LU86938A1 - MONOCLONAL ANTIBODIES AGAINST PSEUDOMONAS AERUGINOSA FLAGELS - Google Patents

Description

52.676

_ _ λ λ "Τ GRAND-DUCHY OF LUXEMBOURG

Ö y 0 T ^ Minister of July 3, 1987 for the Economy and the Middle Classes

É ^ irl Department of Intellectual Property Title issue ........................................ .... S3P LUXEMBOURG

^ ï

Patent Application I. Application

The so-called society; GENETIC SYSTEMS CORPORATION, 3005 First - (2)

Avenue, SEATTLE, Washington 98121, United States of America, - represented by Monsisur - Jacques de Muyser, - acting in --------------- - quality of agent .... ....— ----- --------- -------------------- ----------- --------------------------------- (depositor) this three July 1900 eighty-seven__________________________ - - - (4) at ........ 3 p.m., at the Ministry of the Economy and the Middle Classes, in Luxembourg: 1. this request for obtaining a patent for an invention concerning: "Monoclonal antibodies against .. flagella of pseudomonas (5) aeruginosa. " ________________________________________________ -..............................—. .........................................— ........ ....................- 2. the description in French ...................... of the invention in triplicate; 3. // drawing plates, in three copies: 4. receipt of the fees paid to the Registration Office in Luxembourg, July 1, 198 7 '5. the delegation of power, dated ....... .............................. -................... ........... the ..................................: 6. the successor document (authorization): declares (s) assuming responsibility for this declaration, that Lies) inventor (s) is (are): (6) see designation ...... separate: - do not mention -------- claims (s) for the above patent application the priority of one (or more) application (s) of (7) ....... patents ........ .................................................. ............................................... filed (s) ) §Âi (8hux ...... United States of America on (9) 1. July 3, 1986, December 24, 19S6fet 3. May 15 -1987 ...........

under N ° (10) 1,8 31.98.4 ........................ 2. 9 46, 554 .............................................. ..... and 3, 048.14 3 ...................

in the name of (ll) invsr ± 3urs: 1. 1 + 3, 2, 1 + 3 and - 3.-1, 2 + ...... 3 ............ ...

elect (elect) domicile for him / her and, if designated, for his / her representative, in Luxembourg ...................... — ...... ........

.............. 35, ..... boulevard ...... Royal ..................... .................................................. .................................................. .................................................. .....-......... (12) requests (s) the grant of a patent for the invention for the subject described and represented in the abovementioned appendices, with postponement of this grant to. .............................. 6 ................... .................................................. ................... - month. (13) / mW)

The above application for a patent for invention has been filed with the Ministry of Economy and the Middle Classes, Servicefle la Propriété Intellect epttÆIie ”îTruxembourg. dated July 3, 1987 / <ev / **> \\ l *. V \\ Pr. The Minister of Economy and the Middle Classes, at 3 p.m. 1 i; j P · d.

\ \ î "iy The head of the intellectual property department, ^ A hSIXT ________ f" ύ EXPLANATIONS RELATING TO THE FORMULAmÎTT ^ ÇÇfCf * \ (! »if he> had it''Application for certificate of addition to the patent principle a ia main patent application No .....-..... of ........ ~ ilunscnreiesnora.pTenoir.proiess.v ·.

'... · *' acicsse of the applicant, when the latter is a private individual or your company name. legal form, location of the registered office, when the applicant is a legal person - <31 uu-cnit · .. L · t- _________i 1.! i_____________ _____ri ¥ s _t "_ -β.Ί .-. „Λ. '.nictoiii .n / militi ils • nuinvitn * .. 52.676

<, CLAIM OF PRIORITY

of patent application / ïiK5G3gxièl <Scà'3dtt) jl & I SK 'In the United States' of America: ..

! '’, July 3, 1986 (No. 881.98.4), v; December 24, 1986 (No. 946.554) and May 15, 1987 (No. 048.143).

"- ~ · '' '; ? - s Description Brief filed in support of a request for

PATENT

at

Luxembourg

on behalf of: GENETIC SYSTEMS CORPORATION

SEATTLE, Washington 98121 (United States of America) for: "Monoclonal antibodies against flagella of pseudomonas aeruginosa." • Ί * MONOCLONAL ANTIBODIES AGAINST PSEUDOMONAS AERUGINOSA FLAGELS.

FIELD OF THE INVENTION.

The present invention relates to the application of immunological techniques for obtaining new materials useful for the diagnosis and treatment of bacterial infections and, more particularly, to the production and application of human monoclonal antibodies which are capable of recognizing flagella of Pseudomonas aeruginosa.

BACKGROUND OF THE INVENTION.

Gram-negative disease and its most serious complications, for example bacteremia and endotoxemia, cause significant morbidity and mortality in humans. This is true, in particular, of the Gram-negative organism Pseudomonas aeruginosa, 'which has been increasingly associated with bacterial diseases, especially with nosocomial infections, during the past fifty years.

For the past few decades, antiobiotics have been the therapy of choice for the fight against Gram-negative diseases. The high and continuing morbidity and mortality from Gram-negative bacterial infection are, however, indicative of the limitations of antibiotic therapy, particularly with respect to P, aeruginosa. (See, for example, Andriole "V.G.," Pseudomonas Bacteremia î Can Antibiotic Therapy Improve Survival? ", J. Lab. Clin. Med ,, / 1978 /, 94: 196-199). This prompted the search for other prevention and treatment methods.

One method that has been considered is the strengthening of the host's immune system by active or passive immunization. For example, it has been observed that active immunization of humans or experimental animals with vaccines consisting of whole bacterial cells or purified bacterial endotoxins originating from P, aeruginosa leads to the development of specific opsonic antibodies directed essentially, against determinants of recurrent oligosaccharide units of lipopolysaccharide molecules (LPS) located on the outer membrane of P. aeruginosa (see Pollack, M., Immunoglobulins: Characteristics and Uses of Intravenous Preparations, Alving, BM and Pinlayson, JS, ed ., pages 73-79, US Department of Health and Human Services, 1979). Such antibodies, whether actively generated or passively transferred, have been shown to be protective against the lethal effects of P. aeruginosa infection in various animal models (Pollack, supra) and in some preliminary research in humans (see Young, LS and Pollack, M., P. aeruginosa, Sabath, L., ed., pages 119-132, Hans Huber, 1980).

The above work suggests that immunotherapeutic approaches could be used to prevent and treat bacterial disease due to P. aeruginosa, for example by administering pooled human immunoglobulins that contain antibodies against the infecting strain (s). Human immunoglobulins are defined here as the fraction of human plasma that is enriched for antibodies, among which are specific antibodies against strains of P. aeruginosa. Due to some limitations in the use of human immunoglobulin components, this approach to the treatment of P. aeruginosa disease remains under study (see, for example, *> 3

Collins, M.S. and Roby, R.E., Am. J. Medt / 76 (3A): 168-174, ZT1984J7) ”and so far there are no commercial products available containing these components.

One such limitation associated with immunoglobulin compositions is that they consist of collections of samples from a thousand or more donors, these samples having been preselected based on the presence of particular anti-Pseudomonas antibodies. . This pooling results in averaging the individual antibody titers, which at best results in modest increases in the titer resulting from the desired antibodies.

Another limitation is that the screening process itself requires continuous, expensive sorting of the donor population to ensure product uniformity. Despite these efforts, the immunoglobulins obtained can still have considerable variability from one batch to another and among products from different geographic regions.

Another limitation inherent in immunoglobulin compositions is that their use results in the simultaneous administration of large amounts of foreign protein substances (which may include viruses such as those recently shown to be associated with the syndrome. of Acquired Immunodeficiency or AIDS), which can cause harmful biological effects. The combination of low titers of desired antibodies and high content of foreign substances can often limit the amount of specific immunoglobulins below the optimum and therefore beneficial to the patient.

In 1975, Köhler and Milstein exposed their A 3b 4 fundamental discovery that certain mouse cell lines could be fused with mouse spleen cells to create hybridomas, each of which secretes antibodies of unique specificity. ie monoclonal antibodies (Köhler, G. and Milstein, C., Nature, 256: 495-497 With the advent of this technology, it has become possible, in some cases, to produce large quantities of antibodies murine selectively specific for a particular determinant or determinants on antigens. Later, using technologies developed later, it became possible to produce human monoclonal antibodies (see, for example, United States patent United States of America No. 4,464,465, which is cited here for reference).

It is recognized that in some situations, mouse monoclonal antibodies or compositions of these antibodies can give rise to drawbacks when used in humans. For example, it has been reported that mouse monoclonal antibodies used in experimental studies for the treatment of a certain human disease can elicit an immune response which makes them ineffective (see, Levy, RL and Miller, RA, Ann. Rev . Med., 34: 107-116 / 71983 ^ 7). However, with recent advances in recombinant DNA technology, such as the production of chimeric mouse / human monoclonal antibodies, these difficulties can be reduced. Also, methods for the production of human monoclonal antibodies are currently available (see, Human Hybridomas and Monoclonal Antibodies, Engleman, E.G. et al., Ed., Plenum Publishing Corp. £ 1985J7 "which is cited herein by reference).

Using Λ Λ 5 hybridoma and / or cell transformation technology, many research groups have reported the production of protective monoclonal antibodies against P. aeruginosa infections. Monoclonal antibodies have been produced, which are reactive with various epitopes of P. aeruginosa, including specific surface epitopes of single serotype and multiple serotypes, such as those found in the lipopolysaccharide molecules (LPS) of the bacteria (see, for example, the pending United States of America patent applications Nos. 734,624 and 807,394 assigned to the Applicant, both of which are cited herein by reference). Protective monoclonal antibodies specific for exo-toxin A of P. aeruginosa have also been produced (see, for example, U.S. Patent Application No. 742,170, assigned to the Applicant, which is cited here in reference).

The use of monoclonal antibodies specific for the LPS region of P. aeruginosa, or exotoxins of the bacteria, can provide sufficient protection in some situations, but it is generally preferable to have a broader protective capacity. For example, in the prophylactic treatment of possible infections in humans, it would be preferable to administer one or more protective antibodies against several strains of P. aeruginosa. Likewise, in therapeutic applications where the serotype (s) of the infecting strain (s) is not known, it would be preferable to administer an antibody or a combination of antibodies effective against most if not all clinically important serotypes of P , aeruginosa, ideally by providing reactive antibodies in traditional serotyping schemes.

One aspect of the physiology of P. aeruginosa Κ Λ 6 which has been shown to contribute to the virulence of the organism is motility, which is a capacity resulting essentially from the presence of a flagellum (see, Montie, T . et al., Z ”l982_7, Infect, and Immun., 38: 1296-1298). P. aeruginosa is characterized by the fact that it has a single flagellum at one end of its stick structure. Burned mouse model studies have shown that a greater percentage of mice survived when strains of P. aeruginosa with no movement were inoculated in experimental burns than with strains with movement. (McManus, A. et al., Z * 1980_7, Burns, 6: 235-239 and Montie, T. et al., £ l982 £, Infect, and Immun., 38 s 1296-1298). Other studies on the pathogenesis of P. aeruginosa have claimed that animals immunized with preparations of flagellum antigen are protected when they are burned and infected with strains endowed with movement of the bacteria (see, Holder, I. and al., / ”1982J7” Infect, and Immun., 35: 276-280).

Importantly, the flagella of P. aeruginosa have been studied by serological methods and described as falling into two major antigenic groups designated by H1 and H2 by B. Lanyi (1970, Acta Microbiol. Acad, Sci. Hung., 17:35 -48) and by type a and type b by Ansorg, R. (1978, Zbl. Bafct. Hyg., I. Abt. Orig. A, 242 ï 228-238). The serological typing of flagella by the two laboratories showed that H1 flagella (Lanyi, B., supra) or type b flagella (Ansorg, R., supra) were serologically uniform, that is to say that 'no subgroup has been identified. This serologically uniform flagellar type is hereinafter called type b. The other major antigen, H2 (Lanyi, N., supra) or flagellar type a (Ansorg, R., supra) contained five subgroups. This ". * 7 antigen is said below flagellar type a, with five subgroups ag, a ^, a2, a3 and a4 · The five subgroups of type a are expressed in various combinations on different strains of P. aeruginosa carrying type a, with the exception of the aQ antigen. The ag antigen was found on all type a flagella, but with a variable degree of expression among the strains.

A serotyping scheme based on the major heat-stable somatic antigens of P. aeruginosa is called the Habs scheme, which has recently been incorporated into the International Antigenic Typing System. (See, Liu, Int. J. Syst. Bacteriol., 33: 256, Π1983J.) The Habs reference strains of the flagellar types of P, aeruginosa were characterized by immunofluorescence with polyclonal sera by R. Ansorg (1978, Zbl Bakt. Hyg., I. Abt. Orig, A, 242: 228-238) or by coagglutination on a slide (Ansorg, R et al, 1984, J. Clin. Microbiol., 20: 84-88). Habs 2, 3, 4, 5, 7, 10, 11 and 12 are strains carrying type b flagella and Habs strains 1, 6, 8 and 9 carry type a flagella. A large number of P. aeruginosa strains could therefore probably be recognized by a small number of monoclonal antibodies specific for flagellar proteins.

Consequently, there is a significant need for monoclonal antibodies capable of reacting with epitopes on flagellar proteins, and in some cases also providing protection against multiple serotypes of P. aeruginosa. In addition, some of these antibodies may be suitable for the prophylactic and therapeutic treatment of P. aeruginosa infections, as well as for the diagnosis of such infections. The present invention t * 8 meets these needs.

SUMMARY OF THE INVENTION.

The subject of the invention is new cell lines which can produce monoclonal antibodies capable of binding to flagella present on most strains of the bacteria P. aeruginosa. The monoclonal antibodies react specifically with epitopes on the flagellar proteins of P. aeruginosa and can distinguish between type a flagella and type b flagella of bacteria. In addition, it relates to a pharmaceutical composition suitable for the treatment of a human being susceptible to infection or already infected with P. aeruginosa and containing a prophylactic or therapeutic amount of at least one monoclonal antibody or binding fragment thereof. capable of reacting with flagella of P. aeruginosa strains, the composition preferably also comprising a physiologically acceptable excipient. The composition may also contain any or more of the following: additional monoclonal antibodies capable of reacting with P. aeruginosa exotoxin A; monoclonal antibodies capable of reacting with serotypes of determinants on the LPS of P. aeruginosa; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from human blood plasma, the plasma of which may be from a human having high levels of immunoglobulins reactive with P, aeruginosa; and one or more antimicrobial agents. In addition, it relates to the clinical uses of monoclonal antibodies, including the production of diagnostic kits.

DESCRIPTION OF SPECIFIC EMBODIMENTS.

The present invention provides novel cells capable of producing I * 9 monoclonal antibodies and compositions comprising such antibodies, which compositions are capable of selectively recognizing flagella present on several strains of P. aeruginosa, while individual antibodies typically recognize a type of flagella of P. aeruginosa. The cells in question have identifiable chromosomes in which the DNA of the germ line originating from them or from a precursor cell has rearranged itself to code for an antibody having a type of binding for an epitope on a flagellar protein common to certain strains of P. aeruginosa. For type a flagellar proteins, pan-reactive monoclonal antibodies can be produced? and for type b flagellar proteins, antibodies which are pan-reactive or react with at least 70% of the strains carrying flagella are included. These monoclonal antibodies can be used in a variety of ways, including for diagnosis and therapy.

The monoclonal antibodies thus provided are particularly useful in the treatment or prophylaxis of a serious disease with P. aeruginosa. The surface proteins of P. aeruginosa flagella would be accessible for direct contact with antibody molecules, thus likely inhibiting the motility of the organism and / or facilitating other favorable effects for infected hosts.

The preparation of monoclonal antibodies can be accomplished by immortalizing a cell line capable of expressing nucleic acid sequences encoding antibodies specific for an epitope on the flagellar proteins of multiple strains of P. aeruginosa. The immortalized cell line can be a mammalian cell line which has been transformed by oncogenesis, transfection, mutation or the like. Such cells include myeloma lines, lymphoma lines, or other cell lines capable of supporting expression and secretion of the immunoglobulin or a binding fragment thereof, in vitro. The immunoglobulin or fragment may be an immunoglobulin of natural origin from a mammal other than the mouse or man which are the preferred sources, produced by transformation of a lymphocyte, in particular of a splenocyte, by means of 'a virus or by fusion of the lymphocyte with a neoplastic cell, for example a myeloma, to produce a hybrid cell line. Typically, the splenocyte will be obtained from an animal immunized against flagellar antigens or fragments thereof containing an epitopic site. Immunization protocols are well known and can vary widely while remaining effective. (See, Golding, Monoclonal Antibodies: Principles and Practice, Academie Press, N.Y., C 1983 ^ 7, which is cited here with reference).

Hybrid cell lines can be cloned and examined by standard techniques, and antibodies capable of binding to flagellar determinants of P. aeruginosa can be detected in cell supernatants. Appropriate hybrid cell lines can then be grown on a large scale or injected into the peritoneal cavity of a suitable host for ascites production.

In one embodiment of the present invention, the cells are transformed human lymphocytes which produce human monoclonal antibodies, preferably protective in vivo against accessible epitopes specific for at least one flagellar protein. Lymphocytes can be obtained from human donors who are or have been exposed to the appropriate strains of P. aeruginosa carrying flagella. A preferred method of cell transformation is described in detail in U.S. Patent No. 4,464,465, which is cited herein by reference.

Because they contain the antibodies of the present invention, which are known to be specific for flagellar proteins, the following experiment supernatants can, in some cases, be examined by competitive analysis with the monoclonal antibodies in question, such as means to identify additional examples of anti-flagellar monoclonal antibodies. Therefore, hybrid cell lines can be readily produced from a variety of sources, based on the availability of specific antibodies for particular flagellar antigens present.

Alternatively, when hybrid cell lines, which produce antibodies specific for the epitopic types in question, are available, these hybrid cell lines can be fused with other neoplastic B cells, which other B cells can serve as recipients for genomic DNA encoding the receptors. While neoplastic rodent, particularly murine, B cells are most commonly used, other mammalian species can be used, such as lagomorphic, bovine, ovine, equine, porcine, avian, etc.

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

The cell lines of the present invention may find use other than the direct production of monoclonal antibodies. The cell lines can be fused with other cells (such as human myelomas suitably labeled for a drug, mouse myelomas or human lymphoblastoid cells), to produce hybridomas and thus ensure the transfer of genes coding for monoclonal antibodies. Alternatively, cell lines can be used as sources of the chromosomes encoding immunoglobulins, which can be isolated and transferred to cells by techniques other than fusion. In addition, the genes encoding the monoclonal antibodies can be isolated and used according to recombinant DNA techniques for the production of specific immunoglobulins in a variety of hosts. In particular, by preparing cDNA libraries from messenger RNA, a single cDNA clone, coding for immunoglobulin and free of introns, can be isolated and placed in suitable prokaryotic or eukaryotic expression vectors and then transformed into a host for final mass production. (See, generally, U.S. Patents 4,172,124; 4,350,683; 4,363,799; 4,381,292 and 4,423,147. See also, Kennett et al., Monoclonal Antibodies, Plenum, NY , /71980.7, and the references cited therein, all these sources being mentioned here with reference).

More specifically, according to hybrid DNA technology, the immunoglobulins or fragments of the present invention can be produced in bacteria or yeast. (See, Boss et al., Nucl.

* * 13

Acid. Res., 12: 3791 and Wood et al., Nature, 314: 446, each cited here by reference). For example, messenger RNA transcribed from genes encoding the heavy and light chains of monoclonal antibodies produced by a cell line of the present invention can be isolated by differential hybridization of cDNA using the cDNA of BALB / c lymphocytes. other than the clone in question. The non-hybridizing mRNA will be rich for messages encoding the desired immunoglobulin chains. If necessary, this process can be repeated and then increased to the desired levels of mRNA. The collected ARMm composition can then be subjected to reverse transcription to provide a mixture of cDNA enriched in the desired sequences. RNA can be hydrolyzed with an appropriate ribonuclease and single-stranded DNA can be double-stranded with DNA polymerase I and random acting initiators, for example randomly fragmented calf thymus DNA. The resulting double stranded DNA can then be cloned by insertion into an appropriate vector, for example into viral vectors such as lambda vectors, or into plasmid vectors (such as pBR322, pACYC184, etc.). By developing probes based on known sequences for the constant regions of the heavy and light chains, the cDNA clones having the gene coding for the desired light and heavy chains can be identified by hybridization. Then, the genes can be excised from the plasmids, manipulated to remove superperfluent DNA upstream from the initiation codon or DNA from the constant region, and then introduced into a vector suitable for transformation of a host and the final expression of the gene.

Suitably mammalian hosts * * 14 (e.g. mouse cells) can be used to process the chain (e.g. to join heavy and light chains), to obtain intact immunoglobulin, and to secrete leader sequence-free immunoglobulin, if desired. Alternatively, single-cell microorganisms can be used to produce the two chains, in which case further manipulation may be required to remove the DNA sequences encoding the processing and secretory leader signals, providing an initiation codon at the 5 'end of the sequence encoding the heavy chain. In this way, the immunoglobulins can be prepared and primed to be assembled and glycosylated in cells other than mammalian cells. If desired, each of the chains can be truncated to retain at least the variable region, which regions can then be manipulated to provide other immuno- λ ·.

globulins or fragments specific for flagellar epitopes.

The monoclonal antibodies of the present invention are particularly useful because of their specificity for antigens against virtually all of the variants of P. aeruginosa presently known. In addition, some of the monoclonal antibodies are protective in vivo, allowing incorporation into pharmaceuticals such as combinations of antibodies against bacterial infections.

The monoclonal antibodies of the present invention can also find a wide variety of uses in vitro. For example, monoclonal antibodies can be used for the typing of microorganisms, for the isolation of specific strains of P. aeruginosa, for the selective elimination of P. aeruginosa cells in a heterogeneous mixture 15 "ft of cells, etc.

For diagnostic purposes, the monoclonal antibodies can be either labeled or unlabeled. Typically, diagnostic tests require detection of the formation of a complex by the binding of the monoclonal antibody to the flagellum of the organism P. aeruginosa. When not labeled, antibodies find use in agglutination tests. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for an immunoglobulin. Alternatively, the monoclonal antibodies can be directly labeled. A wide variety of markers can be used, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. Many types of immunoassays are available and, for example, some include those described in US Patents 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074 and 4,098,876, all of which are cited herein by reference.

The monoclonal antibodies of the present invention are usually used in enzyme immunoassays, where the antibodies in question, or second antibodies of a different species, are conjugated to an enzyme. When a sample containing P. aeruginosa of a certain serotype, such as human blood or a lysate thereof, is combined with the antibodies in question, a bond is formed between the antibodies and the molecules having the desired epitope. These cells can then be separated from unbound reagents, and a second antibody * · »16 (labeled with an enzyme) can be added. Next, the presence of an antibody-enzyme conjugate specifically bound to cells is determined. Other conventional techniques well known to those skilled in the art can also be used.

Kits may also be provided for use with the subject antibodies to detect P, aeruginosa in solutions or the presence of flagellar antigens of P. aeruginosa. Thus, the subject monoclonal antibody composition of the present invention can be presented, usually in a lyophilized form, either alone or in conjunction with specific additional antibodies against other Gram-negative bacteria. Antibodies, which can be conjugated to a marker or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g. bovine serum albumin, etc. Generally, these substances will be present in amounts less than about 5%, based on the amount of active antibody, and usually present in a total amount of at least about 0.001%, based on the concentration of antibody. Frequently, it will be desirable to include an inert diluent or excipient to dilute the active ingredients, in which case the excipient may be present in an amount of from about 1% to 99% by weight of the total composition. When a second antibody capable of binding to the monoclonal antibody is used, it will usually be present in a separate vial. The second antibody is typically conjugated to a marker and presented in a formulation analogous to the antibody formulations described above.

The monoclonal antibodies, in particular the ιί 17 human monoclonal antibodies, of the invention, can also be incorporated as components of pharmaceutical compositions containing a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of this invention with a pharmaceutically excipient effective. A pharmaceutical carrier should be any compatible non-toxic substance suitable for administering monoclonal antibodies to a patient. Sterile water, alcohol, fats, waxes and inert solids can be used as excipients. Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) can also be incorporated into the pharmaceutical composition. Such compositions may contain a single monoclonal antibody, so as to be specific for strains of a flagellar type of P, aeruginosa. Alternatively, a pharmaceutical composition can contain two or more monoclonal antibodies to form a "cocktail". For example, a cocktail containing monoclonal antibodies against both types of flagella or against groups of various strains of P. aeruginosa (for example different serotypes) would be a universal product with activity against the vast majority of clinical isolates of this particular bacteria .

The molar fraction of the various constituent monoclonal antibodies will usually not differ by more than a factor of 10, and more usually will not differ by more than a factor of 5, and will usually be in a molar proportion of about lsl-2 relative to each of the other antibody components.

The monoclonal antibodies of the present invention can also be used in combination with existing blood plasma products, such as commercially available gamma globulins and immunoglobulins used in the prophylactic or therapeutic treatment of P, aeruginosa disease in humans . Preferably, for immunoglobulins, the plasma will be obtained from human donors having high levels of immunoglobulins reactive with P. aeruginosa. (See generally: "In-travenous Immune Globulin and the

Compromised Host ", Amer._J; _Med., 76 (3a), March 30, 1984, pages 1-231, which is cited here for reference).

The monoclonal antibodies in question can be used in the form of compositions administered separately, given together with antibiotic or antimicrobial agents. Typically, antimicrobial agents may include an antipseudomonal penicillin (e.g. carbenicillin) in combination with an aminoglycoside (e.g. gentamicin, tobramycin, etc.), but many other agents (e.g. cephalosporins) well known in the art. Tradesman can also be used.

The monoclonal antibodies and their pharmaceutical compositions of the invention are particularly useful for oral or parenteral administration. Preferably, the pharmaceutical compositions are administered parenterally, that is to say subcutaneously, intramuscularly or intravenously. A subject of the invention is therefore compositions for parenteral administration which comprise a solution of the monoclonal antibody or a cocktail thereof dissolved in an acceptable excipient, preferably an aqueous excipient. A variety of aqueous excipients can be used, for example water, buffered water, 0.4% saline, 0.3% glycine solution, etc. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional and well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH adjusting agents and buffers, toxicity adjusting agents, etc., for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in these formulations can vary widely, i.e. from less than about 0.5%, usually from about 11 or from at least about 1% up to 15 or 20% by weight maximum, and will be selected primarily based on fluid volumes, viscosities, etc., to be preferred for the particular mode of administration chosen.

Therefore, a typical pharmaceutical composition for intramuscular injection could be prepared to contain 1 ml of sterile buffered water and 50 µg of monoclonal antibody. A typical composition for intravenous infusion could be prepared to contain 250 ml of sterile Ringer's solution, and 150 of monoclonal antibody. The methods for preparing parenterally administrable compositions are known or obvious to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science, 15th ed. Mack Publishing Company, Easton, Pennsylvania, (1980), which is cited here with reference.

The monoclonal antibodies of the invention can be lyophilized for storage and reconstituted in a suitable excipient before use. This technique has been shown to be effective with conventional immunoglobulins and freeze-drying (and reconstitution) techniques known in the art can be applied. It is obvious to those skilled in the art that freeze-drying and reconstitution can lead to varying degrees various loss of activity of the antibody (for example, with conventional immunoglobulins, IgM antibodies tend to have a greater loss of activity than IgG antibodies) and that use concentrations may need to be adjusted for The compensation.

The compositions containing the monoclonal antibodies of the invention or a cocktail thereof can be administered for the prophylactic and / or therapeutic treatment of P. aeruginosa infections. In a therapeutic application, the compositions are administered to a patient already infected with one or more serotypes of P. aeruginosa in an amount sufficient to cure or at least partially stop the infection and its complications. An amount adequate for this purpose is defined as a "therapeutically effective dose". The effective amounts for this will depend on the severity of the infection and the general condition of the patient's immune system, but will generally range from about 1 to about 200 mg of antibodies per kg of body weight. , but doses of 5 to 25 mg per kg are more common. It should be borne in mind that the products of the invention can, in general, be used in serious pathological conditions, that is to say life threatening situations, or potentially life threatening conditions, especially bacteremia and endotoxemia due to P. aeruginosa.

In prophylactic applications, the compositions containing the antibody of the invention or a cocktail thereof, are administered to a patient not yet infected with P. aeruginosa to increase the patient's resistance to such possible infection. Such an amount is defined as a "prophylactically effective dose". In this use, the precise amounts again depend on the patient's state of health and the general level of immunity, but are generally from 0.1 to 25 mg per kg, especially from 0.5 to 2, 5 mg per kg.

Single or multiple administrations of the compositions can be carried out, the doses and the administration program being selected by the attending physician. In all cases, the pharmaceutical formulations must provide a quantity of the antibody or antibodies of the invention sufficient for the effective curative or prophylactic treatment of the patient. EXPERIENCES.

EXAMPLE 1 The example illustrates the process applied to prepare a murine monoclonal antibody which specifically binds to flagella of P, aeruginosa.

BALB / c mice three months old were immunized intraperitoneally eight times with viable bacteria P. aeruginosa, immunotype Fisher 1 and immunotype Fisher 2 (ATCC n ° 27312 and n ° 27313), every one to two weeks, for a total of nine weeks. The initial doses of bacteria were 8 x 10® and 1 x 10? organisms per mouse for the Fisher 1 immunotype and Fisher 2 immunotype of P. aeruginosa, respectively, and the dose was increased 30 to 60 times during immunizations.

Three days after the last injection, the spleen of a mouse was aseptically removed and a single cell suspension was prepared by gentle rotation of the organ between the frosted ends of two sterile glass slides. Mononu cells ("* 22 key spleen cells were combined in a 4: 1 ratio with mouse myeloma cells in log phase (NSI-1, acquired from Dr. C. Milstein, Molecular Research Council, Cambridge, England) ) and fused to create hybridomas according to the method described by Tarn et al., (1982, Infect. Immun., 36: 1042-1053). The final hybrid cell suspension was diluted to a concentration of 1.5 × 10 ^ cells per ml in HAT-RPMI hybrid medium (RPMI 1640 / ^ Gibco, Grand Island, NYJ7 containing 15% heat-inactivated calf fetal serum, 1 mM sodium pyruvate, 100Jlq / ml of penicillin and streptomycin, hypoxanthine 1.0 x 10 "4 m, aminopterin 4.0 x 10" ^ M and thymidine 1.6 x 10 "5 m), supplemented with 2.0 x 10 ^ cells thymus BALB / c freshly prepared per ml, as feeder cells.

The mixture was placed on plates (200 yuliters per well) in 96-well plates (No. 3596, Costar, Cambridge, MA). The cultures were fed by removal and replacement of 50% of the volume of each well with fresh HAT-RPMI hybrid medium, every two or three days. The culture supernatants were tested for the presence of anti-P antibodies. aeruginosa by fixed enzyme immunosorption assay (ELISA) when cell growth reaches approximately 40% confluence in the wells, usually within 7-10 days.

The supernatants of the cultures of the hybrid cells were tested simultaneously on preparations of external membranes originating from each of the two immunizing bacteria. External membrane preparations were isolated by a modification of the technique of Tarn et al. (1982, Infect, Immun., 36 s 1042-1053), which is cited here with reference. 23 bacteria (Fisher 1 immunotype and Fisher 2 immunotype of P. aeruginosa) were inoculated in a triptycase-soybean broth (TSB) and cultured from 16 to 18 hours at 34 ° C with aeration in a bath stirred by a gyratory agitator. . The bacteria were harvested by centrifugation and washed twice with phosphate buffered saline (PBS, 0.14 M NaCl, 3 mM KCl, 8 mM Na2HP04-7H20, 1.5 mM KH2PO4, pH 7.2) containing 150 trypsin inhibitor units (TJ.IT) of aprin-tinin per ml (Sigma, St. Louis, MO).

The pellet of the final centrifugation was resuspended in 0.17 M triethanolamine and 20 mM disetic edetic acid (EDTA), and then homogenized on ice for ten minutes. The debris was centrifuged from the homogenate at 14,900 g and discarded, and the supernatant was centrifuged again as above. The pellet was rejected again and the membranes were centrifuged out of the supernatant at 141,000 g for one hour. The supernatant was discarded and the membranous pellets were stored overnight at 4 ° C in 10 ml PBS containing 75 I.U. aprotinin per ml. The next day, the pellets were resuspended by shaking and then divided into aliquots and stored at -70 ° C. The protein content of each of the aliquots was determined by the method of Lowry et al. (1951, J. Biol. Chem., 193: 265-275).

The antigen plates for the ELISA tests were prepared as follows. The outer membrane preparations were diluted to 5 µg protein per ml in PBS and 50 µl of solution were placed on a plate in each well of 96-well plates (Linbro # 76-031-05, Flow Laboratories, Inc., McLean, VA), covered and incubated overnight at 37 ° C. Unbound antigen was removed from * 24 plates and 100 µl of bovine serum albumin (BSA) solution in PBS was added to each well, then plates were incubated for 1 hour at 37 ° vs.

After removal of the unabsorbed BSA, the culture supernatants (50 ytditres) from each well of the fusion plates were replicated on the plate in the corresponding wells of the antigen plates and incubated for 30 minutes at 37 ° C. The unbound antibody was removed from the wells and the plates washed three times with 100 µl-iters per well of a 1% (w / v) BSA-PBS solution. Then 50 µl per well of appropriately diluted biotinylated goat anti-mouse IgG (Tago, Inc., Burlingame, CA) was added to each well and incubated for 30 minutes at 37 ° C. The plates were washed three times, as described above, and then 50 µl of a preformed biotinylated horseradish avidinetperoxidase complex (Vectastain ABC Kit, Bector Laboratories, Inc., Burlingame, CA), prepared as directed by the manufacturer , were added to the wells. After 30 minutes at ambient temperature, the Vectastain reagent was removed from the wells, the wells were washed as above and then 100 μlAiter per well of o-phenylenediamine substrat substrate, 8 mg / ml in citrate buffer 0, 1 My pH 5.0, mixed with an equal volume of 0.03% H2O2 (v / v) J7 have et® added. The substrate was incubated for 30 minutes at room temperature in the dark and the reactions were then stopped by the addition of 3N H2SO4 at the rate of 50 μl per well.

The hybridomas secreting monoclonal antibodies which bind to one or the other of the two antigen preparations were located by measuring the absorbance at 490 nm of the colorimetric reactions “* 25 in each well, on a Dynatech Model 580 reader. MicroELISA (Alexandria, VA). The cells in a well, designated as Pa3 IVC2, produced an antibody which bound only to the Fisher 2 immunotype plate. This well was studied in more detail as described below. The monoclonal antibody and the clonal cell line of this well are both identified by the designation Pa3 IVC2 in the text below. The Pa3 IVC2 cells from the original well were minicloned and cloned by limiting dilution techniques, as described by Tam et al., (1982, Infect. Immun ", 36: 1042-1053).

Ascites containing a high titer monoclonal antibody was prepared in CB6 mice (generation F ^ of a BALB / c female x a C57BL / 6 male) according to the methods described by Tam et al. (1982, Infect. Immun., 36: 1042-1053). Males of 2 to 3 month old CB6 mice were injected intraperitoneally with 0.5 ml of Pristane (2,6,10,14-t-tramethylpentadecane, Aldrich Chemical Co., Milwaukee, WI) 10 to 21 days before an intraperitoneal injection of Pa3 IVC2 cells in the logarithmic phase in RPMI. Each mouse was injected with 0.5-1 x 10 ^ cells in 0.5 ml. After approximately two weeks, the accumulated fluid was removed from the mice every two or three days. The antibody concentration in ascites was determined by agarose gel electrophoresis (Paragon, Beckman Instruments, Inc., Brea, CA) and all ascites that contained 5 mg / ml or more of antibodies were pooled , fractionated into aliquots and frozen at -70 ° C. Characterization of the molecular target linked by the monoclonal antibody.

The culture supernatant of the cloned cell line Pa3 IVC2 was tested by ELISA, as “* 26 described above, on preparations of external membranes originating from each of the seven immu-notypic Fisher strains of P. aeruginosa (ATCC 27312 -27318), P. aureofaciens (ATCC 13985) and Klebsiella pneumoniae (ATCC 8047), all prepared as described above. The Pa3 IVC2 antibody bound to the Fisher 2, 6 and 7 immunotypes of the outer membrane preparations of P. aeruginosa and not to the other Fisher immunotypes, nor to P. aureofaciens or K. pneumoniae.

The specific antigen recognized by the antibody Pa3 IVC2 was identified by radioimmune precipitation. In summary, this analysis involves the incubation of radiolabelled antigens with the antibody Pa3 IVC2 and a particulate source of protein A which leads to the formation of insoluble antibody: antigen complexes. These complexes are washed to remove any non-specifically bound antigen and then the complexes are dissociated and separated on polyacrylamide gel. The predominant radioactive species found in the gel are identified in this way as being the corresponding antigen (s) to which the antibody Pa3 IVC2 binds.

Aliquots (25 Jig) of soluble preparations of external membranes of Fisher aerotypes 2, 3, 4 and 5 of P. aeruginosa were radiolabeled in solid phase with 125i using Iodo-gen (Pierce Chemical Co., Rockford, IL) (Fraker and Speck, 1978, Biochem. Biophys. Res. Commun., 80: 849-857? Markwell and Fox, 1978, Biochemistry, 17: 4807-4817). This technique causes iodization of the exposed tyrosine residues of most, if not all, of the proteins contained in the outer membrane preparations.

To decrease the non-specific binding of antigens from the outer membrane to the antibody * »27

Pa3 IVC2, the radiolabelled preparations (5 × 10 6 counts per minute per test) were first incubated for one hour at 4 ° C. with normal BALB / c mouse serum (final dilution 1:40). The culture supernatant Pa3 IVC2 (0 # 5 ml) containing the antibody Pa3 IVC2 was then added to each sample of external membrane. After incubating the antigen and antibody for one hour at 4 ° C, the protein A source, IgGSORB (0.095 ml per sample) (The Enzyme Center, Inc., Boston, MA) was added and incubated for another 30 minutes at 4 ° C (Kessler, SW, 1975, J.

Immunol ,, 115: 1617-1622). IgGSORB was prepared according to the manufacturer's instructions and, just before use, non-specific reactions were prevented by blocking potentially reactive sites with culture medium, by washing the IgGSORB twice with RPMI hybrid (hybrid medium RPMI-HAT without HAT).

The IgGSORB antigen-antibody complexes were centrifuged at 1,500 g for 10 minutes at 4 ° C, washed twice with RIPA phosphate buffer (10 mM phosphate, pH 7.2, 0.15 M NaCl, Triton X 100 1.0 % (v / v), sodium deoxycholate 1.0% (w / v), sodium dodecyl sulfate (SDS) 0.1% (w / v) and aprotinin 1.0% (v / v); two times with highly saline buffer (0.1 M Tris-HC1, pH 8.0, 0.5 M LiCl, 1% beta-mercaptoethanol (v / v); and once with lysis buffer (Tris-HCl 0 , 02 M, pH 7.5, 0.05 M NaCl and Nonidet P-40 0.05% (v / v) (Rohrschneider et al., 1979, Proc. Natl. Acad. Sci., USA, 76: 4479 The antigen bound to the complex was released by incubation with buffer of a sample / 7 Tris-HCl 0.125 M, pH 6.8, SDS 2% (w / v) 77% beta-mercaptoethanol 2% ( v / v) and 20% glycerol (v / v) at 95 ° C for ten minutes and collected in the supernatant after centrifugation at 1,500 g for 10 minutes.

The supernatant samples were then *, 28 applied to 14% polyacrylamide gels containing SOS prepared according to the method of B. Lugtenberg et al. (1978, FEBS Lett., 58: 254-258), as modified by Hancock and Carey (1979, J. Bacteriol., 140: 902-910, which is cited here for reference), and the antigens have been separated in the gel by electrophoresis overnight under constant voltage of 50 V. After fixing the gel in methanol 401 (v / v), acetic acid 10% (v / v) and glycerol 5% (v / v) overnight, it was dried on Whatman 3 MM paper using a Biorad gel dryer (Richmond, CA). The dried gel was covered with a plastic wrap and exposed to Kodak X-AR film for 18 hours at room temperature.

The results of this experiment illustrated that Pa3 IVC2 binds to a single antigen of the outer membrane preparation of the Fisher 2 immunotype of P, aeruginosa, but to no antigen present in the other outer membrane preparations. The molecular weight (PM) of the antigen in the gel was approximately 53,000 daltons, as determined by comparing its mobility with that of reference proteins labeled with 14C (phosphorylase B, PM 92.500; BSA, PM 69.000; ovalbumin, PM 46,000; carbonic anhydrase, PM 30,000; cytochrome C, EM 12,000) (New England Nuclear, Boston, MA) which were separated in the same gel. The molecular weight of this antigen is correlated with the molecular weight of flagel-line, the protein included in the flagella of P. aeruginosa, as reported by Montie et al. (1982, Infect. Immun., 35: 281-288), which is cited here by reference.

In addition, Pa3 IVC2 was examined by ELISA using Habs 1 to 12 strains of P. aeruginosa (A.T.C.C. 33348-33359) which were fixed with K * 29 ethanol on 96-well microtiter plates. The antigen plates were prepared as follows.

Overnight culture broths of each organism were centrifuged, the pellet was washed twice with PBS and then resuspended in PBS to an optical density of 0.2 units for absorbance. 660 nm. The diluted bacteria were placed on a plate in wells (50 yuliters per well) and then centrifuged at 1,500 g for 15 minutes at room temperature. The PBS was aspirated and then ethanol (95%) was added to the wells for 15 minutes at room temperature. After the ethanol was removed from the wells, the plates were air dried and then covered and stored at 4 ° C until needed.

The results of the ELISA tests, recorded as described above, showed that Pa3 IVC2 binds to the Habs strains 2, 3, 4, 5, 7, 10, 11 and 12 fixed by ethanol. This specificity model indicated that Pa3 IVC2 bound to the type b flagella of P. aeruginosa (Ansorg, R., 1978, Zbl. Bakt. Hyg., I.,

Abt. Orig. A, 242: 228-238? Ansorg, R. et al., 1984, J. Clin. Microbiol., 20s 84-88, both cited here for reference). If this specificity is attributed to monoclonal PaC IVC2, the reference strains of P. aeruginosa, immunotype Fisher 2, immunotype Fisher 6, and immunotype Fisher 7 carry type b flagella. From previous experimental data, it was concluded that Pa3 IVC2 specifically binds to type B flagellin from P, aeruginosa.

Protective activity of Pa3 IVC2 in vivo.

Animal experiments were carried out to determine whether the monoclonal antibody Pa3 IVC2 would protect a mouse injected with multiple lethal doses 50 (LD50) of live P. aeruginosa * bacteria. The model chosen was the burned mouse model (Collins, M.S. and Roby, R.E., 1983, J. Trauma, 23: 530-534, which is cited here for reference). Groups of mice were severely burned, according to the authors' protocol, and then immediately injected with 5-10 LD50 of the Fisher 7 immunotype. The monoclonal antibody was administered intraperitoneally as high-grade ascites (0.2 ml intraperitoneally) before the burn and injection. No increase in the number of survivors was observed in animals treated with Pa3 IVC2 compared to those who did not receive antibodies.

EXAMPLE 2 Example 2 illustrates the process for the preparation of a murine hybrid cell line producing a murine monoclonal antibody against type b flagellin of P. aeruginosa, which is protective in vivo.

Adult female BALB / c mice were first injected intraperitoneally with the viable Fisher 6 immunotype of P. aeruginosa (ATCC 27317) (8 x 106 organisms), and two weeks later they were injected with the viable Fisher 5 immunotype of P. aeruginosa (ATCC 27316) (4 x 10 6 organisms). During the next two-week period, viable P. aeruginosa Fisher 5 and Fisher 6 immunotypes were administered simultaneously as two weekly injections. The dose for each organism has been increased so that the final dose is 10 times the initial dose. A final injection of preparations (50 μg of protein) of external membranes of the immunotype Fisher 6 of P. aeruginosa prepared according to the method of R.E.W. Hancock and H. Nikaido (1978, J, Bacteriol ,, "" 31 136: 381-390) was given four days after the last injection of viable bacteria. Three days after the last immunization, the spleen was removed from a mouse and the spleen cells were prepared for hybridization as described in Example 1.

The supernatants of the hybridoma cultures were tested for the presence of anti-P antibodies. aeruginosa by ELISA test on day 10 after the fusion according to the method indicated in Example 1, except that the antigen, for the ELISA plates, consisted of viable bacteria immobilized in the wells of the 96-well microtiter plates. The plates were prepared as follows.

50 ^ liters of poly-L-lysine (PLL) (lyicg / ml in PBS) (Sigma ηβ P-1524, St. Louis, MO) were added to each well of the 96-well plates (Linbro) and incubated for 30 minutes at room temperature. The non-adsorbed PLL was removed and the wells were washed three times with PBS. Bacterial cultures grown overnight in TSB were washed once with PBS and then resuspended in PBS until Aq q nm = ^, 2 OD 50ytt / liter of the bacterial suspensions were added to each well of the plate and allowed to bond at 37 ° C for one hour. Unbound bacteria were removed from the plates with three washings of the wells using Tween saline / ^ NaCl 0.9¾ (w / v), Tween-20 0.05% (v / v) J.

Nonspecific binding of antibodies was blocked by the addition to the wells of 200 yoliters per well of blocking buffer £ "PBS containing dried lean milk 5% (w / v), antifoam A 0.01% (v / v) (Sigma,

St. Louis, MO) and thimerosal 0.01% (v / v) ^ 7 and By incubation for one hour at room temperature. Excess blocking buffer was expelled and the wells

* V

32 were washed three times with saline Tween, as described above.

The culture supernatants (50 ^ liters) were replicated in the corresponding wells of the test plates and incubated at room temperature for 30 minutes. Culture supernatants were removed from the plates, washing the wells five times with Tween saline.

A second stage antibody, conjugated to an enzyme (goat anti-mouse IgG conjugated with horseradish peroxidase + IgM) (Tago, Inc., Burlingame, CA), was diluted in PBS containing 0.1 Tween-20 % (v / v) and 0.2% BSA (w / v), according to the titrations carried out previously, and then 50 μl of the reagent were added to each well and incubated for 30 minutes at room temperature. Excess reagent has been expelled; the wells washed five times in saline Tween; and 100 ^ liters per well of the o-phenylenediamine substrate were added and incubated for 30 minutes, as described in Example 1. The reactions were stopped as in Example 1, and then read for absorbance at 490 nm on a Bio-Tek EL-310 Automated EIA plate reader.

According to the methods described above, the supernatants of fusion cultures were tested for the presence of antibodies which bind to the Fisher 1, 2, 3 or 4 immuno types of P. aeruginosa, but not to the control plates. prepared by the same PLL and blocking technique, but without bacteria. The supernatants containing the antibody which bound to any of these four Fisher immunotypes were tested a second time using each of the seven, Fisher immunotypes of the bacteria separately. The antibody present in the supernatant of a well, PaF4 IVE8, only bound to the Fisher 2, 6 and 7 immunotypes of * * 33 P. aeruginosa. The cells of the PaP4 IVE8 well were cloned by limiting dilution methods, as described in Example 1. The monoclonal antibody and the clonal cell line of this well are both identified by the designation PaF4 IVE8 in the text below. after. Ascites containing high titer monoclonal antibody was produced as described in Example 1, except that BALB / c mice were used instead of CBeF ^.

Specificity of PaF4 IVE8.

One test carried out to identify the antigen linked by the monoclonal antibody PaF4 IVE8 was indirect immunofluorescence on bacterial organisms. Each of the seven reference P. aeruginosa Fisher immunotypes plus an unflagged strain of P. aeruginosa (PA103, ATCC No. 29260, Leifson, 1951, J. Bacteriol., 62: 377-389) and Escherichia coli (GSC A25) were grown overnight at 37 ° C in TSB. The bacteria were centrifuged and then washed twice with PBS. Each strain was resuspended in PBS to an optical density of 2.2 at 660 nm.

The bacterial suspensions were then further diluted 1: 150 and 20 µl samples were placed in single cells on Carlson slides (Carlson Scientific Inc., Peotone, IL) and dried on the blade at 40 ° C. The culture supernatants (25 ^ liters) of PaF4 IVE8 were incubated on the bacterial samples dried on the slides in a humidified room at room temperature for 30 minutes. Unbound antibody was removed from the slides by immersing the slides in distilled water.

After drying of the slides, an anti-mouse goat IgG conjugated with isothiocyanate of 1.34 fluorescein (ITCF) plus an IgM (25 </ & liters per well of a 1:40 dilution in PBS) (Tago, Burlingame, GA) were incubated on the slides for 30 minutes at room temperature in a humidified chamber in the dark. The slides were washed again in distilled water, dried and then covered with a coverslip mounted with glycerol in PBS (9: 1). The slides were observed with a fluorescence microscope.

Fluorescent staining was observed only on Fisher aerotypes 2, 6 and 7 of P. aeruginosa and was observed to be a sinusoid (line) emanating only from one end of the organisms. This is consistent with the morphology and location of the unique polar flagellum of these bacteria.

The reaction of PaF4 IVE8 with flagella was confirmed by immunoblotting analysis. The antigens of the outer membrane of the P. aeruginosa Fisher 6 immunotype (see example 1) were separated by electrophoresis in a 14% polyacrylamide gel containing SDS as described in example 1, except that electrophoresis was conducted for five hours at a constant amperage of 80 mA. Precolored molecular weight markers (lysozyme PM 14.300; beta-lactoglobulin PM 18.400; alpha-chymotrypsinogen PM 25.700; ovalbumin PM 43.000; bovine serum albumin PM 68.000; phosphorylsase B PM 97.400 and myosin PM 200.000) (BRL, Gaithersburg, MD) been included in the same polyacrylamide gel.

The antigens were transferred from the polyacrylamide gel onto a nitrocellulose membrane (MNC), (0.45yom, Schleicher and Schuell, Inc., Keen, NH) in Tris-glycine-methanol buffer (Towbin et al., £ 1979.7 , Proc. Hatl. Acad. Sci., USA, 76: 4350- * * 35 4354) containing 0.05% SDS (w / v) overnight at 4 ° C at constant amperage of 200 mA. After transfer, the MNC was incubated in 0.05% (v / v) Tween-20 in PBS (PBS-Tween) (Batteiger, B, et al., 1982, J. Immunol. Meth., 55: 297-307) for one hour at room temperature. For this step and all the following, the tray containing the MNC was placed on a shaker platform to ensure the distribution of the solution throughout the MNC.

After one hour, the PBS-Tween solution was decanted and ascites PaF4 IVE8 (diluted 1: 1000 in PSB-Tween) was added and incubated with MNC for one hour at room temperature. The MNC was then washed five times, each 5 minutes, with PBS-Tween to remove the unbound antibody. Anti-mouse goat IgG conjugated with alkaline phosphatase plus IgM (Tago, Inc.) was diluted according to the manufacturer's instructions and incubated with MNC for one hour at room temperature. The MNC was washed five times as described above, then the substrate containing bromochloroindolyl phosphate and p-nitrotetrazolium blue (Sigma, St. Louis, MO), prepared as described by Leary et al. (1983, Proc. Natl. Acad. Sci., USA, 80: 4045-4049), was added and incubated 10-20 minutes at room temperature. The reaction was stopped by washing the substrate with distilled water.

The results of this experiment showed that PaF4 IVE8 specifically binds to a single antigen with a molecular weight of 53,000 daltons in the preparation of external membranes. The results of the indirect immunofluorescence test and the immuno-blotting demonstrate that PaF4 IVE8 binds to flagella of P. aeruginosa.

The type of flagellum that PaF4 IVE8 recognizes has 36 * “was determined by ELISA. Habs 1-12 strains (ATCC # 33348-33359) were each linked to wells of 96-well microtiter plates (Linbro) with PLL, and ELISA was performed as described earlier in this example . The source of the antibody PaF4 IVE8 was the culture supernatant. Positive reactions were noted in the wells containing the Habs 2, 3, 4, 5, 7, 10, 11 and 12 strains, therefore indicating that PaP4 IVE8 binds to type b flagella. The in vivo protection data are presented below in Example 4.

EXAMPLE 3 Example 3 illustrates the process for the preparation of a murine hybrid cell line producing a P. aeruginosa anti-flowering monoclonal antibody of type a, which is protective in vivo.

The source of lymphoid cells for fusion was a spleen from an immunized BALC / c mouse, which had been injected four times intraperitoneally for 6 weeks with purified flagella type a (10 to 20yt * g protein) from Habs 6 and 8 strains (ATCC No. 33353 and 33355). The flagella were purified according to the method of T.C. Montie et al. (1982, Infect. Immun., 35: 281-288, which is cited here with reference) except that the final centrifugation of the flagella was carried out at 100,000 g for one hour rather than at 40,000 g for three hours. A second modification adopted for some operations was to detach the flagella from the bacteria for 30 seconds in a blender rather than for 3 minutes. (Allison et al., 1985, Infect. Immun., 49 ï 770-774).

The protein concentrations of each preparation were determined with the Bio-Rad protein test (Bio-Rad, Richmond, CA) and the presence "* 37 of contaminating lipopolysaccharide (LPS) was established by measuring the KDO content (KarXhanis , YD, et al., 1978, Anal. Biochem., 85: 595-601). The molecular weights of flagella proteins were determined by comparing their migration in a polyacrylamide gel with SDS to the migration of proteins labeled dde reference (BRL) (see Example 2). The molecular weight of the flagellin of the Habs 6 strain was 51,700 daltons and that of the flagellin of the Habs 8 strain was 47,200 daltons. These values are in agreement with those obtained by J.S. Allison et al. (1985, Infect. Immun., 49: 770-774, which is cited here for reference).

The fusion of splenocytes from mice immunized against flagellin with NS-1 myeloma cells was carried out three days after the last immunization, as described in examples 1 and 2. When the hybrid cells grew up to approximately 40% of confluence (day 7), the culture supernatants were replicated in corresponding wells of three different antigen plates: the Fisher 1 immunotype of P. aeruginosa linked to PLL (see example 2 for the preparation) and the Habs 6 and Habs 9 strains fixed with formalin.

The bacteria for the formalin-bound antigen plates were grown, washed and diluted as described for the PLL-bound antigen plates. Diluted bacteria (0.2 optical density unit for absorbance at 660 nm) were added to the individual wells (50 ^ liters per well) of Linbro 96-well microtiter plates, and the plates were then centrifuged at 1,200 g for 20 minutes at room temperature. The supernatants were removed from the wells and 75 yoliters of a 0.2% (v / v) formalin solution in PBS was added to each well * Λ 38 and incubated for 15 minutes at room temperature. After formalin was removed from the wells, the plates were air dried and stored at 4 ° C until needed. Formalin does not alter the antigenicity of flagella, as indicated by the capacity of antiflagella antisera to agglutinate organisms treated with formalin (Lanyi, B., 1970, Acta

Microbiol. Acad. Sci., Hung., 17 s 35-48). The immunotype Fisher 1 strain of P. aeruginosa was included as a control, because this strain is not flagellated, as indicated by staining with a biting dye (Manual of Clin. Microbiol., 1985, Lennette, ed. Amer. Soc Microbiol., Wash., DC, p. 1099). The hybrid cells in well FA6 IIG5 produced an antibody which bound to Habs 6 and Habs 9 (two strains carrying flagella type a), but not to the Fisher 1 immunotype.

Cells from well FA6 IIG5 were subcultured and cloned, as described in the previous examples. The monoclonal antibody and the clonal cell line originating from this well are both identified by the designation FA6 IIG5 in the text below. Ascites was produced in BALB / c mice as described in Example 2.

Specificity of FA6 IIG5.

The specificity of the antibody FA6 IIG5 was determined by indirect immunofluorescence and immuno-blotting. Indirect immunofluorescence was carried out essentially as described in Example 2, with the following modifications.

Cultures of bacteria grown overnight on trypticase-soy agar at 30 ° C were collected from the plates with cotton swabs and resuspended in PBS to an absorbance value at 660 nm 0.2 OD unit * 439 formalin £ at a final concentration of 0.37% (v / v) in PBsJ7 was added to the suspension with stirring. After incubation at room temperature for 15 minutes, the bacteria were diluted in PBS in a proportion of 1:12 and 20 μliters of this suspension were placed in individual wells of Carlson slides. After drying, the slides were prepared for observation as described in Example 2. The source of antibody was formed from culture supernatants from the cell line FA6 IIG5.

The fluorescent staining by the antibody FA6 IIG5 was observed on the only strains of P. aeruginosa carrying flagella type a, but on none of those carrying type b. The fluorescent image observed was a sinusoidal line indicating that FA6 IIG5 binds to flagella. The fluorescent signal was increased by treatment of the bacteria with formalin, but this treatment was not required to visualize the flagellar staining with the antibody.

Immunoblotting was carried out as described in Example 2. The sources of type a flagella antigens were the purified flagellar preparations (see the present example). The antigens were separated in 10% polyacrylamide gels containing SDS / 3Laernmli # U.K., 1970, Nature (London), 227: 680-685J7 and transferred to MNC. The preparations of FA6 IIG5, either the culture supernatant or the diluted ascites 1: 1,000 were reacted with MNC, and the reaction detected with an appropriate reagent conjugated to an enzyme and an enzyme substrate, as described in Example 2. Immunoblotting illustrated that FA6 IIG5 binds specifically to PM flagellin, 51,700 from Habs 6 and to PM flagellin 47,200 40 * "from Habs 8.

Confirmation that FA6 IIG5 reacts only with flagellum type a and not type b, was obtained by ELISA, for which purposes Habs 1-12 strains were linked individually with PLL to the wells of 96-well microtiter plates Linbro. The antigen only bound to Habs strains 1, 6, 8 and 9, which are the only strains carrying type a flagella among the twelve (see, Ansorg, R. et al., 1984, J. Clin. Microbiol, , 20: 84-88, and cited here for reference). The in vivo protection studies are presented in Example 4 below.

EXAMPLE 4 Example 4 illustrates the protection of mice passively immunized with the antibodies PaF4 IVE8 and FA6 IIG5 against infection by P. aeruginosa on a model of burned mice.

The anti-flowering monoclonal antibodies were tested on a burned mouse model according to the method of M.S. Collins and R.E. Roby (1983, J. Trauma, 23: 530-534, which is cited here with reference). For the protection studies, all the antibodies were purified by protein A-Sepharose chromatography (Ey, P.L. et al., 1978, Immunochemistry, 15: 429-436, which is cited here in reference) and dialyzed against PBS buffer. The type flagella strain used in animal tests was P. aeruginosa PA220 (acquired from Dr. James Pennington, Boston, MA) and the type b flagella strain was the immunotype

Fisher 2 of P. aeruginosa (A.T.C.C. No. 27313) for reference.

40 g of purified monoclonal antibody were given, intravenously by mouse, one to two hours before the burn and the infectious injection. Immediately after the burn, the animals received f · 41 under pressure ulcer 0.5 ml of cold PBS containing the bacteria injected. The dose injected was approximately 10 LD50 for each organism. The results of the animal tests are presented in Tables I and II.

* 42

TABLE I

Study of protection by a monoclonal antiflagella type a antibody on a burned mouse model · * ·.

Percentage of survival per day ^ Treatment 123 456 789 FA6 IIG5, antiflagella a 100 100 100 100 100 90 90 80 80

PaF4 IVE8, antiflagella b 100 20 10 10 10 10 10 10 10

Non-specific monoclonal antibody anti-LPS 100 40 30 20 10 10 10 10 10 PBS, without bacteria 80 80 80 80 80 80 80 80 80 1. The mice were infected under pressure ulcer with approximately 10 LD50 of P. aeruginosa PA220.

2. The percentage is based on the survival of mice in groups of ten animals, except for the control group receiving PBS only, which consisted of five mice. The days are after burning and infective injection.

* * 43

TABLE II

Study of protection by a monoclonal antiflagella type b antibody on a burned mouse model - * -.

Percentage of survival per day ^ Treatment 12 3 456 789

PaF4 IVE8, antiflagelle b 100 100 90 90 90 90 90 90 90 90 FA6 IIG5, antiflagelle a 100 20 10 10 10 10 10 10 10

Non-specific anti-LPS monoclonal antibody 100 20 20 20 20 20 20 20 20 PBS, without bacteria 100 100 100 100 100 100 100 100 100 1. The mice were infected by pressure ulcer with approximately 10 LD50 of the immunotype Fisher 2 of P aeruginosa.

2. The percentage of survival is based on the number of surviving mice per group of ten, except for the control group receiving PBS only, which consisted of five mice. The days are after burning and infective injection.

44

Very significant survival was observed in mice treated with the anti-a antibody or the anti-b antibody and then infected by injection of the corresponding antigen. Conversely, 80-90% of the untreated but injected mice, or of the animals treated with a non-reactive antiflagellar monoclonal antibody or a non-specific anti-LPS antibody, would die. The inability of the type A antiflagella antibody to protect mice against a lethal injection of Fisher 2 immunotype of P. aeruginosa carrying type B flagella, and the inability of the Type B antiflagelle antibody to protect mice mice against a lethal injection of P. aeruginosa PA220 type a, corroborated in vivo the specificity of the antibodies observed in vitro. The survival of burned but uninfected mice indicated that the burn itself was not lethal.

EXAMPLE 5 Example 5 demonstrates the considerable cross-reactivity of PaF4 IVE8 and FA6 IIG5 with clinical isolates of P. aeruginosa, which proves the clinical utility of these antibodies in the immunotherapy of P. aeruginosa infections.

Clinical isolates have been obtained from hospitals and clinics. The isolates were collected from various collection sites, including blood, wounds, respiratory system, urine and ears. A total of 157 isolates were examined.

PaF4 IVE8 specifically linked to 34 clinical isolates (22%), while the antibody for flagella type a or FA6 IIG5 linked to 102 clinical isolates (65%), for a total of 136 157 isolates (87%). Among the 21 strains which were not recognized by either of the two antibodies, 19 were not flagellated, as indicated by staining with a η * 45 "biting dye. Therefore # the two antibodies in combination bind with 136 of 138 (98%) of the flagellated clinical isolates, confirming previous reports (see, Ansorg, R., 1978, Zbl. Bakt, Hyg. I Abt. Orig. A, 242 s 228-238, which is cited here for reference). EXAMPLE 6 Example 6 illustrates the process for the production of human monoclonal antibodies which bind to type b flagella of P. aeruginosa.

A peripheral blood sample collected from an individual immunized with a high molecular weight polysaccharide preparation (Pier et al., 1984, Infect. Immun., 45: 309) served as a source of B cells. The mononuclear cells were separated of blood by standard centrifugation techniques on Ficoll-Paque £ Boyum (1968) Scand. J. Clin. Lab. Invest., 21: 77J7 and washed twice in a calcium phosphate and magnesium free phosphate buffered saline (PBS).

The mononuclear cells were stripped of T cells according to a procedure modified to form rosettes E. In short, the cells were first resuspended until a concentration of 1 × 10? cells per ml in PBS containing 20% fetal calf serum (SFV) at 4 ° C. 1 ml of this suspension was then placed in a 17 x 100 mm polystyrene round bottom tube and 1 x 10 ^ sheep red blood cells treated with 2-amino-isothiouronium bromide (TEA) were added thereto. a 10% (v / v) solution in Dulbecco medium modified according to Iscove (Iscove medium), Madsen and Johnson (1979), J. Immun. Methods, 27: 61 ^. The suspension was mixed gently for 5-10 minutes at 4 ° C and the cells in rosette E were then separated by centrifugation on Ficoll-Paque H * 46 for 8 minutes at 2,500 g at 4 ° C. Rosette E negative peripheral blood mononuclear cells (CMSPE “) presenting in a band at the interface were collected and washed once in Iscove medium and resuspended in the same medium containing 15% SFV (v / v), L-glutamine (2 mmol per liter), penicillin (100 International Units per ml), streptomycin (100 fn, g / ml), hypoxanthine (1 x 10 “4 m) , 11 aminopterin (4 x 10 “? M) and thymidine (1.6 x 10“ 5 M). This medium is hereinafter called HAT medium.

The cell-driven transformation of CMSPE “was accomplished by co-cultivating these cells with a transforming cell line. The transforming cell line was a human lymphoblastoid cell line positive for the Epstein-Barr nuclear antigen (EBNA) derived by ethyl methanesulfonate mutagenesis (EMS) of the GM 1500 lymphoblastoid cell line, followed by selection in the presence of 30 ml of 6-thioguanine to make cells deficient in hypoxanthine-gua-nine-phosphoribosyltransferase (HGPRT) and therefore sensitive to HAT. This cell line is called cell line 1A2 and was deposited with the American Type Culture Collection (A.T.C.C.) on March 29, 1982 under the number A.T.C.C. CRL 8119. 1A2 cells in the logarithmic growth phase were resuspended in HAT medium and then combined with CMSPE with a proportion of 15 1A2 cells by CMSP. The cell mixture was plated on 30 round bottom 96 well microtiter plates (Costar 3799) at a concentration of 32,000 cells per well in a volume of 200 µliter per well, and incubated at 37 ° C in a humidified atmosphere containing 6% CC> 2 · the cultures were fed on days 5 and 47 8 after placing on the plate by replacing half of the supernatant with fresh HAT medium. Sixteen days after plating, 100% of the wells contained proliferating cells and, in most wells, the cells were of sufficient density for the collection and examination of supernatants for anti-P antibodies. aeruginosa.

The supernatants were examined for the presence of anti-P antibodies. aeruginosa using the SLISA technique as described in Example 2, with the following modifications. A collection of the seven reference Fisher immunotype strains (ATCC No. 27312-27318) (AggQ = 0.2 DO unit) was linked to 96-well flat-bottom microtiter plates (Immulon II, Dynatech), pretreated with poly-L-lysine, incubated and washed, as described in Example 2. After blocking the non-specific binding sites and washing the plates, 50 μl of PBS containing 0.1% (v / v) of Tween -20 and 0.2% BSA (w / v) were added per well. The culture supernatants (50 μl) were then replicated on a plate in the corresponding wells of test plates and in control plates which had been treated with PLL and blocked, but free of bacteria. After incubation and washing, second stage antibodies conjugated to an enzyme (50 ytLLitres per well), antihuman goat IgG conjugated with horseradish peroxidase, and antihuman goat IgM or appropriate dilution in PBS containing 0.1% Tween-20 (v / v) and 0.2% BSA (w / v), were added to the wells and the assay was completed, as described in Example 2 .

Supernatants containing the antibody which bound to the Fisher immunotype pool, but not to the control plate, were tested a second time i 4.

48 using each of the bacteria from the seven Fisher immunotypes separately. The antibody present in the supernatant of a well, 20H11, bound only to the Fisher aerotypes 2, 6 and 7 of P. aeruginosa. Cells were repeatedly subcultured at decreasing cell densities until all wells with growth secreted antibody. The cell line and the monoclonal antibody (isotope IgM) are both identified by the designation 20H11 in the text below.

A second transformation was carried out, in which the source of the B cells was the peripheral blood of a patient suffering from cystic fibrosis, known to have had a chronic infection with P. aeruginosa. The CMSPEs "were prepared, as described above, and cocultivated with the transforming cell line, 1A2, in a proportion in 72 cells 1A2 by CMSPE". The cell mixture was plated on 15 round bottom 96 well microtiter plates at a concentration of 7.4 x 14 cells per well and cultured as above.

The supernatants were tested by ELISA for the presence of anti-P antibodies. aeruginosa sixteen days after the transformation was placed on a plate. The test was carried out as described for the preceding transformation, except that the collection of P. aeruginosa strains used for the initial examination was composed of the reference immunotype Fisher F2, F4, F6 and F7 strains (ATCC No. 27313, 27315, 27316 and 27317), in addition to three clinical isolates from the Genetic Systems Corporation Organism Bank (GSCOB) which had different LPS immunotypes and types of flagella. The clinical isolate PSA 1277 (GSCOB) carries type a flagella and the Fisher 1 LPS immunotype? * t 49 the second PSA G98 isolate (GSCOB) carries type a flagella and the Fisher 3 LPS immunotype; and the third # PSA F625 (GSCOB) carries type b flagella and the Fisher 5 LPS immunotype. This mixture of reference strains and clinical isolates is called a flagellate P. aeruginosa pool. The supernatants containing the antibody which bound to the plaques containing the flagellated P. aeruginosa cluster, but not to the control plates covered with PLL, were tested by ELISA a second time on the individual strains of the cluster. One well, 3C1, bound to the reference strains F2, F6 and F7 and to the clinical isolate F625.

Cloning of the 3C1 cell lines was accomplished by first subculturing the cells in two rounds of low density subcultures, first at 20 cells per well of 96-well plates, then by 2-culture cells per well. Formal cloning of the cells producing the specific antibody was carried out by placing the cells at a density of approximately 1 cell per well on 72-well Terasaki plates (Nunc no. 1-36538) in a volume of 10 ^ olitres per well of HAT medium free of aminopterin (HT medium). The plates were placed in an incubator for 2 to 3 hours to allow the cells to sediment at the bottom of the wells and then examined under the microscope by two agents for the search of wells containing the single cell. The wells were fed daily with HT medium and, when growth was sufficient, the cells were transferred to a 96-well round bottom plate. All wells with growth were tested by ELISA on P. aeruginosa strains carrying type b flagella and all were found to produce the appropriate antibody. The cell line and the monoclonal antibody 50 (isotype IgM) are both identified by the designation 3C1 in the text below.

The antigen identified by 20H11 and 3C1 was formed by flagella, as indicated by indirect immunofluorescence and 11immunoblotting. The techniques were carried out basically as described in Examples 2 and 3. For the indirect immunofluorescence test, strains of P. aeruginosa carrying flagella type b, Fisher F2, F6 and F7 reference immunotypes (ATCC No. 27313 , 27317 and 27318) and a strain carrying flagella type a, reference Fisher 4 immunotype (ATCC No. 27315) were prepared as described in Example 3. The type of flagella of the reference strains was determined by typing with the murine monoclonal antibodies PaF4 IVE8 and FA6 IIG5. The slides were prepared for observation as described in Example 2. The sources of the two antibodies were culture supernatants, and the reagent conjugated to 1iITcf was goat anti-human Ig conjugated to 11 ITCF ( polyvalent) (Tago, Burlingame, CA) in 1: 100 dilution in PBS containing 0.5% (w / v) of bovine gammaglobulins (Miles Scientific, Cat., n ° 82-041-2, Naperville, IL) and 0.1% (w / v) sodium azide as an antiputrid.

The fluorescent staining by the antibodies 20H11 and 3C1 was observed only with the strains of P, aeruginosa carrying flagella type b and not with the strain carrying flagella type a, the reference Fisher 4 immunotype. The fluorescent image observed was a sinusoidal linear image emanating from one end of the bacteria, indicating that the antibodies were binding to the flagella of the bacteria.

L1 immunoblotting was carried out as described in Example 2. The type b flagella purified from <t 51 from the reference Fisher 2 immunotype strain of P. aeruginosa (ATCC No. 27313) and the type a flagella purified to from the reference Habs 6 and Habs 8 strains (ATCC no 33353 and 33355) were prepared as described in Example 3. The antigens were separated on a 10% polyacrylamide gel (see Example 3) and transferred to a MNC. Culture supernatants containing 20H11 or 3C1 antibodies, culture supernatant containing human non-specific antibody and culture media were incubated with MNC and the reaction was detected with anti-human goat Ig conjugated with alkaline phosphatase (polyvalent) (Tago, Burlingame, CA) diluted in PBS containing 0.05% (v / v) Tween-20. The enzyme substrate was prepared as described in Example 2. Immunoblotting showed that the two antigens bind to the flagellin protein of PM 53.000 of the immunotype Fisher 2 and not to the flagellin protein of PM 51.700 of Habs 6, nor to flagellin protein of PM 47,200 from Habs 8. No reaction was observed either with the non-specific human antibody or with the culture media.

Additional confirmation that 20H11 and 3C1 antibodies only bind to type b flagella and not type a was obtained by ELISA, for which purposes Habs 1-12 strains were individually bound with PLL from a well of 96-well Immulon microtiter plate. Antibodies only bound to Habs strains 2, 3, 4, 5, 7, 10, 11 and 12, which are strains carrying type b (Ansorg et al., (1984) J. Clin. Microbiol., 20 s 84). EXAMPLE 7 Example 7 illustrates methods for the production of a human monoclonal antibody which binds to type a flagella of P, aeruginosa.

<b 52

A peripheral blood sample collected from an individual immunized with a high molecular weight polysaccharide preparation (Pier et al. (1981) Infect. Immun., 34: 461) served as a source of B cells. The mononuclear cells were separated from the blood and then rid of T cells as described in Example 6. The cells were then frozen in SFV containing 10% dimethylsulfoxide in a vapor vessel of liquid nitrogen. At a later date, the cells were thawed quickly at 37 ° C, washed once in Iscove medium and resuspended in HAT medium. Cell-driven transformation was accomplished by cocultivating CMSPE "with 1A2 cells in a proportion of 30 1A2 cells by CMSPE". The cell mixture was plated on 30 96-well tissue culture plates at a concentration of 62,000 cells per well. The cells were fed on the seventh day after placing on the plate by replacing half the volume with HAT medium. Cell proliferation was observed in 100% of the wells on the fourteenth day after the placing on plate and supernatants were removed from the wells and tested at this time.

The supernatants were tested by ELISA for the presence of anti-P antibodies. aeruginosa using the pool of flagellated P. aeruginosa and PLL treated plates as a control as described in Example 6. Supernatants containing antibodies which bound to the flagellated pool, but not to the PLL control plates, were retested on individual bacteria strains from the flagellated pool. One well, 21B8, contained an antibody which bound to PSA 1277, PSA G98 and the reference immu-notype Fisher 4, which are the three "" 53 strains of the flagellated pool which carry type a flagella.

Cloning of the cell line 21B8 was accomplished as described in Example 6 for the cell line 3C1 with the following modifications in the formal cloning step. After the wells of the Terasaki plates were scored for the presence of a single cell only, each cell was transferred from the Terasaki plate to an individual well of a round bottom 96-well culture plate, in a volume of 100 yulitres of HAT medium free of aminopterin (HT medium). HAT-sensitive non-transforming lymphoblastoid cells were included in all wells at a density of 500 cells per well as feeder cells. Five days after plating, 100 ^ liters of HAT medium was added to the wells to selectively kill the feeder cells. The wells were further fed on days 7 and 9 after placing on the plate by replacing half of the supernatant with HAT medium. The cells were then fed with HT medium until the cells were of sufficient density to detect the presence of an antibody by ELISA. All wells with growth produced antibody which bound to strains of P. aeruginosa carrying flagella type a. The cell line and the monoclonal antibody (isotype IgG ^) are both identified under the designation 21B8 in the text below.

The antigen identified by 21B8 was formed from flagella, as indicated by indirect immunofluorescence and immunoblotting (see Example 6 for descriptions of techniques). The fluorescent staining by the antibody 21B8 was observed only with the reference immunotype Fisher 4 strain of 54 *, P. aeruginosa (ATCC No. 27315) which carries type a flagella, and not with the reference immunotype 2 strain of P, aeruginosa (ATCC No. 27313) which carries type a flagella. The fluorescent image observed was a sinusoidal linear image from one end of the bacteria indicating that the antibody was binding to the flagella of the bacteria.

Immunoblotting was carried out as described in Example 2. Purified type a flagella from the reference Habs 6 strain of P. aeruginosa (ATCC No. 33353) and type b flagella from the Fisher immunotype strain 2 reference of P. aeruginosa (ATCC No. 27313) were prepared as described in Example 3. The antigens were separated on a 10% polyacrylamide gel (see Example 3) and transferred to an MNC. cultures containing either 21B8 or a non-specific human antibody, and the culture media were reacted with MNC and the reaction was detected with anti-human goat Ig conjugated to alkaline phosphatase (polyvalent) and a substrate of enzyme as described in Examples 2 and 6. Immunoblotting illustrated that the antibody 21B8 bound only to the flagellin protein of EM 51.700 from Habs 6 and not to the flagellin protein of PM 53.000 of the Fisher 2 immunotype. None reaction was neither observed nor with the non-specific human antibody, nor with the culture media.

EXAMPLE 8 Example 8 illustrates the protection of mice passively immunized with human anti-flowering antibodies 20H11, 3C1 and 21B8 against the infectious injection of P. aeruginosa on a burned mouse model.

The human anti-55 flagella monoclonal antibodies were tested on a burned mouse model (see example 4). Antibodies 21B8 and 20H11 were prepared by precipitation of culture supernatants obtained from the respective cell lines with ammonium sulphate (final concentration 50%) (Good et al., Selected Methods in Cellular Immunoloqy, Mishell, BB and Shiigi, SM ed., WJ Freeman & Co., San Francisco, CA, 1980, 279-286). The precipitate was dissolved in PBS, dialyzed against PBS overnight at 4 ° C and then filtered sterile before administration to animals. The source of the 3C1 antibody and that of the non-specific anti-LPS antibody used as negative control in this study were culture supernatants. The positive control for each study was the appropriate purified murine monoclonal antibody PaF4 IVE8 or FA6 IIG6.

The strain carrying flagella type a used in animal tests was the clinical isolate PSA A522 (GSCOB) which expresses the immunotype Fisher 1 LPS and the strain with flagella type b was the clinical isolate PSA A447 (GSCOB) which expresses the Fisher 6 LPS immunotype. Human antibodies (0.45 ml) were premixed with bacteria (more than 5 DL ^ qq in 0.05 ml) and inoculated under pressure ulcer immediately after the burn was administered. The results of the animal tests are presented in Tables III, IV and V.

TABLE III

i * 56

Study of the protection by the human monoclonal antibody 21B8 antiflagella type a in the burned mouse model - * -.

Percentage of survival per day1

Treatment 123 456 789 FA6 IIG5, murine antiflagella a1 100 100 100 100 88 88 88 88 88 21B8, human antiflagella a 100 100 100 100 100 100 100 100 100 20H11 human antiflagella b 100 00000000 PBS / 100 25 12 12 12 12 12 12 12 1. The mice were infected under pressure ulcer with more than 5 DL ^ qo of P. aeruginosa PA220.

2. The percentage of survival is based on the number of surviving mice per group of eight animals. The days are after burning and infective injection.

The purified antibody (10ytcg in 0/45 ml PBS) was premixed with the bacteria and administered under pressure ulcer after the burn and the infective injection.

""

Study of the protection by 11 human monoclonal antibodies 20H11 antiflagella type b in the burned mouse model- * ·.

57

TABLE IV

Percentage of survival per day2

Treatment 123456789

PaF4 IVE8, murine, antiflagella b3 ioo 100 100 100 100 100 100 100 100 20H11, human antiflagella b 100 100 100 100 100 100 100 100 100 21B8 human antiflagella a 100 0 0 0 0 0 0 0 0

Media 80 00000000 1. The mice were infected with bedsores by more than 5 DL ^ qo ^ PSA A447 clinical isolate of P. aeruginosa.

2. The percentage of survival is based on the number of surviving mice per group of five animals. The days are after burning and infective injection. 1 The purified antibody (10 µg in 0.45 ml PBS) was premixed with bacteria and administered under pressure ulcer after the burn and infective injection.

TABLE V

1.

Study of the protection by the human monoclonal antibody 3C1 antiflagella type b in the burned mouse model · * ·.

58

Percentage of survival per day ^

Treatment 123456789

PaP4 'IVE8, murine, anti-flowering b3 χοο 100 100 100 100 100 100 100 100 3C1, human anti-flowering b 100 100 80 80 80 80 80 80 80

Non-specific monoclonal antibody anti-LPS 100 00000000 1. The mice were infected under pressure ulcer with 5 DL100 of PSA A447.

2. The percentage of survival is based on the number of surviving mice per group of five animals. The days are after burning and infective injection.

3. The purified antibody (40 jug) was administered intravenously two hours before the burn and the infective injection.

* * 59

Significant survival was observed in the mice treated with the antiflagelle type a antibody or with one or the other of the antiflagelle type b antibodies and then infected by injection of the corresponding antigen. Conversely, 88-100% of the untreated but injected mice, or those treated with a non-reactive antiflagellar antibody or with a non-specific anti-LPS antibody, would die. As has been observed with murine monoclonal antibodies (see Example 4), human anti-flowering antibodies specifically protect against lethal injection only for organisms carrying the corresponding type of flagella, i.e. the human anti-flowering antibody of type a protects against a lethal injection of the organism carrying flagella of type a and not of type b, and that the antiflagella antibodies of type b protect the mice injected with strains carrying flagella of type b, but not of type at.

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

Clinical isolates of P. aeruginosa (115) obtained from hospitals and clinics and originally isolated from burned wounds and blood have been identified as carrying flagella type a or type b, typing with murine monoclonal antibodies FA6 IIG5 or PaP4 IVE8 (see examples 2, 3 and 5). Fifty-five of the clinical isolates were identified as carrying type a flagella by their reaction with the murine monoclonal antibody FA6 IIG5, and 59 were identified as carrying type b by their reaction with the murine monoclonal antibody PaF4 IVE8.

The cross-reactivity of the human clonal mono- * -¾ 60 antiflagelle type a, 21B8, was considerable because the antibody recognized 54 of 56 clinical isolates carrying type a flagella (96%). The cross-reactivity of 20H11 with isolates carrying type b flagella was also considerable because 20H11 recognized all 59 isolates (100%). In contrast, the other monoclonal antiflagella type b antibody, 3C1, bound only to 43 of 59 isolates (731). These results demonstrate that 20H11 binds to a pan-reactive epitope (i.e. an epitope present on at least about 95% of the flagellate strains of P. aeruginosa), while 3C1 binds to an epitope not present on all type b flagellin molecules. Although the type b flagella antigen appears serologically uniform when analyzed with polyclonal antisera (Lanvi, B., supra, and Ansorg R., supra), the cross-reactivity models of 20H11 and 3C1 show surprising that the flagella type b comprises at least two separate epitopes which can be identified by monoclonal antibodies,

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

EXAMPLE 10 Example 10 illustrates methods for the production of another exemplary human monoclonal antibody which binds to type b flagella of P. aeruginosa, as well as the protective activity of the antibody against infection by P. aeruginosa on a burnt mouse model.

A transformed cell line was prepared and cloned essentially as described in <* 61 Example 7, except that the transformed cell mixture was plated on 20 96-well tissue culture plates at a concentration of approximately 2250 CMSPE- per well. The supernatants were initially tested by ELISA on the flagellated pool as described in Example 6, except that the reference Fisher 2 and 4 immunotype strains were absent from the pool. The positive wells were then tested on each strain of the flagellated collection comprising the immunotypes Fisher 2 and 4. The cell line finally isolated and the monoclonal antibody (isotype IgG) are both identified by the designation 12D7 in the text below. .

The human monoclonal antibody 12D7 revealed considerable cross-reactivity with type a isolates, recognizing 54 of the 56 clinical isolates carrying type a flagella tested (96%). The protective activity of the 12D7 monoclonal antibody is shown in Table VI. The protection studies were carried out essentially as described in Example 4, except that the dose injected was 1 DL ^ qq and that the strain injected was 1624, which is a clinical isolate expressing the Fisher 2 LPS immunotype and the type has flagella.

Study of the protection by the human monoclonal antibody 12D7 antiflagella type a in the burned mouse model1.

λ .► 62

TABLE VI

Percentage of survival per day ^

Treatment 123456789 12D7, human, antiflagelle a 100 100 100 100 100 100 100 100 100 2H9, human anti-Fisher 2 LPS 90 90 90 90 90 90 90 90 90 11G5 murine antiflagelle a 100 100 100 100 100 100 100 100 100 15F4 human, antiflagella b 100 37.5 25 25 25 25 25 25 25 1. The mice were infected under pressure ulcer with 1 DL ^ qq (950 CFU) of P. aeruginosa.

2. The percentage of survival is based on the number of surviving mice per group of ten animals, except for the group 15F4 where N = 8. The days are after burning and infective injection.

3. The purified antibody (SOyug in 0.5 ml PBS) was administered intraperitoneally before the burn and infectious injection.

* + 63 EXAMPLE 11. Example 11 illustrates the production of a human monclonal antibody reactive with type b flagella of P, aeruginosa and the protection of mice passively immunized by this antibody against infection by injection of P. aeruginosa on burnt mouse model.

A transformed cell line was prepared and cloned essentially as described in Example 10, except that the ratio of 1A2 to B cells for transformation was approximately 60: 1 and the mixture of transformed cells was plated in 15 plates at a concentration of approximately 1930 CMSPE “per well. Similarly, the cells were tested on a collection of P. aeruginosa strains comprising G98 (Fisher 3 immunotype, type a flagella) and 1739 (a clinical isolate of the Fisher 5 immunotype, type b flagella) and confirmed on a different collection of strains of P, aeruginosa: 1277, G98, 1739 and the immunotypes

Fisher F2, F4, F6 and F7. The finally isolated cell line and the secreted monoclonal antibody (isotype IgG ^) are both designated under the name 2B8 in the text below.

An immunofluorescence test carried out with 2B8 was positive on a strain. flagella type b immunotype Fisher 2, but negative on a reference strain of flagella type a, immunotype Fisher 4. In the test of clinical isolates 59/59 (100%), flagellates type b were found to be positive.

The protective activity of 2B8 is shown in Table VII. The protection studies were carried out as described in Example 10, except that the clinical isolate F164 (immunotype Fisher 4, flagella type b) was used for infection by injection.

* é 64

TABLE VII

Study of the protection by the human monoclonal antibody 2B8 antiflagella type b in the burned mouse model1.

Percentage of survival per day ^ Treatment 123 456 789 2B8, human, antiflagella b3 100 89 89 89 89 89 89 89 89

PaF4 IVE8, murine antiflagella b4 100 100 100 100 100 100 90 90 90 90 VH3, murine anti-Fisher 2 LPS4 90 20 20 20 20 20 20 20 20 1. The mice were infected under pressure sores with 1 DL100 (105 CFU) of P aeruginosa.

2. The percentage of survival is based on the number of surviving mice per group of ten animals, except for group 2B8 where N = 9. The days are after burning and infective injection.

3. The purified antibody (50 µg in 0.5 ml PBS) was administered intraperitoneally before the burn and infectious injection.

4. The purified antibody (5 μl in 0.5 ml of PBS) was administered intravenously two hours before the burn and the infectious injection.

-i I * 65 EXAMPLE 12, - Example 12 demonstrates the production of another human monoclonal antibody reactive with P. aeruginosa with flagella type b and the protection of mice passively immunized by this antibody against infection by injection of P aeruginosa on a burnt mouse model. .

A transformed cell line was prepared and cloned essentially as described in Example 7, with the following exceptions. A different individual was immunized / _Pier et al., (1984) 45: 309.27 and served as a source of B cells, and the number of CMSPE ”was approximately 2,000 cells per well for plating. The examination was carried out on the Fisher immunotypes 1 to 7 pooled. The finally isolated cell line and the secreted monoclonal antibody (isotype IgG ^) are both designated under the name 14C1 in the text below.

In the clinical test, 59/59 (100%) of the flagellate type b isolates tested positive for 14C1. The protection studies were carried out as described in Example 11 above and are indicated in Table VIII.

TABLE VIII

t ch 66

Study of the protection of the human monoclonal antibody 14C1 antiflagella type b in the "burned mouse" model.

Percentage of survival per day ^ Treatment3 123 456 789 14C1, human, antiflagella b 100 100 100 100 100 90 90 90 90

PaF4 IVE8, murine antiflagella b 100 100 100 100 100 100 100 100 100 human VH3 anti-Pisher 2 LPS 100 40 20 20 20 20 20 20 20 1. The mice were infected under pressure with 1 DL ^ oo (90 CFU) P. aeruginosa F164.

2. The percentage of survival is based on the number of surviving mice per group of ten animals, except for the PaF4 IVE8 group where N = 9. The days are after burning and infective injection.

3. The purified antibody (50 μg in 0.5 ml of PBS) was administered intraperitoneally before the burn and the infectious injection.

<67

It is apparent from the above that the cell lines of the present invention constitute means for producing monoclonal antibodies and fragments thereof which are reactive with flagella of P. aeruginosa and offer cross protection against various strains of P aeruginosa. Surprisingly, many monoclonal antibodies reactive with different epitopes on each type of flagella have been isolated. The antibodies of the present invention make it easier and cheaper to produce prophylactic and therapeutic compositions for use against infections caused by most strains of P. aeruginosa. In addition, cell lines provide antibodies which find applications in immunoassays and other well-known procedures.

The mouse hybridoma lines PaF4 IVE8 and FA6 IIG5 cited in Examples 2 and 3 were deposited at A.T.C.C. June 20, 1986 under the references HB9129 and HB9130, respectively.

The human lymphoblastoid cell lines 20H11 and 21B8 cited in Examples 6 and 7 were deposited at A.T.C.C. December 23, 1986 under the references CRL 9300 and CRL 9301, respectively.

The human lymphoblastoid cell lines 12D7, 2B8 and 14C1 cited in Examples 10, 11 and 12 were deposited at A.T.C.C. May 12, 1987 under references CRL 9422, CRL 9423 and CRL 9424, respectively.

Although the present invention has been described in various details by way of illustration and examples for the sake of clarity and understanding, it is obvious that it is capable of various variants and modifications without going beyond its ambit.

Claims (36)

1. Composition characterized in that it comprises a monoclonal antibody or a binding fragment thereof capable of reacting specifically with the flagella of the bacteria Pseudomonas aeruginosa. 2. Composition according to claim 1, characterized in that the monoclonal antibody or the binding fragment thereof inhibits the motility of bacteria, 3. Composition according to any one of claims 1 and 2, characterized in that the monoclonal antibody or the binding fragment thereof is protective in vivo. 4. Composition according to any one of the preceding claims, characterized in that it comprises a monoclonal antibody capable of reacting specifically with an epitope of a flagellar protein of Pseudomonas aeruginosa. 5. Composition according to claim 4, characterized in that the monoclonal antibody is capable of blocking the binding with the epitope of monoclonal antibodies produced by the cell lines designated under the access numbers A.T.C.C. HB9129, HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. 6. Cell line characterized in that it produces a monoclonal antibody capable of reacting specifically with flagella type a or type b of Pseudomonas aeruginosa. 7. Cell line according to claim 6, characterized in that the monoclonal antibody is protective in vivo against Pseudomonas aeruginosa. 8. Cell line according to any one of claims 6 and 7, characterized in that it is a hybrid cell line. 9. Cell line according to any one of claims 6 to 8, characterized in that it produces human monoclonal antibodies. 10. Cell line according to claim 6, characterized in that it is one of those designated under the access numbers A.T.C.C. HB9129, HB9130, CRL 9300, CRL 9301, CRL 9422, CRL 9423 and CRL 9424. 11. Method for the production of monoclonal antibodies specific for the flagellar proteins of Pseudomonas aeruginosa and capable of treating or preventing infections with Pseudomonas aeruginosa, characterized in that it comprises the culture of at least one of the following cell lines claim 10 and the collection of these antibodies. 12. Pharmaceutical composition useful for the treatment of a human being susceptible to bacteremia and / or septicemia, characterized in that it comprises a prophylactic or therapeutic amount of monoclonal antibodies produced according to claim 11. 13. Monoclonal antibody of the binding fragment thereof, characterized in that it is reactive with an epitope capable of binding to a monoclonal antibody produced according to claim 11. 14. Composition characterized in that it comprises a monoclonal antibody or a binding fragment thereof reactive with an epitope capable of binding to a monoclonal antibody produced according to claim 11. 15. Composition comprising one or more monoclonal antibodies, characterized in that a first "of these monoclonal antibodies is capable of reacting with x t" 70 an epitope of flagella type a or type b of Pseudomonas aeruginosa. 16. Composition according to claim 15 / characterized in that a second of these monoclonal antibodies is capable of reacting with a type of flagella that is not reactive with the first monoclonal antibody. 17. Composition according to any one of claims 15 and 16, characterized in that the first antibody is a human monoclonal antibody, 18. Composition according to any one of claims 15 to 16, characterized in that at least one of the monoclonal antibodies is protective in vivo. 19. Composition according to any one of claims 1 to 5 and 14 to 18, characterized in that it additionally comprises a fraction of gamma globulin originating from the plasma of human blood and / or an antibiotic agent. 20. Pharmaceutical composition, characterized in that it comprises a composition according to claim 19 and a physiologically acceptable excipient. 21. Pharmaceutical composition useful for the treatment or prevention of a Pseudomonas aeruginosa infection, characterized in that it comprises a monoclonal antibody reactive with a flagellar protein of Pseudomonas aeruginosa and protective in vivo, an antimicrobial agent, a gamma globulin fraction from human blood plasma and a physiologically acceptable excipient. 22. Pharmaceutical composition according to claim 21, characterized in that the antibody is a human monoclonal antibody and that the gamma globulin fraction coming from human blood plasma is collected from human beings having elevated levels * 71 of reactive immunoglobulins with Pseudomonas aeruginosa bacteria and / or products thereof. 23. Pharmaceutical composition characterized in that it comprises at least two monoclonal antibodies, each of which reacts specifically with a different type of flagellar protein from Pseudomonas aeruginosa and is capable of treating or preventing infections with Pseudomonas aeruginosa. 24. Pharmaceutical composition according to claim 23, characterized in that at least one of the monoclonal antibodies is a human monoclonal antibody. 25. Pharmaceutical composition according to any one of claims 23 and 24, characterized in that it further comprises at least one human monoclonal antibody capable of reacting with at least one serotypic determinant on a lipopolysaccharide molecule of Pseudomonas aeruginosa and / or a monoclonal antibody reactive with exotoxin A. 26. Composition characterized in that it comprises a human monoclonal antibody reacting specifically with an epitope present on the flagella of Pseudomonas aeruginosa. 27. Composition according to claim 26, characterized in that the epitope is present on type a flagella or type b flagella, but not on both. 28. Composition according to claim 26, characterized in that the epitope is shown by at least about 70% of type b flagella. 29. Composition according to any one of claims 26 and 28, characterized in that the epitope is shown only by flagella type b. 30. Composition according to claim 29, characterized in that the antibody is pan-reactive. * -4 72 31. Pharmaceutical composition useful for the treatment of a human being susceptible to bacterial infections, characterized in that it comprises a prophylactic or therapeutic amount of a monoclonal antibody capable of binding to the flagellum of Pseudomonas aeruginosa, in combination with a or more of the following: a prophylactic or therapeutic amount of a monoclonal antibody capable of reacting with exotoxin A from Pseudomonas aeruginosa; a monoclonal antibody capable of reacting with at least one serotypic determinant on a lipopolysaccharide molecule from Pseudomonas aeruginosa; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from human blood plasma showing elevated levels of immunoglobulins reactive with Pseudomonas aeruginosa bacteria and / or products thereof; or an antimicrobial agent. 32. Composition of monoclonal antibody, characterized in that the monoclonal antibody has the same specificity as any of the monoclonal antibodies designated under the names FA6 IIG5, Pa3 IVC2, PaF4 IVE8, 20H11 and 21B8. 33. A monoclonal antibody composition according to claim 32, characterized in that the monoclonal antibody is conjugated to a label capable of providing a detectable signal. 34. A monoclonal antibody composition according to claim 33, characterized in that the label is a fluorescent substance or an enzyme. 35. Method for determining the presence of Pseudomonas aeruginosa in a sample, characterized in that it comprises the combination of this sample with a monoclonal antibody reactive with the flagella of Pseudomonas aeruginosa and the detection of the formation of complexes . 36.- Kit useful in detecting the presence of Pseudomonas aeruginosa bacteria, characterized in that it comprises a composition of monoclonal antibody comprising at least one monoclonal antibody, in which this antibody reacts with a flagellar protein of a specific type of these bacteria, and markers providing a detectable signal covalently linked to this antibody or linked to second antibodies reactive with each monoclonal antibody.