DK172840B1 - Monoclonal antibodies to Pseudomonas aeruginosa flagella, pharmaceutical preparations containing such antibodies, and c. - Google Patents

Description

in DK 172840 B1

The present invention relates to the use of immunological technologies to produce new materials useful in the diagnosis and treatment of bacterial infections, and more particularly to the production and use of monoclonal antibodies capable of recognizing Pseudomonas aeruginosa flagella.

In particular, the invention relates to monoclonal antibodies or binding fragments thereof which are capable of specifically reacting with a flagellar protein epitope of Pseudomonas aeruginosa and inhibiting the motility of the bacterium, which monoclonal antibody or binding portion thereof is protected in vivo. Compositions including pharmaceutical compositions comprising at least one monoclonal antibody of the invention are also provided. The invention also relates to cell lines for the production of antibodies of the invention and a kit for use in detecting the presence of Pseudonomas aeruginosa. Other aspects of the invention relate to methods for producing monoclonal antibodies of the invention and methods for determining the presence of Pseudomonas aeruginosa 1 in a sample.

The closest prior art in the art is disclosed in U.S. Pat.

No. 4,443,549, which discloses antibodies directed against Pseudomonas aeruginosa 20 pili, and Chemical Abstracts, Vol. 96 (1982) No. 65,355, which discloses a Pseudomonas aeruginosa flagella antiserum. The present invention differs from the prior art in that the monoclonal antibodies of the invention are capable of specifically reacting with a flagellar protein epitope of Pseudomonas aeruginosa, thereby inhibiting the motility of the bacterium, providing a protective effect in vivo.

Gram-negative diseases and the most serious complications thereof, e.g. bacteremia and endotoxemla, are the cause of significant morbidity and mortality in human patients. This is especially true of the Gram-negative organism Pseudomonas aeruginosa, which has been increasingly associated with bacterial infections, in particular nosocomal infections, over the last 50 years.

Over the past decade, antibiotics have been the preferred therapy for the control of Gram-negative diseases. However, the continuing high morbidity and mortality associated with Gram-negative bacterial diseases v1-35 see the limitations of antibiotic therapy, especially with respect to P. aeruginosa (see, e.g., VG Andriole, "Pseudomonas Bacteremia: Can Antibiotic Therapy Improve Survival?", J Lab Lab Clin Med (1978), 2 DK 172840 B1 24: 196-199). This has led to the search for alternative methods of prevention and treatment.

One method that has been considered is the enhancement of the host's immune system by active or passive immunization. For example. It has been observed that active immunization of humans or experimental animals with whole cell bacterial vaccines or purified bacterial endotoxins from P. aeruginosa leads to the development of specific opsonic antibodies directed primarily at determinants of the repeating oligosaccharide units of the lipopoly-saccharide molecules (IPS) located on the outer cell membrane 10 of P. aeruginosa (see M. Pollack, Immunoglobulins: Characteristics and Uses of Intravenous Preparations. BM Alving and JS Finlayson, eds., pp. 73-79, U.S. Department of Health and Human Services, 1979) . Such antibodies, whether actively generated or passively transmitted, have been shown to protect against the lethal effects of P. aeruginosa infection 1 in a variety of animal models (Pollack, supra) and in some preliminary studies with humans (see LS Young and M. Pollack, Pseudomonas aeruginosa, L. Sabath, ed., Pp. 119-132, Hans Huber, 1980).

The above reports suggest that immunotherapeutic methods could be used to prevent and treat bacterial diseases caused by P. aeruginosa, such as by administering pooled human immunoglobulins containing antibodies to the infecting strain (s). Human Immunoglobulins are defined herein as the fraction of fractionated human plasma that is enriched with antibodies, among which there are specific antibodies against strains of P. aeruginosa. As a result of certain natural limitations in the use of human immunoglobu-1in components, this method of treatment for P. aeruginosa diseases remains under investigation (see, e.g., M.S. Collins and R.E.

Roby, Am. J. Med .. 76 (3A): 168-174 (1984)), and no commercial products available utilizing these components are yet available.

A limitation 1 compound of Immunoglobulin in preparations is that they consist of collections of samples from 1000 or more donors which have been pre-selected for the presence of certain anti-Pseudomonas antibodies. This correlation leads to an average of Individual antibody titers which at best results in 1 moderate 35 increases in the resulting titers for the desired antibodies.

Another limitation lies in the fact that the pre-selection process 1 itself requires costly, continuous screening of the donor reunions for ensuring product uniformity. Despite these efforts, immunoglobu-1 in the products can continue to vary considerably from batch to batch and from products from different geographical areas.

A further limitation built into the immunoglobulin preparations is that their use results in the concomitant administration of large amounts of foreign proteinaceous substances (which may include viruses such as those recently detected in Acquired Immune Deficiency Syndrome or AIDS), with the potential to induce adverse biological effects. The combination of low titers 10 of desired antibodies and high levels of foreign substances can often limit, to sub-optimal levels, the amount of specific and thus beneficial immunoglobulins that can be administered to the patient.

In 1975, Kohier and Mil stein reported their preparatory discovery that certain mouse cell lines could be merged with mouse spleen cells to produce hybridomas, each of which would secrete antibodies of a single specificity, ie. monoclonal antibodies (G. Kohier and C. Milstein,

Nature. 256: 495-497 (1975)). With the advent of this technology, it became possible in some cases to produce large amounts of extremely specific murine antibodies against a particular determinant or determinants of antigens. Subsequently, using later developed technologies, it became possible to produce human monoclonal antibodies (see e.g.

It is recognized that mouse monoclonal antibodies or preparations of such antibodies in certain situations may present problems for use in humans. For example. For example, it has been reported that mouse monoclonal antibodies used in assays to treat certain human diseases can induce an immune response that renders them ineffective (R.L.

Levy and R.A. Miller, Ann. Rev. Med., 34: 107-116 (1983)). However, with recent advances in recombinant DNA technology such as the production of chimeric mouse / human monoclonal antibodies, these problems can be overcome. Furthermore, there are presently methods for producing human monoclonal antibodies (see Human Hybridomas and Monoclonal Antibodies. EG Engleman et al., Eds. Plenum Publishing 35 Corp. (1985), which is hereby incorporated in its entirety as part of the present invention. description).

Using hybridoma and / or cell transformation technology, a number of groups have reported the production of monoclonal antibodies that protect against P. aeruginosa infections. Monoclonal antibodies have been produced which react with various epitopes of P. aeruginosa, including single- and multi-serotype specific surface epitopes, such as those found in the bacteria's LPS molecules (see e.g.

U.S. Patent Nos. 5,281,396 and 4,122,906, which are hereby incorporated by reference in their entirety as part of the present disclosure). In addition, protective monoclonal antibodies specific for P. aeruginosa exotoxin A have been prepared (see, e.g., U.S. Patent Application no.

10 742,170, which is hereby incorporated by reference in its entirety.

Although the use of monoclonal antibodies specific for the LPS region of P. aeruginosa, or the exotoxins of the bacteria, may provide adequate protection in certain situations, a wider protection ability 1 is generally preferred. For example. in prophylactic treatment for potential Infections in humans, it would be preferable to administer an antibody or antibodies that protect against a variety of P. aeruginosa strains. Similarly, in therapeutic applications where the serotype or types of the infecting strain (s) are unknown, it would be preferable to administer an antibody or combinations of antibodies effective against most, preferably all, the clinically important P. aeruginosa serotypes. , ideally by providing antibodies that respond across traditional serotyping schemes.

One aspect of P. aeruginosa physiology that has been shown to contribute to the virulence of the organism is motility, an ability primarily due to the presence of a flagellum (see T. Montie et al., (1982)).

Infect, and Immun. 38: 1296-1298). P. aeruginosa is characterized by single flagellum at one end of its rod-shaped structure. Model studies 30 of burned mice have shown that a greater percentage of mice survived when non-motile P. aeruginosa strains were inoculated in experimental burns than when motile strains were used (A. McManus et al. (1980), Burns. 6: 235 -239 and T. Montie et al. (1982), Infect and Immun. 3fi-1296-1298). Other studies on the pathogenesis of P. aeru-35 glnosa have led to the claim that animals immunized with flagella antigen preparations were protected in case of combustion and infection by motile strains of the bacteria (see I. Holder et al ., (1982), 5 DK 172840 B1

Infect, and Immune. 35: 276-280).

Of great importance is that P. aeruginosa flagella have been studied using serological methods and have been reported to fall into two major antigenic groups called H1 and H2 by B. Lanyi 5 (1970, Acta Microbiol. Acad Sci. Hung., 17: 35-48) and type a and type b by R. Ansorg (1978, Zbl. Bakt. Hyg., I. Abt. Orig. A, 242: 228-238). Serological typing of flagella by both laboratories revealed that H1 flagella (B. Lanyi, supra) or flagella type b (R. Ansorg, supra) were serologically similar, that is, no subgroups 10 have been identified. This serologically uniform flagellum type will be referred to as type b. The second major antigen H2 (B. Lanyi, supra) or type a flagella (R. Ansorg, supra) contained five subgroups.

This antigen will be referred to as flagella type a, and the five subgroups as aQ, aj, a2, a3 and a ^. The five subgroups of type a are expressed in varying combinations on different strains of type a carrying P. aeruginosa with the exception of the antigen aQ. The a ^ antigen was found on all type a flagella, though the degree to which it was expressed varied among strains.

A serotyping scheme based on the heat stable major somatic antigens of P. aeruginosa is called a Habs scheme, which scheme has recently been incorporated into the International Antigenic Typing System Scheme (see Liu, Int. J. Syst. Bacterol., 33: 256 (1983)). The flagella types of P. aeruginosa Hope reference strains have been characterized by 25 Immunofluorescence with polyclonal sera by R. Ansorg (1978, Zbl. Bakt.

Hyg., I. Abt. Orig. A, 242: 228-238) or by preparative as-co-agglutination (R. Ansorg et al., 1984, J. Clin. Microbiol. 20: 84-88).

Habs strains 2, 3, 4, 5, 7, 10, 11 and 12 are flagella type b-bearing strains, and Habs strains 1, 6, 8 and 9 carry type s flagella. Therefore, a large number of P. aeruginosa strains may be recognized by a small number of monoclonal antibodies specific for flagellar proteins.

Therefore, there is a significant need for monoclonal antibodies capable of responding with epi topper to flagellar proteins 35 and, in some cases, also to provide protection against multiple serotypes of P. aeruginosa. Furthermore, some of these antibodies should be suitable for use as prophylactic and therapeutic treatments against P. aeruginosa Infections and for the diagnosis of such infections.

The present invention meets these needs.

In accordance with the invention, novel cell lines are provided which can produce monoclonal antibodies which are capable of binding to flagella present on most strains of P. aeruginosa bacteria. The monoclonal antibodies specifically react with epitopes on flagella protelens of P. aeruginosa and they can differentiate between type a and type b flagella on the bacterium. Further, compositions comprising monoclonal antibodies of the invention, or 10 binding fragments thereof, are provided for use in a method of treating a human susceptible to Infection or already infected with P. aeruginosa, which comprises administering a prophylactic or therapeutic amount of at least one monoclonal antibody or binding fragment thereof which is protective in vivo and capable of specifically reacting with a flagellar protein epitope from P. aeruginosa strains, which composition preferably also comprises a physiologically acceptable carrier. The composition may also contain one or more of the following: additional monoclonal antibodies capable of reacting with P. aeruginosa exotoxin A, monoclonal antibodies capable of reacting with serotype determinants on LPS of P. aeruginosa, a human blood plasma gamma globulin fraction, a human blood plasma gamma obul n fraction, where the plasma may be obtained from a human exhibiting elevated levels of P. aeruginosa reactive immunoglobulins, and one or more antimicrobial agents. Furthermore, clinical applications of the monoclonal antibodies are provided, including the preparation of diagnostic kits.

In accordance with the present invention, there are provided novel cells capable of producing monoclonal antibodies and compositions comprising such antibodies which are capable of selectively recognizing flagella present on a variety of P. aeruginosa starmias, where individual antibodies typically recognize one type of P. aeruginosa flagella. The cells in question have identifiable chromosomes in which the germ-line DNA of these or a precursor cell is rearranged to encode an antibody with a binding point for an epitope on a flagellar protein found among certain P. aeruginosa strains. For type a flagellar proteins, pan-reactive monoclonal antibodies can be prepared, and for type b flagellar proteins 1n- 7, antibodies that are pan-reactive or react with at least ca.

70¾ of the flagellum-bearing strains. These monoclonal antibodies can be used in a variety of ways, including for diagnosis and therapy.

The monoclonal antibodies thus obtained are particularly valuable in the treatment or prophylaxis of serious diseases caused by P. aeruginosa. The surface proteins on flagella of P. aeruginosa are available for direct contact with the antibody molecules, which is therefore likely to inhibit the motility of the organism and / or promote other effects favorable to the infected hosts.

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

The immortal in serous cell line may be a mammalian cell line that has been transformed by oncogenesis, by transfection, mutation or the like. Such cells include myelomaly nine cells, lymphoma lines, or other cell lines capable of supporting the expression and secretion of 1mmunoglobulin network or a binding fragment thereof in vitro. The immunoglobulin or fragment may be a naturally occurring mammal other than the preferred mouse or human sources produced by transformation of a lymphocyte, in particular a splenocyte, by a virus or by fusion of the lymphocyte with a neoplastic cell, e.g. a myeloma, to produce a hybrid cell line. Typically, the splenocyte will be obtained from an animal immunized against flagellar antigens or fragments thereof containing an epitopic point. Immunization protocols are well known and can vary considerably and yet be effective (see Golding, Monoclonal Antibodies: Principles and Practice. Academic Press, N.Y.

(1983), which is hereby incorporated as part of the present disclosure.

The hybrid cell lines can be cloned and screened according to conventional techniques and antibodies capable of binding to P. aeruginosa flagellar determinants are detected in the cell supernatants.

The appropriate hybrid cell lines can then be grown in culture on a large scale or injected into the peritoneal cavity for a suitable host to produce ascites fluid.

In one embodiment of the present invention, cells are transformed human leukocytes producing human monoclonal antibodies, preferably in vivo protective, against available epitopes specific for at least one flagellar protein. The lymphocytes can be obtained from human donors that are or have been exposed to the appropriate 5 flagellum-bearing strains of P. aeruginosa. A preferred cell-driven transformation process is described in detail in U.S. Pat.

4,464,465, which is hereby incorporated by reference into the present disclosure.

By having the antibodies of the present invention available which are known to be specific for the flagellar proteins, in some cases the supernatants of subsequent experiments can be screened in a competition sample with the subject monoclonal antibodies as a means of identifying further examples of antibodies. -flagellar monoclonal antibodies. Thus, hybrid cell lines can be readily prepared from a variety of sources based on the availability of the available antibodies specific for the particular flagellar antigens.

Alternatively, where hybrid cell lines are available that produce antibodies specific for the epitopic sites concerned, these hybrid cell lines may be fused with other neoplastic B cells, where such other B cells may serve as recipients of genomic DNA encoding for the receptors. Although rodent, especially murine, neoplastic B cells are most commonly used, other mammalian species may be used such as lagomorphs, cattle, sheep, horses, pigs, poultry or the like.

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

The cell lines of the present invention can find uses other than for the direct preparation of the monoclonal antibodies. The cell lines can be fused with other cells (such as suitable drug-labeled human myeloma, mouse myeloma or human lymphoblastoid cells) to produce hybridomas, thus providing for the transmission of the genes encoding the monoclonal antibodies. Alternatively, the cell lines can be used as a source for the chromosomes encoding the Immunoglobulin, which can be isolated and transferred to cells by techniques other than fusion. Furthermore, the genes encoding the monoclonal antibodies can be isolated and used in accordance with recombinant DNA technology to produce the specific immunoglobulin in a variety of hosts. In particular, in the preparation of cDNA libraries from messenger RNA, a single cDNA clone encoding the immunoglobulin and without introns can be isolated and placed into appropriate prokaryotic or eukaryotic expression vectors and subsequently transformed into a host for final mass production (see generally, U.S. Patent Nos. 4,172,124, 4,350,683, 4,363,799, 4,381,292, and 4,423,147; and Kennett et al., 10 Monoclonal Antibodies. Plenum, New York (1980), and the references cited therein, all of which are incorporated herein by reference).

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

Res. 12: 3791, and Wood et al., Nature. 314: 446, both of which are incorporated herein by reference). For example. for example, the messenger RNA transcribed from the genes encoding the light and heavy chains of the monoclonal antibodies produced by a cell line of the present invention can be isolated by differential cDNA hybridization using cDNA from other BALB / c lymphocytes than that clone. The non-hybridizing mRNA will be rich in the messages encoding the desired immunoglobulin chains.

As needed, this process can be repeated to further increase the desired 25 mRNA levels. The extracted mRNA preparation can then be reverse-transcribed to form a cDNA blotting that is enriched with the desired sequences. The RNA can be hydrolyzed with an appropriate RNase and the ssDNA made double-stranded with polymerase I DNA and random primers, e.g. randomly fragmented calf thymus DNA. The resulting 30 dsDNA can then be cloned by Introduction 1 to a suitable vector, e.g. virus vectors such as lambda vectors or plasma vectors (such as pBR322, pACYC184, etc.). By developing probes based on known sequences for the light and heavy chain constant regions, the cDNA clones having the gene encoding the desired light and heavy chains can be identified by hybridization. Thereafter, the genes can be cut from the p ismids, manipulated to remove redundant DNA before the initiation codon or constant region DNA, and then inserted into a suitable vector for the trans-formation of a host and final expression of the gene.

Conveniently, mammalian hosts (e.g., mouse cells) can be used to treat the chain (e.g., assembly of the heavy and light chains) to produce an intact immunoglobulin and, if desired, secretion of the immunoglobulin without the leader sequence. Alternatively, unicellular microorganisms may be used to form the two chains, where further manipulation may be necessary to remove the DNA sequences encoding the secretory leader and processing signals while providing an initiation codon at the 5 'end. of the sequence which 10 codes for the heavy chain. In this way, the immunoglobulins can be prepared and processed so that they are pooled and glycosylated in cells other than mammalian cells. If desired, each of the chains can be truncated to retain at least the variable region, which can then be manipulated to produce other immunoglobulins or fragments specific to the flagellar epitopes.

The monoclonal antibodies of the present invention are particularly useful because their specificity for antigens against almost all known P. aeruginosa variants. Furthermore, some of the monoclonal antibodies are protective in vivo, enabling them to be incorporated into pharmaceutical products such as antibody combinations for use in methods to protect against bacterial infections.

Monoclonal antibodies of the present invention can also be widely used in vitro. By way of example, the monoclonal antibodies may be used for the typing of microorganisms, for the isolation of specific P. aeruginosa strains, for the selective removal of P. aeruginosa cells in a heterogeneous mixture of cells or the like.

For diagnostic purposes, the monoclonal antibodies may be either labeled or unlabeled. Typically, diagnostic studies involve detection of the formation of a complex through the binding of the monoclonal antibody to the flagellum of the P. aeruginosa organism. The unlabeled antibodies are applicable for agglutination studies. Furthermore, unlabeled antibodies can be used in combination with other labeled antibodies (other antibody) which are reactive with the monoclonal antibody such as antibodies specific for immunoglobulin. Alternatively, the monoclonal antibodies can be directly labeled. A wide variety of labels can be used as radionuclides, fluorescents, enzymes, enzyme substrates, enzyme factors, enzyme inhibitors, ligands (especially haptens), etc. Numerous immunoassays are available and, for example, include some described in U.S. Pat. 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, all of which are incorporated herein by reference.

In general, the monoclonal antibodies of the present invention are used in enzyme immunoassays in which the antibodies or "other" antibodies of another species are conjugated to an enzyme. When a sample containing P. aeruginosa of a particular serotype such as human blood or a lysate thereof is combined with the subject antibodies, binding between the antibodies and the molecules exhibiting the desired epitope occurs. Such cells can then be separated from the unbound reagents and another antibody (labeled with an enzyme) added. Then, the presence of the antibody-enzyme conjugate, which is specifically bound to the cells, is determined. Other conventional techniques well known to those skilled in the art may also be used.

Combination kits can also be provided for use with the subject antibodies to detect P. aeruginosa in solutions or the presence of P. aeruginosa flagellar antigens. Thus, the monoclonal antibody preparation of the invention may be provided, usually in a lyophilized form, either alone or in association with additional antibodies specific for other Gram negative bacteria. The antibodies that can be conjugated to a label or unconjugated are contained in the sets of buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g. bovine serum albumin 25 or the like. Generally, these materials will be present in an amount of less than ca. 5% by weight based on the amount of active antibody and usually present in a total amount of at least about 0.001% by weight, again based on the antibody concentration. Often, it will be desirable to add an inert excipient or excipient to dilute the 30 active ingredients, where the expat may be present in an amount of from approx. 1 to 99% by weight of the total composition. When another antibody capable of binding to the monoclonal antibody is used, this will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody preparations described above.

The monoclonal antibodies, in particular human monoclonal antibodies, of the invention may also be incorporated as components of pharmaceutical preparations containing a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of the invention together with a pharmaceutically effective carrier. A pharmaceutical carrier should be any compatible non-toxic substance suitable for delivering the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes and inert solids can be used as the carrier. Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Such preparations may contain a single monoclonal antibody so that they are specific for strains of one flagellar type of P. aeruginosa. Alternatively, a pharmaceutical composition may contain two or more monoclonal antibodies to form a "cocktail".

For example. for example, a cocktail containing monoclonal antibodies against both types of flagella or groups of the different P. aeruginosa strains (e.g., different serotypes) would be a universal product with activity against the majority of the clinical isolates of this particular bacterium.

The mole ratio of the various monoclonal antibody components will usually not differ by more than one factor of 10, more usually not by more than one factor of 5, and will usually be in a mole ratio of approx. 1 to 1-2 of each of the other antibody components.

The monoclonal antibodies of the present invention may also be used in combination with existing blood plasma products such as commercially available gamma globulin and immunoglobulin products used for the prophylactic or therapeutic treatment of P. aeruginosa disease in humans. Preferably, for the Immunoglobulins, the plasma will be obtained from human donors exhibiting elevated levels of Immunoglobulin reactive with P. aeruginosa (see generally the Compendium "Intravenous Immune Globulin and the Compressed Host", Amer. J. Med.

76 (3a), March 30, 1984, pp. 1-231, which is hereby incorporated by reference).

The subject monoclonal antibodies may be used as separately administered compositions administered in combination with antibiotics or antimicrobial agents. Typically, the antimicrobial agents may contain an anti-pseudomonal penicillin (e.g., carbenicillin) in association with an aminoglycoside (e.g., gentamicin, tubramycin, etc.), with numerous additional agents (e.g., cephalosporins) well known to those skilled in the art. also used.

13 DK 172840 B1

The monoclonal antibodies and pharmaceutical compositions thereof according to the invention are particularly useful for oral or parenteral administration. Preferably, the pharmaceutical compositions may be administered parenterally, subcutaneously, intramuscularly or intravenously. The invention thus provides compositions for parenteral administration comprising a solution of the monoclonal antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g. water, buffered water, 0.4% saline, 0.3% glydine and the like. These solutions are sterile and generally free of 10 particulate matter. These compositions can be sterilized by conventional well known sterilization technique. The compositions may contain pharmaceutically acceptable excipients as needed to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like. sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these preparations can vary considerably, i. from less than approx.

0.5%, usually at or at least approx. 1%, to as much as 15 or 20% by weight, and will be selected primarily based on liquid volume, viscosity, etc., preferably according to the particular selected mode of administration.

Thus, a typical intramuscular injection pharmaceutical composition can be prepared to contain 1 ml of sterile, buffered water and 50 mg of monoclonal antibody. A typical intravenous infusion preparation can be prepared to contain 250 ml of sterile Ringer's solution and 150 mg of monoclonal antibody. Current methods for the preparation of parenterally administrable compositions are well known or obvious to those skilled in the art and are described in greater detail 1 e.g. Remington's Pharmaceutical Science.

15th paper, Mack Publishing Company, Easton, Pennsylvania (1980), which is hereby incorporated as part of the present disclosure.

The monoclonal antibodies of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins, and lyophilization and reconstitution techniques known in the art can be used. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g.

In conventional Immunoglobulins, IgM antibodies appear to have greater activity loss than IgG antibodies) and it may be necessary to adjust application levels to compensate.

14 DK 172840 B1

The compositions containing the present monoclonal antibodies or a cocktail thereof may be administered for the prophylactic and / or therapeutic treatment of P. aeruginosa infections. In therapeutic use, the compositions are administered to a patient already infected with one or more P. aeruginosa serotypes, an amount sufficient to cure or at least partially cure the Infection and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose". Amounts effective for this use depend on the severity of the infection and the general condition of the patient's own immune system, but range from 1 to about 1 in general. 1 to approx. 200 mg antibody per kg body weight at doses ranging from 5 to 25 mg / kg as they are more commonly used. It should be recalled that the materials of the present invention can generally be used in serious illnesses, i.e. life-threatening or potentially life-threatening situations, especially bacteremia and endotoxemla, p.g.a. P. aeruginosa.

For prophylactic applications, preparations containing the subject antibody or a cocktail thereof are administered to a patient not already infected with P. aeruginosa to increase the patient's resistance to such a potential infection. Such amount is defined as a "prophylactically effective dose". For this use, the precise amounts again depend on the patient's state of health and general Immune T1 levels, but generally range from 0.1 to 25 mg / kg, especially 0.5 to 2.5 mg / kg.

Single or multiple administrations of the compositions can be performed with dose levels and patterns selected by the treating physician.

In all cases, the pharmaceutical compositions should provide an amount of the antibody or antibodies of the invention sufficient to effectively treat or prevent the patient.

30

Example i

Example 1 shows the methodology used to prepare a murine monoclonal antibody that binds specifically to P. aeruginosa flagella.

35 3-month-old BALB / c mice were immunized intraperitoneally 8 times with viable P. aeruginosa Fisher immunotype 1 and Fisher Immunotype 2 (ATCC Nos. 27312 and No. 27313) bacteria every 1 to 2 weeks for a total of 9 weeks.

DK 172840 B1

The initial doses of bacteria were 8x10B and 1x107 organisms per mouse, respectively. P. aeruginosa Fisher immune type I and Fisher immune type 2, and the dose was increased 30 to 60 times 1 during the immunizations.

Three days after the last injection, the spleen was removed aseptically from 1 5 mice and a single cell suspension was prepared by gently rotating the organ between the matted ends of two sterile slides. Mononuclear spleen cells were combined in a 4: 1 ratio with log phase mouse myeloma cells {NSI-1, obtained from Dr. C. Milstein, Molecular Research Council, Cambridge, England) and fused to form hybridomas in accordance with that of Tam et al. described procedure (1982, Infect.

Immun., 36: 1042-1053). The final hybrid cell suspension gel was diluted to a concentration of 1.5 x 10 4 cells per ml in RPMI hybrid HAT (RPMI 1640 (Gibco, Grand Island, NY)) containing 15% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 100 / µg / ml penicillin and strep- -4 -7 5 tomycin, 1.0 x 10 M hypoxanthine, 4.0 x 10 M aminopterin and 1.6 x 10 M thymidine) containing 2.0 x 10 6 per ml freshly preserved BALB / c thymocytes as feeding cells.

The mixture was plated (200 #il / well) in 96-well plates (No. 3596, Costar, Cambridge, MA). Cultures were born by removal and replacing 50% of the volume of each well with fresh RPMI hydride HAT every 2 to 3 days. Culture supernatants were investigated for the presence of anti-P. aeruginosa antibodies by the enzyme-linked immunosorbant assay (ELISA), as cell growth had reached ca. 40% confluence in the wells, usually within 7 to 10 days.

The culture supernatants of the hybrid cells were examined simultaneously on outer membrane preparations from each of the two immunized bacteria. Outer membrane preparations Isolated by a modification of the method of Tam et al. (1982, Infect. Immun. 36: 1042-1053), which is hereby incorporated by reference. Bacteria (P. aeruginosa Fisher immunotype 1 and Fisher 1mmu-type 2) were seeded in trypticase soy substrate (TSB) and grown for 16-18 hours at 34 ° C under aeration on a rotary shaking bath. The bacteria were recovered by centrifugation and washed twice with phosphate-buffered saline (PBS, 0.14 M NaCl, 3 mM KCl, 8 mM Na2 HPO4-7 H2O, 1.5 mM KH2PO4, pH 7.2) containing 150 trypsin inhibitory units 35 (TIU) aprotinl per ml (Sigma, St. Louis, MO).

The tablet from the last centrifugation was resuspended in 0.17 M triethanolamine, 20 mM disodium ethylenediaminetetraacetic acid (EDTA), and then homogenized on ice for 10 minutes. Debris was pelleted from the homogenate at 14,900 x g and discarded and the supernatant was centrifuged again as above. The tablet was discarded again and the membranes pelleted from the supernatant by centrifugation at 141,000 x g for 1 hour. The supernatant was discarded and the membrane tablets were stored overnight at 4BC in 10 ml PBS containing 75 T.I.U. aprotinin per ml. The following day, the tablets were resuspended by vortex treatment and then divided and stored at -70 ° C. The protein content of each was determined by the method of Lowry et al. (1951, J. Biol. Chem. 193: 265-275).

The ELISA antigen plates were prepared as follows. The outer membrane preparations were diluted to 5 μg per ml protein in PBS and 50 μΐ of the solutions were plated in each well of 96-well plates (Linbro no.

76-031-05, Flow Laboratories, Inc., McLean, VA), sealed and incubated overnight at 37 ° C.

After unfolding of unabsorbed BSA, culture supernatants (50 μΐ) were replicated from each well of the fusion plates in the corresponding wells of antigen plates and incubated for 30 minutes at 37 ° C. The unbound antibody was flipped out of the wells and the plates were washed 3 times with 100 μΐ 1% (w / v) BSA-PBS per well. Next, 50 μΐ per 20 well of appropriately diluted biotinylated goat anti-mouse IgG (Tago, Inc., Burlingame, CA) was added to each well and incubated for 30 minutes at 37 ° C.

The plates were washed 3 times as described above and then 50 µl of a pre-made avidin: biotynilized horseradish peroxidase complex (Vectastaln ABC Kit, Vector Laboratories, Inc., Burlingame, CA), prepared according to the manufacturer's specifications, was added to the wells. After 30 minutes at room temperature, the Vectastain reagent was tipped out of the wells, the wells were washed as above and then 100 μΐ per well of substrate, o-phenylenediamine (0.8 mg / ml in 0.1 M citrate buffer, pH 5.0, mixed with an equal volume of 0.03% (vol./vol.) ^ and). Substrate 30 was incubated for 30 minutes at room temperature 1 dark and the reactions were then terminated by the addition of 50 μΐ / well of 3N HgSO4.

Hybridoma cells secreting monoclonal antibodies that bind to one of the two antigen preparations were localized by measuring the absorbance at 490 nm of the colorimetric responses in each well of a Dynatech Model 580 MicroELISA reader (Alexandria, VA). The cells in one well, called Pa3 IVC2, produced antibody that bound only to the Fisher immunotype 2 antigen plate. This well was studied further as described below.

in 17 DK 172840 B1

The monoclonal antibody and clonal cell line from this well are both identified by the term Pa3 IVC2 in the following text. Pa3 IVC2 cells from this master well were mini-cloned and cloned by limiting dilution technique as described by Tam et al. (1982, Infect.

5 Immun. 36: 1042-1053).

Ascite fluid containing high titer monoclonal antibody was prepared in CB6 Fj mice (BALB / c (female) x C57BL / 6 (male) Fj) according to those of Tam et al. described procedures (1982, Infect. Immun. 36: 1042-1053).

Two- to three-month-old male CB6 Fj mice were injected intraperitoneally with 0.5 ml of Pristane (2-, 6-, 10-, 14-tetramethylpentadecane,

Aldrich Chemical Co., Milwaukee, WI) 10-21 before Intraperitoneal injection with log phase Pa3 IVC2 cells in RPMI. Each mouse was injected with 0.5 to 1 x 10 6 cells in 0.5 ml. After approx. For 2 weeks, the accumulating fluid was removed from the mice every two to three days. The concentration of antibody 15 in the ascites fluid was determined by agarose gel electrophoresis (Paragon, Beckman Instruments, Inc., Brea, CA) and all ascites containing 5 mg / ml or more antibody were collected, divided into aliquots and frozen at -70 ° C.

Characterization of the molecular target bound by the monoclonal antibody

Culture supernatant from the cloned Pa3 IVC2 cell line was assayed by ELISA as described above on outer membrane preparations of all seven P. aeruginosa Fisher immunotype strains (ATCC No. 27312-27318), 25 P. aureofaciens (ATCC No. 13985) and Klebsiella pneumoniae (ATCC no.

8047), all prepared as described above. Antibody Pa3 IVC2 bound to the outer membrane preparations of P. aeruginosa Fisher immunotypes 2, 6 and 7, and not to other Fisher immunotypes, P. aureofaciens or K. pneumonia ae.

The specific antigen identified by antibody Pa3 IVC2 was identified by radloimmunoprecipitation. Briefly, this assay implies that radiolabeled antigens are incubated with Pa3 IVC2 antibody and a protein source of protein A which results in the formation of insoluble antibody: antigen complexes. These complexes are washed to remove any non-specifically bound antigen and then the complexes are dissociated and separated into a polyacrylamide gel. Oe dominant radioactive substances present in the gel are thereby identified as the corresponding antigens to which antibody Pa3 IVC2 binds.

Portions (25 µg) of soluble P. aeruginosa Fisher Immunotypes 125 2, 3, 4 and 5 outer membrane preparations were radiolabeled in solid phase with I using Iodo gene (Pierce Chemical Co., Rockford, IL) (Fraker 5 and Speck, 1978, Biochem. Biophys. Res. Commun. 80: 849-857, Markwell and Fox, 1978, Biochemistry. 17: 4807-4817). This procedure resulted in elimination of exposed tyrosine residues on most, if not all, of the proteins contained in the outer membrane preparations.

To reduce non-specific binding of the outer membrane antigens 10 to antibody Pa3 IVC2, the radiolabelled preparations (5 x 10 6 counts per minute per sample) were first incubated for 1 hour at 4 ° C with BALB / c normal serum (1:40 final dilution). ). The Pa3 IVC2 culture supernatant (0.5 ml) containing the Pa3 IVC2 antibody was then added to each outer membrane sample. After incubating antigen and antibody for 1 hour at 4 ° C, the protein A source, IgGSORB (0.095 ml per sample) (The Enzyme Center, Inc. Boston, MA) was added and incubated for an additional 30 minutes at 4 ° C (SW Kessler, 1975, J. Immunol. 115: 1617-1622). IgGSORB was prepared according to the manufacturer's specifications and immediately prior to use, non-specific reactions were blocked by blocking potentially reactive sites with culture medium by washing IgGSORB twice with RPMI hydride (RPMI hybrid HAT medium without HAT).

The antigen-antibody-IgGSORB complexes were pelleted at 1500 xg for 10 minutes at 4 ° C, washed twice with phosphate RIPA buffer (10 mM phosphate, pH 7.2, 0.15 M NaCl, 1.0% (vol "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 salt 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. Natl.

30 Acad. Sci., USA, 76: 4479-4483). Antigen bound to the complex was released by incubation with sample buffer (0.125 M Tris-HCl, pH 6.8, 2% (w / v) SDS, 2% (v / v) beta-mercaptoethanol and 20% (v / v). vol.) glycerol) at 95 ° C for 10 minutes and collected in the supernatant after centrifugation at 1500 xg for 10 minutes.

The supernatant samples were then added to 14% polyacrylamide gels containing SDS prepared according to the method of B. Lugtenberg et al.

(1978, FEBS Lett., 58: 254-258, which is hereby incorporated by reference), and the antigens are separated into the gel by electrophoresis overnight at 50 V constant voltage. After fixing the gel in 40% (v / v) methanol, 10% (v / v) acetic acid and 5% (v / v) glycerol overnight, it was dried on Whatman 3 mm paper via a Biorad gel dryer (Richmond, CA).

The dried gel was covered with thin plastic film and exposed to "Kodak X-AR" film for 18 hours at room temperature.

Results from this experiment showed that Pa3 IVC2 bound to only one antigen in the outer membrane preparation of P. aeruginosa Fisher immunotype 2 alone, and not to any antigen present in other outer membrane preparations. The molecular weight (MW) of the antigen in the gel was approx. 53,000 daltons as determined by comparing its mobility with the mobility of 14 C-labeled protein standards (phosphorylase B, 92,500 MW, BSA, 69,000 MW, ovalbumin, 46,000 MW, carbonic anhydrase, 30,000 MW, cytochrome C, 12,000 MW) (New England Nuclear, Boston, MA), which was separated in the same gel. The molecular guard of this antigen was similar to the molecular guard of flagel-lin, the protein comprising flagella of P. aeruginosa as reported by Montie et al. (1982, Infect. Immun. 35-281-289), which is hereby incorporated by reference.

Furthermore, Pa3 IVC2 was tested by ELISA using P. aeru-20 ginosa Hope strains 1-12 (ATCC No. 33348-33359), which were fixed with ethanol to 96-well microtiter plates. The antigen plates were prepared as follows.

Night-old substrate cultures of each organism were pelleted, washed twice with PBS and then resuspended in PBS to a 60β60 of 0.2 O.D. units. The diluted bacteria were plated in 1 well (50 μΐ per well) and then centrifuged at 1500 x g for 15 minutes at room temperature. The PBS was aspirated and then ethanol (95%) was added to the wells for 15 minutes at room temperature. After removing ethanol one from the wells, the plates were air dried and then covered and stored at 4 ° C until used.

Results of the ELISA experiments performed as described above showed that Pa3 IVC2 bound to ethanol-fixed Habs strains 2, 3, 4, 5, 7, 10, 11 and 12. This pattern of specificity indicated that Pa3 IVC2 bound to type b flagella by P. aeruginosa (R. Ansorg, 1978, Zbl. Baked. Hyg., I.

35 Abt. Orig. A, 242: 228-238, R. Ansorg et al., 1984, J. Cl in. Microbiol ..

20: 84-88, both hereby incorporated by reference). Based on this specificity assessment of monoclonal Pa3 IVC2, P. aeruginosa bears the reference strains, Fisher immunotype 2, Fisher immunotype 6 and Fisher immunotype 7, type b flagella. From the previous experimental data, it has been concluded that Pa3 IVC2 binds specifically to P. aeruginosa flagellin type b.

5

In vivo protective activity of Pa3 IVC2

Animal experiments were performed to determine whether monoclonal antibody Pa3 IVC2 would protect a mouse infected with multiple LD 2 values of live P. aeruginosa bacteria. The model of choice was the burned mouse-10 model (M.S. Collins and R.E. Roby, 1983, J. Trauma. 23: 530-534, which is hereby incorporated by reference). Groups of mice were severely burned according to the author's prescription and were then immediately exposed to 5-10 LD 2 values of Fisher immunotype 7. Monoclonal antibody was administered intraperitoneally as high-titration sites (0.2 ml intraperitone-15al) before combustion and attack. No increase in the number of survivors observed in Pa3 IVC2 treated animals compared to the animals that did not receive antibody.

Example 2 Example 2 illustrates the methodology for producing a murine hybridoma cell line that produces a murine monoclonal antibody against P. aeruginosa flagellin type b that is protective in vivo.

Adult female BALB / c mice were first injected intraperitoneally with live P. aeruginosa Fisher immune type 6 (ATCC No. 27317) (8x10 * organisms), followed two weeks later by an injection of live P. aeruginosa Fisher immunotype 5 (ATCC No. 27316) (4 x 10 8 organisms). For the subsequent two week period, live P. aeruginosa Fisher Immunotype 5 and Fisher Immunotype 6 were administered together in two weekly injections. The dosage of each organism was increased such that the final dose was 10 times greater than the initial dose. A final injection of P. aeruginosa Fisher immunotype 6 outer membrane preparations (50 µg protein) prepared according to the method of R.E.W. Hancock and H. Nikaido (1978, J. Bacterlol.

136: 381-390) was given 4 days after the last live bacterial injection. Three days after the last immunization, the spleen was removed from one 35 mice and the spleen cells were prepared for hybridization as described in Example 1.

Culture supernatants of the hybridoma cells were examined for the presence of anti-β. aeruginosa antibodies by ELISA on day 10 after fusion according to the procedures of Example 1 except that the antigen to the ELISA plates was live bacteria immobile in the wells of 96 well microtiter plates. The plates were prepared as follows.

5 50 µΊ poly-L-lysine (PLL) (1 µg / ml in PBS) (Sigma No. P-1524, St.

Louis, MO) was added to each well in 96-well plates (Linbro) and incubated for 30 minutes at room temperature. Unadsorbed PLL was flipped out and the wells were washed 3 times with PBS. Bacterial cultures grown overnight in TSB were washed once with PBS and then resuspended in PBS to 10 0-D-g60nm * 50 μΐ of the bacterial suspensions added to each well of the plate and allowed to bind at 37 ° C for 1 hour. Unbound bacteria were removed by tilting the plates and the wells were then washed three times with saline "Tween" (0.9% (w / v) NaCl, 0.05% (v / v) "Tween-20").

Non-specific binding of the antibodies was blocked by the addition of 200 μΐ per well of blocking buffer (PBS containing 5% (w / v) skimmed milk powder, 0.01% (v / v) "Antifoam A" (Sigma, St Louis, MO) and 0.01% (w / v) thimerosal) to the wells and incubation for 1 hour at room temperature. Excess blocking buffer was poured out and the 20 wells were washed 3 times with saline "Tween" as previously described.

Culture supernatants (50 μΐ) were replica plated in the corresponding wells of the sample plates and incubated at room temperature for 30 minutes. The culture supernatants were removed by tilting the plates and wells washed 5 times with saline "Tween".

An enzyme-conjugated second-stage antibody (horseradish peroxidase-conjugated goat anti-mouse IgG + IgM) (Tago, Inc., Burlingame, CA) was diluted in PBS containing 0.1% (v / v) "Tween-20 and 0.2% (w / v) BSA according to previously determined titrations, and then 50 μΐ of reagent was added to 30 each well and incubated for 1 30 minutes at room temperature. Excess reagent was removed, the wells washed 5 times 1 saline "Tween" and 100 μΐ / well o-phenylenediamine substrate added and incubated for 30 minutes as described in Example 1. Reactions were terminated as in Example 1 and then read at nm on a " Bio-Tek EL-310 "automated EIA plate reader.

Using the methods described above, the culture supernatants of the fusion were examined for the presence of antibodies that bind to P. aeruginosa Fisher immunotypes 1, 2, 3 or 4, but not to control plates prepared by the same PLL and blocking procedure, but without bacteria. Supernatants containing antibody that bind to one of these four Fisher immunotypes were tested again using 5 of each of the 7 Fisher immunotype bacteria separately. Antibody present in the one well supernatant, PaF4 IVE8, bound only to P. aeruginosa Fisher immunotypes 2, 6 and 7. Cells from well PaF4 IVE8 were cloned by limiting dilution methods as described in Example 1. The monoclonal antibody and the clonal cell line from this well is both identified by the term PaF4 IVE8 in the following text. High monoclonal antibody-containing ascites fluid was generated as described in Example 1 except that BALB / c mice were used instead of CB6Fj.

Specificity of PaF4 IVF8 One test performed to identify the antigen that is bound by PaF4 IVE8 monoclonal antibody was indirect immunofluorescence on bacterial organisms. Each of the 7 reference Fisher immunotypes of P. aeruginosa plus a non-flagellated strain of P. aeruginosa (PA103, ATCC No. 29260, Leifson, 1951, J. Bacteriol .. 62: 377-389) and Escherichia coli 20 (GSC A25) was grown overnight at 37 ° C in TSB. The bacteria were pelleted by centrifugation and then washed twice in PBS. Each strain was resuspended in PBS to a 0D-660nm * 2> 2.

The bacterial suspensions were then further diluted 1: 150 and 20 µl samples were placed in individual wells on Carlson slides (Carlson Scientific Inc., Peotone, IL) and dried on the plate at 40 ° C. Culture supernatants (25 µl) of PaF4 IVE8 were incubated on the dried bacterial samples on the plates in a humidified chamber at room temperature for 30 minutes. Unbound antibody was washed off the sample plates by dipping them in distilled water.

After drying the sample plates, fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse IgG + IgM (25 µl per well of a 1:40 dilution in PBS) (Tago, Burlingame, CA) was incubated on the sample plates 1 30 minutes at room temperature 1 a humidified chamber 1 dark. The test plates were washed again in distilled water, dried and then covered with a 35 tire strip provided with glycerol in PBS (9: 1). The test plates were observed with a fluorescence microscope.

Fluorescent labeling was only observed on P. aeruginosa Fisher 1m- in DK 172840 B1 23 monotypes 2, 6 and 7 and was observed as a snusoidal pattern (line) starting from only one end of the organisms. This is consistent with the morphology and location of the individual polar flagellum of these bacteria.

The reaction of PaF4 IVE8 with flagella was confirmed by immunoblot analysis. Outer membrane antigens of P. aeruginosa Fisher immuno-type 6 (see Example 1) were separated by electrophoresis in a 14% polyacrylamide gel containing SDS as described in Example 1 except that the electrophoresis was performed for 5 hours at 80 mA constant power supply.

10 Labeled molecular weight markers (lysozyme, 14,300 MW, beta-lactoglobulin, 18,400 MW, alpha-chymotrypsinogen, 25,700 MW, ovalbumin, 43,000 MW, bovine serum albumin, 68,000 MW, phosphorylase B, 97,400 MW and myosin, 200,000 MW ) (BRL, Gaithersburg, MD) was included in the same polyacrylic amide gel.

Antigens were transferred from the polyacrylamide gel to a nitrocellulose membrane (NCM), (0.45 µm, Schleicher & Schuell, Inc., Keen, NH) in a Tris-glycerin-methanol buffer (Towbin et al. (1979), Proc. Natl Acad.

Sci., U.S.A., 76: 4350-4354) containing 0.05% (w / v) SDS overnight at 4 ° C at a constant power supply p & 200 mA. After transfer, NCM was incubated 0.05% (v / v) "Tween-20" in PBS (PBS "Tween") (B. Batteiger et al., 1982, J. Immunol. Meth. 55: 297-307) for 1 hour at room temperature. In this step and in all subsequent steps, the tray containing NCM is placed on a rocking substrate to ensure distribution of the solution over the entire NCM.

After 1 hour, the PBS "Tween * solution was poured off and PaF4 IVE8 assayed (diluted 1: 1000 in PBS" Tween ") was added and incubated with NCM for 1 hour at room temperature. The NCM was then washed 5 times, 5 times. minutes each time, with PBS "Tween" to remove unbound antibody. Alkaline phosphatase-conjugated goat anti-mouse IgG + IgM (Tago, Inc.) was diluted 1ht.

30 manufacturer's specifications and Incubated with the NCM for 1 hour at room temperature. The NCM was washed 5 times as described above and the substrate containing bromochloroindolyl phosphate and nine trobiottetrazolium (Sigma, St. Louis, MO) prepared as described by Leary et al. (1983,

Proc. Natl. Acad. Sci., U.S.A., 80: 4045-4049), was added and incubated for 10-20 minutes at room temperature. The reaction was terminated by washing the substrate away with distilled water.

The results of this experiment showed that PaF4 IVE8 specifically binds to a single antigen with a molecular weight of 53,000 daltons in the outer membrane preparation. The results of the indirect immunofluorescence assay and immunoblotting show that PaF4 IVE8 binds to the flagella of P. aeruginosa.

The flagellum type recognized by PaF4 IVE8 was determined by ELISA. Hope strains 1-12 (ATCC No. 33348-33359) were each bound to the wells of 96 well mi crotid therapy (Linbro) with PLL, and ELISA was performed as described earlier in this example. The source of PaF4 IVE8 antibody was culture supernatant. Positive reactions were observed in wells 1n-10 containing Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, indicating that PaF4 IVE8 binds to type b flagella. The in vivo protective data are shown below in Example 4.

Example 3 Example 3 shows the methodology for preparing a murine hybridoma cell line that produces a monoclonal antibody reactive with anti-β. aeruginosa flagellum type a, which is protective in vivo.

The lymphoid cell source for the fusion was a spleen from an immunized BALB / c mouse that had been injected four times intraperitoneally 20 over a 6-week period of purified type α flagella (10-20 µg protein) from Habs strains 6 and 8 ( ATCC Nos. 33353 and 33355). Flags were purified according to the method of T.C. Montie et al. (1982, Infect. Immun. 35: 281-288, which is hereby incorporated by reference) except that the final centrifugation of flagella was done at 100,000 x g for 1 hour instead of 25,000 x g for 1 hour. Another modification used for certain procedures was to cut the flagella from the bacteria for 30 seconds in a blender instead of for 3 minutes (Allison et al., 1985, Infect. Immun. 49: 770-774).

Protein concentrations for each preparation were determined by the Bio-Rad 30 protein sample (Bio-Rad, Richmond, CA) and the presence of contaminating lipopolysaccharide (LPS) was detected by measuring the KDO content (YD Karkhanis et al., 1978, Anal. Biochem .. 85: 595-601). The molecular weights of the flagella proteins were determined by comparing their migration 1 an SDS-polyacrylamide gel with the migration of standard protein markers 35 (BRL) (see Example 2). The molecular weight for Hope's 6 flagellin was 51,700 daltons and for Hope's 8 f1 agel linen it was 47,200 daltons. These values are consistent with those of O.S. Allison et al. (1985, Infect. Immune 25 DK 172840 B1 49: 770-774), which is hereby incorporated by reference.

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 hybridoma cells grew to ca. 40% confluency 5 (day 7), replica-plated culture supernatants in corresponding wells on 3 different antigen plates, PLL-bound P. aeruginosa Fisher immunotype 1 (see Example 2 for preparation) and formally fixed Hope 6 and Hope 9.

The bacteria for the formally fixed antigen plates were cultured, washed, and diluted as described for PLL bound antigen plates. Diluted bacteria (0.2 0.0 units at A ^ q) were added to individual wells (50 µl per well) in Linbro 96-well microtiter plates, and the plates were then centrifuged at 1200 x g for 20 minutes at room temperature. The supernatants were flipped out of the wells and 75 µl of 0.2% 15 (v / v) formalin in PBS was added to each well and incubated for 15 minutes at room temperature. After the formalin was tilted out of the wells, the plates were air dried and stored at 4 ° C until used. Formalin did not alter the antigenicity of the flagella as demonstrated by the ability of anti-flagella antisera to agglutinate formally treated organisms (B. Lanyl, 1970, Acta Microbiol. Acad. Sci., Hung., 17: 35-48). P. aeruginosa Fisher Immunotype 1 strain was included as control because this strain was non-flagellated as shown by etching stain (Manual of Cl in. Microbiol. 1985, Lennette, ed. Amer.

Soc. Microbiol., Wash., D.C., p. 1099). Hybrid cells 1 the well, which was designated FA6 IIG5, produced an antibody that binds to Hope 6 and Hope 9 (both flagella type α-bearing strains) but not Fisher immunotype 1.

Cells from well FA6 IIG5 were sub-cultured and cloned as described in previous examples. The monoclonal antibody and clonal cell line 30 of this well are both identified by the designation FA6 IIG5 in the following text. Ascites were produced in BALB / c mice as described in Example 2.

Specificity of FA6 1165 The specificity of the antibody FA6 IIG5 was determined by indirect immunofluorescence and immunoblotting. Indirect immunofluorescence was performed essentially as described in Example 2 with the following modifications.

26 DK 172840 B1

Bacterial cultures grown overnight on p & trypticase soy agar at 30 ° C were removed from the cotton swab plates and resuspended in PBS to one of 0.2 O.D. units. Formalin (0.37% (v / v) in final PBS concentration) was added to the suspension with vortexing. After 5 incubation at room temperature for 15 minutes, the bacteria were diluted 1:12 in PBS and 20 μΐ of this suspension was placed in individual wells on Carlson slides. After drying, the slides were prepared for consideration as described in Example 2. The antibody source was culture supernatant from the FA6 IIG5 cell line.

10 Fluorescence labeling of the FA6 IIG5 antibody was stratified only by P.

aeruginosa strains carrying type a flagella and none of those carrying type b. The observed fluorescence pattern was a sinusoidal line pattern indicating that FA6 IIG5 bound to the flagella. The fluorescence signal was enhanced by treating the bacteria with formalin, but the treatment was not necessary to visualize flagellar skin labeling with the antibody.

Immunoblotting was performed as described in Example 2. The sources of flagella type a antigens were the purified flagellar preparations (see this example). Antigens were separated in 10% polyacrylamide gel containing 20 SDS (U.K. Laemmli, 1970, Nature (London), 227: 680-685) and transferred to an NCM. Preparations of FA6 IIG5, either culture supernatant or ascites, diluted 1: 1000, reacted with the NCM and the reaction was detected with an appropriate enzyme-conjugated reagent and enzyme substrate as described in Example 2. The immunoblot showed that FA6 IIG5 specifically bound to 51,700 MW for Hope 6 and 47,200 MW flag line for Hope 8.

Confirmation that FA6 IIG5 reacted only with flagella of type a and not of type b was obtained by ELISA, where Hope's strains 1-12 were individually bound by PLL to the wells of Linbro 96-well microtiter plates. The antibody only bound to Hope strains 1, 6, 8 and 9, which are the only 30 strains out of the 12 that carry type a flagella (see R. Ansorg et al. 1984, J. Clin. Microbiol. 20 : 84-88, which is hereby incorporated by reference). In vivo protection studies are described in the following Example 4.

Example 4

Example 4 shows the protection of mice passively immunized with antibodies PaF4 IVE8 and FA6 IIG5 against the attack of P. aeruginosa in the mouse model burned 27 DK 172840 B1.

The monoclonal anti-flagell antibodies were tested in the burnt mouse model according to the method of M.S. Collins and R.E. Roby (1983, Jj.

Trauma. 23: 530-534, which is hereby incorporated by reference). For the protection studies, all antibodies were purified by protein "A-Sepharose" chromatography (P.L. Ey et al., 1978, Immunochemistry, 15: 429-436, which is hereby incorporated by reference) and dialyzed in PBS buffer. The strain used in the animal experiments carrying type a flagella was P. aeruginosa PA220 (from Dr. James Pennington, Boston, MA), and the strain of fla b gels of type b was the reference P. aeruginosa Fisher immunotype 2 (ATCC No. 27313).

40 µg of purified monoclonal antibody was administered per mouse intravenously 1 to 2 hours before incineration and attack. Immediately after combustion, the animals received 0.5 ml of cold PBS subeschart containing the attack bacteria. The dose of attack was approx. 10 LD 2 is for each organism.

The results of the animal experiments are shown in Tables I and II.

28 DK 172840 B1

IabfiLi

Protective study for an anti-flaoella type a monoclonal. antibody in the burnt mouse model1 5 2

Percent Survival Per Day Treatment 123456789

Anti-flagella a, 10 FA6 IIG5 100 100 100 100 100 90 90 80 80

Anti-flagella b,

PaF4 IVE8 100 20 10 10 10 10 10 10 10

Non-specific anti-LPS monoclonal antibody 100 40 30 20 10 10 10 10 10 15 PBS, no bacteria 80 80 80 80 80 80 80 80 80 1 Mice were attacked subeschart by ca. 10 LD, -n's of P. aeruginosa PA220.

2

The percent value is based on surviving mice in 10 animal groups with 20 except for the pure PBS control group, which consisted of 5 mice.

Days are after burn and attack.

Table II

5 29 DK 172840 B1

Protective study of anti-flagella type b monoclonal antibody in the burned mouse model * 2

Percent Survival Per Day Treatment 123456769

Anti-flagella b, 10 PaF4 IVE8 100 100 90 90 90 90 90 90 90

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

Non-specific anti-LPS monoclonal antibody 100 20 20 20 20 20 20 20 20 15 PBS, no bacteria 100 100 100 100 100 100 100 100 100 1 Mice were attacked subeschart by ca. 10 LD50s of P. aeruginosa

Fisher Immunotype 2.

2

The percentage survival was based on the number of mice that survived 20 per group of 10 mice, with the exception of the pure PBS control group, which consisted of 5 mice. Days are after burn and attack.

Very significant survival was observed in mice treated with the anti-a antibody or anti-b antibody and then exposed to the corresponding antigen. Conversely, 80-90% of untreated but infected mice or animals treated with a non-matching ant1-flagellar monoclonal antibody or non-specific anti-LPS antibody died. The inability of anti-flagella type a antibody to protect mice against a lethal attack 30 of P. aeruginosa Fisher immunotype 2 with flagella of type b, and the inability of anti-flagella type b antibody to protect mice against a lethal Type A infestation attacks P. aeruginosa PA220 confirmed the in vivo specificity of the antibodies observed in vitro. Survival in burned but not infected mice Indicated that burning itself was not lethal.

30 DK 172840 B1

Example 5

Example 5 shows the significant cross-reactivity of PaF4 IVE8 and FA61165 with clinical isolates of P. aeruginosa, indicating the clinical utility of these antibodies in immunotherapy of P. aeruginosa infections.

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

PaF4 IVE8 specifically bound to 34 clinical isolates (22%), while the 10 flagella type a antibody, FA6 IIG5, bound to 102 clinical isolates (65%) corresponding to a total of 136 out of 157 isolates (87%). Out of the 21 strains that were not recognized by any antibody, 19 were non-flagellated as shown by pickling staining. Therefore, antibodies in combination bound 136 to 138 (98%) flagellated clinical isolates, confirming previous reports (see R. Ansorg, 1978, Zbl. Bact. Hyg., In Abt.

Orig. A, 242: 228-238, incorporated herein by reference).

Example 6

Example 6 demonstrates methods for producing human monoclonal antibodies that bind to P. aeruginosa type b flagella.

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 a source of B cells. Mononuclear cells were separated from the blood by standard centrifugation technique on a "Ficoll-Paque" (Boyum (1968), 25 Scand. J. Cl in Lab. Invest. 21:77) and washed twice in calclum / mag-nesium-free. phosphate-buffered saline (PBS).

The mononuclear cells were liberated for T cells using a modified E rosetting procedure. Briefly, cells were first resuspended to a concentration of 1 x 10 7 cells / ml in PBS containing 20% fetal calf serum (FCS) at 4 ° C. 1 ml of this suspension was then placed in a 17 x 100 mm polystyrene round-bottomed test tube to which 1 x 109 2-amino-isothiouronium bromide (AET) treated red blood cells from sheep were added from a 10% (v / v) solution in Iscove's modified Dulbecco's medium (Iscove's medium) (Madsen and Johnson 35 (1979), J. Immun. Methods. 27:61). The suspension mixed very gently for 5-10 minutes at 4 ° C and the E-rosetted cells were then removed by centrifugation on "Ficoll-Paque" for 8 minutes at 2500 x g at 4 ° C. E-rosette negative peripheral blood mononuclear cells (E'PBMC) bound at the interface were collected, washed once in Iscove's medium and resuspended in the same containing 15% (v / v) FCS, L-glutamine 2 mmol / 1). , penicillin (100 IU / ml), streptomycin (100 pg / ml), hypoxanthine 5 (1 x 10 7 M), aminopterin (4 x 10 7 H) and thymidine (1.6 x 10 5 M). This medium is then called HAT medium.

Cell-driven transformation of E'PBMC was proceeded by co-culturing these cells with a transforming cell line. The transforming cell line was an Epstein-Barr nuclear antigen (EBNA) positive human lymphoblastoid cell line derived by ethyl methanesulfonate (EMS) mutagenesis of the lymphoblastoid GM 1500 cell line followed by selection in the presence of 30 pg / ml 6- thioguanine to make cells deficient on hypoxanthine-guanine phosphoriboxyltransferase (HGPRT) and thus HAT-sensitive.

This cell line is dominated by the 1A2 cell line and deposited with the American Type Culture Collection (ATCC) on March 29, 1982 under ATCC no.

CRL 8119. 1A2 cells 1 logarithmic growth phase were suspended in HAT medium and then combined with E'PBMCs at a ratio of 15 1A2 cells per PBMC. The cell mixture was plated in 30 round bottom 96-well microtiter plates (Costar 3799) at a concentration of 32,000 cells / well 20 l a volume of 200 µl / well and incubated at 37 ° C in a humidified atmosphere containing 6% CO 2. Cultures were born on days 5 and 8 after irradiation by replacing half of the supernatant with fresh HAT medium. At 16 days after irradiation, 100% of the wells contained proliferating cells and in most of the wells the cells were of sufficient density 25 to remove and test supernatants for anti-P. aeruginosa antibodies.

Supernatants were screened for the presence of anti-P. aeruginosa antibodies using ELISA technique as described in Example 2 with the following modifications. A pooling of the 7 Fisher immunotype reference strains (ATCC No. 27312-27318) (Αβ60 - 0.2 O.D. units) was bound to flat-bottomed 96-well microtitters (Immulon II,

Dynatech), pre-treated with poly-L-lysine, was incubated and washed as described in Example 2. After blocking of non-specific binding sites and leaching of the plates, 50 µl of PBS containing 35% 0.1% (v / v) was added. "Tween-20" and 0.2% (w / v) BSA per well. Culture supernatants (50 µl) were then replicated in the corresponding test plate wells and in control pads treated with PLL and blocked but not containing bacteria. After incubation and leaching, enzyme-conjugated second-stage antibodies (50 ml per well), horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-human IgM were appropriately diluted in PBS containing 0.1% (v / v). vol.) "Tween-20" and 5 0.2% (w / v) BSA to the wells and sample were completed as described in Example 2.

Supernatants containing antibody that bound to the pooled Fisher immunotypes but not to the control plate were tested again using each of the 7 Fisher immunotype bacteria separately. Antibody present in the one-well supernatant, 20H11, bound only to P. aeruginosa Fisher immunotypes 2, 6 and 7. The cells were repeatedly subcultured with decreasing low cell densities until all wells with growth secreted antibody. The cell line and the monoclonal antibody (IgM isotype) are both identified by the designation 20H11 in the subsequent text.

Another transformation was made where the source of B cells was the peripheral blood of a cystic fibrosis patient known to have a chronic P. aeruginosa infection. E'PBMCs were prepared as described above and co-cultured with a transforming cell line 1A2 at a ratio of 72 IA2 cells per E'PBMC. The cell mixture was plated in 15 round-bottom 96-well scraps in terraps at a concentration of 7.4 x 10 6 cells per well and grown as above.

Supernatants were examined by ELISA for the presence of anti-P. aeruglnose antibodies 16 days after the transformation was effected. The test was performed as described for the previous transformation except that the pooled P. aeruginosa strains used for the initial screening were composed of Fisher immunotype reference strains F2, F4, F6 and F7 (ATCC Nos. 27313, 27315, 27316 and 27317), and three clinical isolates from the Genetic Systems Corporation organism bank (GSC0B), which had 30 different LPS immunotypes and flagella types. The clinical isolate PSA 1277 (GSC0B) carries type a flagella and Fisher immunotype 1 LPS, the second Isolate PSA G98 (GSC0B) carries type a flagella and Fisher immunotype 3 LPS, and the third, PSA P625 (GSC0B), carries type b flagella and Fisher Immunotype 5 LPS. This mixture of reference strains and clinical isolates is hereafter referred to as the P. aeruginosa flagella assemblage. Supernatants containing antibody that bound to the plates containing the P. aeruginosa flagel junction but not to the PLL-coated conjugate plates were examined by ELISA once again on the individual strains of the junction. A well, 3C1, linked to reference strains F2, F6 and F7 and to the clinical in the sun at F625.

Cloning of the 3C1 cell line was done by first subculturing the cells 5 for two low-density subcultures, first with 20 cells per well of 96-well plates followed by culture at two cells per well. Formal cloning of the specific antibody-producing cells was performed by irradiating the cells at a density of ca. 1 cell / well in 72-well Terasaki plates (Nunc No. 1-36538) in a volume of 10 µl / well 10 HAT medium without the aminopterin component (HT medium). The plates were placed in an incubator for 2-3 hours for the cells to settle on the bottom of the wells, and then a microscopic evaluation of two individuals for wells containing a single cell was performed. The wells were born daily with HT medium and when the outgrowth was sufficient, the cells were transferred to a 96-well round bottom plate. All outgrowth wells were examined by ELISA on P. aeruginosa strains with type b flagella, and all were found to produce the appropriate antibody. The cell line and the monoclonal antibody (IgM isotype) are both identified by the term 3C1 in the following text.

The antigen identified as 20H11 and 3C1 were flagella as shown by indirect immunofluorescence and immunoblotting. The studies were done basally as described in Examples 2 and 3. For the indirect immunofluorescence test, P. aeruginosa strains with type b flagella were prepared, reference Fisher Immunotypes F2, F6 and F7 (ATCC Nos. 27313, 27317 and 2527318) and a strain of type a flagella, reference Fisher Immunotype 4 (ATCC No. 27315) as described in Example 3. The flagella type of the reference strains was determined by typing with the murine monoclonal antibodies PaP4 IVE8 and FA6 1165. The slides were prepared for consideration as described 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) diluted 1: 100 in PBS containing 0.5% (vol./ vol.) bovine gammagi obul in milk (Miles Scientific, Cat. No. 82-041-2, Naperville, IL) and 0.1% (w / v) sodium as a preservative.

35 Fluorescence labeling of the 20H11 and 3C1 antibodies was stratified only with P. aeruginosa strains bearing type b flagella and not with the type a flagella bearing strain, reference Fisher immunotype 4. The observed 34 DK 172840 B1 fluorescence pattern was a sinusoidal bar pattern derived from one end of the the bacterium, indicating that the antibodies bound to the bacterial flagella.

Immunoblotting was performed as described in Example 2. Purified type 5 b flagella from P. aeruginosa reference strains Fisher immune type 2 (ATCC No. 27313) and purified flagella type a from reference strains Hope 6 and Hope 8 (ATCC Nos. 33353 and 33355) were prepared as described. in Example 3. Antigens were separated in a 10% polyacrylamide gel (see Example 3) and transferred to an NCM. Culture supernatants containing 20H11 or 3C1 10 antibodies, culture supernatant containing a non-specific human antibody, and culture media were incubated with the NCM and the reaction detected with alkaline phosphatase-conjugated goat anti-human Ig (polyvalent) (Tago, Burlingame, CA) diluted in PBS containing 0.05% (v / v) "Tween-20". Enzyme substrate was prepared as described in Example 2. The immunoblot illustrated that both antibodies bound to the 53,000 MW flagel-1in protein for Fisher immunotype 2 and not to the 51,700 MW flagel lin protein for Hope 6 or to 47,200 MW flagel lin protein for Hope. 8th

No reaction was performed with neither the non-specific human antibody nor the culture medium.

20 Further confirmation that antibodies 20H11 and 3C1 bound only to type b flagella and not to type a were obtained by ELiSA, where Habs strains 1-12 individually bound to the wells of "Immulon" 96-well mi crotiter plates. The antibodies bound only to Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, which are type B-bearing strains (Ansorg et al.

25 (1984), J. Cl in. Microbiol .. 20:84).

Example 7

Example 7 shows methods for preparing a human monoclonal antibody that binds to P. aeruginosa type a flagella, A peripheral blood sample from a high molecular weight polysaccharide preparation Immunized individual (Pier et al. (1981), Infect. Immun., 34: 461) served as a source of B cells. The mononuclear cells are 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 sul foxide in a liquid nitrogen vapor tank. At a later stage, the cells were rapidly thawed at 37 ° C, washed once in Iscove's medium and resuspended in HAT medium. Cell-driven transformation is accomplished by co-culture! 35 DK 172840 B1 of the E'PBMCs with 1A2 cells relative to 30 IA2 cells per E'PBMC. The cell mixture was plated in 30 96-well tissue culture plates at a concentration of 62,000 cells / well. Cultures were born on the 7-day after irradiation by replacing half the volume with HAT medium. Cell proliferation was observed in 100% of the wells on the 14-day post ironing, and supernatants were removed from the wells and examined at this time.

Supernatants were examined by ELISA for the presence of anti-P. aeruginosa antibodies using the flagellated P. aerugi-10 nosa conjugate and PLL-treated plates as control as described in Example 6. Supernatants Containing antibodies that bound to the flagellated conjugate but not to the PLI control plates were re-examined for the individual bacterial strains in the flagellated junction. One well, 21B8, contained antibody that binds to PSA 1277, PSA 15 G98, and reference Fisher Immunotype 4, which are the three strains of the flagellated gene carrying type a flagella.

Cloning of the 21B8 cell line was performed as described in Example 6 for the 3Cl cell line with the following modifications in the formal cloning step. After the wells 1 Terasaki plates were evaluated for the presence of only a single cell, each cell was transferred from the Tera-saki plates to an individual well in a 96-well round-bottom culture plate 1 for a volume of 100 µl HAT medium without the aminopterin component. (HT medium). Nontransforming HAT-sensitive lymphoblastoid cells were included in all wells at a density of 500 cells / well as feeding cells. 5 25 days after ironing, 100 μΐ HAT medium was added to the wells to selectively kill the feeding cells. Wells were again born on days 7 and 9 after ironing out, replacing half of the supernatant with HAT medium.

Next, the cells were born with HT medium until the cells were of sufficient density to detect the presence of antibody at 30 ELISA. All outgrowths produced antibody that bound to flagella type a bearing P. aeruginosa strains. The cell line and the monoclonal antibody (IgG isotype) are both identified by the designation 21B8 in the following text.

The antigen identified as 21B8 was flagella as shown by 1n-35 direct immunofluorescence and immunoblotting (see Example 6 for a description of the techniques). Fluorescence labeling of the 21B8 antibody was observed only with P. aeruginosa reference strain Fisher immunotype 4 (ATCC no.

36 DK 172840 B1 27315) carrying type a flagella and Not with P. aeruginosa reference strain immunotype 2 (ATCC No. 27313) bearing type b flagella. The observed fluorescence pattern was a sinusoidal streak pattern originating from one end of the bacterium as an indication that the antibody bound 5 to the bacterial flagella.

Immunoblotting was performed as described in Example 2. Purified type a flagella from P. aeruginosa reference strain Hope 6 (ATCC No. 33353) and purified flagella type b from P. aeruginosa reference strain Fisher Immunotype 2 (ATCC No. 27313) were prepared as described in Example 3 Antigens were separated in a 10% polyacrylamide gel (see Example 3) and transferred to an NCM. Culture supernatants containing either 21B8 or a non-spiked human antibody and culture medium were reacted with the NCM and the reaction was detected with alkaline phosphatase-conjugated goat anti-human Ig (polyvalent) and enzyme substrate as described in Examples 2 and 6.

The immunoblot showed that the 21B8 antibody bound only to the 51,700 MW flagellin protein of Hope 6 and not to the 53,000 HW flagellin protein of Fisher Immunotype 2. No reaction was observed with neither the nonspecific human antibody nor the culture medium.

EXAMPLE 9

Example 8 shows protection of mice that are passively immunized with human anti-flagella antibodies, 20H11, 3C1, and 21B8, against P. aeruginosa 1 infestations in the burned mouse model.

The human anti-flagella monoclonal antibodies were tested in the 25 burned mouse model (see Example 4). 21B8 and 20H11 antibodies were prepared by precipitating culture supernatants formed by the respective cell lines, with ammonium sulfate (50% final concentration) (Good et al., Selected Methods in Cellular Immunology. B.B. Mishell and S.M.

Shiigi, eds., W.J. Freeman & Co., San Francisco, CA, 1980, 279-286).

The precipitate was made soluble in PBS, dialyzed against the PBS overnight at 4 ° C, and then sterilized before administration to animals.

The source of antibody 3C1 and the non-specific anti-LPS antibody used as negative control in this study was culture supernatant. As a positive control for each study, the appropriately purified murine monoclonal antibody, PaP4 IVE8 or FA6 IIG5, was included.

The flagella type a carrier strain used in the animal experiments was the clinical isolate PSA A522 (GSC0B), which expresses Fisher immunotype 1 37 DK 172840 B1 LPS, and the flagella type b strain was clinical isolate PSA A447 (GSCOB), which expresses Fisher Immunotype 6 LPS . The human antibodies (0.45 ml) were premixed with the bacteria (more than 5 LD 2's in 0.05 ml) and inoculated subeschart immediately after the incineration. The results of the animal experiments are shown in Tables III, IV and V.

Table III

Protection study for the human anti-flagella type a monoclonal antibody 21B8 in the burnt mouse model1 2 15 Percent survival per day

Treatment 123456789

Murine anti-flagella a, FA6 IIG53 100 100 100 100 88 88 88 88 88 20 Human anti-flagella a, 21B8 100 100 100 100 100 100 100 100 100

Human anti-flagella b, 20H11 100 00000000 PBS 100 25 12 12 12 12 12 12 12 25 ___ 1 Mice were attacked subeschart with more than 5 lDj00s of P.

aeruginosa PSA A522.

2

The percentage survival was based on the number of mice surviving per group of 8 animals. Days are after burn and 30 attacks.

3 Purified antibody (10 µg 0.45 ml PBS) was premixed with the bacteria and administered subeschart after combustion and infestation.

5

Protection studies for the human anti-flannel cell monoclonal antibody 20H11 in the burned mouse model * 38 DK 172840 B1

Tjbe3-.IV

2

Percent Survival Per Day Treatment 123456789

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

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

Human anti-flagella a, 21B8 100 00000000 15 Media 80 00000000

Mice were attacked subeschart with more than 5 LDin0s of P. aeruginosa, clinically in the sun at PSA A447.

2

The percentage survival was based on the number of mice that survived 20 per group of 5 animals. Days are after burn and attack.

3 Purified antibody (10 µg in 0.45 ml PBS) was premixed with the bacteria and administered subeschart after combustion and infestation.

Protection study for the human anti-flavoid type b monoclonal antibody 5f 3C1 in the burned mouse model * 39 DK 172840 B1

Table V

2

Percent survival per day Treatment 123456789 10 Murine anti-flagella b,

PaF4 IVE83 100 100 100 100 100 100 100 100 100 100

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

Non-specific anti-LPS

15 monoclonal antibody 100 00000000 * Mice were attacked subeschart with more than 5 LD ^ 's of PSA A447.

2 The percentage survival was based on the number of mice surviving per group of 5 animals. Oage is after burn and 20 attacks.

3

Purified antibody (40 µg) was administered intravenously 2 hours before combustion and attack.

Very significant survival was observed in mice treated with the anti-flagella type a antibody or 1 of the 2 anti-flagella type b antibodies, and then exposed to the corresponding antigen. Conversely, 88-100% of the untreated but infected noses or those treated with a non-matching anti-flagellar monoclonal antibody or non-specific anti-LPS antibody died. As observed for the murine monoclonal antibodies (see Example 4), the human anti-flagella antibodies only protect against lethal attack of the individual, the organisms carrying the corresponding flagella type, ie. that human anti-flagella type a antibody conferred protection with lethal attack of the flagella type a carrying organism and not type b, whereas anti-flagella type b antibodies protected 35 mice that were attacked by flagella type b carrying strains but not type a carrying organisms.

40 DK 172840 B1

Example 9

Example 9 shows the cross-reactivity of the human anti-flagella antibodies 20H11, 3C1 and 21B8 with P. aeruginosa clinical isolates.

P. aeruginosa clinical isolates (115) obtained from hospitals and 5 clinics and primarily isolated from burns and blood were identified for flagella type a or type b, by typing with the murine monoclonal antibodies FA6 IIG5 or PaF4 IVE8 (see Examples 2, 3 and 5). 55 of the clinical isolates were identified as carrying type a flagella by reaction with the murine monoclonal antibody, 10 FA6 IIG5, and 59 were identified as type b bearing by their reaction with the murine monoclonal antibody, PaF4 IVE8.

The cross-reactivity of the human monoclonal anti-flagella type a antibody, 21B8, was extensive, with the antibody recognizing 54 of the 56 flagella type a carrying clinical isolates (96%). The cross-reactivity of 15 20H11 with isolates bearing type b flagella was also extensive, with 20H11 recognizing all 59 isolates (100%). In contrast, the second monoclonal anti-flagella type b antibody 3C1 bound to only 43 of the 59 isolates (73%). These results show that 20H11 binds to a pan-reactive epitope (i.e., an epitope present in at least about 95% of 20 flagellated P. aeruginosa strains), whereas 3C1 binds to an epitope that is not present. on all type b flagel1 in n-molecules. Although the flagella type b antigen is serologically uniform by analysis with polyclonal antisera (B. Lanvi, supra and R. Ansorg, supra), the cross-reactivity patterns of 20H11 and 3C1 surprisingly show that the type b flagella have at least two separate epitopes, which can be identified by monoclonal antibodies.

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

30

Example 10

Example 10 shows methods for preparing another example of human monoclonal antibody that binds to P. aeruginosa type b flagella, as well as the protective activity of this antibody against P. attack.

35 aeruginosa in the burned mouse model.

A transformed cell line was prepared and cloned essentially as described in Example 7 except that the transformed cell mixture was plated onto 20 96-well tissue culture plates at a concentration of ca. 2250 E'PBMC per well. Supernatants were initially tested with ELISA on the flagellated pool as described in Example 6 except that Fisher immunotype 2 and 4 reference strains were missing in the pool. Positive wells were subsequently examined on each of the strains of the flagellated collection, including Fisher immunotypes 2 and 4. The finally isolated cell line and the monoclonal antibody (IgG isotype) are both identified by the term 12D7 in the subsequent text.

10 The human monoclonal antibody 12D7 showed extensive cross-reactivity with anti-flagella type a isolates, recognizing 54 of the 56 flagella type a carrying clinical isolates tested (96%). The protective activity of the monoclonal antibody 12D7 is shown in Table VI. The protection studies were performed essentially as described in Example 4 except that the attack dose was 1 LDjqq and the attack strain was 1624, a clinical isolate expressing Fisher immunotype 2 LPS and type a flagella.

42 DK 172840 B1

Table VI

Protective Study of the Human Anti-Flaoella Type A Monoclonal Antibody 12D7 in the Burned Mouse Model * £ 5

Percent Survival Per Day Treatment3 123456789

Human anti-flagella a, 10 1207 100 100 100 100 100 100 100 100 100

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

Murln anti-flagella a, IIG5 100 100 100 100 100 100 100 100 100 15 Human anti-flagella b, 15F4 100 37.5 25 25 25 25 25 25 25 1 Mice were attacked subeschart with 1 LD100 (950 CFU) of P.

aeruginosa 1624.

2 20 Percent survival was based on the number of mice surviving per group of 10 animals except for the 15F4 group, where N - 8 days are after combustion and attack.

3

Purified antibody (50 µg in 0.5 ml PBS) was administered i.p. 2 hours before combustion and attack.

25

Example 11

Example 11 shows the preparation of a human monoclonal antibody reactive with b type flagella P. aeruginosa, as well as the protection of passively immunized mice with this antibody against attack of 30 P. aeruginosa in the burned mouse model.

A transformed cell line was prepared and cloned in substantially as described in Example 10 except that the 1A2 to B cell transformation ratio was approx. 60: 1 and the transformed cell mixture was plated on 15 plates at a concentration of ca. 1930 E'PBMC 35 per well. In addition, the cells were examined for a pooling of P. aeruginosa strains comprising G98 (Fisher 3 immunotype, flagella type a) and 1739 (a clinical isolate of Fisher 5 immunotype, flagella type b) and subjected to a different pool. of P. aeruginosa strains: 1277, G98, 1739 and Fisher immunotype F2, F4, F6 and F7. The finally isolated cell line and secreted monoclonal antibody (IgGj in soot type) are both referred to by the term 2B8 in the following text.

An immunofluorescence assay performed with 2B8 was positive on a fla gella type b, Fisher 2 immunotype strain but negative on a flagella type a, Fisher immune type 4 reference strain. When tested on clinical islets, 59/59 (100%) of flagellated 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 clinical isolate F164 (Fisher immunotype 4, flagella type b) was used for the attack.

Protective Study for the Human Anti-Flauna Type B Monoclonal Antibody 2B8 in the Burned Mouse Model * 44 DK 172840 B1

Table VII

2

Percent survival per day Treatment 123456789 10 Human anti-flagella b, 2B83 100 89 89 89 89 89 89 89 89

Murln anti-flagella b,

PaF4 IVE84 100 100 100 100 100 100 90 90 90

Murln anti-Fisher 2 LPS, 15 VH34 90 20 20 20 20 20 20 20 20 * Mice were attacked subeschart with 1 LDjqq (105 CFU) of P.

aeruginosa F164.

2

The percentage survival was based on the number of mice that survived 20 per group of 10 animals except for the 2B8 group, where N - 9 days are after combustion and attack.

3

Purified antibody (50 μl in 0.5 ml PBS) was administered i.p. 2 hours before combustion and attack, a

Purified antibody (5 μς in 0.5 ml PBS) was administered 1st p.p. 2 hours 25 before combustion and attack.

Example 12

Example 12 shows the preparation of another human monoclonal antibody reactive with flagella b type P. aeruginosa, as well as the protection of passively immunized mice with this antibody against P. aeruginosa attack in the burned mouse model.

A transformed cell line was prepared and cloned essentially as described in Example 7 with the following exception. Another individual was immunized (Pier et al. (1984) 45: 309) and served as a B-35 cell source, and the smear level of E'PBMC was about 2000 cells per well. The screening was performed on pooled Fisher 1-7 immunotypes. The finally isolated cell line and secreted monoclonal antibody are referred to hereinafter as 14C1.

In clinical testing, 59/59 (100%) of the flagella type B isolator tested tested positive with 14C1. The protection studies were performed as described in Example 11 above and the results are shown in Table VIII.

5

Table VIII

Protective study of the human anti-flagella type b monoclonal antibody 14CI in the burnt mouse model * 10 2

Percent Survival Per Day Treatment3 123456789

Human anti-flagella b, 15 14C1 100 100 100 100 100 90 90 90 90

Murine anti-flagella b,

PaF4 IVE8 100 100 100 100 100 100 100 100 100 100

Murine anti-Fisher 2 LPS, VH3 100 40 20 20 20 20 20 20 20 20 _______ * Mice were attacked subeschart with more than 1 LDjQ0s (90 CFU) by P. aeruginosa F164. island

The percentage survival was based on the number of mice surviving per group of 10 animals except the PaF4 IVE8 group, 25 where N = 9. Days are after combustion and attack.

3

Purified antibody (50 Mg in 0.5 ml PBS) was administered l.p. 2 hours before combustion and attack.

From the foregoing, it will be appreciated that the cell lines of the present invention provide means for producing monoclonal antibodies and fragments thereof that are reactive with P. aeruginosa flagella and cross-protective against various P. aeruginosa strains. Surprisingly, a number of monoclonal antibodies reactive with different epitopes were soldered to each type of flagella. The ant1 substances of the present invention allow prophylactic and therapeutic preparations to be more economically and more readily prepared for use against infections caused by most P. aeruginosa strains. Furthermore, the cell lines provide antibodies which are useful in immunoassays and other well known procedures.

!

Claims (32)

A monoclonal antibody or binding fragment thereof, characterized in that the antibody or binding fragment thereof is capable of specifically reacting with a flagella lower protein epitope of Pseudomonas aeruginosa and inhibiting the motility of the bacterium which is a monoclonal antibody or binding portion thereof. protective in vivo. 2. Monoclonal antibody or binding fragment thereof according to claim 10 1, characterized in that the antibody or binding fragment thereof is capable of blocking binding to said flagellar protein epitope of a monoclonal antibody produced by cell lines designated ATCC accession numbers HB9129. , HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. 15 The monoclonal antibody or binding fragment thereof according to claim 1 or 2, characterized in that the antibody or binding fragment thereof has the same binding specificity for a flagellated leather protein epitope of Pseudomonas aeruginosa as any monoclonal antibody produced by cell lines with designations ATCC accession numbers HB9129, HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. Monoclonal antibody according to any one of claims 1 to 3, characterized in that the antibody is produced by cell lines designated ATCC accession numbers HB9129, HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. The monoclonal antibody or binding fragment thereof of claim 1, which antibody or binding fragment thereof has the same binding specificity as any of the monoclonal antibodies designated FA6 IIG5, Pa3 IVC2, PaF4 IVE8, 20H11 and 21B8. A monoclonal antibody according to any one of the preceding claims, characterized in that the antibody is conjugated to a label capable of providing a detectable signal. DK 172840 B1 The monoclonal antibody of claim 6, characterized in that the label is a fluorescent substance or enzyme. A composition, characterized in that it comprises one or more monoclonal antibodies, wherein a first of the monoclonal antibodies is a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof which is present in capable of responding with an epitope of type a or type b flagella of Pseudomonas aeruginosa. 9. A composition according to claim 8, characterized in that another of said monoclonal antibodies is capable of reacting with a type of flagella which is not reactive with the former monoclonal antibody. A composition according to claim 8, characterized in that the epitope is present on type a or type b flagella, but not on both. 11. A composition according to claim 8, characterized in that the first antibody is a human monoclonal antibody. A composition according to claim 8, characterized in that the epitope is exhibited by at least approx. 70¾ of type b flagella. A composition according to claim 8, characterized in that the epitope is exhibited solely by type b flagella. 25 A composition according to any one of claims 8 to 13, characterized in that the antibody is pan-reactive. The composition of any of claims 8 to 14, A pharmaceutical composition, characterized in that it comprises a composition according to any one of claims 8 to 15 as well as a physiologically acceptable carrier. DK 172840 B1 A pharmaceutical composition useful in the treatment or prevention of a Pseudomonas aeruginosa infection, characterized in that it comprises a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof which is reactive with 5 flagellar protein of Pseudomonas aeruginosa and protective in vivo, an antimicrobial agent, a gammagi obulin fraction from human blood plasma, and a physiologically acceptable carrier. Pharmaceutical composition according to claim 17, characterized in that the antibody is a human monoclonal antibody and that the gamma globulin fraction of human blood plasma is obtained from individuals exhibiting elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa bacteria and / or products. thereof. A pharmaceutical composition, characterized in that it comprises at least two monoclonal antibodies according to any one of claims 1 to 7, or binding fragments thereof, each antibody or binding fragments thereof each specifically reacting with a different type of Pseudomonas aeruginosa flagellar protein. and is capable of treating 20 or preventing Pseudomonas aeruginosa infections. Pharmaceutical composition according to any one of claims 17 to 19, characterized in that at least one of the monoclonal antibodies is a human monoclonal antibody. 25 A pharmaceutical composition according to any one of claims 17 to 20, characterized in that the composition further comprises at least one human monoclonal antibody capable of reacting with at least one serotypic determinant to an Upopolysaccharide molecule of Pseudomonas 30 aeruginosa and / or a monoclonal antibody reactive with exotoxin A. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof, for use in a method of treating a human susceptible to infection or already infected with Pseudomonas aeruginosa. DK 172840 B1 A cell line, characterized in that it produces a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof capable of specifically reacting with type 5 or type b Pseudomonas aeruginosa flagella. The cell line of claim 23, characterized in that the cell line is a hybrid cell line. The cell line of claim 23 or 24, characterized in that it produces human monoclonal antibodies. Cell line according to any one of claims 23 to 25, characterized in that it has an ATCC accession number selected from the group 15 consisting of HB9129, HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. Sxt for use in detecting the presence of Pseudomonas aeruginosa bacteria, characterized in that it comprises a monoclonal antibody preparation containing a) at least one monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof, or binding fragment thereof, reacts with a type-specific flagellar protein of the bacterium, and b) labels that provide a detectable signal covalently bound to the antibody or bound to other antibodies reactive with each monoclonal antibody. Use of a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof, in the manufacture of a medicament for treating or preventing a Pseudomonas aeruginosa infection in an individual. A method of producing a monoclonal antibody according to any one of claims 1 to 8, or a binding fragment thereof, comprising culturing in at least one of the cell lines of claim 26 and extracting the antibodies. DK 172840 B1 A composition characterized in that it comprises a monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof which is reactive with an epitope capable of binding to a monoclonal antibody produced by the method of claim 29. 30 characterized in that it further comprises a gamma globulin fraction from human blood plasma and / or an antibiotic agent. A monoclonal antibody according to any one of claims 1 to 7, or a binding fragment thereof, characterized in that it is reactive with an epitope capable of binding to a monoclonal antibody produced by the method of claim 29 . 32. A method for determining the presence of Pseudomonas aeruginosa in a sample, characterized by combining the sample with a monoclonal antibody according to any one of claims 1 to 7, 15 or a binding fragment thereof, and demonstrating complex formation.