SE506421C2 - Monoclonal antibodies to Pseudomonas aeruginosa and preparations containing them, test kit containing the preparation, and cell lines expressing the antibodies - Google Patents

Abstract

Cell lines have been produced that secrete monoclonal antibodies which bind to the flagellar proteins of selected Pseudomonas aeruginosa strains. Some of these antibodies have been found to be protective against lethal challenges of P. aeruginosa. Pharmaceutical compositions containing these antibodies, can be used singly or in combination with other monoclonal antibodies, blood plasma fractions and antimicrobial agents, to form prophylactic and therapeutic compositions.

Description

10 15 20 25 30 35 506 421 l - J construction measures and treatment.

One method that has been considered is the strengthening of the host animal immune system by active or passive immunization. Sa for example, it has been found that active immunization of human or experimental animals with bacterial whole cell vaccines or purified bacterial endotoxins from Q. aeruginosa leads to the development of specific opsonic antibodies directed primarily at determinants of the atherosclerotic future oligosaccharide units in the lipopolysaccharide units (LPS) molecules located on the outer cell aeruginosa (see Pollack, M., Immunoglobu- lins: Characteristics and Uses of Intravenous Prepara- Alving, B.M. 79, U.S. Pat. Department of Health and Human Services, 1979). the membrane of E. tions, and Finlayson, J.S., ed., p. 73- Such antibodies, although actively generated, have or transmitted passively, proved to protect against the lethal the effects of an E. aeruginosa infection in a variety and at some preliminary L.5. and Pollack, 119- animal models (Pollack, sugra) studies with humans (gg Young, M., Pseudomonas aeruginosa, Sabath, L., eds. sid. 32, Hans Huber, 1980).

The above reports show that immunotherapeutic methods could be used to prevent and trade bacterial diseases caused by E. aeruginosa, sasom by administration of pooled human immunoglobulins, which contain antibodies to the infecting strain or the infecting strains. Human immunoglobulins is defined in the present context as the part a. fractionated human plasma enriched in antibodies, among which are represented specific antibodies against strains of E. aeruginosa. As a result of some inherent limitations in the use of human immuno- globulin components is still this method of action of diseases caused by E. aeruginosa objects 10 15 20 25 30 35 soaí421 for examination (see, for example, Collins, M.S. and Roby, R.E., Am. J. Med., 70 (3A): 168-174, (1984), and for at present there are no commercial products available; available that utilize these components.

Such a restriction associated with immunoglobulin preparation is that they consist of pools of samples from a thousand number or more donors, in which samples have been selected advance with respect to the presence of special anti-Pseudofl monas antibodies. This pooling leads to an Å mean titers of individual antibody titers, which " which best results in moderate increases in it resulting titer of the desired antibodies.

Another limitation is that the preselection process itself requires expensive continuous "screening" of the donor pool to ensure consistent product quality. Despite these strictures, immunoglobulin products can still show considerable variations from batch to batch and among products from different geographical areas.

Another such limitation that is inherent in immunoglobulin preparations are that their use W results in co-administration of large amounts of avv foreign proteinaceous substances (which may include I viruses such as the viruses that have recently been shown to be associated with acquired immune deficiency syndrome <ê: quired lmmune Deficiencv Syndrome or AIDS), with potential to produce adverse biological effects.

The combination of low titers of desired antibodies and a high content of foreign substances can often limit, to suboptimal levels, the amount of specific and thus beneficial immunoglobulins that can be administered to the patient. Year 197? Hohler and Milstein reported their seminal 10 15 20 25 30 35 506 421 discovered that certain mouse cell lines could be fused (fused) with mouse spleen cells to create hybridomas, which each could secrete antibodies with one even specificity, i.e. monoclonal antibodies (Hohler, G. and Milstein, C., Nature, 256: 495-497 (1975)).

With the advent of this technology, it became possible to i some cases produce large quantities of highly specific murine antibodies to a particular determinant or determinants of antigens. Then it came under use of later developed technology possible to produce human monoclonal antibodies (see, e.g. U.S. 4,464,4ó5, which is incorporated herein by reference by reference thereto).

It is understood that in some situations monoclonal mouse antibodies or preparations of such antibodies can cause problems when used on humans. Sa for example, it has been reported that monoclonal mouse bodies, which have been used in experimental studies for treatment of certain human diseases, can cause one immune responses that make them ineffective (Levy, R.L., and Miller, R.A., Ann. Reef. Med., 34: 107-116 (1983)). With recently advances in recombinant DNA technology, such as preparation of chimeric mouse / human anti-human monoclonal bodies, however, these problems can be eliminated. Methods for the production of human monoclonal antibodies is also now available (see, Human Hybridomas and Monoclonal Antibodies, Engleman, 5.6., Et al., Eds., Plenum Publishing Corp. (1985), which is incorporated herein by reference description by reference thereto).

Using hybridoma and / or cell transformation technology, a number of groups have reported the production of monoclonal antibodies, which protect against E. aeruginosa infections. Monoclonal antibodies have prepared, which are reactive with respect to various 10 15 20 25 30 35 SÛ6421 Ul epitopes of fi. aerugihosa, including specific single-river and multipelserotype vtepitopes. as it is found in 1 :, LPS molecules in bacteria (see for example the American: patent applications 734,624 and 807,394, both of which are incorporated herein by reference live with this description by reference thereto).

Protective monoclonal antibodies have also been prepared. bodies specific for E. agggggggga; ß -exotouin A (see, for example, U.S. Patent Application- 742, 170, which is incorporated herein by reference by reference thereto).

Although the use of monoclonal antibodies, that is specific for the LPS region of E. aergginosa or the exotonins of the bacteria, may provide adequate protection in certain situations, it is in general preferred to have a broader protection option. Said for example it would in prophylactic treatments with regarding potential infections in humans be preferred to administer an antibody or antibodies protective against a diversity E. aeruginosa strains. Likewise, it would at therapeutic applications, where the serotype or the serotypes of the infecting strain and those infecting strains not known (a), may be preferred to administer an antibody or a combination of antibodies, which are effective against most, if not all, of the clinically important E. aeruginosa serotypes. It 7 ideal would be to provide antibodies, which are reactive independently of traditional serotyping scheman.

One aspect of E. aeruginosa physiology that has been shown to be contribute to the virulence of the organism, motility is a stomach derived mainly from the presence of a flagellum Infect. and lmmun., 38: l29b-1298). E. aeruginosa excelsï 10 15 20 25 30 35 506 421 of displaying a single flagellum in one spirit of its made structure. Model studies with fire mice have shown that a larger percentage of mice survived when non- mobile E. aeruginosa strains were inoculated into experimental burns. damage when mobile trunks were used. (McManus, A. et al. (1980), Burns, 6: 235-239 and Nontie. T .. et al. (1982), Infect. and Immun., §§: 1296-1298). change sub- searches regarding the pathogenesis of E. aeruginosa have demonstrated that animals immunized with flagellar antigen preparations, were protected when burned and infected with mobile strains of the bacteria (gg, Holder, I. et al. (1982), Infect. and Immun., 35: 276-280).

What is important is that E. aeruginosa flagella have studied by serological methods and has reported to fall within two main antigenic groups. as H1 and H2 are designated by E. Lanyi (1970, Acta Microbiol.

Acad. Sci. Hung .. 17: 35-48) and type a and type b of Ansorg, R. (1978, Zbl. Bakt. Hyg., I. Abt. Ürig. A, 242: 228-238). Serological typing of flagella of bada laboratories showed that Q; flagella (Lanyi. B., sugra) or type b flagella (Ansorg. R., sugra) var serologically homogeneous, i.e. no subgroups have not identified. This serologically homogeneous flagella type is hereinafter referred to as type b. The second main the antigen, H2 (Lanyi. B .. ggggg) or flagellum of type a (Qnsorg, R., sugra). contained five subgroups. This antigen is hereinafter referred to as type a and flagella the five subgroups as as, a ,, az, as and aa. The five subgroups of type a are expressed in varying combinations ions of different strains of E. aeruginosa. which bears type a, with the exception of the antigen ao. The antigen ao was found on all flagella of type a. although the degree of its expression varied among the strains. väfmê- m I-l 'U TJ Ij | ll | U A serotonicity schedule. which bases 10 15 20 25 30 35 506 421 stable somatic major antigens of E. aeruginosa, referred to as the Habs scheme. which has recently been incorporated with the schedule belonging to the International Antigenic Typing System (see Liu, Int. J. Syst. Bacteriol., 33: 256 (1983).) The flagellar types of the Habs reference strains of fi. aeruginosa has been characterized by immunofluorescence stage with polyclonal sera by R. Ansorg (1978, Zbl.

Baked. Hyg., I. Abt. Orig. A, 242: 228-238) or by 1984, Q.

Clin. Hicrobiol., 20: 84-BB). Habs tribes 2. 3, 4, 5, slide coagglutination (Ansorg, R. et al., ll and 12 ar strains. carrying flag¿s of type¿ b, and Habs strains 1, 6, 8 and 9 carry flagella of type a. A large number of the strains of E. aeruginosa could thus be recognized by a small number of monof Vclonal antibodies, specific for pa flagellar proteins.

Consequently, there is a significant need for monoclo + nal antibodies capable of reacting with epitopes on flagellar proteins and in some cases even with ability to provide protection against multiple serotypes of E. aeruginosa. Furthermore, some of these antibodies would be suitable for use in prophylactic and therapeutic treatment of E. aeruginosa infections as well as in the diagnosis of such infections. Present invention meets these needs.

SUMMARY OF THE INVENTION New cell lines are provided, which can produce monoclonal antibodies capable of binding to flagella present on most strains of E. aeruginosa bacteria. The monoclonal antibodies reacts specifically with epitopes on flagellar proteins in E. aeruginosa and can distinguish between flagella of type a 10 15 w ' 25 30 35 506 421 and type b of the bacteria. Furthermore, one is provided way of treating a person who is exposed to of or already infected with E. aeru inosa, by administration of a prophylactic or therapeutic amount of a formulation containing at least one monoclonal antibody or binding fragment thereof capable of react with the flagella of E. aeruginosa strains, wherein the formulation preferably also comprises a physiological one acceptable carrier. The preparation may also contain one or several of the following: additional monoclonal antibodies capable of reacting with E. aggggiggäa exotoxin A; monoclonal antibodies capable of reacting with serotype determinants on LPS of E. aeruginosa; a gamma- globulin fraction from human blood plasma; a gamma-globulin- fraction from human blood plasma, where the plasma can be obtained from people who show elevated levels of immuno- globulins reactive with E. aeruginosa; and one or several antimicrobial agents. Furthermore, status of diagnostic test kits.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, there is provided new cells capable of producing monoclonal antibodies and preparations comprising such antibodies bodies, which preparations can selectively recognize them aeruginosa- wherein individual antibodies typically sense flagella present on a variety of E. strains, again a type of E. aggggiggga flagella. Present cells have identifiable chromosomes, where microbline DNA from them or a precursor cell has been rearranged for coding of an antibody with a binding site for an epitope pa a flagellar protein, which is common to some E. aeruginosa strains. For flagellar proteins of type a pan-reactive monoclonal antibodies can be prepared; and for type b flagellar proteins, antibodies are included. 10 15 20 25 30 35 506 »421 which are pan-reactive or react with at least about 70% of the flagellar-bearing tribes. These monoclonal anti- 7 bodies can be used in a variety of different ways inside ¥ attan4 the diagnosis and therapy.

The monoclonal antibodies provided thereon methods are especially useful in treatment or prophylaxis of serious diseases caused by &. aeruginosa. aerugi ~ Qgâå are available 4ör direct contact with antibody molecules The surface proteins on the flagella of E. the refrigerators, which would thus probably inhibit the organism and / or substantiate other effects fi which are favorable to the infected vertebrates.

The production of monoclonal antibodies can provide by immortalization of a cell line with ability to express nucleic acid sequences encoding anti- bodies, which are specific to an epitope on them the lagellar proteins of multiple strains of E. aerugi- sniff. The immortalized cell line may be a mammalian cell cell line, which has been transformed by oncogenesis, by trans4ection, mutation or the like. Such cells inside ~ takes myeloma lines, lymph + omelines or other cell lines with the ability to substantiate eupression and excretion of the immunoglobulin or binding fragment thereof LQ vitro. The immunoglobulin or fragment thereof may be a naturally occurring immunoglobulin from another mammals and the preferred mouse or human calves, which produced by transformation of a lymphocyte, in particular an eplenocyte, by means of a virus or by emulation of the lymphocyte with a neoplastic cell. for example, a myeloma, for the production of a hybrid cell line. the splenocyte is obtained from an animal, which Typical have been immunized against flagellar antigens or ¥ fragments: hence containing an eotopic place. immunization ~ 10 15 20 25 30 35 506 421 10 Protocols are well known and can vary considerably but still be effective. (see, Golding, Nonoclonal Antibodies: Principles and PracticgL Academic Press, N.Y. (1983), som incorporated into this description by reference thereto.) The hybrid cell lines can be cloned and screened in in accordance with conventional techniques and antibodies in the the overlying cell fluids can be detected, which have stomach to bind to flagellar E. aeruginosa determinant ter. The appropriate hybrid cell lines in question can then be obtained grow in large-scale culture or injected into the peritoneal tail of a suitable host animal for the production of ascites liquid.

According to one embodiment of the present invention, the cells transformed human lymphocytes, which produce human monoclonal antibodies, preferably protective ig ¿¿¿G, against available epitopes, which are specific to at least one flagellar protein. The lymphocytes can be obtained from human donors, who are exposed to or have exposed to the appropriate flagellar strains questioned by E. aeruginosa. A preferred cell-controlled transport formation process is described in detail in U.S. Pat. 4,4ó4,4ó5, som is hereby incorporated by reference.

By accessing the antibodies of the present invention invention, which are known to be specific to those flagellar proteins, in some cases the overlying ones the liquids in subsequent experiments are subjected to screening in a competition analysis with the present monoclonal anti- bodies as a means of identifying further examples on anti-flagellar monoclonal antibodies. Thus can Hybrid cell lines are easily produced from a variety of sources based on the availability of the present antibodies, which are specific for special flagellar antigens. 10 15 20 25 30 35 _which are known for each animal species. ses, ll When hybrid cell lines are available, which produce anti bodies specific for the present epitopic sites, alternatively, these hybrid cell lines can be fused with other neoplastic B cells, where such B cells can serve as recipients of genomic DNA, which kzdar for especially the receptors. Although neoplastic rodent, Steering wheel B cells are what are commonly used, can other mammalian species are used, such as hardy animals, cattle, father, horse, pig, bird or the like.

The monoclonal antibodies may belong to any one preferably of the immunoglobulin classes or subclasses, such as Ig fi, IgD, Igâ, IgE, or subclasses of IgG, In general, they can the monoclonal antibodies are used intact or as sasom Fv, Fab, F (ab *) 2, binding fragments, but usually show intact.

The cell lines of the present invention may be found use other than for the direct production of the mono-f the clonal antibodies. The cell lines can be fused together other cells (as appropriately drug-labeled Ü human myeloma, mouse myeloma or lymphoblastoid hcman cells) m for the production of hybridomas and thus provided transfer of the genes encoding the anti-i monoclonal the bodies. Alternatively, the cell line can be used as a keel to the chromosomes encoding the immunoglobulins, ä W which can be isolated and transferred to cells by technology other than fusion. Furthermore, the genes that encode the monoclonal antibodies are isolated and used in å in accordance with recombinant DNA technology for the production of f the specific immunoglobulin in a variety of vertebrates. q By preparing CDNA libraries from buddarar RNA can especially a single cDNA clone is isolated, which Lodar for V immunoglobulin and is free of introns, and * is introduced toe » 421 10 15 20 25 30 35 506 421 12 appropriate prokaryotic or eukaryotic expression vectors and then transformed into a host animal for (see generally, U.S. 4,172,124; and 4,423,147. final bulk production 4,350, ó83; 4,363,799; 4.38l, 292; See also, Kennett and Monoclonal Antibodies, Plenum, New York (1980) al., and references therein, all of which incorporated into this description by reference thereto.) In accordance with hybrid DNA technology, more specifically the immunoglobulins or fragments of the present invention invention is prepared in bacteria or yeast. (See, Boss, et al., Nucl. Acid. Res., 12: E791 and Wood et al., Nature 314: 44ó, both of which are incorporated herein by reference by reference thereto.) Thus, for example, -RNA, which is transcribed from the genes that encode them the light and heavy chains of the monoclonal antibodies produced by a cell line of the present invention finning, isolated by differential cDNA hybridization using cDNA from BALB / c lymphocytes other than present clone. mRNQ, which does not hybridize, is rich in the messages encoding the desired immunoglobulin the chains. As needed, this process can be repeated for to further increase the desired mRNA levels. The stripped mRNA preparation can then be transcribed conversely to provide a cDNA mixture, which is enriched with respect to the desired sequences. RNA can then hydrolyzed with an appropriate RNase and ssDNA can double-stranded with DNA polymerase I and random initiators, for example, randomly fragmented calf tv DNA. The the resulting dsDNA can then be cloned by insertion into a suitable vector, for example viral vectors such as lambda pACYCl84. -g fi r vectors or plasmid vectors (such as pBRu¿_. etc.). By developing samples based on known sequences for the constant regions of the light and heavy the chains can be such cDNA clones that contain that gene 10 15 20 25 30 35 5o6§421 which encodes the desired light and heavy chains identi-f by hybridization. Then the genes can excrete fi from the plasmids, are manipulated to remove over-: fluid DNA upstream of the initiation codon or constant region DNQ and then inserted into a suitable vector for 7 transformation of a host cell and finally expression of genes.

Suitably, host mammalian cells (e.g., mouse cells) may be used for machining the chain (for example, joining it heavy and light chains) for the production of an intactf immunoglobulin and further secrete the immunoglobulin freely from the leader sequence. if so desired. Alternatively, one can V use unicellular microorganisms to produce dee two chains, where further manipulation may be required before to remove the DNA sequences that encode it secretarial leader and process signals while de- provides an initiation codon at the 5 'end of the Ä sequence encoding the heavy chain. Get this way can immunoglobulins are prepared and processed so that they can; combined and glycosylated in cells other than mammalian fiber cells. If desired, each of the chains can be truncated said that they maintain at least the variable region, å which can then be manipulated to provide other immunoglobulins or fragments, which are specific for the flagellate epitopes.

The monoclonal antibodies of the present invention are particularly useful because of their specificity * antigens in virtually all of them at present aeruginosa variants. the antibodies are also protective Lg vivo, known E. Some of the monoclonal which allows its incorporation into pharmaceutical products, such as antibody combinations for bacterial infections.

Monoclonal antibodies of the present invention can 10 15 20 25 30 35 506 421 14 also find widespread use in vitro. Said for example the monoclonal antibodies can be used for typing microorganisms. for the isolation of specific E. aeruginosa for selective removal of E. strains, aeruoinosa- cells in a heterogeneous mixture of cells or the like.

For diagnostic purposes, the monoclonal antibodies may either be labeled or unlabeled. Typically includes diagnostic analysis methods detection of the formation of a complex by binding the monoclonal antibody to the flagellum of E. I unmarked aeruginosa organisms. form, the antibodies find use in agglutination -analyzes. Furthermore, unlabeled antibodies can be used in combination with other labeled antibodies (secondary bodies) which are reactive with the monoclonal antibody the body, such as antibodies specific for immunoglobulin.

Alternatively, the monoclonal antibodies can be labeled Immediately. A variety of markers can be used, such as radio nuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. Numerous types of immunoassay methods exist available and by way of example reference is made to the perhaps Patents 3, B17,827, 3,8SO.752. 3,901,654, 3, 35,074, 3, 84, S33, 3,996,345, 4,034,074 and 4,098, B76, all of which are incorporated herein by reference. display in addition.

Usually the monoclonal antibodies according to present invention in enzyme immunoassay. where lying antibodies or secondary antibodies from one another species is conjugated to an enzyme. When a test. as contains E. aeruginosa of a certain serotype, such as human blood or lysed thereof. combined with the present anti- bodies bind between the antibodies and those molecules that exhibit the desired epitope. Dwlika cells can then be separated from unbound reagents 10 15 20 25 30 35 5o @ 421 and a secondary antibody (labeled with an enzyme) put. Thereafter, the presence of the antibody is determined. enzyme-conjugate that is specifically bound to the cell.

Other conventional techniques, which are well known to those skilled in the art. man, can also be exploited.

Test kits can also be provided for use with present antibodies to detect E. aeruginosa in solutions or the presence of flagellar E. ag; gQ¿gg§a-ant¶- gener. Thus, the present monoclonal antibody- Å preparation according to the present invention is provided usually in lyophilized form, either separately or in addition together with additional antibodies specific for with respect to other gram-negative bacteria. The antibodies ,,, which may be conjugated to a marker or be unconjugated, in the test batches of buffer substances, such as Tris, phosphate, carbonate, etc, stabilizers, biocides, inert proteins, for example bovine serum albumin or similar.

In general, these materials are present¿ in an amount less than about 5% by weight, based on the amount of: active antibody, and is usually present in a total amount of at least about 0.001% by weight, calculated on the body concentration. It is often desirable to introduce an inert excipient or excipient for dilution of the active ingredients, wherein the excipient may be present in an amount of from about 1 to 99 weight fl of the total preparation. When a secondary antibody medq able to bind to the monoclonal antibody used des, this is usually present in a separate ampoule. The; the secondary antibody is typically conjugated to a marker and is prepared in a manner analogous to that described above antibody preparations.

The monoclonal antibodies, especially monoclonal human antibodies, according to this invention may also face fi lived as components of pharmaceutical preparations, which 10 15 20 25 30 35 506 421 16 contains a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of this invention A pharmaceutical finning and a pharmaceutically effective carrier. pharmaceutical carrier should be a compatible non-toxic substance, suitable for the administration of the monoclonal antibodies the bodies of the patient. Sterile water. alcohol, fats, waxes and inert solids can be used as carrier. Pharmaceutically acceptable adjuvants (buffer agents, dispersants) can also be incorporated into it the pharmaceutical preparation. Such preparations may contain hold a single monoclonal antibody specific for strains of a flagellar type of E. aeruginosa. Alternatively a pharmaceutical preparation may contain two or more monoclonal antibodies to form a "cocktail". Sa for example, is a cocktail, which contains monoclonal antibodies against both types of flagella or against groups of the various E. aeruginosa strains (e.g., e.g. different serotypes), a universal product with activity against the vast majority of its clinical isolates special bacteria.

The molar ratio between the different monoclonal antibody compounds the components usually do not differ by more than one factor 10, more commonly not by more than one factor 5, and mol- > _ \ the ratio is usually about 1: 1-e with respect to de the other antibody components.

The monoclonal antibodies of the present invention finning can also be used in combination with existing blood oil products. as commercially available gamma globulin and immunoglobulin products. which was used in the prophylactic or therapeutic treatment of E. aeruginosa diseases in humans. For immunoglobuli- The plasma is preferably obtained from human donors, 1 which show elevated levels of immunoglobulins, which are reactive with respect to Q. aeruginosa. (Too general 10 15 20 25 30 35 soe 421 17 É _ U “Intravenous Immunef With., information is referred to the compendium Globulin and the Compromised Host, "Amer. J. 7ó (3a), 1984, March 30, p. 1-23, the contents of which are live with this description by reference thereto.) The present monoclonal antibodies can be used as separately administered preparations, which are added together antibiotics or antimicrobials. Typically, they can antimicrobial agents include an anti-pseudomonal penicillin (for example carbenicillin) together with an amino glycoside (for example gentamycin, tobramycin, et:.>, but numerous additional agents (e.g. cephalosporins), which are well known to those skilled in the art can also be used.

The monoclonal antibodies and pharmaceutical preparations the uses thereof of this invention are particularly reversible for oral or parenteral administration. Before- In addition, the pharmaceutical preparations may be administered race barenterally, i.e. subcutaneously, intramuscularly or intravenously. Thus, this invention provides preparations for parenteral administration, which contain take a solution of the monoclonal antibody or a cocktail thereof dissolved in an acceptable carrier. preferred show an aqueous grain. A variety of aqueous carrier can be used. for example water, buffered water, 0.2 saline, 0.32 glycine and the like. These solutions are sterile and generally free of particulate matter shaped material. The preparations can be sterilized by conventional well-known sterilization techniques. These preparations may contain such pharmaceutically acceptable excipients required to approximate physiological conditions, such as pH adjusting agents and buffer agents, tomicity adjusting agents and the like, e.g. show sodium acetate, sodium chloride. potassium chloride. calcium- chloride, sodium lactate, etc. the concentration of antibody in these preparations can vary within wide limits, i.e. i 10 15 20 25 30 35 506 421 18 weight, usually at least about 1 weight. from less than about 0.5 weight to as much as 15 or 20 and the choice of which it is based primarily on liquid volumes. viscosities etc., depending on the mode of administration chosen.

Thus, a typical pharmaceutical preparation for intravenous muscular injection is prepared containing 1 ml of sterile buffered water and 50 mg monoclonal antibody. A typical preparation for intravenous infusion can be prepared by 250 ml of sterile Ringer's solution and 150 mg of mono- clonal antibody. Current methods for producing parenterally administrable preparations are known or obvious to the person skilled in the art and described in more detail Pharmaceutical Science, 15th for example in Reminqton's _uppl., Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference. display in addition.

The monoclonal antibodies of this invention may lyophilized for storage and reconstituted in an appropriate carrier before use. This technology has proven to be effective with conventional immunoglobulins and within technology known lyophilization and reconstitution technology Can be used. It is obvious to those skilled in the art that lyophilization and reconstitution can lead to varying degrees loss of antibody activity (for example with conventional tional immunoglobulins, IgM antibodies tend to show greater loss of activity than Igü antibodies) and for compensation that the levels of use must be adjusted hence.

The formulations containing the present monoclonal antibodies or a cocktail thereof, may be administered for prophylactic and / or therapeutic treatment of admini- E. aeruginosa infections. For therapeutic use the preparations are transferred to a patient who already has 10 15 20 25 30 so§í421 19 infected with one or more E. aeruginosa serotypes, kg an amount sufficient to cure or at least partially prevent the infection and its complications. One amount. adequate to achieve this, 5 defined as a "therapeutically effective dose". Quantities, which are effective for this purpose, depend on the infections degree of liability and the general condition of the patient's own immune system but generally varies kilo body weight ,; about 1 to about 200 mg of antibody per with doses of 5-25 mg per kilogram usually used. Nan must keep in mind that the materials according to this invention can generally be used for serious disease condition, i.e. life-threatening or potentially life-threatening the situations, especially bacteremia and endotonemia to followed by E. aeruginosa.

In prophylactic applications, the preparation is administered containing the present antibody or a cocktail thereof, to a patient who does not already have by E. aeruginosa in order to enhance the patient's resistance to such a potential infection. And then 7 amount is defined as a "prophylactically effective dose". this use also depends here on the exact amounts of pa the patient's state of health and general immunity levels generally ranges from 0.1 to 25 mg / kg, especially V 0.5-2.5 mg / kilo.

Single or multiple administrations of the preparations can be performed with dose levels and dosing patterns as required sued by the attending physician. Under all circumstancesjwm the pharmaceutical preparations should provide an amount of the antibody or antibodies thereof invention sufficient for effective treatment or prophylaxis of the patient. 10 15 20 25 30 506 421 DISCLAIMER EXAMPLE 1 Example 1 disables the methodology used produce a monoclonal antibody from rat animals. which binds specifically to E. aeruginosa flagella.

Three-month-old BALB / c mice were immunized intraperitoneally. eight times with viable E. aeruginosa Fisher immunotype 1 and Fisher immunotype 2 (A.T.C.C.% 27312 and # 273l3) every two weeks for a total of nine weeks. The initial doses of the bacteria were 8 N 10 * and 1,107 organisms per mouse of E. aeruginosa Fisher immunotype 1 and Fisher tiil ao-faiaigt immunotype 2 and the dose was increased ZO- during the course of immunization.

Three days after the last injection, the spleen was removed from a mouse aseptically and a single cell suspension was made by careful rotation of the organ between them matte ends of two sterile slides. Enkarniga spleen cells were combined in a ratio of 4: 1 with (NSI-1, obtained from Dr. C.

England) mouse myeloma log phase cells Milstein. Molecular Research Council. Cambridge. and was infused to produce hybridoma according to the procedure + arande described by Tam et al. (1? 82, Infect. lmmun., 3rd: 1042-1053). The final hybrid cell suspension the ion was spat on to a concentration of 1.5 u 10 ° cells per ml in RPMI-hybrid HAT (RPMI 1640 (Gibco, Grand Island, NY) containing 1-2 heat-inactivated calf ¥ ostereerum, 1 mM sodium tested, lüüpg / ml penicillin and strepto- mycin, 1.0 r lO ** M hypouantin. 4.0 10 "? M aminopterin and l.o 10 "(M tvmidin)., which included 2.0 x 10 ° per 10 15 20 25 30 35 so @ §421 ml of freshly prepared BALB / c thymocytes as feeder cells.

The landing was spread (200 ul per compartment) on plates with 96¿W compartment (# 3596, Costar, Cambridge, MA). The hultures were fed by removing and replacing 50% of the volume in each compartment with fresh RPMI-hybrid-HAT every other or every_¿lf third day. overlying culture fluids were analyzed with \ aeruginosa antibodies 0 when ceiiš regarding the presence of anti- E. by enzyme-linked immunosorbent assay in the compartments, the plant nade night about 40% confluence van-V within 7-10 days.

The overlying culture fluids from the hybrid cells were analyzed simultaneously on outer membrane preparations from var and one of the two immunizing bacteria. Outer membrane fi preparations were isolated by a modification of the method: as reported by Tam et al. (1982. Infect. Immun., 3b: 1042-1053), which is incorporated herein by reference The bacteria aeruginosa Fisher- reference thereto. (E. immunotype 1 and Fisher immunotype 2) were inoculated into trypticase soy culture medium (TSBV and allowed to grow lb-18 hours at Q) 34 ° C during aeration in a rotary shaker bath. Bacterial? was harvested by centrifugation and washed twice with phosphate buffered saline (PBS, 0.14 M NaCl. 3 mh KCl, 8 mM Na 2 HPO 4 -7 H 2 O, 1.5 m ISO 'trypsin inhibitors (T.I.U.) contained aprotinin in per ml (Sigma, St. Louis, M0).

The pellet from the final centrifugation is resuspended. was dissolved in 0.17 M triethanolamine, 20 mM disodium ethylene (EDTA) and was thus homogenized -diamine tetraacetic acid after on ice for 10 minutes. The cell fragment pelleteradee from the homogenate at 14,900 x g and discarded the supernatant was centrifuged using as above.

The pellet was discarded anyo and the membranes were oelletized from the supernatant by centrifugation at 10 15 20 25 30 35 so6 421 l40,000 x g in one hour. The supernatant was discarded and the membrane pellets were stored overnight at 4 ° C in 10 ml PBS, ml. The next day, the pellets were resuspended by vortexing. which contained 75 T.I.U. aprotinin per formation and then aliquoted and stored at -70 ° C. The protein content of each pellet was determined by the method set forth by Lowry et al. (1951, Q.

Eiol. Chem., 193: 20-25-275). the antigen plates for ELISA were prepared as follows.

The outer membrane preparations were diluted to 5 pg per ml of protein in PBS and 50 μl of the solutions were spread in each compartment on one plate with 96 compartments (Linbro # 76-031-05, Flow Laboratories, Inc., McLean, VA), was sealed and incubated over at night at 37 ° C. Unbound antigen was removed from the plate. 100 ul 5% bovine serum albumin (BSA) i and (weight / volume) PBS was added to each compartment and the plates were incubated for one hour at Z7 ° C.

After removal of unabsorbed BSA, the replicate was plated supernatants (SO2) from each compartment of the fusion in the corresponding compartments on the antigen plates and 37 ° C. the plates was incubated for 30 minutes with the unbound antibody was removed from the compartments and the plates were washed three times times by 100 μl 1% (w / v) BSQ-PBS per compartment. Where- after, 50 μl per compartment was diluted appropriately Inc .. Burlingame, Vi d biotin-derived goat anti-mouse IgU (Tago, CH) 17 ° C. above and then added to the compartments 50ul of a pre- to each compartment and incubated for 30 minutes a The plates were washed three times as described avidin: biotinylated horseradish peronidase (Vectastain ABC Hit, Vector Laboratories. Inc., CQ), -complex Eurlingame, which had been produced in the manufacturer's specifications. After 30 minutes at room temperature The Vectastain reagent was removed from the compartments, the compartments were washed as above and then 100 were added 10 15 BBQ lf J W m1 per compartment of substrate, o-phenylenediamine (0.8 mg / ml i 0.1 M citrate buffer, pH 5.0, mixed with an equal volume 0.03 volume H2D =). minutes at room temperature in the dark and the reactions was then broken by adding SO p1 EN H2 SO4 per compartment.

Hybridoma cells secreting monoclonal antibodies . \ :, a. which bind to either of the two antigen preparations, l was located by measuring the absorbance at 490 nm avà rr the colorimetric reactions 1 each with one "Dynatech Model 580 NicroELlSA reader" (Alexandria, VA), å ä The cells in antibody, was examined in more detail sasom -immunotype 2. This compartment described below.

The substrate was incubated during Z fi š a compartment with the designation Pa3 IVC2 produced which bound only to the antigen plate with Fishefá 10 15 20 25 30 35 506 421 24 The monoclonal antibody and the clonal cell line from this compartment both are identified by the designation Paï IVC2 in the following text. Paš IVC2 cells from the mother compartment minicloned and cloned by the spruce bed techniques described by Tam et al. (1982, lnfect.

Immun., 36: 1042-1053). which contains monoclonal (BÅLB / C Askitesvatska. antibody with high titer, was produced on CBb F; mice (female mice) C57BL / 6 (male mice> F1) according to the procedure described by Tam et al. (1982, Infect. Immun., 36: 1ü42- 1053 Male to two to three months old male CBb F received injections intraperitoneally with 0.5 ml of pristan (2. 6, 10. 14-Tetramethylpentadecane. Aldrich Chemical Co ..

Milwaukee, NI) 10-21 days before intraoeritoneal injection with log-phase Paš IVC2 cells in RPMI. Each mouse received one injection of 0.5-1 107 cells in 0.5 ml. After about two weeks, the accumulated fluid was removed from the mice every other or every third day. The female concentration of anti- body in the ascites fluid was determined by agarose gel Beckman Instruments, Inc .. Brea, EA) trophes (Paragon. and all ascites containing 5 mg / ml or more antibody was pooled, aliquoted and frozen at -70 ° C.

Characterization of the molecular object template bound by the monoclonal antibody supernatant from the cloned Pai IVC2 cell the line was analyzed by ELISA as described above water membrane preparations from all seven Fisher immuno- aeruginosa (A.T.C.C. 27312-2? Z1B). E. aureofaciens (A.T.C.C. 13? 85) (A.T.C.C. 8047). tvp stems of E. and bs lebsiella pneumoniae all prepared as above. Anti- kroooen Pai IVCI band to the water membrane preparations of 6 and 7 of E. aeruginoea but not to the other Fisher immotvperna, E. agg§gi¿; ¿§Q§. or Fisher immunotypes 2.

Q. gneumoniae. 10 15 20 25 30 35 , Fisher immunotypes 2, _, 506 to 421 The specific antigen identified by the antibody Pa3 IVC2 was identified by radioimmunoprecipitation-Å » In short, this method of analysis involves incubating radiolabeled antigens with the antibody Paï IVC2 and a special source of protein A, resulting in the formation of insoluble antibody: antigen complex. These complexes are washed to remove any non-specific bound antigen and then the complexes are dissociated and separated in a polyacrylamide gel. The dominant radioactive substances found in the gel are identified thereby as the corresponding antigen or antigens to which antibody or antibody Paï IVC2 binds.

Aliquots (25 pg) of soluble outer membrane preparations from -v ~ l 4 and 5 of E. aeruoinosa labeled radioactive in solid phase with 1251 using W of Iodo gene (Pierce Chemical Co., Rockford, IL) (Fraker and Speck, 1978, Biochem. Bioghys. Res. Commun., 80: 84? -L , 857; Markwell and Fox, 1978, Biochemistry, 17: 4807-4817) i This procedure resulted in iodination of exposed tyrosinate residues of most, if not all, proteins ingested in the outer membrane preparation.

To reduce non-specific binding of outer membrane antigen gene for the antibody Paš IVC2 was first incubated (5 x radiolabeled preparations 10 * pulses per minute Analyze) for one hour at 4 ° C with normal BALB / c serum (final dilution 1:40). overlying Paš IVC2 culture f containing the antibody Pa3 IVC2 was added After liquid (0.5 ml) then to each of the outer membrane samples. incubation of antigen and antibody for one hour at At 4 ° C, the source was added to protein A, IgG50RB (ü.Û95 ml per sample) (The Enzyme Center, Inc., Boston, MA) and was incubated for an additional 30 minutes at 4 ° C (fi essler, {> S.W., 1? 75, J. Immunol., 115: 1bl7-1622). IgGSORB was set according to the manufacturer's specifications and soon before use, non-specific reactions were performed 10 15 20 25 30 35 for ten minutes and recovered in 506 421 26 by blocking potentially reactive sites with culture medium by washing IgG5DRB twice with RPMI hybrid HATRED).

(RPMI hybrid HAT medium excluding The antigen-antibody-IQBSDRB complexes were pelleted at 1500 x g for ten minutes at 4 ° C, washed twice with phosphate-RIPA buffer (10 mM phosphate, pH 7.2, 0.15 M NaCl, 1.0 v / vMZ Triton X-100, 1.02 (w / v) sodium deoxycholate, 0.12 (w / v) sodium dodecyl sulfate (SDS) and 1.0 volume of aprotinin); two times with high salt buffer 1 volvml beta- -mercaptoethanol; (0.02 M Iris-Hcl, and once with lys buffer 0.5 M Naßl, 1979, pH 7.5, 0.05 volume Nonidet P-40) (Rohrschneider et al., Proc. Natl. Acad.

U.S.A., 76: 4479-4483. olemet, (0.125 M Tris-HCl, Sci., Antigen, which was bound to com- was released by incubation with agitation buffer pH 6.8, 2% SDS, 2 vol at 95 ° C (weight / volume) beta-mercaptoethanol and 20 volumes of glycerol) the overlying one the liquid after centrifugation at 1500 x g for ten minutes.

The overlying liquid orbs were then apolizated on 14% polyacrylamide gels, which contained SDS and had produced set by (1978, FEBS Lett., (1? 79. J. Bacteriol., the method specified by B. Lugtenberg et 5B: 254-258) 140: 902-? 10). al. modified by Hancock and Carey as an infer- live with this description by male reference dartíll) gel by electrophoresis After vinegar and the antigens were secreted in overnight at a constant voltage of 50V. fixation of the gel in 40 volmk methanol, 10 volmmï svra and 5 volvmï glvcerol overnight it was dried on Whatman 3 MM oaoper via a Biorad gel tower (Richmond. CA).

The dried gel was covered with unloaded film and exoned for Kodak X-AR film for 18 hours at room temperature.

The results of this experiment showed that Paš IVC2 bound to 10 15 _20 25 30 35 5oe 421 27 only one antigen in the outer membrane preparation of only F * - Fisher immunotype s of E. aeruginosa and not to anything other antigen present in the other outer membrane preparations rates. The molecular weight (MV) of the antigen in the gel was approximately 53000 daltons, when determined by comparison of its _ mobility with the mobility of ** C-labeled protein standard eggs MV 92500; BSA, MV 69000: E NV 3013043; (phosphorylase B. ovalbumin, MV 46000; 12000) (New England Nuclear, carbonic anhydrase, cytochrome C, MV Boston, MA), which was separated in the same gel. The molecular weight of this antigen correlated with the molecular weight of flagellin, aeruginosa, (1982, Infect. be incorporated into this description by reference thereto. the protein that makes up W the flagella of E. sasom have been reported by¿ Montie et al. lmmun., 35: 281-288), as inward Furthermore, Paß IVC2 was examined by ELISA use of Habs strains 1-12 of E. aeruginosa; 96-compartment microtiter plates. which were +xerted with ethanol pa The antigen plates are prepared were as follows. -Ä Cultures of each organism, which had taken hold ' at night. pelleted, washed twice with PBS and was resuspended in PBS to an Aaèa value of 0.2 0.D. units. The secreted bacteria were plated in åachv (50 ul per compartment) and then centrifuged at PBS to the unions 1500 x g for 15 minutes at room temperature. (95%) After was aerated and then ethanol was added for 15 minutes at room temperature. the ethanol had been removed + the compartments were air dried the plates and then thanked and stored at 4 ° C until they were to be used.

The results of the ELISA test, performed as described above. showed that Paï IVC2 bound to the ethanol4ixated Habs strains 2, 3, 4, S, 7, 10. 11 and 12. This speci- ficity patterns suggested that Paä IVCE tied to 1978.

E. aeruginosa- + 1age1lerna type b (Ansorg, R., 10 15 20 25 30 35 506 421 28 Zbl. Baked. Hyg .. I. Abt. Orig. A, 242: 22 -238: Ansorg, R. et al., 1984, J. Clin. Microbiol .. 20: 84-BB. which both are incorporated into this description by reference thereto). Based on this specification determination with with respect to pa monoclonal PaI IVC2 carries E. aeruginosa- reference strains Fisher immunotype 2. Fisher immunotype 6 and Fisher ~ immunotype 7 type b flagella. Of the foregoing experimental data, one can conclude that Pa? IVC2 binds specifically to E. aggggiggâg flagellin type b.

IN VIVO ACTIVITY BY Päï IVC2 Animal experiments were performed to determine whether the monoclonal the antibody Paš IVC2 would protect a mouse, which had exposed to multiple LD = ° doses of live the model was the model me., 1983,. g.

E. aeruginosa bacteria. The selected M.S., Trauma, 23: 530-534, which is incorporated herein by reference with burnt mice (Collins. and Roby, by reference thereto). Groups of mice were exposed a serious burn according to the authors' protocol and was then immediately exposed to 5-10 LD = ° doses of Fisher Immunothio 7. Monoclonal antibody was administered intraperitoneally as high titerarchitis (0.2 ml intraperitoneally nealt) before causing burns and infection.

No increase in the number of survivors was found in Paš IVC2-treated animals compared to those not received any antibody.

EXAMPLE 2 Examples show the methodology for preparation ax one rat animal hybridoma cell line. which produces a monoclonal wheeled antibody to type b E. aeruginosa flagellin. which protects ig vivo.

Female adult BALB / c mice first received injections intraperitoneally with viable E. aeruginosa Fisher (ATCC No. 27317) (W x followed -immune type g 10 * organisms). 10 15 20 25 30 35 '29 two weeks later by an injection of viable E. aeruginosa Fisher immunotype 5 (ATCC No. 27316) (4 x During the following two-week aeruginosa Fisher- lü * organisms). the period was administered vigorously E. immunotype 5 and Fisher immunotype 6 together in two indes injections per week. The dosage of each organism was increased said that the final dose was ten times greater than the W initial dosage. A final injection of outer membrane ~ preparation of E. aeruginosa Fisher immunotype 6 (50 ug protein), which had been prepared according to the method used and H. (1978, 1). was given four days after it stated by R.E.N. Hancock Nikaido sacteriui., 13e = 3e1-390), last the injection of viable bacteria. Three days later the last immunization, the spleen was removed from a mouse and the spleen cells were prepared for hybridization as well described in Example 1. r overlying culture fluids from the analyte-H hybridoma cells were observed with respect to the presence of anti-E. aeruginosa- antibodies by ELISA on the tenth day after fusion according to the procedures set forth in Example 1, with except that the antigen for the ELISA plates was viable. strong bacteria, which had been immobilized in the compartments to the 96-compartment microtiter plates. The plates were prepared that follows.

Fifty microliters of poly-L-lysine (PLL) (1 μg / ml in PBS) (Sigma # P-1524, St. Louis, MO) was added to each compartment in 96-compartment plates (Linbro) and incubated for 30 minutes at room temperature. Non-adsorbed PLL removed nadee and the trays were washed three times with PBS. Bacteria-V cultures, which had been allowed to grow overnight in TSB, were washed once with PBS and then resuspended in PBS to one D.D.ase nm value of 0.2. Fifty microliters of the bacterial suspensions were added to each compartment of the plate and allowed to bind at 37 ° C for one hour. Unbound back-I terries were removed and then the trays three were washed times with brine- Tween (0.9Z (w / v) NaCl, sne 421 10 15 20 25 30 35 506 421 “M 0.05 volume Z Tween-20).

Non-specific binding of the antibodies was blocked by addition of 200 μl / compartment of blocking buffer (PBS) fat free Louis, MO) containing 5% (w / v) (Sigma, St. powdered milk, 0.01 volume Z Antifoam A and 0.01% (weight / volume) thimerosal) to the compartments and incubation below one hour at room temperature. excess blocking buffer was removed and the compartments were washed three times with saline-Tween as described above. supernatants (50 μl) were replicated in corresponding compartments on the assay plates and incubated at room temperature for 30 minutes. The overlying cultivars the liquids were removed and the compartments were washed five times saline-Tween.

An enzyme-conjugated second-stage antibody (horseradish peroxygen) das-conjugated goat anti-mouse IgS + IgM) (Tago, Inc., Burlingame, CA) was diluted in PBS containing 0.1 v / v Tween-20 and 0.22 (w / v) BSA as before determined titrations and then 50 μl was set off reagents to each compartment and incubation was performed minutes at room temperature. the excess reagent is removed nades; the compartments were washed five times with brine-Tween; and 100 μl of o-phenylenediamine substrate per compartment was added and incubation took place for 30 minutes as described in Example 1. The reactions were stopped as indicated in Example 1 and then reading took place at A490 nm on a Bio-Tek EL-310 Automated EIA Flate Reader.

Using the methods described above, the overlying the culture fluids from the merger with respect to pa as bound to E. aeruginosa but not to the presence of antibodies, -u Fisher immunotypes 1, 2. o or 4, the control plates. which had been produced with the same PLL and blocking process but without bacteria. overlying which bond to any of liquids. which contained antibody, 10 15 20 25 30 35 31 soag421 these four Fisher immunotypes, a second gan was analyzed. using each of the seven Fisher immunotypes Antibody present in the over- ll PaF4 IVE8 tape only 6 and 7. the bacteria separately. lying liquid from a compartment, to e.

Cells from the FaF4 IVEB compartment were cloned by border diluent. aeruginosa Fisher immunotypes 2, methods as described in Example 1. The monof clonal antibody and the clonal cell line from this Å compartments are identified both by the designation PaF4 IVE8 in foal-V jande text. Askitesvatska containing high titer of monof clonal antibody was produced as described in Example I except that BALB / c mice were used instead CBóF ;.

SPECIFICITY OF E_E4 IVEB An assay, performed to identify the antigen bound by the monoclonal antibody PaF4 IVE8, var indirect immunofluorescence in bacterial organisms. Be ooh one of the seven Fisher reference immunotypes of E. aeruoin nose plus a non-flagellar strain of E. aeruginosa (PAIOE, _ :: A.T.C.C. 29260, Leifson, 1951, J. Bacteriol., 62: 377-38%) and Esgner-ichia eggs 37 ° E and TSB. The bacteria were pelleted by centrifugation and then washed twice in PBS. grow overnight at Each strain was resuspended in PBS to a 0.D.a. ° nm value avi P- Fl 32 fy: The bacterial suspensions were then further diluted in slope 1: 150 and orover of 20 ul were placed in the indifg video compartments on Carlson slides (Carlson Scientificé Inc., Peotone. IL) and dried on the slide at 4Ü'C¿ supernatants (25 μl) of PaF4 IVE8 incubated row on the dried bacterial samples on the slides in one humidity chamber at room temperature for 30 minutes. in Unbound antibody was washed away from the slides by dipping these in distilled water. fa 10 15 20 25 30 35 506 421 After the slides were dried, they were incubated (FITC) -conjugated goat anti-mouse PBS) fluorescein isothiocyanate -Igß + IgM (25 ul per compartment i CA) a spade of 1:40 i (Tago, Burlingame. On slides for 30 minutes at room temperature in a humid chamber in the dark. The slides were washed anyo in distilled water and was dried and then thanked with a tack glass.

PBS (9: 1).

AS mounted with glycerol in the slides was then studied under a fluorescence microscope.

Fluorescence staining was observed only on Fisher immunoassays. types 2, 6 and 7 of E. aeruoinosa and were found be a sinusoidal pattern (line). which was based on only one spirit of the organisms. This Year in accordance origin with the morphology of and its location single polar flagella of these bacteria.

The reaction between PaF4 IVE8 and flagella was confirmed by immunoblotting analysis. Outer membrane antigens from _E. aeruginosa Fisher immunotype 6 (see Example 1) separated by electrophoresis in a 14% polyacrylamide gel, which contained SDS. as described in Example 1. with except that the electrophoresis was run for five hours at a constant current of SO mëmp. Pre-colored molecular weight markers (lysozyme, MV 14300; beta-lactog MV 18400; bumin. MV 43000; B, MV 97400; MD) alpha-chymotrypsinogen. M 25700: oval- MV 58000: (BR-L, bulin, bovine serum albumin. phosphorylated and myosin. MV 200000 Gaithersburg, was incorporated with the same polyacrylamide gel.

Antigens were transferred from the polyacrylamide gel to a nitrocellulose membrane, (NCM). (0.45 um. Schleicher & Schuell. Keen. NH) 1 (1979), and Tris-glycine-methanol buffer Natl. Acad. U.5.A., SDB, Inc ..

(Towbin et al. Proc. Sci. 7ó: 4350-4354). containing 0.052 (w / v) above overnight at 4 ° C at a constant current of 200 mA.

After the transfer, NCM was incubated in 0.05 volm of Tween-20 in PBS E .. et 1982, Q.

(PBS-Tweeny (Batteiger, 10 15 20 25 30 35 506 421 Immunol. Meth., 55: 297-307) for one hour at room temperature. For this step and all subsequent steps the tray containing NCM was placed on a shaker plate. form to take care of the distribution of the solution over the wholeww NCM.

After one hour, the PBS-Tween solution and PaF4 were poured off IVEB ascites (diluted 1: 1000 in PBS-Tween) incubated with NCM for one hour at room temperature. was added and NCM was then washed five times, five minutes each gang, with PBS-Tween to remove unbound antibody. Alkaline phosphatase-conjugated goat anti-mouse IgG + 1gM specifications and incubated with NCM for one hour (Tago, Inc.) was diluted according to the manufacturer at room temperature. NCM was washed five times as usual »Described above and a substrate containing bromochloro-g indolyl phosphate and nitroblatt tetrazolium (Sigma.

St. Louis, MD) and had been prepared as described by Leary et al. (1983, Natl. Acad. U.S.A., eo-4045-4049), Froc. Sci., was added and incubated for 10-20 minutes sub- at room temperature. The reaction was stopped by the filtrate was washed with distilled water. showed that PaF4 IVEB band specifically to a single antigen with a molecular weight of The results of this experiment 53000 daltons in the outer membrane preparation. The results of the_ indirect immunofluorescence analysis and immunoblotting shows that PaF4 IVES binds to the flagella of E. aerugi- HDSo.

The type of flag that PaF4 IVEB recognized was determined by ELISA. Habs strains 1-12 (A.T.C.C. # 33348-Ešïíë) bands each to the compartments in microtiter plates of 55 compartments (Linbro) with PLL and ELISA was performed as described earlier in this example. The source of the antibody PaF4 g \ IVES was the supernatant. Positive reactions: which contained the Habs tribes E, 3. 4, indicating that PaF4 IVEE binds found in trades, 5, 7, 10, 11 and 1 :, 10 15 20 25 30 35 5oeg421 55-4 to type b flagella. Protection data determined LQ vivo is shown below in Example 4.

EXAMPLE 3 Example 3 shows the methodology for preparing a steering wheel. animal hybridoma cell line, which produces the monoclonal antibody which is reactive with anti-E. aeruginosa-fla- gels of type a and protects Lg vivo.

The lymphoid cell source for the fusion was a spleen from one immunized BALB / c mouse that had received injections four times intraperitoneally over a six-week period of purified flagella type A from Habe- (â.T.C.C. (10-20 pg protein) 333353 and 33355). strains 6 and B The flagella were purified according to the method indicated by T.C. (1982, 35: 28-228, incorporated into this description by reference Montie et al. Infect. Immun., Som in addition) except that the final spin one of the flagella was performed at 100,000 x g for one hour and not at 40,000 x g for three hours. A second modification tion made in certain proceedings was that the flagella from the bacteria were subjected to shear for 30 seconds in a mixer instead of under three 1985, Infect. lmmun .. 49: 770- minutes. (Allison et al. 774).

The protein concentrations of each preparation were determined by (Bio-Rad. Richmond. CA) (LFS) Bio-Rad Protein Analysis and the presence of contaminating lipopolysaccharide was measured by measuring the HDD content (Harkhanis, Y.D .. et 85: 595-601).

Anal. Biochem., Molecular Weights al .. 1978, of the flagellar proteins were determined by comparing an SDS-polyacrylamide gel with the migration Molecular- their migration in in protein standard markers (BRLs) (see Example I). the weight for Habs e-flagellin was 51700 daltons and for Habs E-flagellin 47200 dalton. These values agree with those obtained by J.S. Alllison et al. (1985. 10 15 20 25 30 35 49: 770-774, which is incorporated herein by reference writing by reference thereto).

Infect.Immun., Fusion of splenocytes from flagellin-immunized mice P and NS-1 myeloma cells were performed three days after the last one the immunization as described in Examples 1 and 2. When the hybridoma cells grew to a confluence of about 40% (day 7) replicated overlying culture fluids in corresponding compartments of three different antigen plates. PLL-bound T E. aeruginosa Fisher immunotype 1 (see Example 2 for prepaf formalin-fixed Habs 6 and Habs 9.

The bacteria for the formalin-fixed antigen plates fic fi u grow. and antigen plates. Diluted bacteria at A, °) was added to the individual compartments (50 μl per compartments) at Linbro microtiter plates with 96 compartments and q the plates were then centrifuged at 1200 x g for 20 h minutes at room temperature. The supernatants of? was removed from the compartments and 75 μl of 0.2 v / v 2 formalin in; PÉS was added to each compartment and incubated for 15 hours minutes at room temperature. After the formalin had removed from the compartments, the plates were air-dried and stored at 4 ° C prior to use. Formalin did not change the antigenicity of the flagella, as evidenced by ability of anti-flagellar antisera to agglutinate formalin-treated organisms (Lanyi. B .. 1970, äcta Hicrobiol. Acad. Sci., Hung .. 17: 35-48). Strains E. aeru¶, giggäa Fisher-: mmunotype 1 was incorporated as a control after as stated by V Microbiol .. 1985, Soc. Wash., D.C., p. the compartment designated Fàé IIG5 as this strain was non-flagellar, Manual of Clin.

Lennette. red. ñmer. Microbiol .. 1099). Hvbrid cells in produced an antibody, which bound to Habs 6 and Habs Å (both strains bearing flagella of type a) but not to, Fisher immunotype 1.

Cells from fack_FAó IIG5 were subjected to subculture and 506 421 was washed and diluted as described for PLL bundw (0.2 cam units 10 15 20 25 35 36 was cloned as described in the previous example. The mono- clonal antibody and the clonal cell line thereof compartments are identified both by the designation Fâó IIE5 in the following the text. Askites were produced in BALB / c mice as described in joke in Example 2.

Specificity of FA6 IIG5 The specificity of the antibody F 2 IIE5 was determined by indirect immunofluorescence and immunoblotting. Indirectly immunofluorescence was performed essentially as described in Example 2 with the following modifications.

Bacterial cultures. which had been allowed to grow overnight on trypticase-soy agar at 30 ° C, was removed from the plates _with cotton tops and resuspended in PBS to one 9660 value of 0.2 0.D. units. Formalin (0.37 volvmï final concentration in PBS) was added to the suspension below vortex formation. After incubation at room temperature below For 15 minutes, the bacteria were diluted in a ratio of 1:12 PBS and EH ul of this suspension were placed in the individual dual compartments on Carlson slides. After drying, prepare the slides were subjected to microscopy as described in Example 2. The source of antibody was overlying culture fluid from the Fêé IIG5 cell line.

Fluorescent staining of the Fâó IIG5 antibody only with E. aeruginosa strains bearing flagella of type a but not with any of those who received type b. The observed fluorescence pattern was a sinusoidal format line pattern. which showed that Fâb IIG5 tied to the flagella. The fluorescence signal was amplified by action of the bacteria with formalin but the treatment was not required to visualize the flagellar the staining with the antibody. Example 2.

Immunoblotting was performed as described in a hall to flagella type a antigens where they were purified 10 15 go 25 30 35 506 421 37 flagellar preparations (see this example). äntigenen separated in 10% polyacrylamide gels containing SDB (Laemmli. U.K., 1970, Nature 227: óBO-685) and transferred to an NCM. Preparation of Fëó 1155, supernatant or ascites in the spade (London), either 1: 1000, was allowed to react with NCM and the reaction was shown medi a suitable enzyme-conjugated reagent and enzyme substrate 1 as described in Example 2. The immunoblotting assay showed that Fab 1165 path spe = 1 + 1kt to fiageiiin etc. 51700 av Habs 6 and flagellin MV 47200 by Habs B.

Confirmation that Fâb IIG5 reacted only with flagellum type a and not type b were obtained by ELISA, where Habs strains 1-12 were tied individually with PLL to the unions in Linbro 96-compartment microtiter plates. The antibody bound only to Habs strains 1, 6, 8 and 9, which are the only ones of the twelve strains bearing type a flagella, (see Qnsorg, R., et al., 1984, J. Clin. Microbiol., 20: 84-88, which is incorporated herein by reference thereto). Precautionary studies gg vivg ashless are exemplified in example 4; below.

EXAMPLE 4 Example 4 disables the protection of mice that are passive immunized with antibodies FaF4 IVEB and Fâó IIGS against infection of E. aeruginosa in the model with fire mouse.

The anti-flagellar monoclonal antibodies were tested in model with fire mouse according to the method described Robv (1983, J. joke by M.S. Collins and R.E. Trauma, or- = 1o-5: 4, .'.-- n uæ incorporated into this specification by reference thereto). For equivocal studies, all anti + § were purified bodies by protein A-Sepharose chromatography (Ey, P.L. et al., 1978. Immunochemistry, 15: 42? -43é. as in- be embedded with this description by male reference thereto). and dialyzed in PBS buffer. The tribe that upobar lagels of tvp a and used in the animal mince search were, 10 15 20 25 35 506 421 ie E. aeru inosa PAEZO (from Dr.

MQ) E. agruginosa Fisher immunotype 2 (A.T.C.C.

James Pennington, FIF Boston, and the flagellar type b strain was the reference strain 27313).

Forty micrograms of purified monoclonal antibody was added each mouse intravenously 1-I hours ¥ öre the achievement of burn and infection. the damage had been done, the animals received 0.5 ml containing the infecting bacteria, sarskorpan.

The results III each organism. of Tables I and TABLE I. -flagelltyo a hrs I l -b Treatment 1 Q fi t i -f 1 agël 1 100 1 ÛÜ IC-'Ü a, Fab 1165 100 Anti-flagell 100 20 b, PaF4 Ivee Non-specific anti-LP5 mono- clonal anti- body 100 PBS. no baking rier The infection dose was approx Percentage with the model with burned mouse * naii Pas, during 10 LD = o-doses for the animal experiments are shown in survival by day * 100 90 10 80 50 w w 90 BO 10 10 10 Immediately after burning Protection study of a monoclonal antibody of anti- STAY 10 10 30 01 * Mice were infected during with approx IÜ LDQQ ' 10 15 20 25 30 35 so6 421 doses of E. aeruginnsa PA220. 2Percentage is based on the survival of mice in groups of ten animals, with the exception of the control group with only Pas. 0 after causing burns and infection. which consisted of five mice. The days are numbered Table II. Skvdds study of a mouth clonal antibody of anti-flagellar type b in the model with burnt mouse * Treatment 1 Percentage survival per day2MÅm hrs) fl -b f.

O* \ | N 6 Anti-flagell b, PaF4 IVES 100 100 90 Q0 90 90 90 90 Anti-flagell a, Fab 1155: oo: o 10 10 10 10 lo lo aåxø Non-specific anti-LPS mono- clonal anti- body 100 20 20 20 20 20 20 20 20 V20 PS5, inga bacteria 100 100 100 100 100 100 100 100 100 _100 * Mice were incubated under the scar with about 10 LD = ° - 'ä -doses of E. aeruginosa Fisher immunotype e. The percentage is based on the survival of mice in 10 15 20 25 30 35 506 421 groups of ten animals with the exception of the control group with only PBS which consisted of five mice. The days are numbered after causing burns and infection.

Very significant survival was found in mice. as had been treated with anti-a-antibody or anti-b-anti- body and subsequently infected with the corresponding antigen. IN in contrast, 80-90% of the untreated men died infected mice or animals. which had been treated with a non-matching anti-flagellar monoclonal antibody or non-specific anti-LPS antibody. Üförmagan hos the antibody of anti-flagellar type a to protect moss from one lethal infection of E. aeruginosa Fisher immunotype 2, which carries type b flagella, and indigestion of the antibody of anti-flagella type b to protect mice from a lethal infection tion of E. aeruginosa PAEEO. which bears tvp a, confirmed Due to the specificity of the antibodies that had stated LQ vitro. survival in burnt mice. as not had been infected. showed that the burn itself was not letal.

EXAMPLE 5 Example 5 ash damages the extensive cross-reactivity of FaF4 IVE8 and FQ6 IIG5 with clinical E. aeruginosa isolates, demonstrating the clinical utility of these bodies in immunotherapy of E. aeruginosa infections. clinical isolates were obtained from hospitals and clinics.

The isolates originated from a variety of isolation sites O including blood, sar. rsspirati nsorganen. urine and ears.

A total of 157 isolates were examined.

PaF4 IVE8 bound specifically to 34 clinical isolates (222), while the antibody of flagellvp a, Fäo IIG5, nd to ba 102 clinical isolates (65%) for a total of 136 of 15 * isolates (87%). of the 21 tams that were not recognized by either III 10 15 20 25 30 35 so @ 421 '41 the antibody was 19 non-flagellar, as shown by genomšf dyeing with beet paint. Therefore, both antibodies bound t combination to 136 of 138 (98%) of flagellar clinical isolate. which confirms previous reports (see, R.

Ansorg, 1978, Zbl. Baht. Hyg., I Abt. Drig. A, 242: 228-; 238, which is incorporated herein by reference. addition). @ »L EXAMPLE 6 Example 6 neutralizes methods for producing mono clonal human antibodies, which bind to E. aeruginosaw flags of type b.

A peripheral blood sample from an individual who had immunized with a high molecular weight polysaccharide preparation: (Pier et al., 1984 Infect. Immun., 45: 309), served somia source of B cells. Mononuclear cells were separated from ä the blood by standard spin technique on Ficoll-Faque: scanu. J. ciin. Lab. Invgšg., 21-77> and 4'l ' was washed twice in calcium / magnesium-free phosphate buffer saline (PBS).

The mononuclear cells were freed from T cells below use of a modified E-rosetting technique. In Å In short, this meant that the cells were first resuspended to a concentration of 1,107 cell ml in PBS sloping 20% fetal calf serum susoension was then introduced in a 17 x 100 mm round bottomed bolystren tube, to which was added 1 x 10 '2-aminone -isothiouronium bromide KAET) -treated red blood cells 7 from father from a 10% (volvmï) solution in Iscove's modi-1 celebrated Dulbecco medium (Iscoves medium) (Madsen and Johnson <1? 79) Q¿ Immun. Methods, 27:61). The suspension mixed mvcket gently for S-10 minutes at 4 ° C and the E-rosetted cells were then removed by centrifugation oa Ficoll-Paque for B minutes at 2500 x g at 4 ° C. E-rosette-negative mononuclear peripheral 10 15 20 25 30 35 506 421 042 blood cells (E * PBMC), which collected at the spruce surface. manufacturing was taken and washed once in Iscove's medium and re- was suspended in the same containing 15 v / v% FCS.

L-glutamine (2 mmol / l, penicillin (100 IU / ml), streptomycin (100 μg / ml), hvpoxanthin (10 10 N), aminopterin (4 x 10-VM) and thymidine (1.6 x 10 6 ° M). This medium is denoted in it following as HAT medium.

Cell-mediated transformation of E'PBMC was accomplished by co-culture of these cells with a transforming cell line.

The transforming cell line was an Epstein-Barr nuclear antigen (EBNA) -oositive lymphoblastoid human cell line, as derived from ethyl methanesulfonate (EMS) mutagenesis thereof lymphoblastoid cell line GM 1500, followed by selection in presence of 30 μg / ml 6-thioguanine to confer the cells deficiency of hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) and thus make the cells HAT-sensitive. This cell line is referred to as the 1A2 cell line and has been deposited at the American Type Culture Collection (A.T.C.C.) on D? March 1982 under the designation A.T.C.C. CRL 8119. Logarithmic growth phase 1A2 cells were suspended in HAT medium and then combined with E'PBMC in a retrieval of fifteen IAS cells per PBMC. The cell mixture was coated on thirty round-bottomed microtiter plates with 96 compartment (Costar 3799) at a concentration of 32000 cells / compartment and in a volume of 200 μl per compartment and incubated at 37 ° C in a humidified atmosphere containing 6% CO 2. cultures were fed oa days 5 and 8 after smearing through replacement of half the supernatant with fresh HAT medium. Sixteen days after smearing were noted that 100% of the compartments contained proliferating cells and that in most of the compartments the cells had sufficient density for removal and analysis of the overlying ones the water shoes with respect to anti-E. aeruginosa anti- -bodies.

The supernatants were subjected to screening regarding the presence of anti-E. aeruginosa antibodies 10 15 20 25 35 4: using the ELISA-tech-Å described in Example 2 niken but with the following modifications. A pool of the seven Fisher immunotype reference strains (A.T.C.C. No. 27312- 2731e> bands: iii fiatboctnade ' 96-compartment microtiter plates (Immulon II, Dvnatech), som had been pretreated with poly-L-lysine, incubated and was washed as described in Example 2. After blocking! of non-specific binding sites and washing of i the plates were charged with 50 μl of PBS containing 0.1 volume of Tween-year -20 and 0.22 (w / v) BSA to each compartment. överligganšgp the culture broths (SO p1) were then replicated in motif corresponding compartments on the analysis plates and on control plates ,, j_ which had been treated with PLL and blocked but which did not contained bacteria. After incubation and washing were added: enzyme-conjugated second-stage antibodies (50 ml per compartment>, _ horseradish peroxidase-conjugated goat anti-human Igß and goat anti-human IgM, which had been appropriately diluted PBS containing 0.1 ESA, described in example ¿. vøiymx Twesn-20 and 0.22 (weight / voiymfa to the unions and the analysis was thus completed supernatants. which contained antibody as band to the pool of Fisher immunotypes but not to the control plate, was analyzed a second time using each of the seven Fisher immunotype bacteria separately Antibody present in the supernatant from one 20Hi1. n : l bound only to the Fisher immunotypes 2, 6 " aeruginosa. repeatedly with decreasing low cell densities to compartment, and 7 of E. The cells were subjected to subculture its all compartments with growth secreted antibody.

The cell line and the monoclonal antibody (Ig fi isotype) are both identified by the designation 2OH11 in the following.

A second transformation was performed, where the source of the B- -cells derived from the peripheral blood of a patient with: lr cystic fibrosis and which was known to have a chronic E. H aeruginosa infection. E'PBMC was produced as §_ s0¿ 421 10 15 20 25 30 35 506 421 44 described above and co-cultured with the transforming cell line. 1A2, at a ratio of 72 IAS cells per E'PBMC. The cell mixture was spread in fifteen round bottoms 96-compartment microtiter plates at a concentration of 7.4 10 * cells per compartment and cultured as above. overlying liquids were analyzed by ELISQ with regarding the presence of anti-E. aeruginosa antibodies sixteen days after the transformation had been ironed out.

The analysis was performed as described for the previous transformation. mation except that the pool of E. aeruginosa strains. which was used for the initial screening. consisted of Fisher immunotype reference strains F2. F4. Fb and F7 (A.T.C.C. Nos. 27313, 27315, 27316 and 27317) and three clinical isolates from Genetic Systems Corporation Organism Bank (GSCDB), which had different LPS immunotypes and flagellar types. The clinical isolate PSA 1277 (ESCÜB) carries type a flagella and Fisher immunotype 1 LPS; the other the isolate PSA G98 (BSCÜB) carries type a and flagella LPS; and the third isolate PSA Fó25 (GSCÜB) carries type b and Fisher immunotype 5 flagella Fisher immunotype 3 LPS. This mixture of reference strains and clinical isolate is referred to as the flagellar E. aeruginosa pool. supernatants, which contained antibody. which band to plates containing the flagellar E. aeruginosa- the pool but not to the FLL-coated control plates, was analyzed by ELISA a second time with respect to pa A compartment, ECI. band and to the individual trunks in the pool. to the reference strains F2. Fb and F7 the clinical ka isolatet Fó25.

Cloning of the cell line EC! was performed by first subcultured the cells in two rounds with subculture of teams density. first with 20 cells per compartment in the 96-well plates compartment. followed by culture with 2 cells per compartment. Formal cloning of the specific antibody-producing cells was performed by plating the cells at a density of about 1 cell / compartment in 72-compartment Terasaki plates (Nunc UI 10 15 _20 25 30 35 506 421 # 1-36538) in a volume of 10 μl / compartment of HAT medium, as lacked the aminopterin component (HT medium). The plates -v was placed in an incubator for 2-d hours to allow; deposition of the cells at the bottom of the compartments and under- Ä was then searched microscopically by two persons with those in compartments that contained a single cell. The compartments were fed daily with HT medium and when the growth was sufficient the cells were transferred to a 96-well round bottom plate compartment. All outgrown compartments were analyzed by ELISA with respect to E. aeruginosa strains, which carried flagella of type b, and all were found to produce the appropriate one the antibody in question. The cell line and the monoclonal antibody the body is identified both by the designation ECI i (IgM isotype) the following text.

The antigen identified by 2OH11 and 361 was flagella as evidenced by indirect immunofluorescence and immunoblotting. These methods were mainly performed as described in Examples 2 and 3. aeruginosa strains, reference Fisher immunotypes 27313, 27317 and 27318) fluorescence analysis was prepared E. carried flagella type b, F2, Fb and F7 (A.T.C.C. Nos. And a strain of unarmed flagella of type a, reference Fisher- (A.T.C.C. No. 27315), The flag type of the heferin strains was determined by -immunotype 4 as described in Example 3. typing with the rat monoclonal antibodies PaF4 IVE3 and FA6 IIG5. l tion as described in Example 2. The sources of bathing the antibodies were overlying culture fluids and that The FITC-conjugated reagent was FITC-conjugated; Burlingame, Cê) in predicted 0.52 (multi-valued) PBS, -human-Ig & Tago, (weight / volume) 82-041-2, 1: 100 in which contained -gammaglobulins (Miles Scientific, Cat. No.

Naperville, IL) and 0.12 (w / v) sodium azide as preservative.

Fluorescence staining of the antibodies 2OH11 and ÉC1 obeer-f only with E. aeruginosa strains, which received For indirect immuno- as The slides were prepared for examina-Å bovine 506 421 46 ílageller of type b, and not with the strain that bore flagella of type a, namely the reference Fisher immunotype 4. The observed fluorescence pattern was a sinusoidal patterns derived from one spirit of the bacteria. which showed that the antibodies bound to the bacterial flagella. 10 15 20 25 30 35 so§, 421 47 ¿¿' Immunoblotting was performed as described in Example 2 aeruginosa-re ¥ erens- 27313), and Purified type b strokes from E. strains Fisher immunotype 2 (A.T.C.C. no. purified type A flagella from the reference strains Habs 6 and Habs 8 (A.T.C.C. vvwcv no. and 33355) were prepared as described in Example 3. Antigens were separated into one 10% polyacrylamide gel (see Example 3) and transferred to an NCM. supernatants containing anti- 3C1, containing a non-specific human antibody and culture fl the bodies 2OHl1 or overlying culture fluids media were incubated with NCM and the reaction was paused with 1 an alkaline phosphatase-conjugated goat anti-human Ig (fl CA) Enzyme substrates were prepared as follows where) (Tago, Burlingame, diluted in PBS containing 0.05 volume fi Tween-20. described in Example 2. The immunoblotting assay showed that bath the antibodies bound to the ílagellin protein of Fisher _ .. -immunotype 2 with the molecular weight Sauuü and not to The lagel1in protein of Habs 6 with the molecular weight 51700 ochï nor to the flagellin protein of Habs 8 with molecular šï. weight 47200. No reaction was observed with either qen non-specific human antibody or culture media.

Further confirmation that the antibodies 2OH11 and H 361 bands only for flagella of type b and not for type @, _ obtained by ELISA, in which Habs strains 1-12 bands were individually duel with PLL to ëacken in Immulon microtiter plates in with 96 compartments. The antibodies bound only to the Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, which are strains that support fl ». <1ae4> J. clan. microbini., 2o = a4>. “ï type b (Ansorg et al.

EXQMPLE 7 Example 7 neutralizes methods + initialization of a human monoclonal antibody, which binds to E. aeruoinosa ~ flags of type a.

A periodic blood test + ran a person, who had immunized with a high molecular weight polysaccharide preparation (Pier et al. 10 15 20 25 30 35 506 421 48 (1981) Infect. lmmun., 34: 46l), served as the source of E-cells. The mononuclear cells were separated from the blood and was then freed from T cells as described in Example 6. The cells were then frozen in FCS containing 10% dimethyl sulfoxide in a tank with liquid nitrogen. By one later, the cells thawed rapidly at 37 ° C, was washed once in lscove medium and resuspended in HQT medium. Cell-controlled transformation is accomplished by co-culture of E 'PBMC with IAZ cells in a ratio of 1A2 cells per E ”PBMC. The cell mixture was plated in 30 tissue culture plates with 96 compartments in a concentration of 62000 cells per compartment. The lizards were fed on the seventh the day after the ironing by replacing half the volume with HAT medium. Cell proliferation was found in 100% of the trades on the fourteenth day after ironing and supernatants were removed from the compartments and was analyzed at this time.

The supernatants were analyzed by ELISA aeruginosa antibodies by using the flagellar E. aeruginosa pool regarding the presence of anti-E. and PLL-treated plates as control, as described in examples and supernatants, which contained anti- bodies, which bind to the flagellar pool but not to the PLL control plates, were analyzed using the indi- vidual bacterial strains in the flagellar pool. One ZIBB, , .Å .._. compartment, contained antibody that bound to PSA salmon /, PSA G98 and Reference Fisher immunotype 4, which are the three strains in the flagellar pool that carry flagella off type a.

Cloning of cell line 2158 is accomplished as described in Example 6 for the cell line 3Cl with the following modifications in the formal cloning step. After the compartments in The Terasaki plates had been examined for pa the presence of a single cell. each cell was transferred from The Terasaki plate to an individual compartment 1 a round bottom cultivation solatta with 95 compartments in a volume of 100 μl HAT 10 15 20 25 35 49 505 421 -medium. which lacked the aminopterin ~ component (HT medium).

Non-transforming. HAT-sensitive lymphoblastoid cells _: was incorporated into all compartments at a density of 500 V cells / compartments as feeder cells. Five days after ironing 100 μl of HAT medium was added to the compartments to selectively kill the food cells. The compartments were fed anyo on days 7 and 9 after smearing by replacing half of it the supernatant with HAT medium. The cells were fed there_ after with HT medium until the cells had sufficient density to detect the presence of antibody medium ELISA. All compartments with outgrown produced antibody. 1 as bands to E. aeruginosa strains bearing flagella: of type a. The cell line and the monoclonal antibody (IgG1 isotype) is both identified by the designation 2188 i the following text.

The antigen identified by 2138 was flagella as could be shown by indirect immunofluorescence and immunoblotting (see Example 6 for a more detailed description) of the technologies). Fluorescence staining of the antibody 2188 was found only with the E.aeruginosa reference strain Fisher immunotype 4 (A.T.C.C. No. 27315). which bears aeruginosa reference 27313). flagella of type a. and not with E. strain immunotype 2 (A.T.C.C. No. bearing flagella of type a. The observed fluorescence pattern¿ was a sine line pattern moving from one end of ä the bacteria, which showed that the antibody bound to bakf terials flagella.

Immunoblotting was performed as described in Example 2.

Purified flagella of type a from E. aeruginosa reference-H strain Habs 6 (A.T.C.C. No. 33353) and purified flagella fi type b from the E. aerudinosa reference strain Fisher immuno- type 2 (A.T.C.C. No. 27313) was prepared as described. in Example 3. Antigens were separated in a 10% polyacrylic and was transferred to a Nam. 1 amide gel (see Example 3) overlying cultivation waters, containing either 21BB'šl or a non-specific human antibody. and culture media: 10 15 20 25 30 35 506 421 was reacted with NCM and the reaction was demonstrated with a calic phosphatase-conjugated goat anti-human Ig (multiple) and enzyme substrates as described in Examples 2 and 6.

The immunoblotting assay showed that the antibody ZIBB only bound to the flagellin protein of Habs 6 by molecular weight 51700 and not to the ílagellin protein of Fisher immunotype 'fl c with a molecular weight of 53000. No reaction was observed with the non-specific human antibody or culture medium the media.

EXAMPLE E Example 8 disables the protection of mice that have passive immunized with anti-flagellar human antibodies 2OH11, 3C1 and 2188 against infection with the E. model aeruginosa i with fire mouse.

The anti- + monoclonal human antibodies was tested in the model with a burned mouse (see example 4). Antibodies 21BB and IOH11 were prepared by precipitation. of supernatants, which had been discarded respective cell lines, with ammonium sulphate (final concentration 50%) Mishell.

(Good et al., Selected Methods in Cellular Immunolooy, B.B. 5.M .. W.J. 27? -286). and Shiigi, Freeman & Co., iaao, ed., San Francisco, CA, The precipitate soluoilizes in PBS, dialyzed against PBS overnight at 4 ° C and sterile was then filtered before administration to animals.

Hallan to antibody SBI and the used non-specific the anti-LPS antibody as a negative control in this sub- search was overlying cultivation water. As positive control + is each examination used the lamoliga purified rat monoclonal antibody PaF4 IWEE or P4 Fëó IG5. fi IT m is used O S, S E U The tribe that carried ilageller of ty o ECU H ~~ _> 4'_ TU ff IN) a in animal experiments it was clinical isolate (GSCÜE), the strain with flagella of type o was the clinical isolate F. which expresses Fisher immunotype 1 LP. and 10 15 2.0 25 30 35 soel421 which expresses Fisher immune type 6 LPS. (0.45 ml) 0.05 ml) FSA A447 (GSCOB), The human antibodies were premixed with the bacteria (more than 5 LD. @ ° doses in and inoculated during ear ~ ekdrpan immediately after the fire has been commits. The results of the animal multiplication atgee in the tables III, IV and V.

TABLE III. body 21B8 of anti-flagell type al in the model with burnt ~ Protection study of the mdnoclonal human anti- mouse Percentage survival per day: Treatment 1 2 T 4 5 6 7 8 9 Steering wheel- 100 100 100 100 88 88 BB BB V88 -anti-flagell a, FA6 IIGSS HLI m years! _ an t i "1 O O I O O 1 OO 1 OO 1 OO 1 OO 1 OO 1 OO -flagell a, 2138 Hccman-anti- mc »c» c »o c» I c »o c» o -flagell D, 2OH11 PBS 100 25 12 M hrs D-l hrs b * n smile hrs bi IJ I- fl rJ * Möeeen inëekteradee during earrekdrpan with more than 5 LD, °° fi doses of E. aeruginnea PSA A522.

* The percentage survival is based on the number 506 421 mice that survived per group of eight animals. The days refer to after causing burns and infection. Purified antibody (10 μg in 0.45 ml PBS) + was mixed with 5 bacteria and was administered under the scab after causing burns and injections.

TABLE IV. Protection study of the human monoclonal Body 2OH11 of anti-flagell type b in the model with burnt mouse* ______________________________________________________________ __1 Percentage survival per day: 15 Treatment 1 I T 4 5 6 7 E Q RattdJurs anti- -flagell b, 20 PaF4 IVEB = 100 100 100 100 100 100 100 100 100 100 Human-anti- -flagell b, 20H 1 1 100 100 1 00 1 00 1 00 100 100 1 00 1 00 2 5 Human-anti- -flagell a, 2 1 BB 1 00 0 0 0 0 0 0 0 0 0 3 0 Mgg 1 Ey- 30 0 0 0 0 0 0 0 0 0 * The moss was infected under the sarekorpan with more than 5 LD1 °° - doses of the clinical E. aeruoinosa isolate FEA Q447. 35 * The percentage survival is based on the number mice that survived per gruoo about five animals. Yes after the onset of burns and infection. 10 15 20 25 30 35 _-flagell b, U U so§0421 Purified antibody (10 pg in 0.45 ml PBS) was premixed with the bacteria and was administered under the scab after causing burns and injection.

TABLE V. Conservation study of the human monoclonal antibody data 301 of anti-flagellum type b in the model with a burned mouse _____________.._.__.._..._______..________._____..___.._-__.__...___..._ ~ '_. _... .i _: Percentage survival per day * hrs M b U 0 w W m Treatment 1 RattdJurs anti- -flagell b, FaF4 IVE8 $ 100 100 100 100 100 100 100 100 100 100 Human-anti- 301 100 100 80 80 SO BO BO 80 EG Non-specific anti-monoclonal ~ LPS Antibody 100 O O O O O O O O 10 L ______________________________________________________ - .., ...... 1 * The mice were infected doses of PSA A447. under the sarcorpa with more than 5 LDiøø? Ä * The percentage survival is based on the number groups of five animals. The days refer to after \ f causing injury and injection. Purified antibody (40 ug> was administered intravenously tvaw hours before the onset of burns and infection.

A very significant survival was observed in mice, 10 15 20 25 30 35 lsoe 421 which had been treated with the anti-flagella type antibody a or any of the two antibodies of anti-flagellar type b after infection with the corresponding antigen. Umvvant dog BB-100% of the untreated but infected mice or those who had been treated with a non-matching anti- flagella monoclonal antibody or non-specific antibody -LPS antibody. As could be ascertained with those the rat monoclonal antibodies (see Example 4) protect the anti-4lagellar human antibodies specifically against lethal infection only by the organisms that carry the equivalent flagelltvp, i.e. the human antibody of anti-flagellar TV a provided protection against lethal infection of the organism that carried flagellar type a and not type.b and the antibodies of anti-flagella type b protected mice, which had been infected with strains bearing ëlagell type b but not organisms supporting type a. _} EMPEL 9 Example 9 neutralizes the cross-reactivity of the -flagellar human antibodies 2OH11, 3C1 and BIBB with clinical E. aeruginosa isolates.

Clinical E. aeruginosa isolates (115). which had been obtained from hospitals and clinics and isolated mainly from burns and blood, were identified as emerging + type a or type b layers by typing with the the rat monoclonal antibodies FA6 IIG5 or PaF4 Fifty-five of the clinical IVEB (see Examples 2, I and 5). isolate was identified as bearing ilageller by tyo a 3 by reaction with the steering wheel monoclonal antibody Get IIGS and 5? was identified as unopoarande tvo b by their reaction with the rat monoclonal anticoar PaF4 IUEE.

The horse reactivity nos the human monoclonal antibody off ti- anti-flagell type a, was unmatched by an kropoen kande again 54 of the Eb clinical: solat fl ëoïl as 10 15 _20 25 30 35 UI UI so§ 421 carried flagella of type a. The parent reactivity nos 2OH11 with the isolates carrying flagella type b were also re- 7 comprehending by 2OH11 recognizing all S9 isolates (1002). In contrast, the remaining monoclonal nal antibody of anti-flagellar type b, 3C1, to only 43 of the 59 isolates (73%). These results show that EOHI1 binds to a pan-reactive epitope (i.e. an epitope> present on at least about 95% of the flagellar> E. aeruginosa strains), while ECI binds to an epitope which is not present on all flagellin molecules of type bla Although the flagella-type antigen is serologically homDgentš “ when analyzed with polyclonal antisera (Lanvi, B., supra), for 2OH11 and 3C1 surprising that the flagella of type b su ra, and Ansprg, R., show the cross-reactivity patterns has at least two separate epitopes that can be identified by monoclonal antibodies.

The extensive cross-reactivity of the antibodies 2138 and 2OH11 with clinical P. aeruginosa isolate shows it special clinical utility of these antibodies aeruginosa infections. in immunotherapy of E.

EXAMPLE 10 Example 10 disables methods for + framing of a further examples of a human monoclonal antibody, which binds to E. b. type b aeroginosa ilagelles, as well This antibody's protective activity against infection with E. aeruginosa in the model with a burned mouse.

A transformed cell line was prepared and cloned into »U essentially as described in example /, with the exception of that the transformed cell mixture was spread on 20 tissue culture plates with 96 tack in a concentration of about 2250 E'PBMC per compartment. supernatants analyzed initially by ELISA on the flagellar pool, with the exception that Fisher- as described in Example 6, Ü -immunotype reference strains e and 4 were missing in the pool. 10 15 20 25 30 35 506 421 Positive compartments were then analyzed on each of strains in the flagellar pool including Fisher immuno- type 2 and 4. The finally isolated cell line and the identified bada monoclonal antibody (IQG isotype) with the designation 12D7 in the following text.

The human monoclonal antibody 12D7 showed extensive cross-reactivity with anti-flagella type a and jug isolates again 54 of the 56 clinical isolates tested (96%), which The protection activity of the mono- Table VI. carried flagella of type a. clonal antibody 12D7 is shown in Skvddsstudier- were carried out essentially as described in Example 4, with except that the infecting dose was 1 Lüiom and that the infecting strain was 1624, a clinical isolates expressing Fisher immunotype 2 LPS and flagella vav typ a.

TABLE VI. Protection study of the human monoclonal the body 1207 of anti-flagell type a in the model with burnt mouse* Percentage survival per day * Treatment: 1 I I 4 5 6 "8 9 Human-anti- -flagell a, 120? 100 100 100 100 100 100 100 100 100 100 Human-anti- -Fisher 2 LP5. : H9 90 90 90 90 90 90 90 90 90 90 90 Steering wheel -anti-ïlagell a. 1165 100 100 100 100 100 100 100 100 100 100 10 15 20 25 30 35 sne 57 Human-anti- -flagell b, 15F4 100 37.5 25 25 25 25 25 25 25 ___-__.-____________.__.________.__.___.__.___________________.__.,.,., Q ..

* The mice in + were echoed under the crust with 1 Lüioo (950 CPU) by E. aeruginosa 1624.

* The percentage survival is based on the number surviving mice per group of ten animals, with the exception of group 15F4 where N = 8. The days are after arrival of burn and infection. 0.5 ml PBS) intraperitoneally two hours before the onset of burns. Purified antibody (50 pg) was administered injury and infection.

EXAMPLE 11 Example 11 ash damages the preparation of a monoclonal human antibody, which reacts with E. aeruginosa of flagellum type b, and the protection of mice that have been passively immunized with this antibody against infection with E. aeruginosa at the model with a burned mouse.

A transformed cell line was prepared and cloned into essentially as described in Example 10, with the exception of that the transformation ratio 1A2 to B-cell was approximately? 6Û: 1 and the trans + normalized cell mixture was plated on fifteen plates in a concentration of about 1930 E'PBMC per compartment.

E. strains comprising G98 3, ilagell type a) and 1739 {layer type b) The cell was also analyzed on a pool of aeru inosa- (Fisher immunotype (a clinical isolate -immunotvp 5, and was confirmed on another M " pool of E. aeruginosa strains: 1277. GQB, I73§ and Fisheršv P4, Fa and F ?. -immunotypes F2, The finally isolated the cell line and the secreted monoclonal antibody 42 1 by Fisher1§ï 506 A421 (lgßl isotype) are both identified by the designation 288 i the following text.

An immuno + fluorescence assay, performed with EEE, was positive on a Fisher immunotype strain 2 of flagellum type b men negative on a Fisher immunotype reference strain 4 of ¥ 1agell- type a. When tested on clinical isolates, 59/5 reacted? (1002) of flagell type b isolate positive.

The protective activity of 288 is shown in Table VII.

The protection studies were performed as described in Example 10, with the exception of clinical isolate F1b4 (Fisher immunotype 4, flagellar TV b) was used for the infection.

TABLE VII. Protection study of the human monoclonal the body EEE of anti-flagellum type b in the model with burnt mouse* Percentage survival per day Treatment 1 2 3 4 S é 7 B 9 Human-anti- -flagell b, 2B8 = 100 89 89 89 89 89 B9 8? B? 89 Steering wheel -anti-flagell b.

PaF4 IVEB “100 100 100 100 100 100 90 90 90 90 Steering wheel -anti-Fisher 2 Lpg! VH3 ~ gg: Q 20 20 20 “Û 20 20 70 .__ * The moss was infected under the crust with 1 Lßiøo (105 10 15 20 25 30 35 sø§ 421 CFU) of E. aeruginosa F1b4.

* The percentage survival is based on the number surviving mice per group of ten animals, with the exception of: group 288 where N = 9. The days refer to the aya coming burn and infection. Purified antibody (50 μg in 0.5 ml PBS) was administered intraperitoneally two hours before the onset of burns. injury and infection.

Purified antibody (5 pg in 0.5 ml PBS) was administered intraperitoneally two hours before the onset of burns injury and infection.

EXAMPLE 12 Example 12 detoxifies the preparation of another mono-g global human antibody, which reacts with E. aeruginosa avÉ flagel1type_b, and protection of mice. who have been immunized passively with this antibody against infection with E. aerugingea at the model with burned mouse.

A transformed cell line was prepared and cloned into 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 cell source and smear The level of E “PBMC was approximately 2099 cells per compartment.

The screening was performed on ooola Fisher immunotypes 1-7.-É It finally isolated the cell line and secreted it monoclonal antibodies (lgß, -isotvp) are identified both ¿ with the designation 14C1 in the following text.

In clinical cases, 59/59 (1002) of isolates were of flagelltvp b positive with 14C1. The safety studies were also performed as described for example 1 Table VIII. 11 above and the results are shown 10 15 20 25 30 35 _-Fisher 2 506 421 60 TABLE VIII. body 1401 of anti- + lage1ltype b in the model with burnt Conservation study of the human monoclonal mouse* ll u ß L m w m n Treatment ”1 Human-anti- -flagell b, 14C1 100 100 100 100 100 90 90 90 90 90 Steering wheel -anti-flagell b, PaF4 IVE8 100 100 100 100 100 100 100 100 100 100 Human-anti- LPS, VHB 100 40 20 20 20 20 20 20 20 _________._.___________.________._______________________.___- < * Mice were infected under the scab with more than 1 LD1 °° (90 CFU) of E. aeruginosa F1ó4.

* The percentage survival is based on the number surviving mice per group of ten animals, with the exception of or open PaF4 IVE8 where N = ?. u The days refer to after causing burn and infection.

Purified antibody (50 pg in 0.5 ml PBS) was administered intraperitoneally two hours before the onset of the fire injury and injection.

From the above it appears that the cell lines according to The present invention provides means for producing status of monoclonal antibodies and fragments thereof, 10 sug 421 61 which are reactive with E. aeruginosa flagella and cross- protective against various E. aeruginosa strains. A number of monoclonal antibodies have surprisingly been isolated, which be reactive with different epitopes on each type of flagellum.

The antibodies of the present invention allow a V more economical and easier production of pro + ylactic and therapeutic preparations for use against infections caused by most Q. aeruginosa strains-L na. Furthermore, the cell lines provide antibodies, which finds use in immunoassays and other well known procedures.

Claims (25)

soe, 421 70 15 20 25 30 35 61 PATENTKRAV A monoclonal antibody or binding fragment thereof characterized in that it is capable of specifically reacting with a flagellar protein epitope of Pseudomonas aeruginosa bacteria to inhibit the motility of the bacteria which monoclonal antibody or binding fragment thereof protects in vivo against Pseudomonas aerugection. Monoclonal antibody according to claim 1, characterized in that it is capable of blocking the binding to the epitope of monoclonal antibodies produced by cell lines designated ATCC deposit numbers HB 9129, HB 9130, CRL 9300 and CRL 9301. Monoclonal antibody according to claim 1, characterized in that it has the same binding specificity to a flagellar protein epitope of Pseudomonas aeruginosa as any of the monoclonal antibodies with ATCC deposit numbers HB 9129, HB 9130, CRL 9300 and CRL 9301. ' A monoclonal antibody according to claim 3, characterized in that it is conjugated to a marker capable of providing a detectable signal. Monoclonal antibody according to claim 4, characterized in that the marker is a fluorescent agent or an enzyme. Preparation of monoclonal antibodies, characterized in that a first of these monoclonal antibodies is capable of reacting with a flagellar protein flagella or flagella type b of Pseudomonas aeruginosa to inhibit the motility of the bacteria and that the monoclonal antibody protects Ponudinosa in vivo. -infection. 10 75 _20 25 30 35 es Formulation according to claim 6, characterized in that a second of said monoclonal antibodies is capable of reacting with a type of flagella which is not reactive with said first monoclonal antibody. isp Formulation according to claim 6, characterized in that said first antibody is a human monoclonal antibody. 6 A formulation comprising one or more monoclonal antibodies or binding fragments thereof as defined in claim 1, 6 or 8 and a gamma globulin fraction from human blood plasma and / or an antibiotic agent. á 'z Formulation according to claim 6, characterized in that the epitope is present in at least about 70% of the flagella of type b. ll. Preparation according to Claim 6, characterized in that the epitope is presented only by the type b flagella. Formulation according to Claim 11, characterized in that the antibody is pan-reactive. Pharmaceutical formulation, characterized in that it comprises one or more monoclonal antibodies or binding fragments thereof as defined in any one of claims 1, 6 or 8 and a physiologically acceptable carrier. 14. Pharmaceutical Preparation Useful for Treatment If prophylaxis of a Pseudomonas aeruginosa infection is known to react with an aeruginosa to protect in vivo, it comprises a monoclonal antibody as a flagellar protein epitope of Pseudomogaff to inhibit bacterial motility. and as against Pseudomonas aeruginosa infection Ni: agent, a gamma globulin fraction from human antimicrobial blood plasma and a physiologically acceptable carrier. 506 421 10 15 20 25 30 35 64 Pharmaceutical preparation according to Claim 14, characterized in that the antibody is a human monoclonal antibody and in that the gamma globulin fraction from human blood plasma has been obtained from humans with elevated levels of immunoglobulins which react with Pseudomonas aeruginosa bacteria or products thereof. Pharmaceutical preparation comprising at least two monoclonal antibodies, characterized in that each of the antibodies specifically reacts with a flagellar protein epitope in different types of flagellar Pseudomonas aeruginosa protein to inhibit the motility of the bacteria and can treat or prevent Pseudomonas aeruginosa. infections and that they protect in vivo against Pseudomonas aeruginosa infection. Formulation according to Claim 16, characterized in that at least one of the monoclonal antibodies is a human monoclonal antibody. Formulation according to claim 16, characterized in that it comprises at least one human monoclonal antibody capable of reacting with at least one serotypic determinant on a lipolysaccharide molecule of Pseudomonas aeruginosa and / or a monoclonal antibody which reacts with endotoxin A. Cell line, characterized in that it produces a monoclonal antibody capable of specifically reacting with a flagellar protein type of Pseudomonas aeruginosa flagella of type a or type b to inhibit the motility of bacteria and that the monoclonal antibody protects in vivo against Pseudo - monas aeruginosa infection. Cell line according to Claim 19, characterized in that it is a hybrid cell line. 10 15 20 25 30 Qi? Cell line according to claim 19, characterized in that it produces human monoclonal antibodies. Cell line according to Claim 19, characterized in that it is one of the cell lines ATCC Deposit Nos. HB 9129, HB 9130, CRL 9300 and CRL 9301. Methods of producing monoclonal antibodies which are specific for flagellar proteins of Pseudomonas aeruginosa and inhibit bacterial motility and can treat or prevent Pseudomonas aeruginosa infections, characterized in that at least one of the cell lines according to claim 22 is cultured and the antibody is recovered. 24. A method of detecting the presence of Pseudomonas aeruginogaiiï in a sample, characterized in that the sample is combined with a monoclonal antibody into a flagellar protein epitope of Pseudomonas aeruginosa and which inhibits bacterial activity and protects in vivo against Pseudomonas aeruginos . Test kit for use in detecting the presence of Pseudomonas aeruginosa bacteria, characterized in that it comprises a monoclonal antibody preparation containing at least one monoclonal antibody, the antibody reacting with a type-specific flagellar protein epitope of the bacteria to inhibit and protect the bacteria. vivo against Pseudomonas aeruginosa infection, and markers that provide a detectable signal covalently bound to the antibody or bound to other antibodies that react with the monoclonal antibody.