samples were then applied to SDS containing 14% polyacrylamide gel, which was modified according to the method of B. Lugtenberg et al., (1978, FEBS Lett, 58: 254-258) after the modification by Hancock and Carey (1979, J. Bacteriol., 140: 902-910). The antigens were then separated in the gel overnight by electrophoresis at a constant voltage of 50 volts. After fixation of the gel overnight in 4% (v / v) methanol, 10% (v / v) acetic acid and 5% (v / v) glycerin, it was placed on Whatman paper 3MM using a Biorad gel dryer (Richmond, CA) dried. The dry gel was covered with a plastic wrap and exposed to Kodak X-AR film for 18 hours at room temperature.

The results of this experiment showed that Pa3 IVC2 was only bound to an antigen only in the preparation of the outer membranes of P. aeruainosa-Fisher-Immunotvo 2, but not to any of the antigens present in the other preparations of outer membranes. The molecular weight (MW) of the antigen in the gel was approximately 53,000 Daltons, as compared to its mobility with that of 14C protein standards (phosphorylase B, 92,500 MW; BSA, 69,000 MW; ovalbumin, 46,000 MW; carbon anhydrase, 30,000 MW; cytochrome C, 12,000 MW) (New England Nuclear, Boston, MA) separated in the same gel. The molecular weight of this antigen is

9

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796 A5

relied to the molecular weight of flagellin, the protein comprising the P. aeruginosa flagella, as reported by Montie et al., (1982, Infect. Immun. 35: 281-288).

In addition, Pa3 1VC2 was examined by ELISA using the P. aeruginosa-Habs strains 1-12 (ATCC No. 33348-33359) which were fixed with ethanol in 96-well microtiter plates. The anti-gene plates were prepared as follows.

Broth cultures of each organism incubated overnight were pelleted, washed twice with PBS and resuspended in PBS to an Aeeo of 0.2 O.D. units. The diluted bacteria were introduced into wells (50 μl per well) and then centrifuged at 1500 × g for 15 min at clearing temperature. The PBS was suctioned off and 95% ethanol was added to the wells for 15 min 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 was bound to the ethanol-fixed Habs strains 2,3,4,5,7,10,11 and 12. These specificity patterns indicated that Pa3 IVC2 was bound to the type b flagella of P. aeruginosa (Ansorg, R., 1978, Zbl. Bakt. Hyg "I. Dept. Orig. A" 242: 228-238; Ansorg, FL, et al., 1984, J. Clin, Microbiol., 20: 84-88). Due to this assignment of the specificity of Pa3 IVC2 monoclonal, the P. aeruginosa reference strains, Fisher immunotypes 2, 6 and 7 carry type b flagella. From the previous experimental data it could be concluded that Pa3 IVC2 is specifically bound to P. aeruginosa flagellin type-b.

in vivo protective activity of Pa3 IVC2

Animal experiments were performed to determine whether the Pa3 IVC2 monoclonal antibody would protect a mouse infected with multiple LDso units from live P. aeruginosa. The fired mouse model (Collins, M.S., and Roby, R.E., 1983, J. Trauma, 23: 530-534) was selected as the model. Groups of mice were severely burned according to the authors' protocol and were immediately infected with 5-10 LDso units of Fisher immunotype 7. Monoclonal antibodies were administered intraperitoneally as high-titre ascites (0.2 ml intraperitoneally) before combustion and infection. No increase in the number of survivors was observed in the animals treated with Pa3 IVC2 compared to those who did not receive any antibody.

Example 2

This example demonstrates the methodology for producing a murine hybridoma cell line that produces a monoclonal murine antibody to P. aeruginosa flagellin type-b that is protective in vivo.

Adult female BALB / c mice were first injected intraperitoneally with viable P. aeruoinosa-Fisher immunotype 6 (ATCC No. 27317) (8 x 106 organisms). Two weeks later, viable P. aeruainosa-Fisher-immunotvp 5 (ATCC No. 27316) was injected (4x10e organisms). During the following two-week period, viable P. aeruoinosa-Fisher immunotvp 5 and 6 were administered together in a two-week injection. The dose to each organism was increased so that the final dose was ten times greater than the starting dose. A final injection of a P. aeruoinosa-Fisher-Immunotvo 6 outer membrane preparation (50ng protein) made by the method of R.E.W. Hancock and H. Nikaido (1978, J. Bacteriol., 136: 381-390) were given 4 days after the last injection of viable bacteria. 3 days after the last immunization, the mouse spleen was removed and the spleen cell prepared for hybridization as described in Example 1.

Culture supernatants of the hybridoma cells were analyzed on the tenth day post-fusion with ELISA for the presence of anti-P. aeruoinosa antibodies examined. The procedure given in Example 1 was used, with the exception that the antigen for the ELISA plates was a viable bacterium that was immobilized in the wells of a 96-well microtiter plate. The plates were made as follows.

50 µl of poly-L-lysine (PUL) (1 µg / ml in PBS) (Sigma # P-1524, St. Louis, MO) was added to each well of 96-well plates (Llnbro) and 30 min incubated at room temperature. Illy adsorbed PLL was removed and the wells washed three times with PBS. Bacterial cultures grown overnight in TSB were washed once with PBS and then resuspended in PBS until an O.D.660nm = 0.2. 50 μl of the bacterial suspension were added to each of the wells of the plate and left to stand at 37 ° C. for 1 hour. Unbound bacteria were removed by flicking the plates and then washing them three times with saline tween (0.9% (w / v) NaCl, 0.05% (v / v) Tween-20).

Unspecific binding of the antibodies was blocked by adding 200 nl / well of a blocking buffer (PBS, containing 5% (w / v) non-fat dry milk, 0.01% (v / v) Antifoam A (Sigma, St. Louis, MO) and 0.01% (w / v) thimerosal) in the wells and incubated for 1 hour at room temperature

10th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677796 A5

temperature. Excess blocking buffer was removed and the wells washed three times with saline tween as described above.

Culture supernatants (50 μl) were replication-transferred into the corresponding wells of the test plates and incubated at room temperature for 30 min. The culture supernatants were removed and the wells washed five times with saline tween.

An enzyme-conjugated, second step antibody (horseradish peroxidase-conjugated goat anti-mouse IgG and IgM) (Tago, Inc. Burlingame, CA) was treated with PBS containing 0.1% (v / v) Tween-20 and 0.2% (w / v) BSA, diluted according to previous titrations. Then 50 nl of the reagent was added to each well and incubated for 30 min at room temperature. The excess reagent was discarded, the wells washed five times with saline tween and 100 µl of o-phenylenediamine substrate added to each well. Then incubated for 30 min as described in Example 1. The reactions were as in Example 1 , finished and then read at A49onm with a Bio-TEk EL310 Automated EIA-PIate-Reader.

Using the methods described above, the culture supernatants of the fusion were examined for the presence of antibodies which were bound to P. aeruainosa-Fisher immune cells 1, 2, 3 or 4 but not to control plates which had the same PLL and blocking method, but without bacteria had been produced. Antibody-containing supernatants bound to any of these 4 Fisher immunotypes were examined a second time. Each of the 7 Fisher immunotype bacteria was used individually. The antibody present in the PaF4 IVE8 well was only bound to P. aeruainosa-Fisher immunotypes 2, 6 and 7. Cells from the PaF4 IVE8 well were cloned by limiting dilution, as described in Example 1. The monoclonal antibody and the clonal cell line from this well are both designated PaF4 IVE8 in the following text. High titer ascites fluid on monoclonal antibodies was prepared as indicated in Example 1, except that BALB / c mice were used instead of CB6Fi mice.

Specificity of PaF41VE8

One of the investigations to identify the antigen bound by the monoclonal antibody PaF4 1VE8 was the indirect immunofluorescence of bacterial organisms. Each of the 7 reference Fisher immunotypes of P. aeruginosa, plus a non-flagellate strain of P. aeruginosa (PA103, ATCC 29260, Leifson, 1951, J. Bacteriol., 62: 377-389) and Escherichia coli (GSC A25) were grown overnight in TSB at 37 ° C. The bacteria were pelleted by centrifugation and washed twice with PBS. Each strain was resuspended in PBS to an O.D.660nm = 2.2.

The bacterial suspensions were then further diluted 1: 150 and 20 nl samples were placed in individual wells of Carlson's slides (Carlson Scientific Inc., Peotone, IL) and dried on the slide at 40 ° C. Culture supernatants (25 gl) of PaF4 IVE8 were incubated on the dry bacterial samples on the slide in a humid chamber at room temperature for 30 min. Unbound antibodies were washed off the slides by immersing the slides in distilled water. After drying the slides, fluorescein isothiocyanate (FITC) was then conjugated to goat anti-mouse IgG + IgM (25 nl per well of a 1:40 dilution in PBS) (Tago, Burlingame, CA) for 30 min at room temperature incubated in a humid chamber. The slides were washed again with distilled water, dried and covered with a coverslip that was fixed with glycerin in PBS (9: 1). The slides were viewed with a fluorescence microscope.

Fluorescence stains were only observed in P. aeruoinosa-Fisher immunotypes 2, 6 and 7, as a sinusiodal pattern (line), which originated from only one end of the microorganisms. This is consistent with the morphology and location of the single polar flagellum of these bacteria. ^

The response of PaF4 IVE8 to Flageila was confirmed by immunoblot analysis. Outer membrane antigens from P. aeruoinosa-Fisher immunotype 6 (see Example 1) were separated by electrophoresis in SDS containing 14% polyacrylamide as described in Example 1, except that the electrophoresis was for 5 hours. was carried out at a constant current of 80 mA. Pre-colored molecular weight markings (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 ; (BRL, Gaithersburg, MD) had been added to the same polyacrylamide gel.

The antigens were treated with a Tris-glycine-methanol buffer (Towbin et al., (1979), Proc. Nati. Acad. Sci., USA, 76: 4350-4354), which contained 0.05% (w / v) SDS Transfer overnight at 4 ° C and a constant current of 200 mA from the polyacrylamide gel to a nitrocellulose membrane (NCM) (0.45 µm, Schleicher & Schuell, Inc., Keen, NH). After the transfer, the NCMs were dissolved in 0.05% (v / v) Tween-20 in PBS (PBS-Tween) (Batteiger, B., et al "1982, J. Immunol. Meth., 55: 297-307) incubated for 1 hour at room temperature. For this and all subsequent stages, the NCM was placed on a shaking platform to ensure an even distribution of the solution over the entire NCM.

After 1 hour the PBS-Tween solution was poured away and the PaF4 IVE8 ascites (diluted 1: 1000 in PBS-Tween) were added and incubated with the NCM for 1 hour at room temperature. The NCM

11

5

10th

15

20th

25th

30th

35

40

45

50

55

60

CH677 796A5

was then washed five times, 5 minutes each, with PBS-Tween to remove unbound antibodies. Alkaline phosphatase-conjugated goat anti-mouse IgG + IgM (Tage, Inc.) was diluted according to the manufacturer's specifications and incubated with the NCM for 1 hour at room temperature. The NCM was washed five times as indicated above, and the substrate-containing bromochloroindolyl phosphate and nitro blue tetrazolium (Sigma, St. Louis, MO), prepared as described by Leary et al., (1983, Proc. Nati. Acad. Sci., USA , 80: 4045-4049) was added and incubated for 10 to 20 minutes at room temperature. The reaction was stopped by washing the substrate with distilled water.

The results of this experiment show that PaF4 IVE8 was specifically bound to a single antigen with a molecular weight of 53,000 daltons in the outer membrane preparation. Indirect immunofluorescence and immunoblot assay results demonstrate that PaF4 1VE8 binds to the P. aeruginosa flageila.

The Flageila types that PaF4 IVE8 recognized were determined by ELISA, Habs strains 1-12 (ATCC No. 33348-33359) were each bound with PLL in the wells of 96-well microtiter plates (Linbro) and the ELISA Test performed as mentioned earlier in this example. The source of PaF4 IVE8 antibody was culture supernatant. Positive reactions were observed in wells containing Ghe Habs strains 2,3,4,5,7,10,11 and 12. This indicates that PaF4 IVE8 is bound to type b fla gella. The in vivo protection data are presented in Example 4 below.

Example 3

This example demonstrates the methodology for the production of a murine hybridoma cell line that contains a monoclonal antibody that is anti-P. aeruginosa reacts and is protective in vivo.

The source of lymphoid cells for the fusion was the spleen of an immunized BALB / c mouse, which in a 6-week period had four times intraperitoneally purified type a FIagella (10 to 20 ng protein) from Habs strains 6 and 8 (ATCC 33 353 and 33 355). The flagella were made according to the method of T.C. Montie et al. (1982, Infect. Immun. 35: 281-288) except that the final centrifugation of the flagella was performed at 100,000 x g for 1 hour instead of 40,000 x g for 3 hours. In some cases, a second modification was introduced in which the bacteria were sheared off the flaella in a blender in 30 seconds instead of in 3 minutes (Allison et al., 1985, Infect. Immun., 49: 770-774).

Protein concentrations in each preparation were determined using a Bio-Rad protein assay (Bio-Rad, Richmond, CA) and the presence of contaminating lipopolysaccharides (LPS) was determined by measuring the KDO content (Karkhanis, YD, et al., 1987 , Anal. Biochem., 85: 595-601). The molecular weights of the Flageila proteins were determined by comparing their migration in an SDS polyacrylamide gel with those of standard protein labels (BRL) (see Example 2). The molecular weight of Habs 6 Flagellin was 51,700 Daltons and that of Habs 8 Flagellin was 47,200 Daiton. These values agree with those of J.S. Allison et al. (1985, Infect. Immun., 49: 770-774).

The fusion of splenocytes from flagellin immunized mice and NS-1 myeloma cells was performed 3 days after the last immunization as described in Examples 1 and 2. When the hybridoma lines had grown to a confluence of approximately 40% (day 7), the culture supernatants were transferred to the corresponding wells from three different antigen plate replication transfers in which P. aeruoinosa-Pisher-Immunotvp 1 was bound with PLL (cf. , Example 2 for the preparation) and Habs 6 and Habs 9 were fixed with formalin.

The bacteria for the formalin-fixed antigen plates were grown, washed and diluted in the same manner as described for the PLL-bound antigen plates. Diluted bacteria (0.2 O.D. units at Aeeo) were added to individual wells (50 nl per well) of Linbro 96-well microtiter plates and the plates were then centrifuged at 1200 x g at room temperature for 20 minutes. The supernatants were removed and 75 ml 0.2% (v / v) formalin in PBS was added to each well and incubated for 15 min at room temperature. After the formalin was removed, the plates were air dried and stored at 4 ° C until use. Formalin did not affect flagella antigenicity, as evidenced by the ability of anti-flagella antisera to agglutinate organisms treated with formalin (Lanyi, B., 1970, Acta Microbiol. Acad. Sci., Hung., 17:35 -48). The P. aeruainosa-Fisher-Immunotvp 1 strain was included as a control because it is a flagellate-free strain as demonstrated by stain staining (Manual of Clin. Microbiol., 1985, Lennette, publisher Amer. Soc. Microbiol. , Wash., DC, page 1099). Hybrid cells in the well designated Fa6 I1G5 produced an antibody that was bound to Habs 6 and 9 (both flagella-bearing strains) but not to Fisher immunotype 1.

The cells of the FA6 IIG5 wells were further grown and cloned in subcultures as described in the previous examples. The monoclonal antibody and the clonal cell line from this well are referred to in the following text as FA6 IIG5. Ascites were produced in BALB / c mice as described in Example 2.

12

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677796A5

Specificity of FA6IIG5

The specificity of the antibody FA6 HG5 was determined by indirect immunofluorescence and immunoblot examination. Indirect fluorescence was performed essentially as described in Example 2 with the following changes:

Bacterial cultures grown overnight at 30 ° G on trypticase soy agar were removed from the plates with cotton swabs and in PBS to an A660 of 0.2 O.D. Units resuspended. Formalin (final concentration 0.37% (v / v) in PBS) was introduced into the suspension with vortexing. After incubation at room temperature for 15 min, the bacteria were diluted 1:12 in PBS and 20 nl of this suspension were placed in individual wells of Carlson slides. After drying, the slides were prepared for viewing as in Example 2. The antibody source was the culture supernatant from the FA6 IIG5 cell line.

Fluorescence staining by the FA6 IIG5 antibody was only observed in P. aeruoinosa strains carrying type a flagella, but not in those carrying type b. The observed fluorescence pattern was a sinusoidal line pattern, suggesting that FA6 IIG5 was bound to the flagella. The fluorescence signal was enhanced by treating the bacteria with formalin; however, this treatment was not necessary to make the flagella staining visible with the antibody.

The immunoblot test was carried out as in Example 2. The purified flagellar preparations (see this example) were the source of Flagella type a antigens. The antigens were separated in SDS containing 10% polyacrylamide (Laemmli, U.K., 1970, Nature (London), 227: 680-685) and transferred to an NCM. Preparations of FA6 IGG5, either culture supernatant or ascites diluted 1: 1000, were reacted with the NCM and the reaction was detected using a suitable enzyme-conjugated reagent and enzyme substrate, as described in Example 2. The immunoblot assay showed that FA6 IIG5 was specifically bound to the Habs 6 molecular weight 51 700 and Habs 8 molecular weight 47 200.

It was confirmed by ELISA that FA6 1IG5 only reacted with Flagella type a, but not type b. In this test, Habs strains 1-12 were individually bound to the wells of Linbro 96-well microtiter plates with PLL. The antibody was only bound to Habs strains 1, 6, 8 and 9, which are the only Flagella type a bearing strains of the 12 (Ansorg, R., et al., 1984, J. Clin. Microbiol ., 20: 84-88). In vivo protection tests are illustrated in Example 4 below.

Example 4

This example demonstrates the protection of mice passively immunized with the PaF4 IVE8 and FA6IIG5 antibodies against P. aeruginosa infection in the burned-mouse model.

The anti-Flagella monoclonal antibodies were used in the burned mouse model according to the method of M.S. Collins and R.E. Roby (1983, J. Trauma, 23: 530-534). For these studies, all antibodies were purified by Protein A-Sepharose chromatography (Ey, P.L., et al., 1978, Immunochemistry, 15: 429-436) and dialyzed into PBS buffer. The flagella type a carrying strain used in the experimental animals was P. aeruginosa PA220 (from Dr. James Pennington, Boston, MA) and the flagella type b strain was the reference P. aeruainosa-Fisher-immunotvo 2 ( ATCC No. 27313).

Purified monoclonal antibodies were intravenously administered to each mouse 1 to 2 hours before combustion and infection. Immediately after the combustion, the animals received 0.5 ml of cold PBS containing the infectious bacteria. The infection dose was about ten times the LD50 of each organism. The results of the animal experiments are shown in Tables I and II.

Table I

Study of the protective effect of an anti-Flagella type a monoclonal antibody in the burned mouse model1

treatment

% Survivors per day2

1

2nd

3rd

4th

5

6

7

8th

9

Anti-Flagellaa, FA6IIG5

100

100

100

100

100

90

90

80

80

Anti-Flagella b, PaF4 IVE8

100

20th

10th

10th

10th

10th

10th

10th

10th

non-specific anti-LPS

100

40

30th

20th

10th

10th

10th

10th

10th

monoclonal antibody

PBS without bacteria

80

80

80

80

80

80

80

80

80

1The mice were infected subescharly with about ten times the LD50 of P. aeruginosa PA220. The percentages are based on the survival of the mice in groups of 10 animals each, with the exception of the control group, which received only PBS, in which only 5 animals were. Days specified are after burn and infection.

13

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796 A5

Table II

Study of the protective effect of an anti-Flagella type b monoclonal antibody in the burned mouse

Model 1. •

Treatment% survivors per day2

1 2 3 4 5 6 7 8 9

Anti-flageila b, PaF41VE8

100

100

90

90

90

90

90

CO

0

CO

0

Anti-Flageila a, FA6IIG5

100

20th

10th

10th

10th

10th

10th

10 10

non-specific anti-LPS

100

20th

20th

20th

20th

20th

20th

20 20

monoclonal antibody

PBS without bacteria

100

100

100

100

100

100

100

100 100

1Mice were infected subescharly with about ten times the LD50 from P. aeruoinosa-Fisher-immunotvp 2.

The percentages are based on the survival of the mice in groups of 10 animals each, with the exception of the control group, which received only PBS, in which only 5 animals were. The specified days are after burn and infection.

Very significant survival was observed in animals treated with the anti-a or anti-b anti-body and then infected with the appropriate antigen. Conversely, 80 to 90% of the untreated but infected mice or the animals which had been treated with a non-corresponding monoclonal antibody or a non-specific anti-LPS antibody died. The inability of the anti-flagella type a antibody to protect mice against lethal infection with flagella type b-bearing P. aeruoinosa-Fisher immunotvp 2 and the inability of the anti-flagella type b antibody Protecting mice against lethal infection with type a-bearing P. aeruginosa PA220 confirmed in vivo the specificity of the antibodies already observed in vitro. The survival of burned but not infected mice showed that the burn itself was not fatal.

Example 5

Example 5 demonstrates the extensive cross-reactivity of PaF4 IVE8 and FA6 IIG5 with clinical isolates of P. aeruginosa, demonstrating the clinical utility of these antibodies in immunotherapy for P. aeruginosa infections.

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

PaF4 IVE8 was able to bind with 34 clinical isolates (22%), while the "antibody of flagella type a, FA6 IIG5, with 102 clinical isolates (65%) bound, both together a total of 136 out of 157 isolates (87% Of the 21 strains that were not recognized by any of the antibodies, 19 had no flagella, as was demonstrated by staining. As a result, the combination of the two antibodies bound 136 of 138 (98%) of clinical isolates with flagella, which earlier reports confirmed (see R. Ansorg, 1978, Zbl. Bakt. Hyg., I Abt. Orig. A, 242: 228-238).

Example 6

Example 6 demonstrates methods for the production of human monoclonal antibodies which bind to the flagella of P. aeruginosa type b.

A peripheral blood sample from an individual immunized with a high molecular weight polysaccharide preparation (Pier et al., 1984, Infect, Immun., 45: 309) served as the B cell source. The mononuclear cells were separated by conventional blood centrifugation techniques on Ficoll-Paque (Boyum (1968) Scand. J. Clin. Lab. Invest, 21:77) and were washed twice with calcium and magnesium-free, phosphate-buffered saline (PBS).

The mononuclear cells were freed from the T cells using a modified E-rosette procedure. The cells were resuspended to a concentration of 1 × 107 lines / ml in PBS, which contained 20% fetal calf serum (FCS), at 4 ° C. 1 ml of this suspension was then placed in a polystyrene round-bottom tube with the dimensions 7 x 100 mm, to which 1 x 109 red blood cells from sheep were added, which had been treated with 2-amino-isothiouronium bromide (AET), from a 10% (v / v) solution in Dulbecco's medium modified according to Iscove (Iscove's medium) (Madsen and Johnson (1979) J. Immun. Methods., 27:61). The suspension was gently shaken at 4 ° C for 5 to 10 minutes, and then the E-rosette cells were opened by centrifugation

14

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

Ficoll-Paque removed at 2500 g at 4 ° G for 10 min. The E-rosette negative peripheral blood mononuclear cells (E-PBMC) which banded at the interface were collected and washed once in Iscove's medium and in the same containing 15% (v / v) FCS, L- Glutamine (2 mmol / l), penicillin (100 lU / ml), streptomycin (100 ng / ml), hypoxanthine (1 x 10-4 M), aminopterin (4 x 10-7 M) and thymidine (1.6 x 10 ~ s M) contained, resuspended. This medium is referred to below as the HAT medium.

The cell-driven transformation of the E-PMBC was carried out by cocultivating these cells with a transforming cell line. This transforming cell line was an Epstein-Barr nuclear antigen (EBNA) positive human lymphoblastoid cell line, which was obtained by deriving the GM 1500 lymphoblastoid cell line by ethyl methane sulfonate (EMS) mutagenesis and subsequent selection in the presence of 30 ng / ml 6-thioguanine to give the lines a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficit and thus make them HAT sensitive. This cell line was designated the 1A2 cell line and was deposited with the American Type Culture Collection (ATCC) on March 29, 1982 with the accession number ATCC CRL 8119. The 1 A2 cells in the logarithmic growth phase were suspended in HAT medium and then suspended with the E-PBMC in the ratio of fifteen 1A2 cells per PBMC. The cell mixture was distributed on 30 round-bottom 96-well microtiter plates (Costar 3799), with a concentration of 32,000 cells / well in a volume of 200 l per well and at 37 ° C. in a humidified atmosphere which contained 6% CO 2 , incubated. Cultures were added on days 5 and 8 after loading by replacing half of the supernatant with fresh HAT medium. Sixteen days of loading, 100% of the holes contained proliferating cells and in most of the holes the cells were sufficiently dense to remove the supernatants and to anti-P. test aeruainosa antibodies.

The supernatants were screened for the presence of anti-P. aeruainosa antibodies using the ELISA technique as described in Example 2 but with the following changes. A pool of the 7 Fisher immunotype reference strains (ATCC No. 27312-27318) (A660 = 0.2 OD units) was bound to flat-bottom 96-well microtiter plates (Immulon II, Dynatech), with poly-L-lysine pretreated, incubated and washed as in Example 2. After blocking the non-specific binding sites and washing the plates, 50 nl of 0.1% (v / v) Tween-20 containing PBS and 0.2% (w / v) BSA were added per well. The culture supernatants (50 nl) were then slapped into corresponding holes in test plates and control plates which had been treated with PLL and blocked but did not contain any bacteria. After the incubation, enzyme-conjugated antibodies of the second stage (50 ml per well) horseradish-peroxidase-conjugated goat anti-human IgG and goat anti-human IgM were expediently in PBS, which 0.1% (v / v) Tween-20 and 0.2% (w / v) BSA was added to the holes and the test was completed as described in Example 2.

Supernatants containing antibodies bound to the pool of Fisher's immunolypes but not to the control plate were tested a second time, each of the 7 Fisher's immuno-type bacteria being tested separately. An antibody was present only in the supernatant of a hole, 20H11, which was only bound to the P. aeruainosa-Fisher immunotopes 2, 6 and 7. The cells were repeatedly subjected to subcultures with decreasing low cell densities until all the holes that showed growth excreted antibodies. The cell line and the monoclonal antibody (IgM isotype) were designated 20H11 in the following text.

A second transformation was performed in which the source of B-lines was peripheral blood taken from a patient with cystic fibrosis who was known to have a chronic P. aeruainosa infection. The E-PBMC were prepared as described above and co-cultivated with the transforming cell line 1A2 in a ratio of 72 1A2 cells per E-PBMC. The cell mixture was applied to 15 round-bottom microtiter plates with 96 wells with a concentration of 7.4 x 104 cells per well and cultured as described above.

The supernatants were tested by ELISA for the presence of anti P. aeruainosa antibody 16 days after the start of the transformation. The test was performed as described for the previous transformations, except that the pool of P. aeruainosa strains used for the original screening from Fisher immunotype reference strains F2, F4, F6 and F7 (with the ATCC no 27 313, 27 315, 27 316 and 27 317) and three clinical isolates from the Genetic Systems Corporation Organism Bank (GSCOB), which had different LPS immunotypes and flagella types. The clinical isolate PSA I277 (GSCOB) has type a flagella and an LPS of Fisher immunotype 1; the second isolate PSA G98 (GSCOB) has type a flagella and an LPS of Fisher immunotype 3; and the third PSA F625 (GSCOB) has type b flagella and a Fisher immunotype 5 LPS. This mix of reference strains and clinical isolates is called the flagged P. aeruainosa pool. The supernatants, which contained antibodies bound to the plates containing the flagged P. aeruainosa pool, but not to the PLL-coated control plates, were tested a second time by ELISA for individual strains of the pool. A hole 3CI bound the reference strains F2, F6 and F7 and the clinical isolate F625.

The 3CI cell line was cloned by first subculturing the cells in two rounds of a low-density subculture, first with 20 cells per well on a 96-well plate and then

15

5

10th

15

20th

25th

30th

35

40

45

50

55

60

CH677796 A5

finally by cultivation with 2 cells per hole. Formal cloning of the specific antibody producing cells was performed by plotting the cells at a density of about 1 cell / well in 72-well Terasaki plates (Nunc No. 1-36538) with a volume of 10 nl / well with HAT Medium, the aminopteridine component (HT medium) being missing. The plates were placed in an incubator for 2-3 hours to allow the cells to attach to the bottom of the holes and were then checked by 2 people for holes containing only a single cell. HT medium was added to these holes daily, and when the outgrowth was sufficient, the cells were transferred to a 96-well round bottom plate. All growth holes were ELISAed for P. aeruainosa strains. which type B flagella were tested, and it was found that all of them produce the appropriate antibody. The cell line and the monoclonal antibody (IgM isotype) are both referred to as 3CI in the following text.

The antigens designated 20H11 and 3CI were flagella, as demonstrated by indirect immunofluorescence and immunoblotting. The techniques were carried out as essentially described in Examples 2 and 3. The P. aeruainosa strains were used for the indirect immunofluorescence test. which carried type b flagella, the reference Fisher immunotypes F2, F6 and F7 (ATCC No. 27 313, 26 317 and 27 318) and a strain with type a flagella, reference Fisher immunotype 4 (ATCC No. 27 315), as prepared in Example 3. The flagella type of the reference strains was determined by typing with the monoclonal mouse antibodies PaF4 IVE8 and FA6 IIG5. The slides were prepared for observation as in Example 2. The sources of both antibodies were culture supernatants and the FITC-conjugated reagent was FITC-conjugated goat anti-human Ig (polyvalent) (Tago, Burlingame, CA) in a 1: 100 ratio with PBS, which was 0.5% (w / v ) containing bovine gamma globulin (Miles Scientific, Cat. No. 82-041-1, Naperville, IL) and 0.1% (w / v) sodium azide as a preservative.

The fluorescence staining of the antibodies 20H11 and 3CI was only observed in P. aeruainosa strains which contained Flageila of type b and not in Fisher immunotype 4 with Flagella type a. The observed fluorescence pattern was a sinusoidal line pattern, which starts from one end of the bacteria and indicates that the antibodies are bound to the flagella of the bacteria.

Immunoblotting was carried out as in Example 2. Purified flagella type b of P. aeruaino-sa reference strains Fisher immune type 2 (ATCC No. 27 313) and purified Flageila type a of the reference strains Habs 6 and Habs 8 (ATCC No. 33 353 and 33 355) were as in Example 3 produced. The antigens were separated in a 10% polyacrylamide gel (see Example 3) and transferred to an NCM. Culture supernatants containing 20H11 or 3CI antibodies, culture supernatants containing a non-specific human antibody and culture medium were incubated with NCM and the reaction was treated with an alkali, phosphatase-conjugated goat anti-human-Ig (polyvalent) (Tago, Burlingame, CA), which was detected in PBS containing 0.05% (v / v) Tween-20. The enzyme substrate was prepared as in Example 2. The immunoblock showed that both antibodies were bound to the Fisher immunotype 2 flagelline protein with a molecular weight of 53,000 and not to the Habs 6 flagelline protein with a molecular weight of 51,700 or to the Habs 8 flagelline protein with a molecular weight of 47,200. No reaction was observed with the non-specific human antibody or the culture medium.

Additional confirmation that antibodies 20H11 and 3CI were only bound to type b flagella and not to type a was obtained by ELISA, in which the Habs strains 1-12 individually with PLL at the holes of immunoIon-96 -Hole microplates were bound. The antibodies bound only the Habs strains 2,3,4,5,7,10,11 and 12, which are strains that have the flagella type b (Ansorg, et al., (1984) J. Clin. Microbiol. 20:84).

Example 7

Example 7 demonstrates methods for producing a human monoclonal antibody that binds P. aeruginosa with Type a flagella.

A peripheral blood sample of a person immunized with a high molecular weight polysaccharide preparation (Pier et al., (1981) Infect. Immun., 34: 461) served as the B cell source. The mononuclear cells were separated from the blood and then freed from T cells as described in Example 6. The cells were then frozen in FCS containing 10% dimethyl sulfoxide in a liquid nitrogen vapor tank. At a later time, the cells were quickly thawed at 37 ° C, washed once in Iscove's medium and resuspended in HAT medium. The cell-driven transformation was accomplished by coculturing the E-PBMC with 1A2 cells at a ratio of 30 1A2 cells per E ~ PBMC. The cell mixture was applied to 30 96-well tissue culture plates at a concentration of 62,000 cells per well. The cells were carried out on the 7th day after application by replacing half of the volume with HAT medium. Cell proliferation was observed in 100% of the holes on the 14th day after application and the supernatants were removed from the holes and tested at that time.

The supernatants were determined by ELISA for the presence of anti-P. aeruainosa antibodies tested using the flagged P. aeruainosa pool and PLL-treated plates as controls as described in Example 6. The supernatants, which contained antibodies, which were attached to the flagellated pool

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH677 796 A5

bound, but not to the PLL control plates, were again tested for individual bacterial strains of the fiagellated pool. One hole, 21B8, contained an antibody bound to PSA I277, PSA G98 and the reference Fisher immunotype 4, these strains of the fiagellated pool flageila of type a.

The 21B8 cell line was cloned as described in Example 6 for the 3Cl cell line with the following modifications to the formal cloning step. After those holes of the Terasa-ki plates, which contained only a single cell, were counted, each cell from the Terasa-ki plate was inserted into a single hole of a 96-hole round-bottom plate with a volume of 100 I HAT medium, transferred without aminopteridine component (HT medium). Non-transforming, HAT-sensitive lymphoblastoid cells were added to all wells at a density of 500 cells / well as nutrient cells. Five days after application, 100 L of HAT medium was added to the wells to selectively kill the nutrient cells. The holes were again nourished on days 7 and 9 after application 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 the antibody by ELISA. All holes with an outgrowth produced antibodies that bound P. aeruainosa strains with the flagella type a. The cell line and the monoclonal antibody (IgGi isotype) are referred to herein as 21B8.

The antigen designated 21B8 was Flageila, as demonstrated by indirect fluorescence and immunoblotting (see Example 6 for the descriptions of the techniques). The fluorescent staining of the 21B8 antibody was only observed in the P. aeruginosa reference strain of Fisher immunotype 4 (ATCC No. 27 315), which has flagella of type a, and not in the P. aeruginosa reference strain of immunotype 2 (ATCC No. 27,313), which has type a flagella. The observed fluorescence pattern was a sinusoidal line pattern that originated from one end of the bacteria, indicating that the antibody was bound to the flagella of the bacteria.

Immunoblotting was carried out as shown in Example 2. Purified type a flagella from P. aeruginosa reference strain Habs 6 (ATCC No. 33 353) and purified Flageila type b of the P-aeruainosa reference strain of Fisher immunotype 2 (ATCC No. 27 313) were prepared as in Example 3. The antigens were separated in 10% polyacrylamide gel (see Example 3) and transferred to NCM. The culture supernatants containing either 21B8 or a non-specific human antibody and culture medium were reacted with the NCM and the reaction was carried out with an alkaline phosphatase-conjugated goat anti-human Ig (polyvalent) and an enzyme substrate as in the Examples 2 and 6 described, demonstrated. The immunoblot showed that the antibody 21B8 was only bound to the flagelline protein with a molecular weight of 51,700 from Habs 6 and not to the flagelline protein with a molecular weight of 53,000 of the Fisher immunotype 2. No reaction was observed with the non-specific human antibody or the culture medium.

Example 8

Example 8 shows protection in mice which were passively immunized with the human anti-Flageila antibodies 20H11, 3CI and 21B8 against a challenge with P. aeruginosa on the mouse burn site.

The human monoclonal anti-Flagella antibodies were tested on the mouse burn model (cf. Example 4). 21B8 and 20H11 antibodies were produced by precipitating culture supernatants, which were formed by corresponding line lines, with ammonium sulfate (50% final concentration) (Good et al., Selected Methods in Cellular Immunology, Mishell, BB and Shiigi, SM, editor, WJ Freeman & Co., San Francisco, CA, 1980, 279-286). The precipitate was dissolved in PBS, dialyzed against PBS overnight at 4 ° C and then sterile filtered before administration to the animals. The source of the 3CI antibody and the non-specific anti-LPS antibody used as a negative control in the present study was culture supernatant. The appropriate, purified mouse antibody PaF4 IVE8 or FA6IIG5 served as a positive control for each study.

The flageila type a strain used in the animal experiments was the clinical isolate PSA A522 (GSCOB) expressing the LPS of Fisher immunotype 1 and the flagella type b strain was the clinical isolate PSA A447 (GSCOB ), which expresses the LPS of Fisher immunotype 6. The human antibodies (0.45 ml) were pre-mixed with the bacteria (more than 5 LD100 in 0.05 ml) and inoculated sub-sharply immediately after the burn was inflicted. The results of the animal experiments are shown in Tables III, IV and V.

17th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

RR

CH 677 796 A5

Table ili

Protection study of anti-Flagella-type human monoclonal antibody 21B8 on mouse sore wound model 1.

treatment

% Survivors per day: 1 2 3

>

4th

5

6

7

8th

9

Mouse anti-Flagella a, FA6

100

100

100

100

88

88

88

88

88

HG53

Human anti-flageila b, 21B8

100

100

100

100

100

100

100

100

100

Human anti-flagella b, 20H11

100

0

0

0

0

0

0

0

0

PBS

100

25th

12

12

12

12

12

12

12

"• The mice were challenged subscharely with more than 5 LD100 P. aeruginosa PSA A522. 2The percentage of survivors is based on the number of mice that survived in a group of 8 animals. The days are the days after the burn and the Challenge, purified antibody (10 fxg in 0.45 ml PBS) was premixed with the bacteria and administered subscharely after inflaming the burn and challenge.

Table IV

Protection study of the anti-Flagella type b human monoclonal antibody 20H11 on the mouse sore wound model1

treatment

% Survivors per day2 12 3 4

5

6

7

8th

9

Mouse Anti-Flagella b.

100

100

100

100

100

100

100

100

100

PaF4 IVE83

Human anti-flagella b, 20H11

100

100

100

100

100

100

100

100

100

Human anti-flagella a, 21B8

100

0

0

0

0

0

0

0

0

medium

80

0

0

0

0

0

0

0

0

1The mice were challenged subscharely with more than 5 LD100 P. aeruginosa PSA A447 2The percentage of survivors is based on the number of mice that survived in a group of 8 animals. The days are the days after the burn wound and the challenge. 3 Purified antibody (10 (xg in 0.45 ml PBS) was premixed with the bacteria and administered sub-categorically after inflaming the burn and challenge. '

Table V

Protection study of the human, monoclonal antibody 3CI of the anti-flagella type-b on the mouse wound wound model1

Treatment% survivors on day 2

1

2nd

3rd

4th

5

6

7

8th

9

Mouse anti-Flagella b, PaF41VE83

100

100

100

100

100

100

100

100

100

Human anti-flagella b, 3CI

100

100

80

80

80

80

80

80

80

Non-specific anti-LPS monoclonal antibody

100

0

0

0

0

0

0

0

0

1The mice were challenged with more than 5 LD100 PSA A447.

The percentage of survivors is based on the surviving mice in 5 groups of animals. The number

Days represents the number of days after the burn and the challenge.

3The purified antibody (40 jig) was intravenously administered 2 days before the burn and the challenge.

tion administered.

Very significant survival was observed in mice treated with the anti-flagella type a antibody or challenged with the two anti-flagella type b antibodies and then challenged with the appropriate antigen. Conversely, 88 to 100% of the untreated her18 died

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

challenged mice or those treated with an inappropriate anti-flagellar monoclonal antibody or a non-specific LPS antibody. As was observed with the monoclonal mouse antibodies (see Example 4), the human anti-flagella antibodies specifically protect against the lethal challenge only against organisms which carry the corresponding flagella type, i.e. against human antibodies of anti-flagella type a, provided that the lethal challenge was carried out with an organism which had flagella type a and not type b and that mice which had antibodies with anti-flagella Type b were protected when challenged with strains of Flageila type b, but not with an organism of type a.

Example 9

Example 9 demonstrates the cross-reactivity of human anti-Flagella antibodies 20H11, 3CI and 21B8 with clinical isolates of P. aeruginosa.

Clinical isolates from P. aeruginosa (115), obtained from hospitals and clinics and which were isolated primarily from burns and blood, were treated with monoclonal mouse antibodies FA6 IIG5 or PaF4 IVE8 (cf. Examples 2,3 and 5) as organisms typed with flagella of type a or b. Fifty-five of the clinical isolates were identified as wearing Type A flageila by reacting with the FA6 IIG5 mouse monoclonal antibody and 59 were identified as belonging to type B by identifying with the PaF4 IVÉ7 mouse monoclonal antibody .

The cross-reactivity of the anti-flagella type a 21B8 human monoclonal antibody was extensive in that the antibody recognized 54 of 56 clinical isolates bearing flagella type a (96%). The cross-reactivity of 20H11 with the isolates which had type b flagella was also extensive in that the 20H11 recognized all 59 isolates (100%). In contrast, the other anti-flagella type b 3Cl monoclonal antibody bound only 43 of the 59 isolates (73%). These results show that 20H11 binds to a pan-reactive epitope (ie an epitope that was bound to at least about 95% of the P. aeruoinosa strains with Flagella), while 3CI binds to an epitope that is not found in all type b -flagelline molecules is present. Even so, even the flagella type b antigen is serologically uniform when analyzed with polyclonal antisera (Lanvi, B., supra and Ansorg, R. supra), surprisingly, the cross-reactivity patterns of 20H11 and 3Cl show that the flagella type - b has at least two separate epitopes which can be identified by monoclonal antibodies.

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

Example 10

Example 10 demonstrates methods for making another exemplary human monoclonal antibody that binds Flagella type b P. aeruginosa and also has an antibody protective effect against a P. aeruginosa challenge in the mouse burn model.

A transformed cell line was prepared and cloned essentially as in Example 7, except that the transformed cell mixture was applied to 20 96-well tissue culture plates at a concentration of approximately 2250 U ~ PBMC per well. The supernatants were first tested by ELISA in the flagellate pool as described in Example 6, except that the Fisher immunotypes 2 and 4 reference strains were not present in the pool. Positive holes were then tested on each of the strains present in the flagellate pool, including Fisher immunotypes 2 and 4. The finally isolated cell line and the monoclonal antibody (IgG isotype) are both identified in the following text with the designation 12D7.

The human monoclonal antibody 12D7 showed extensive cross-reactivity with anti-flagella type a isolates, recognized 54 out of 56 of the clinical isolates tested, which carried type a flagella. The protective activity of the 12D7 monoclonal antibody is shown in Table VI. Protection trials were carried out essentially as in Example 4, except that the challenge dose was 1 LDioo and the challenge strain 1624, a clinical isolate expressing the Fisher immunotype 2 LPS and being Flagella-type-a.

19th

CH677 796A5

10th

15

30th

35

40

Table VI

Protection study of the anti-FIageila type a 12D7 human monoclonal antibody in the mouse burn model1

Treatment3

% Survivors per day2 12 3 4

5

6

7

8th

9

Human anti-flagella a, 12D7

100

100

100

100

100

100

100

100

100

Human Anti Fisher 2 LPS,

2H9

90

90

90

90

90

90

90

90

90

Mouse anti-Flagella a, 11G5

100

100

100

100

100

100

100

100

100

Human anti-flagella b, 15F4

O

O

37.5

25th

25th

25th

25th

25th

25th

25th

1 The mice were challenged subscharely with more than 1 LDioo (950 CFU) from P. aeruginosa I624. 2The percentages of survivors are based on the number of surviving mice per group of 10 herds, with the exception of the 15F4 group where N = 8. Days are the number of days after the burn wound and challenge.

purified antibody (50 jxg in 0.5 ml PBS) was i.p. Administered 2 hours before the burn and the challenge.

Example 11

2_ Example 11 demonstrates the production of a human monoclonal antibody that was reactive with P. aeruginosa of Flagella type-b and the protection of mice that were passively immunized with this antibody against a challenge with P. aeruginosa on the mouse burn model.

A transformed cell line was made and cloned essentially as described in Example 10, except that the cell transformation ratio of 1A2 to B was about 60 * 1 and the transformed cell line on 15 plates at a concentration of about 1930 E-PBMC per well was applied. The cells were also pooled from P. aeruainosa strains. which contained G98 (Fisher immunotype 3, Flagella type a) and 1739 (a clinical isolate of Fisher immunotype 5, Flagella type b), and tested in another pool containing the P. aeruginosa strains 1277, G98 , 1739 and contained the Fisher immunotypes F2, F4, F6 and F7. The last isolated cell line and the secreted monoclonal antibody (IgGt isotype) are both identified in the following text with the designation 2B8.

An immunofluorescence test was carried out with 2B8, which was positive on the Flagella type b (Fisher immune type 2) strain but was negative on the Flagella type a (Fisher immunotype 4) reference strain. When testing the clinical isolates, 59/59 (100%) of the flagella type b isolates were positive.

The protective activity of 2B8 is shown in Table VII. The protective studies were performed as described in Example 10, except that the clinical isolate F164 (Fisher-lmmu-notyp 4, Flagella-type-b) was used for the challenge.

Table VII

45 Protection study of the human, anti-Flagella type b 2B8 monoclonal antibody on the mouse burn model

Treatment% survivors per day2

1

2nd

3rd

4th

5

6

7

8th

9

50

Human anti-flagella b, 2B83

100

89

89

89

89

89

89

89

89

Mouse anti-Flagella b, PaF4

IVE84

100

100

100

100

100

100

90

90

90

Mouse Anti-Fisher 2 LPS,

55

VH34

90

20th

20th

20th

20th

20th

20th

20th

20th

1The mice were challenged sub-sharply with 1 LDioo (105 CFU) from P. aeruginosa F164.

2 The percentages of survivors are based on the number of mice surviving per group of 10 mice, except that in the 2B8 group N = 9. The days are counted from the time of the burning and the challenge.

purified antibody (50 p.g in 0.5 ml PBS) was i.p. Administered 2 hours before the burn and the challenge.

purified antibody (5 ng in 0.5 ml PBS) was i.p. Administered 2 hours before the burn and the challenge.

65 - - ————

20th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 677 796 A5

Example 12

Example 12 demonstrates the production of another human monoclonal antibody that reacts with Flagella type b P. aeruginosa and the protection of mice that had been passively immunized with this antibody against infection with P. aeruginosa in the mouse fire model.

A transformed cell line was essentially prepared and cloned as described in Example 7, but with the following exceptions. Another individual was immunized (Pier et al., (1984) 45: 309) and served as the B cell source and the loading level of E-PBMC was about 2000 cells per well. Screening was performed on pooled Fisher 1-7 immunotypes. The cell line that was finally isolated and the antibody that was eliminated (IgGM type) are both designated 14CI in the text below.

In the clinical test, 59/59 (100%) of the flagella type b isolate showed a positive reaction with 14CI. The protective effect studies were carried out as in Example 11 and are shown in Table VIII.

Table VIII

Study on the protective effect of anti-Flagella type b monoclonal human antibody 14CI in the mouse fire model1

Treatment3

% Survivors per day2 12 3 4

5

6

7

8th

9

Human anti-flagella b, 14CI

100

100

100

100

100

90

90

90

90

Mouse anti-Flagella b, PaF4

IVE8

100

100

100

100

100

100

100

100

100

Human Anti-Fisher2 LPS,

VH3

100

40

20th

20th

20th

20th

20th

20th

20th

The percentages are based on the survival of the mice in groups of 10 animals with the exception of the PaF4 1VE8 group, in which only 9 animals were present. The days given are after burn and infection.

purified antibody (50 [in 0.5 ml PBS) was i.p. Administered 2 hours before burn and infection.

From the foregoing, it can be seen that the cell lines of the present invention are a means of producing monoclonal antibodies and fragments thereof which react with P. aeruginosa flagella and also offer cross-protection against various P. aeruainosa strains. Surprisingly, a number of monoclonal antibodies were isolated that responded to each type of Flagella with different epitopes. The antibodies of the present invention allow more economical and easier production of prophylactic and therapeutic compositions that can be used to control most P. aeruginosa infections. In addition, the cell lines provide antibodies that can be used in immunoassays and other known methods.

Although the present invention has been presented in some detail for the sake of clarity, it is understood that certain changes are possible within the scope of the claims,

Claims (1)

Claims 1. Monoclonal antibody or binding fragment thereof, characterized in that it is capable of reacting specifically with Pseudomonas aeruoinosa bacterial flakes and is protective in vivo against the bacteria mentioned. 2. Monoclonal antibody or binding fragment thereof according to claim 1, characterized in that the monoclonal antibody is human. 3. Monoclonal antibody or binding fragment thereof according to claim 1, characterized in that it is reactive with an epitope which is present on the flagella of type a or b, but not on both. 4. Monoclonal antibody or binding fragment thereof according to claim 3, characterized in that the epitope is present in at least about 70% of the flagella of type b. 5. A monoclonal antibody or binding fragment thereof according to claim 1, characterized in that it is able to block the binding to the epitope of monoclonal antibodies which are marked by the line lines with the deposit numbers ATCC HB9129, HB9130, CRL9300, CRL9301, CRL9422, CRL9423 and CRL9424 to be produced. 6. monoclonal antibody or binding fragment thereof according to claim 3, characterized in that the epitope is formed only in the flagella type b. 21 5 10th 15 20th 25th 30th 35 40 45 50 55 CH677 796A5 7. Monoclonal antibody or binding fragment thereof according to claim 6, characterized in that it is pan-reactive. 8. A monoclonal antibody or binding fragment thereof according to claim 7, characterized in that it is conjugated with a label which causes a detectable signal. 9. Monoclonal antibody or binding fragment thereof according to claim 8, characterized in that the label represents a fluorescence marker or an enzyme. 10. A cell line for the production of monoclonal antibodies according to claim 1, which antibody can react specifically with a Pseudomonas aeruainosa flaaellum Tvp a or type b. 11. Cell line according to claim 10, characterized in that it is a hybrid cell line. 12. Cell line according to claim 11, characterized in that it produces a human, monoclonal antibody. 13. Cell line according to claim 10, characterized in that it has the deposit number ATCC HB9129, HB9130, CRL9300, CRL9301, CRL9422, CRL9423 or CRL9424. 14. Pharmaceutical composition, characterized in that it has a monoclonal antibody according to one of claims 1 to 9. 15. Pharmaceutical composition according to claim 14, characterized in that it also contains an antimicrobial agent and a human gamma globulin fraction. 16. Pharmaceutical composition according to claim 14, characterized in that the antibody is a monoclonal antibody and the human gamma globulin fraction has an increased content of immunoglobulins which are reactive with Pseudomonas aeruginosa and / or products thereof. 17. Pharmaceutical composition according to claim 14, characterized in that it contains at least two monoclonal antibodies according to one of claims 1 to 9, each of which reacts with a different type of flagellar protein from Pseudomonas aeruginosa and which is capable of treating Pseudomonas aeruoinosa infections or to prevent. 18. Pharmaceutical composition according to claim 17, characterized in that at least one of the monoclonal antibodies is a human, monoclonal antibody. 19. Pharmaceutical composition according to claim 14, characterized in that it also contains a human, monoclonal antibody which can react with at least one serotypic determinant of a liposaccharide molecule of Pseudomonas aeruginosa and / or a monoclonal antibody which is reactive with Exotoxîn A . 20. A method for determining the presence of Pseudomonas aeruginosa in a sample, characterized in that the sample is combined with a monoclonal antibody according to claim 1, which is reactive with Pseudomonas aeruainosa flaoellae and the complex formation is detected. 21. Reagent set for carrying out the method according to claim 20, characterized in that the reagent set contains the following components: a monoclonal antibody composition which contains at least one monoclonal antibody according to one of claims 1 to 9, which is reactive with a type-specific, flagellar protein of the bacterium, and - Labels for detectable signals which are covalently bound to said antibody or to second antibodies which are reactive with each of said monoclonal antibodies. 22