BE1000743A3 - MONOCLONAL AGAINST Pseudomonas aeruginosa flagella. - Google Patents

Abstract

cell lines secreting monoclonal antibodies capable of binding to the flagellar proteins of certain Pseudomonas aeruginosa. Some of these antibodies protect against lethal doses of these bacteria. Pharmaceutical compositions comprising these antibodies in combination with other monoclonal antibodies, blood plasma fractions and antibiotics find prophylactic and therapeutic applications.

Description

       

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    MONOCLONAL ANTIBODIES AGAINST FLAGELLAS FROM --- PSEUDOMONAS AERUGINOSA.



  FIELD OF THE INVENTION.



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



     BACKGROUND OF THE INVENTION.



   Gram-negative disease and its most serious complications, for example bacteremia and endotoxemia, are the cause of significant morbidity and mortality in humans. This is true, in particular, of the organism
Gram-negative 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 regard to
P. aeruginosa. (See, for example, Andriole, V. G., "Pseudomonas Bacteremia: Can Antibiotic Therapy
Improve Survival? ", J. Lab. Clin. Med., / 1978 /,
94: 196-199). This prompted the search for other prevention and treatment methods.



   One process that has been considered is the strengthening of the host immune system by immuni-

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   active or passive. 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 from P. aeruginosa leads to the development of specific opsonic antibodies directed
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 essential1emeuL. against determinants of recurrent oligosacchar of lipopolysaccharide molecules (LPS) located on the outer membrane ----- --due de-i-aeugino & a- rack, mu bulins - Characteristics and Uses of Intravenous Preparations, Alving , B. and Finlayson, J. pages 73-79, U.



  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).
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  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 which 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 certain limitations in the use of human immunoglobulin components, this approach to the treatment of P. aeruginosa disease remains under study (see, for example,

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 Collins, M. S. and Roby, R. E., Am. J.

   Med., 76 (3A): 168- Collins, M.S. and Roby, R.E., Am. J. Med., 76 (3A): 168-174, [1984]), and until now there is no -7y, E no
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 products available containing these commercial components.



   One such limitation associated with immunoglobulin compositions is that they consist of
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 gatherings-chattttans-er! - from a - a thousand or more donors, these samples having been preselected according to the presence of parti-
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 c-fold anti-Psei-tomos. ntMement-led - to --- average individual antibody titers, which at best results in modest increases in the titer resulting from desired antibodies.



   Another limitation is that the preselection 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 batch to batch 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 the desired antibodies and the high content of foreign substances can often limit the quantity of specific immunoglobulins below the optimum and therefore beneficial which can be administered to the patient.

   In 1975 Kohler and Milstein exhibited their

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 fundamental discovery that certain lineages of mouse cows are fustonized-with '' '- - each of which secretes antibodies of a specific immunity, that is to say monoclonal antibodies (Kohler,
G. and Milstein, C., Nature, 256: 495-497 El975¯7).
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  ----- With. . possible, in a few cases, to produce- quantities of selectively-specific murine antibodies ----- a-determinant-by H mining on antigens. Later, using technologies developed later, it became possible to produce human monoclonal antibodies (see, for example, U.S. Patent No. 4,464,465, which is cited herein by reference).



   It is recognized that in some situations, mouse monoclonal antibodies or compositions of these antibodies can cause disadvantages when used in humans. For example,
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 it has been reported monoclonal antibodies for treatment of a certain human disease can elicit an immune response which makes them inoperative (see, Levy, RL and Miller, RA, Ann. Rev. Med., 34: 107-116 / '1983. 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
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 Antibodies, Engleman, E. et al., Ed., Plenum Publishing Corp. 19857, which is here in G. reference).



   Using hybridoma technology

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 and / or cell transformation, many groups of protective antibodies against infection with P. aeruginosa. Monoclonal antibodies have - 1 epopes P. aeruginosa, including surface epitopes specific to-ser-otype-unique-and- a-serotypes -TeIrS -two found in solid cells (LPS) delta-bacteria (see, for example, the isis of Am6 of Nos. 624 and 807,394 ceded by the Applicant, which is where it comes from) both are cited here for reference.) Specific protective monoclonal antibodies for P. aeruginosa exotoxin A have also been produced.
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 (see, for example, United States patent application 742,170 assigned to the Applicant, which is cited by reference).



  The use of monoclonal antibodies specific for the LPS region of P. aeruginosa, or exotoxins of the bacteria, can provide protection
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 sufficient situations, but it is sufficient in qqgL in some generally preferable to have a larger 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 or 1 the serotype 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 the serotypes clinically. important of P. aeruginosa, ideally by providing reactive antibodies in traditional srotyping schemes.



   An aspect of the physiology of P. aeruginosa

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 which has been shown to contribute to the virulence of ----- rorgantsme-et-lar, -qui-est-one-crpacm "" resulting n (see, Montie, T. and 7, 38. aeruginosa does that has a single flagellate with a rod structure - Bes-F-model mtyttlitbrûlée mouse studies have shown that a greater percentage of mice survived when non-moving P. aeruginosa strains were inoculated in experimental burns than if strains with movement were used (McManus, A. et al., / '1980. 7. Burns, 6: 235-239 and Montie, T. et al., 19827, Infect. and Immun., 38: 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 . et al., [1982],
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. Bakt.



     Hyg., 1. Abt. Orig. A, 242: 228-238). The serological typing of flagella by the two laboratories showed that the Hl 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

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 antigen is described below as flagellar type a, with five ----- socf-groopes-ao, -ay, -33 -l "groups u} varied on different strains of P. aeruginosa carrying the antigen was found on all flagella type-a-ma-Ls-with-aaAtM. --- cTg-elessouches.



   A serotyping scheme based on antigens
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 es ----- stable P. aeruginosa is known as the Habs scheme, which was recently incorporated into the International Antigenic Typing System.



  (See, Liu, Int. J. Syst. Bacteriol., 33: 256, '1983 7.) The Habs reference strains of the flagellar types of P. aeruginosa were characterized by immunofluorescence with polyclonal sera by
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 Ailsorg Abt. Orin. A, 242 228-238) or by coagglutination on a slide (1978, Zbl. Bakt. Hyg., 1. (Ansorg, R et al, 1984, J. Clin. Microbiol., 20: 84-88). Habs strains 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 strains . aeruginosa could therefore probably be recognized by a small
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 number of for specific monoclonal antibodies against 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
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 for the prophylactic and therapeutic treatment of P. aeruginosa infections, as well as for the diagnosis of such infections. The present invention

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 respond - SUMMARY- DEIANV13NT mE. m s SUMMARY-IE-L - "- INVENIv The invention of cells which can produce monoclonal antibodies capable of workshop flagelles present on most strains of the bacterium P. aeruginosa.
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  --- r & -a pe Les lie with epitopes on the flagellar proteins of P. aeruginosa- and can distinguish between ype s --- antibodiesbacteria. 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 exotoxin A of P. aeruginosa; 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 immunoglobulin 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 new cells capable of producing monoclonal antibodies

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 and compositions comprising such antibodies, ----- which Hres-compostoits-are-epables-de-eeonnte- - les-sur strains of P. whereas the individual antibodies recognize flagella of P. aeruginosa. The cells in question have chromosomes squeJ - cell DNA for an antibody, a flagellar protein common to certain strains of P. aeruginosa. For flagellar proteins of type a, pan-reactive monoclonal antibodies can be produced, and for flagellar proteins of type b, antibodies that 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 the flagella of P. aeruginosa would be accessible for direct contact with the antibody molecules, thus probably inhibiting the motility of the organism and / or facilitating the other favorable effects for the 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 immoralist cell line can be a mammalian cell line which has been transformed by oncogenesis, transfection, mutation or

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 equivalent.

   Such cells include the lines ----- Rtyetotne-tsTrlgneesdlympnonte7 aurEres celltles expressiön and "" the secretion of the immunoglobulin or a fragment of "liaisoh fragment can between an original immunoglobulin - nureje-dH-mammdrfere nr- ot itonmte - which are the preferred sources, produced by trans mation of a - lymphocyte, particular of a splenocyte, --- au-moyea-dm-vrrus-eu-par-fusion ympheeyte-awec - a cell neoplastic, such as 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. well known and can vary considerably while remaining effective (See, Golding, Monoclonal Antibodies: Principles and Practice, Academic Press, N.

   Y., 1983, which is cited here with reference).



   Hybrid cell lines can be cloned and examined using 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 an appropriate host for the production of ascites.



   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

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   obtained from human donors who are or have been exposed to appropriate strains of P. aeruginosa carrying flagella. A preferred method of cell transformation is described in detail in the patent
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 No. 464,465, which is cited herein by reference.



  -faiDu fäftqüT. Here are the antibodies of the present invention, which are known to be specific for supernatant proteins, supernatants ------
4be examined by competitive analysis with the monoclonal antibodies in question, as a means to identify additional examples of monoclonal antiflagellar 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, these other B cells can serve as recipients for genomic DNA encoding 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, monoclonal antibodies can be

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 used intact or in the form of link fragments, ------ treJrs-jueFvTFtb-F -mais-habdrtrueI. emet-aacs-- - tion can find a use other than direct production cells can be fused with other cells (jteUL & above ejiably des-cells produce deaT coding for monoclonal antibodies. Alternatively, the lines cells 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 immunoglobulins.
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 specific in a variety In particular, by preparing cDNA libraries from host RNA. messenger, 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, N. Y., 1980, and the references cited there, all these sources being mentioned here in 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.

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  Acid. Res., 12: 3791 and Wood et al., Nature, ---- 3t4 446 -, - chacnjn-etanL-ct4-ici "example, er -partir coding for the heavy and light chains of antibodies body mo o s12a r Lm 1--9-b of the present invention, can be isolated by hybria by means of cDNA-from lymphocytesBALB / cautres that the clone The hybrid mRNA not will be rich for sessagQs: desired. If necessary, this process can be repeated to then rise 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 made 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, pACYCl84, 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 suitable vector for transformation of a host and the final expression of the gene.



   Suitably mammalian hosts

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 (by used to treat the chain (for example joining mouse cells) can be heavy and light chains), in order to obtain an intact immunoglobulin, and to secrete the leader-sequence-free immunoglobulin, if desired. Alternatively, single cell microorganisms may be used to produce the two chains, in which case further manipulation may be required to remove the DNA sequences encoding the processing and leader signals
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 - - - ----- -from re-creation, tm providing a co-don the 5 ′ end of the sequence coding for the heavy chain.

   In this way, the immunoglobulins can be prepared and primed so as 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 immunoglobulins 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 removal of P. aeruginosa cells from a heterogeneous mixture

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 cells, etc.



  ----------- Bans-des-butrS-de-iagno & tic-les monoclonaux "marked. Typically, diagnostic tests require ----- d-ddecier-ta-formatmr'anrmptxe- by-the-link-of the antibody to the flagellum of the organism P- are-not-labeled & -U-antibody tination. In addition, the unbranded antibodies-can- anH. can be used in combination with other marked 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 labels 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 U.S. 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 presenting the desired epitope. These. cells can then be separated from unbound reagents, and a second antibody

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   (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.
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 --Aiinsjr, - ta - composition question of the present invention can be presented, usually in a lyophilized form, either alone, or together with additional specific 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.



   Monoclonal antibodies, especially

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   Human monoclonal antibodies, the invention may 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 effective excipient. A pharmaceutical excipient should be any compatible substance
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 non-toxic suitable for administering patient monoclonal antibodies. Water fats, eTTesso. e 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 may contain two or more monoclonal antibodies to form a "cocktail". For example, a cocktail containing monoclonal antibodies against both types of rabies or against groups of various strains of P. aeruginosa (eg different serotypes) would be a universal product with activity against the vast majority 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 1: 1-2 per compared 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 gamma globulins and commercial immunoglobulins.

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 cially available used in the prophylactic or therapeutic of the disease to be treated. aeruginosa in humans. Preferably, for immunoglobulins, the plasma will be obtained from human donors having high levels of immunoglobulins reactive with P. aeruginosa. (See generally: "IntravenousImmuneGlobulinandthe 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 (eg carbenicillin) in combination with an aminoglycoside (eg gentamicin, tobramycin, etc.), but many other agents (eg 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 via the route. parenteral, ie subcutaneous, intramuscular or intravenous.



   The invention therefore relates to 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, physiological saline solution a
0.4%, 0.3% glycine solution, etc. These solu-

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 EMI19.1
 tions are sterile and partYcu resefITsees generally free of material by conventional and well known sterilization techniques.

   The compositions may contain pharmaceutically acceptable auxiliary substances, required to approach physiological conditions,
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 as aroTstemerit-hard-pH buffering agents, toxicity agents, etc., e.g. sodium acetate, sodium, potassium chloride - ----- -eTctjrerum ,, - sodium, etc. The antibody concentration in these formulations can vary widely, i.e. from less than about 0.5%, usually from about 1% or from at least about 1% up to 15 or 20% in maximum weight, 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 g of monoclonal antibody. A typical composition for intravenous infusion could be prepared to contain 250 ml of sterile Ringer's solution, and 150 in of monoclonal antibody. The methods for preparing parenterally administrable compositions are known or obvious to one of ordinary skill in the art.
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 m trade 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 for reference.



   The monoclonal antibodies of the invention can be lyophilized for storage and reconstituted in a suitable excipient before use. This technique has proven effective with conventional immunoglobulins and freeze-drying techniques

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 and re-enactment known in the art can
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 to be of trade to be applied. 1 is that lyophilization and reconstitution can lead to varying degrees of loss of activity of the antibody (for example, with conventional immunoglobulins, IgM antibodies tend to have a
 EMI20.2
 plus plusgrande üe-tles TgG) that Use concentrations may need to be for compensation.



  The-s - activity loss-monoclonal compositions 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 be in the range of about 1 to about 200 mg of antibody per kg body weight but doses of 5 juices
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 than 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,
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 especially and endotoxemia due in particular to bacteremia. aeruginosa.



   In prophylactic applications, the compositions containing the antibody of the invention or a cocktail thereof, are administered to a patient

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 not yet infected with P. aeruginosa to increase
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 --- 6- ssib e. resistance Sune Such a quantity is defined as a "prophylactically effective dose". In this usage, the precise amounts again depend on the patient's state of health and the general level of immunity, but
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 are especially concentrated from 0.5 to 2.5 mg per kg.



  Single or multiple - Tis-pBrorvent-eure-efectuees, ss-et-Ire-program of administration being selected by the attending physician. In all cases, the formulations m e: --gén. In general, from 0.1 to 25 mg per kg, pharmaceuticals must supply 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.



   Onzeg three month old BALB / c mice immunized intraperitoneally eight times with viable bacteria P. aeruginosa, immunotype Fisher 1 and immunotype Fisher 2 (ATCC n * 27312 and nO 27313), every one to two weeks, for one total of nine weeks. Initial doses of bacteria were
8 x 106 and 1 x 107 organisms per mouse for the Immunotype Fisher 1 and the immunotype Fisher 2 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

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 Clear spleen have a proportion 47lwith logarithmic myelomedesounsenhase Molecular Research Council, Cambridge, England) and fused to create hybridomas according to the process described by Tarn et al., --- 36 lspenisn = "this final is diluted to a concentration of 1 , x 106 cells per ml in a mili-eu-hybrid (RPMI --- Ir640 Gibco, -Gramd-fTslarld, having been combined heat-inactivated calf fetal serum, 1 mM sodium pyruvate, 100 g / ml of penicillin and streptomycin,

   1.0 x 10-4 M hypoxanthine, 4.0 x 10-7 M aminopterin and 1.6 x 10-5 M thymidine), supplemented with 2.0 x 106 BALB thymus cells / c freshly prepared per ml, as feeder cells.



   The mixture was placed on plates (200 liters 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 antiP 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 external membrane preparations 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: 1042-1053), which is cited here for reference. Of

  <Desc / Clms Page number 23>

 
 EMI23.1
 bacteria (Fisher 1 immunotype and Fisher 2 immunotype --- d P. aTerugnQfsa. otrt-temoculee triptycase-soya (TSB) and cultured from 16 "IShours T 34" C with aeration in a bath stirred by a gyratory agitator. by centrifugation and washed twice with --tramp & tmee-au-Phosphate saline solution (PBS, NaG- Na2HP04-7HO8iM PO mM, 2) containing 150 trypsin inhibitor units (U.

   I.) apro-tinTnearLJLLJigma, MCL) -. 'H
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 out of the homogenate a
14. 900 g and discarded, and the supernatant was centrifuged again as above. The size was discarded 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 40C in 10 ml PBS containing
75 U. 1. T. of aprotinin per ml.

   The following day, the pellets were resuspended by shaking and then divided into aliquots and stored at -70oC. 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 external membrane preparations were diluted to 5 g of protein per ml in PBS and 50 liters of the solution were placed on a plate in each well of 96-well plates (Linbro nO 76-031-05, Flow Laboratories, Inc., McLean, VA), covered and incubated overnight at 37 ° C. The unbound antigen has been removed from

  <Desc / Clms Page number 24>

 
 EMI24.1
 plates and 100 iiters of albumin serum --bov-krre-- (DSAY-da- "a --ä - ä - incub * eä --- üendärit well, s nt hour at 37OC.



  After elimination of the nonabsorbed BSA, culture supernatants (50 liters) from a solution each pu-i-ts-d & s-plates = de-f-usion were replicated on the plate in the corresponding wells of the antigen plates and incubations. 30 minutes at 37 C. The antibody
 EMI24.2
 non-Li-a-eL- & LuML-its-et-les ves-- three times with 100. liters per well of a 1% (w / v) BSA-PBS solution. Next, 50 liters per well of appropriately diluted biotinylated goat anti-mouse IgG (Tago, Inc., Burlingame, CA) were added to each well and incubated for 30 minutes at 37OC.



  The plates were washed three times, as described above, and then 50 liters of a preformed avidin complex: biotinylated horseradish peroxidase (Vectastain ABC Kit, Bector Laboratories, Inc., Burlingame, CA), prepared according to the manufacturer's instructions, were added to the ApKLejs wells 30 minutes at room temperature, the Vectastain reagent was removed from the wells, the wells were washed as above and then 100 liters per well of 0phenylenediamine substrate [0.8 mg / ml in buffer citrate
 EMI24.3
 0.0 M at pH 5.0, mixed with a volume of H 2 O 2 at 0.03% (v / v)] were 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 laughs per well.



   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.

  <Desc / Clms Page number 25>

 
 EMI25.1
 in well, on a Dynatech Model 580 reader --- HicroELISA- rexandria, These "one-wells, calledatapa3 know an antibody which bound only to the plaque each of the Fisher 2 immunotype antigen. This well was studied in more detail as described below. Antibody
 EMI25.2
 --monocrlonal and the detirtlaira line this well are both identified by the designation Pa3 IVC2 - the text below. The Pa3 cells-orlgnal-wells have been removed-and-cottes-pa. limiting dilution methods, as described by Tarn et al., (1982, Infect. Immun., 36: 1042-1053).



   Ascites containing a high titer monoclonal antibody was prepared in CB6 Fl mice (generation Fi 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). Mice of mice
CB6 Fi ages 2 to 3 months were injected intraperitoneally with 0.5 ml of Pristane (2, 6, 10, 14-tetramethylpentadecane, Aldrich Chemical Co., Milwaukee, - MI) 10 to 21 days before intraperitoneal injection of Pa3 IVC2 cells in the logarithmic phase into
RPMI. Each mouse was injected with 0.5-1 x 107 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, Bookman Instruments, Inc., Brea, CA) and all ascites that contained 5 mg / ml or more of antibodies were pooled, split 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

  <Desc / Clms Page number 26>

 
 EMI26.1
 described above, on preparations of external membranes notypesFfsher. "aeruginosa A. from P. aureofaciens (A. 13985) and Klebsiella from decnaccunedesseptsouchesimmu-pheumoniae (ATCC 8047), all prepared as described above. The antibody Pa3 IVC2 linked to
 EMI26.2
 - Knrier-2 Knnona-ae "external membranes of P. aeruginosa and not to the other immunotypes --- Fisher, nor to P. aureofaciens" or --K-pneMtSuMte- The antigen by the antibody Pa3 IVC2 was identifies 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.



   Ze aliquots 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. Commun. Res., 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 preparations of external membranes.



   To decrease the non-specific binding of antigens to the outer membrane & the antibody

  <Desc / Clms Page number 27>

 
 EMI27.1
 Pa3 the radiolabelled preparations (5 x 106 shots per "ml testontd" first Tncubeespente une
IVC2, (final dilution 1:40). The Pa3 IVC2 culture supernatant (0.5 ml) containing the Pa3 IVC2 antibody was then added to each outer membrane sample. After incubation of antigen and anti
 EMI27.2
 body for one hour at 4OC, protein A source, IgGSORB (0, Center, Inc., -MB-a-t-amte-et-ineubee another 30 minutes (Kessler, S. 1975, J.
095 ml per sample) (The EnzymeImmunol., 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 IgGSORB twice with RPMI hybrid ( RPMI-HAT hybrid medium 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 (phosphate 10 mM, 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); twice with highly saline buffer (0.1 M TrisHC1, pH 8.0, 0.5 M LiCl, 1% beta-mercaptoethanol (v / v); and once with lysis buffer (0.02 M Tris-HCl , pH 7.5, 0.05 M NaC1 and Nonidet P-40 0.05% (v / v) (Rohrschneider et al., 1979, Proc. Natl. Acad. Sci., USA, 76: 4479-4483) .

   The antigen linked to the complex was released by incubation with a sample buffer [Tris-HCl 0.125 M, pH 6.8, SDS 2% (w / v) 7, beta-mercaptoethanol 2% (v / v) and 20% glycerol (v / v) at 950C for ten minutes and collected in the supernatant after centrifugation at 1,500 g for 10 minutes.



   The supernatant samples were then

  <Desc / Clms Page number 28>

 applied to 14% polyacrylamide gels containing SDS prepared according to the method due B. Lugtenberg et al. (1978, FEBS Lett., 58: 254-258), as modified by Hancock and Carey (1979, J
 EMI28.1
 Bacteriol., 140: 902-910, which is cited here with reference), and the antigens were separated in the gel '"by' * e The constant of 50 V. After fixing the gel in '' methanol (v / v), 10% actic acid and Whatman 3 MM paper glycerol 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 bound 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 to that of reference proteins labeled with 14C (phosphorylase B, PM 92. 500;
 EMI28.2
 BSA, PM 69. carbonic anhydrase, PM 30. cytochrome C, PM 12.



  England Nuclear, Boston, MA) which were separated in the same gel. The molecular weight of this antigen is correlated with the molecular weight of flagellin, 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 for reference.
 EMI28.3
 



  In addition, Pa3 IVC2 a by ELISA using Habs 1 to 12 strains of P. aeruginosa (A. 33348-33359) which were fixed with

  <Desc / Clms Page number 29>

 ethanol was examined on 96-well microtiter plates.
 EMI29.1
 



  . t. -p-à-r-gds - c6m-me sult-.



  The prepared antigen tablets were overnight culture broths from each organism were centrifuged, the pellet was washed twice with PBS and then resuspended in PBS to an optical density of 0.2 units.
 EMI29.2
 AbsorbancrrOnmr es - were placed on a plate in wells (50 liters per well) and then centrifuged at 1. for 15 --L-MMMM BS -et-etTSTtttre-- ethanol (95%) a 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 40C until used.



   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 model of specificity indicated that Pa3 IVC2 binds to 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., 20: 84-88, both cited here by reference). If this specificity is attributed to monoclonal IVC2 Pa3, 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 body Pa3 IVC2 would protect a mouse injected with multiple lethal doses (DLgo) of P. aeruginosa bacteria

  <Desc / Clms Page number 30>

 
 EMI30.1
 alive. The chosen one was the mouse model --- biubee Colin-M &.-Et-Roby, -RrE, -18, -J.-Trauma-- - is cited here (reference). gave groups of mice a severe burn, according to the protoleole of the authors, which were injected with 5 to 10 DLo from Fisher 7. The antibody-monocJLona. i-administered 'voier sousform (0, 2 ml intraperitoneally) before the burn and injection. Neither was observed in animals treated with vF was not observed in animals treated with model Pa3 IVC2 compared to those who had not received antibodies.



  EXAMPLE 2.-
Example 2 illustrates the process for the preparation of a murine hybrid cell line producing a monoclonaluride antibody against type b flagellin from 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 g 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.,

  <Desc / Clms Page number 31>

 
 EMI31.1
 : 381-390) atedonnee e-ladernière viable. 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.



   Supernatants from hybridoma cultures
 EMI31.2
 were the presence n-ficcir7ps arif-i - P. aeruginosa by ELISA test on day 10 after the following fusion in the example as 9ene, viable bacteria immobilized in the wells of the 96-well microtiter plates. plates were prepared as follows.



   50 poly-L-lysine (PLL) nitres / mol in PBS) (Sigma n P-1524, St. Louis, MO) were
 EMI31.3
 was added to and incubated for 30 minutes at room temperature. The non-adsorbed PLL was removed and the wells were washed three times with PBS. The bacterial cultures cultivated overnight in TSB were washed once with PBS and then resuspended in PBS until A660 nm 0.2 OD 50 liters of the bacterial suspensions were added to each well of the plate and allowed to settle. bind at 37 "C for one hour. Unbound bacteria were removed from the plates, with three washings of the wells using saline Tween [0.9% NaCl (w / v), 0.05% Tween-20 (v / v) 7.



   Nonspecific binding of antibodies was blocked by the addition to the wells of 200 l itres per well of blocking buffer [PBS containing dried lean milk 5% (w / v), antifoam A 0.01% (v / v) (Sigma, St. Louis, MO) and 0.01% thimerosal (v / v)] and by incubation for one hour at room temperature.



  Excess blocking buffer was expelled and the wells

  <Desc / Clms Page number 32>

 have been washed three times with saline Tween, as
 EMI32.1
 - tt recedeaunentr .------------------------ c-ur Les ageants lJ. ) were replicated in the corresponding wells of the plates - 30 minutes. Culture supernatants were removed from the plates, washing the wells five times - = using saline Tween.
 EMI32.2
 



  An enzyme antibody -GG to horseradish peroxidase + IgM) (Tago, Inc., Burlingame, CA), was diluted in PBS containing Tween-20 0.1% of second step, conjugated a (v / v) and 0.2% BSA (w / v), according to titrations carried out previously, and then glasses of the reagent were added to each well and incubated for 30 minutes at room temperature. The excess reagent was expelled the washed wells 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 reagents
 EMI32.3
 These were repeated in the example 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. 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 in the supernatant of one well, PaF4 IVE8, only bound to the Fisher immunotypes 2,6 and 7 of

  <Desc / Clms Page number 33>

   P. aeruginosa.

   The cells of the PaF4 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 CB6F1.



  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 n 29260, Leifson, 1951, J. Bacteriol., 62: 377-389) and Escherichia coli (GSC A25) have were grown overnight at 37 C in TSB. The bacteria were centrifuged and then washed twice with PBS. Each strain 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.
 EMI33.1
 below.

   Ascites containing highly reproduced monoclonal antibody criticized in Example 1, except BALB / c mice were used instead of CB6F Spé-c - ifi A test carried out to identify the antigen by l he PaF4 IVES monoclonal antibody was indirect immunofluorescence on bacterial organisms.



   Each of the seven reference Fisher immunotypes
P. aeruginosa plus a non-flagella strain of
P. aeruginosa (PA103, A.T.C.C. n 29260, Leifson, 1951,
J. Bacteriol., 62: 377-389) and Escherichia coli (G.S.C. 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- until a
 EMI33.2
 optical density of 2.2 z 660 nm.



   The bacterial suspensions were then further diluted 1: 150 and 20 liter samples were placed in individual cells on Carlson slides (Carlson Scientific Inc., Peotone, IL) and dried on the slide 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 the slides, an anti-mouse goat IgG conjugated with isothiocyanate

  <Desc / Clms Page number 34>

   fluorescein (ITCF) plus an IgM (25 liters per well of a 1:40 dilution in PBS) (Tago, Burlingame, CA) were incubated on the slides for 30 minutes at room temperature in a humidified room 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). 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 the flagella was confirmed by immunoblotting analysis. The antigens of the external membrane of the immunotype Fisher 6 of P. aeruginosa (see example 1) were separated by electrophoresis in a polyacrylamide gel at 14. % containing SDS as described in Example 1, except that the electrophoresis was carried out 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) were included in the same polyacrylamide gel.



  The antigens were transferred from the polyacrylamide gel onto a nitrocellulose membrane (MNC), (0.45 / 4ion, Schleicher and Schuell, Inc., Keen, NH) in Tris-glycine-methanol buffer (Towbin et al., [1979], Proc Natl, Acad. Sci., USA, 76: 4350-

  <Desc / Clms Page number 35>

 
 EMI35.1
 4354) containing SDS 0.05% (w / v) overnight at 40C - -prans - fM-t MNC Teeh-200705% PBS (PBS-Tween) (Batteiger, B. et al., 1982, J .



  Immunol.



   = - - -un-- amperage-cunsta: nt of ambient temperature. For this stage and all the following, the tray containing the MNC was placed on a shaking platform to ensure the distribution of the solution throughout the - MNC.
 EMI35.2
 



  - - After one hour, the PBS-Tweenaété solution - - - 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 immunoblotting demonstrate that PaF4 IVE8 binds to flagella of P. aeruginosa.



   The type of flagellum that PaF4 IVE8 recognizes has

  <Desc / Clms Page number 36>

 
 EMI36.1
 was determined by ELISA. Habs strains 1-12 3-43 - j3 --- 33 5) microtiter well wells with PLL, and ELISA was earlier in this example. The source of the antibody PaF4 IVE8 was the culture supernatant. Of
 EMI36.2
 revisions- have been -noted6- containing Habs strains 2, 3, 4,5, 7, 10,11 and 12, thus indicating that PaF4 IVE8 binds - to type-fl-agellas. The personal protection data is 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 of -type a (10 g protein) from- of strains
Habs 6 and 8 (A. T. C. C. nos 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 for reference) except that the final centrifugation of the flagella was carried out at 100,000 9 for one hour rather than at 40,000 for three hours .

   A second modification adopted for some operations was to detach the flagella from the bacteria during
30 seconds in a blender rather than during
3 minutes. (Allison et al., 1985, Infect. Immun.,
49: 770-774).



   The protein concentrations of each preparation were determined with the Bio-Rad protein test (Bio-Rad, Richmond, CA) and the presence

  <Desc / Clms Page number 37>

 
 EMI37.1
 of lipopolysaccharide (LPS) in Y. O., and "1978, Biochetn., The gross flagellum protein poJLds were determined by comparing their migration in a polyacrylamide gel with SDS to the migration of dde-labeled proteins
 EMI37.2
 referencetSRl- exempl The weight of the flagellin of the Habs 6 strain was 51. of the flagellin of --Habs-8-was-of-4. let's talk. urµ-sorTE-en-- agreement with those obtained by J. S. Allison et al. (1985, Infect. Immun.; 49: 770-774, which is cited here with reference).



   The fusion of splenocytes from mice immunized against flagellin with cells from
 EMI37.3
 NS-1 myeloma was performed three days after the last immunization, as described in Examples 1 and 2. When the hybrid cells grew to approximately 40% confluence (day 7), the supernatants of the
 EMI37.4
 cultures in corresponding wells of three were replicated. * Fisher 1 immunotype of bound P. aeruginosa & PLL (see Example 2 for preparation) and formalin-bound Habs 6 and Habs 9.



   The bacteria for the formalin fixed 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 liters per well) of Linbro 96-well microtiter plates, and the plates were
 EMI37.5
 then been 200 g for 20 minutes & centrifuged at 1. room temperature. Supernatants were removed from the wells and 75 liters of 0.2% (v / v) formalin solution in PBS was added to each well

  <Desc / Clms Page number 38>

   and incubated for 15 minutes at room temperature.



  After removing formalin from the wells, the plates were air dried and stored at 40C 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
 EMI38.1
 Microbiol. Acad. Sci., Hung., 17 35-48). The immunotype Fisher l strain of aeruinosa as a control, as indicated by the coloring a biting tincture (Manual of Clin. Microbiol., Lennette, ed. 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 immunoblotting. Indirect immunofluorescence was carried out essentially as described in Example 2, with the following modifications.



   Cultures of bacteria that grew overnight on trypticase-soy agar at 30 oe were collected from the plates with cotton swabs and resuspended in PBS until an absorbance value of 660 was obtained. 0.2nm OD unit From

  <Desc / Clms Page number 39>

 
 EMI39.1
 formalin a final concentration of 0.37% (v / v) --- in-PBS-in-outee - la-Suspension-sous-- "agttation.-After a - room for 15 minutes, bacteria were diluted in PBS in liters of this suspension were placed in --- individual slides of the blades of the prepared observation described in Example 2. The source iormSLLMeAntsde & -de-La cellular FA6 IIG5.



  Fluorescent staining by the antibody
1: at 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
 EMI39.2
 that TTG imxEl cent was increased by treatment of bacteria with formalin, but this treatment was not required to visualize 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 CLaemmli, U. K., 1970, Nature (London), 227: 680-685] and transferred to an MNC. The preparations of FA6 IIG5, either the culture supernatant or the diluted ascites 1: 1.000 were reacted with the MNC, and the reaction detected with an appropriate reagent conjugated to an enzyme and an enzyme substrate, as described in Example 2. Immunoblotting a illustrates that FA6 IIG5 binds specifically to flagellin from P.

   M. 51. 700 from Habs 6 and flagellin from P. M. 47. 200

  <Desc / Clms Page number 40>

 
 EMI40.1
 from Habs 8.



  - ------ ccmffrmat. only a and type b, "a obtained by ELISA, for which purpose strains e to" Habs teHeesindividuallyYement avecPLLaux wells of 96-well microtiter plates Linbro. The antigen-binds-only-to the Habs and a strains, are the only strains carrying flagella type a among -the R. and a1., 1984, --Clin, L <ii-eti-I reference). The in vivo protection studies are re on-du - fatt-que-FA & -YI & 5 - react - 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 burnt mouse model.



   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 in reference). For the protection studies, all the antibodies were purified by protein A-Sepharose chromatography (Ey, P. L. et al., 1978, Immunochemistr, 15: 429-436, which is cited here by 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 p Fisher 2 immunotype. aeruginosa (A.T.C.C. no 27313) for reference.



   40 g of purified monoclonal antibody were given, intravenously by mouse, one day two hours before the burn and the infectious injection. Immediately after the burn, the animals received

  <Desc / Clms Page number 41>

 0.5 ml of cold PBS containing pressure ulcers
 EMI41.1
 Bact ceesLaTdoseinjectµeStaltaabout 10 DLgo for each organism. The results of the animal tests are presented in Tables I and II.

  <Desc / Clms Page number 42>

 



   TABLE 1 Study of protection by a monocional anti-flagella antibody type a on a burned mouse model1.
 EMI42.1
 
 <tb>
 <tb>



  Percentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> FA6 <SEP> IIG5,
 <tb> antiflagella <SEP> a <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 90 <SEP> 90 <SEP> 80 <SEP> 80
 <tb> PaF4 <SEP> IVE8, <SEP>
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 20 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10
 <tb> Antibodies
 <tb> monoclonal
 <tb> no <SEP> specific
 <tb> anti-LPS <SEP> 100 <SEP> 40 <SEP> 30 <SEP> 20 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10
 <tb> PBS, <SEP> without
 <tb> bacteria <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80
 <tb>
 
 EMI42.2
 1. The mice were infected under pressure ulcer with approximately 10 DLgo 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.

  <Desc / Clms Page number 43>

 



  TABLE II
 EMI43.1
 Study of protection by a monoclonal antibody --- Etüde e. type b antiflageile on burnt mouse model.
 EMI43.2
 
 <tb>
 <tb> of <SEP> the <SEP> protection <SEP> by <SEP> a <SEP> anticprps <SEP> monocionalPercentage <SEP> from <SEP> survival <SEP> by <SEP> day <SEP>
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> PaF4 <SEP> IVE8,
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 100 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90
 <tb> PA6 <SEP> IIG5 <---- <SEP>
 <tb> antiflagella <SEP> a <SEP> 1DO-20 <SEP> r0'10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10-- <SEP>
 <tb> Antibodies
 <tb> monoclonal
 <tb> no <SEP> specific
 <tb> anti-LPS <SEP> 100 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20
 <tb> PBS,

    <SEP> without
 <tb> bacteria <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb>
 
 EMI43.3
 - 1. The mice were infected under pressure ulcer with approximately 10 LD50 of the Fisher 2 immunotype of
 EMI43.4
 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.

  <Desc / Clms Page number 44>

 
 EMI44.1
 



  A very significant survival to --- ctrez-les-sourts-trsttes-avec-rairticorps-anH.-to the antibodyanti-bet TnjectIOn of the corresponding antigen. Conversely, 80-90% of the mice treated with an anti-flagellar monoclonal antibody -ti f - == Ht & tiatif an antibody-nen- & peejyEtteaatri-LPS, would die. : - type a to protect mice against a lethal e asa - po-rt-ante - di-imm of type b flagella, and the inability observed to prevent type b flowers to protect 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 acline isolates of P. aeruginosa, which demonstrates 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 a variety of collection sites, including blood, wounds, respiratory system, urine and ears. A total of 157 isolates were examined.



   PaF4 IVE8 specifically binds to
34 clinical isolates (22%), while the antibody for type a or FA6 IIG5 flagella bound
102 clinical isolates (65%), for a total of 136 of the
157 isolates (87%). Among the 21 strains which were recognized by neither of the two antibodies, 19 were not flagellated, as indicated by staining with a

  <Desc / Clms Page number 45>

 
 EMI45.1
 biting tincture. two antibodies in combmaisoh desT38 clinical isolates (see, Ansorg, R., 1978, Zbl. Bakt, Hyg. I Abt.



  A, 242 228-238, which is cited here for reference).



  EXAMPLE 6 The procedure for the production of human monoclonal antibodies which bind to type b flagella of P. aeruginosa --------- UH-hantiJrton - de-sng in 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 from the blood by standard centrifugation techniques on Ficoll-Paque [Boyum (1968) Scand. J.



  Clin. Lab. Invest., 21:77] and washed twice in a calcium phosphate and magnesium free phosphate buffer (PBS).



   The mononuclear cells were freed from the T cells according to a procedure modified to form rosettes E. In short, the cells were first resuspended to a concentration of 1 × 10 7 cells per ml in PBS containing 20 % fetal calf serum (SFV) at 4 C. 1 ml of this suspension was then placed in a round bottom polystyrene tube of 17 x 100 mm and 1 x 109 sheep red blood cells treated with 2-amino-isothiouronium bromide (AET) from a 10% (v / v) solution in Dulbecco medium modified according to Iscove (Iscove medium) Madsen and Johnson (1979), J. Immun. Methods, 27: 61: 7.

   The suspension was mixed gently for 5-10 minutes at 40C and the cells in rosette E were then separated by centrifugation on Ficoll-Paque

  <Desc / Clms Page number 46>

 
 EMI46.1
 for 8 minutes in mononu- cells 1-6-aire-g- ---- cteaire- u-s. E-negative "(CMSPE *) were collected and washed once in lscove medium and resuspended in the same medium containing 15% SFV (v / v), L-glutamine (2 mmol per
 EMI46.2
 liter), p-en-ici-rtlne with streptomycin (10 / ml), hypoxanthine (1 x 10-4 M), aminopterin (4 x 10-7 M) and - --de- ne- 6 - x-705-M.-ee-miet-eat - (100 Uni-e-és- I ternationales - after mid HAT.



   The transformation with cell drive of CMSPE-a Eto is accomplished by cocultivating these cells with a line of transforming cells. The transforming cell line was an Epstein-Barr nuclear antigen (EBNA) positive human lymphoblastold cell line derived by ethyl methanesulfonate mutagenesis (EMS) from the GM 1500 lymphoblastold cell line, followed by a selection in the presence of 30 g / ml of 6-thioguanine to make the cells deficient-in hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) and therefore sensitive to HAT. This cell line is called cell line 1A2 and was deposited in 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 log growth phase were resuspended in HAT medium and then combined with CMSPE with a proportion of 15 1A2 cells by CMSP. The mixture of cells was placed on a plate in 30 microtiter plates of 96 wells with a round bottom (Costar 3799) a concentration of 32,000 cells per well in a volume of panes per well, and incubated at 370C in a humidified atmosphere containing
 EMI46.3
 2. Crops have 6% C02 'Days 5 and

  <Desc / Clms Page number 47>

   cultures were fed aux8 after plating 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 to collect and examine the supernatants for
 EMI47.1
 ---- "- '" - "The supernatants were for the presence of anti-P. aeruginosa antibodies binding technique-EtrISA crrj. with the following modifications. A collection of the seven reference Fisher immununotype strains (ATC C n 27312-27318) (A660 = 0.2 DO units) 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 non-specific binding sites and washing the plates, 50 titles of PBS containing
0.1% (v / v) of Tween-20 and 0.2% of BSA (w / v) were added per well. The culture supernatants - (50 "miters) 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 step antibodies conjugated to an enzyme liters per well), antihuman goat IgG conjugated with horseradish peroxidase, and antihuman goat IgM or appropriate dilution in PBS containing Tween-20 0.1% (v / v) and 0.2% BSA (w / v),

   were added to the wells and the test 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

  <Desc / Clms Page number 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 2,6 and 7 immunotypes of P. aeruginosa. Cells were repeatedly subcultured at decreasing cell densities until all wells with growth secreted
 EMI48.1
 the antibody.

   The cell line and the monoclonal antibody both identified la-a <eslgration Eextei-apres --------- A second transformation was carried out, in which the source of B cells was the peripheral blood of a patient suffering from cystic fibrosis, known to have had a chronic infection to P. aeruginosa. The CMSPE- were prepared, as described above, and cocultivated with the transforming cell line, 1A2, in a proportion in 72 1A2 cells by CMSPE-. The mixture of cells has
 EMI48.2
 plate on 15 round bottom 96 well microtiter plates at a concentration of 7 x 104 cells-well share and cultured as shown--
4 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 previous 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;

  <Desc / Clms Page number 49>

 
 EMI49.1
 the second isolate PSA G98 (GSCOB). weigh flagella - (GSCOB) v -Tles b and the Fisher 5 LPS immunotype. This mixture of reference strains and "isolates infinitely endangered by P. aeruginosa flagella.

   The supernatants - in the plates containing not on the PLL-covered control plates, were tested once and for all by individual m- & s of the assembly. 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 to 20 cells per well of 96-well plates, then to the 2 cell culture per well. The formal cloning of the cells producing the specific antibody was carried out by placing the cells on a plate at a density of approximately 1 cell per well in 72-well Terasaki plates (Nunc nest 1-36538) in a volume of 10 / alites. 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 samples to find wells containing a single cell. The wells were fed daily with HT medium and, when growth was sufficient, 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 anti

  <Desc / Clms Page number 50>

 
 EMI50.1
 monoclonal body (isotype 19M) two idenifJ. l l7'antJLgene "identifies ii-g alt formed by flagella, as indicated by fluorescence niques were carried out basically as described --- d-ans et 3. Pour-tre-fest-d'i. indirect fluorescence, P. aeruginosa strains carrying type b flagella, Fisher F2 immunotypes, --F6-eFTe c T. -2, -27-e- 27318) and a strain carrying type a flagella, 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 monoclonal antibodies
 EMI50.2
 murine and FA6 IIGS. The slides were prepared for observation as described written in
PaF4 IVE8 example 2.

   The sources of the two antibodies were culture supernatants, and the ITCF-conjugated reagent was anti-human goat Ig conjugated to UCF- (polyvalent) (Tago, Burlingame, CA) in dilution 1: 100 in PBS containing 0.5% (w / v) bovine gamma globulin (Miles Scientific, Cat., N * 82-0441-2, Naperville, IL) and sodium azide 0.1% (w / v ) 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 immu- type Fisher 4 reference. 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.



   The immunoblotting was carried out as described in Example 2. The type b flagella purified at

  <Desc / Clms Page number 51>

 
 EMI51.1
 from the reference strain of "P." afg1. 2r73T3l-e-flageries of the purified type S 8 reference (ATCC na 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 an MNC. The supernatants of cultures containing the 20H11 antibodies or
 EMI51.2
 3C1, the supernatant containing a non-kekix-de-c antibody that incubates with MNC and the reaction to an antihuman 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 the flagellin protein of PM 47. 200 of 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 linked individually with PLL from a well of 96-well Immulon microtiter plate. The 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: 84).



  EXAMPLE 7.-
Example 7 illustrates methods for the production of a human monoclonal antibody which binds to P. aeruginosa type a flagella.

  <Desc / Clms Page number 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 cleared of
 EMI52.1
 -This as described in the example eTTjes were then frozen in SFV containing 10% of dimethyl sulfoxide in a liquid tank.



  -A- da e thawed quickly at 37 C, washed once in Iscove medium and resuspended in medium
HAT. Cell-driven transformation was accomplished by cocultivating CMSPE with 1A2 cells in a proportion of 30 1A2 cells by CMSPE-. The cell mixture was plagued in 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 plating and the supernatants were removed from the wells and fested at this time.



   The supernatants were tested by ELISA for the presence of anti-P antibodies. aeruginosa using the pool of P. aeruginosa flagella and PLL treated plates as a control as described in Example 6. Supernatants containing antibodies which bound to the flagellum pool, but not to the PLL control plates, were retested on individual strains of the flagellum pool. One well, 21B8, contained an antibody which bound & PSA I277, PSA G98 and the reference immunofisher Fisher 4, which are the three

  <Desc / Clms Page number 53>

 
 EMI53.1
 strains of the flagellum collection which carry - "'Leclonage e-Ta née te" accomplished as described in Example 6 for the line of the formal cloning stamp.

   After the wells of the Teasai piaques were moved from Terasaki to a ---- ----- v-idue-l-d2-uue-4e-cul-t-ur -e in a look treks volume 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 were added to the wells to selectively kill the feeder cells. The wells were further fed on days 7 and 9 after plating by replacing half of the supernatant with HAT medium.

   The cells were then fed with rnieu --- HT until the cells were of sufficient density to detect the presence of an antibody by ELISA. All wells with growth produced antibody that bound to strains of P. aeruginosa with flagella type a. The cell line and the monodonal antibody (isotype IgG1) are both identified under the designation 21B8 in the text below.



     The antigen identified by 21B8 was form of 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 Fisher 4 immunotype strain of

  <Desc / Clms Page number 54>

 
 EMI54.1
 P. aeruginosa 27315) which carries - f sd tansorache reference immunotype of. aerugYnosa *. wears type a flagella. The fluorescent image observetaTtuneImagelineairesinusoldaleemanant observed Zt was a sinusoidal linear image emanating from a bound eHres-de-a-bac was bound to the Immunobl. in example 2. Firagelles from- to purified --- from-from - to - suehe - Habs --de - reference-from --- P. aeruginosa (A. T. C.

   C. n * 33353) and type b flagella from the P. aeruginosa reference Fisher 2 immunotype strain (A. T. C. C. n * 27313) were
 EMI54.2
 prepared 3. The repairs as described in p were separated on a 10% polyacrylamide gel (see example 3) and transferred to an MNC. Culture supernatants containing either 21B8 or a non-specific human antibody, and culture media were reacted with MNC and the reaction was detected with anti-human goat Ig conjugated to alkaline phosphatase (polyvalent) and a enzyme substrate as described in Examples 2 and 6.

   Immunoblotting a illustrates that the antibody 21B8 binds only to flagellin protein of PM 51. 700 of Habs 6 and not to flagellin protein of PM 53. 000 of the immunotype Fisher 2. No reaction was observed neither 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.



   Human anti-monoclonal antibodies

  <Desc / Clms Page number 55>

   flagella 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 sulfate (final concentration 50%)
 EMI55.1
 (Good et al., Selected Methods in Cellular Immunology, "MishenTBTBT T" ed7,. Teörü-an and Co "San Francisco, CA, 1980, 279-286).



  -ölubilis PBS, dialyzed against --- rmit-ä-4C-and-then 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 type A flagellum strain used in animal testing was the clinical isolate PSA A522 (GSCOB) which expresses the Fisher 1 LPS immunotype and the type B flagella strain was - 1-1 iso-l-at PSA A447 clinic (GSCOB) which expresses the Fisher 6 LPS immunotype. Human antibodies (0.45 ml) were premixed with bacteria (more than 5 DL100 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.

  <Desc / Clms Page number 56>

 



  TABLE III Study of the protection by the monoclonal antibody Study of the protection by the monoclona antibody of burned mice.
 EMI56.1
 
 <tb>
 <tb>



  Percentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> FA6 <SEP> IIG5, --- mutineer
 <tb> antiflagella <SEP> a3 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 88 <SEP> 88 <SEP> 88 <SEP> 88 <SEP> 88
 <tb> 21B8,
 <tb> human
 <tb> antiflagella <SEP> a <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> 8:11 p.m.
 <tb> human
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> PBS, <SEP> 100 <SEP> 25 <SEP> 12 <SEP> 12 <SEP> 12 <SEP> 12 <SEP> 12 <SEP> 12 <SEP> 12
 <tb>
 1. The mice were infected with bedsores by more
 EMI56.2
 of 5 DLIOO of P. aeruginosa 2. The percentage of survival is low on the number of surviving mice per group of eight animals.

   The days are after burning and infective injection.



  3. The purified antibody (10og in 0.45 ml PBS) was premixed with bacteria and administered under pressure ulcer after the burn and infective injection.

  <Desc / Clms Page number 57>

 
 EMI57.1
 



  TABLE IV --EtüdedelapEpActionparlJLhuman-regional antibody 20H11antiflagellestype human 20H11 antiflagella, ij2e b in the burned mouse model.
 EMI57.2
 
 <tb>
 <tb> b <SEP> danPercentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> PaF4 <SEP>
 murine <tb>,
 <tb> IVE8, <SEP> antiflagella <SEP> b3 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> 8:11 p.m.,
 <tb> human
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> 21B8
 <tb> human
 <tb> antiflagella <SEP> a <SEP> 100 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> Backgrounds <SEP> 80 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 
 EMI57.3
 1.

   The mice were infected by bedsores with more than 5 DIOO PSA A447 from 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.



  3. The purified antibody (10 g in 0.45 ml of PBS) was premixed with the bacteria and administered under pressure ulcer after the burn and the infectious injection.

  <Desc / Clms Page number 58>

 



  TABLE V
 EMI58.1
 - - - - - - --- - Etjudss ionpar] human 3Cl b in the mouse.
 EMI58.2
 
 <tb>
 <tb> beyond <SEP> pfotecPercentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> PaF4 <SEP> IVE8,
 <tb> mur-in7 '"
 <tb> antiflagella <SEP> b3 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> 3C1,
 <tb> human
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 100 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80
 <tb> Antibodies
 <tb> monoclonal
 <tb> no <SEP> specific
 <tb> anti-LPS <SEP> 100 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 1. The mice were infected under pressure sores by
5 OL100 from 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 g) was administered intravenously two hours before the burn and the infective injection.

  <Desc / Clms Page number 59>

 
 EMI59.1
 



  One type a 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-dandruff antibodies specifically protect against lethal injection only for organisms carrying the corresponding type of flagella, i.e. the human anti-dandruff antibody type a protects against a lethal injection of the organism carrying type a flagella and not type b, and that type b antiflagella antibodies protect the mice injected with strains carrying type b flagella, but not type a .



  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 PaF4 IVES (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 mono- antibody

  <Desc / Clms Page number 60>

 
 EMI60.1
 clonal human antiflagella type a, 21B8, was --considerable - 56 - blistering-carrying isolates (96%). 20Hll with isolates carrying types of the fat-that-the-antibody-has-recognized-54 as also the fact that 20Hll recognized all 59 isolates (100%). a 43 of 59 isolates (73%). These results demonstrate that
 EMI60.2
 un-äpi-ton, -e epitoge on at least approximately 95% of the pan-r & act-if- (-cle-st --- ä - say-unflagellate strains of P. aeruginosa), while 3C1 binds an epi tope 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 20Hll and 3C1 show surprisingly, type b flagella comprises at least two separate epi topas 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 the type b flagella of P. aeruginosa, as well as the protective activity of the antibody against infection by P. aeruginosa sure
 EMI60.3
 smile model
A transformed cell line was prepared and cloned essentially as described in

  <Desc / Clms Page number 61>

 
 EMI61.1
 Example 7, except that the mixture of cells - 2250 CMSPE- per well. The supernatants were initially tested by ELISA on the flagellated pool as described in Example 6, except that the strains
 EMI61.2
 - jmmytTrher-tet yTCµ - of the gathering.

   The positive wells were then tested on the stock strain - --eompenant & trypes - Fisher -4T-E <a-lgnee finally isolated cells 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 injected dose was 1 DLIOO and that the injected strain was 1624, which is a clinical isolate expressing the immunotype Fisher 2 LPS and the guy has flagella.

  <Desc / Clms Page number 62>

 
 EMI62.1
 



  TABLE VI Study of the Protection by Human Antibody Antibodies i2D7 Anti-Flaqelles Tsne in the Burned Mouse Model
 EMI62.2
 
 <tb>
 <tb> monoclonalPercentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 ---
 <tb> 12D7,
 <tb> human,
 <tb> antiflagella <SEP> a <SEP> - <SEP> 100 <SEP> -1-00 <SEP> 100 <SEP> -100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP>
 <tb> 2H9,
 <tb> human
 <tb> anti-Fisher <SEP> 2 <SEP> LPS <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90
 <tb> 11G5
 murine <tb>
 <tb> antiflagella <SEP> a <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> 15F4
 <tb> human,
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 37,

    <SEP> 5 <SEP> 25 <SEP> 25 <SEP> 25 <SEP> 25 <SEP> 25 <SEP> 25 <SEP> 25
 <tb>
 The mice were infected with bedsores by
1 DLloo (950 CFU) from P. aeruginosa.



  2. The percentage of survival is low 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 (50 g in 0.5 ml PBS) was administered intraperitoneally before the burn and the infectious injection.

  <Desc / Clms Page number 63>

 
 EMI63.1
 



  EXAMPLE L. Example flagella type b antibodies to P. aeruginosa and the protection of mice passively immunized by this antibody against infection by P. aeruginosa injection on a model of
 EMI63.2
 mouse-burnt .-------- = ------- A cell line was prepared and cloned essentially as in ----- the example -au-que-le-Fappt-de-1A2 üJres-B for the transformation of approximately 60 and that the transformed mixture of transformed cells was placed on a plate 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 carried out on a different collection of P. aeruginosa strains: 1277, G98, 1739 and the immunotypes Fisher-F2, F4, F6 and F7. The finally isolated cell line and the secreted monoclonal antibody (isotype IgGl) are both designated under the name 2B8 in the text below.



   An itumunofluorescence test performed with 2B3 was positive on a flagellum type b strain immunotype Fisher 2, but negative on a reference flagellum type a strain, Fisher immunotype 4. In the test of clinical isolates 59/59 (100%) flagella type b isolates tested positive.



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

  <Desc / Clms Page number 64>

 



  TABLE VII
 EMI64.1
 "Study by human 288 antiflael1es type of burned mouse
 EMI64.2
 
 <tb>
 <tb> Percentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> 2B8,
 <tb> human, <SEP> - <SEP>
 <tb> antiflagella <SEP> b3 <SEP> 100 <SEP> 89 <SEP> 89 <SEP> 89 <SEP> 89 <SEP> 89 <SEP> 89 <SEP> 89 <SEP> 89
 <tb> PaF4 <SEP> IVES,
 murine <tb>
 <tb> antiflagella <SEP> b4 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 90 <SEP> 90 <SEP> 90
 <tb> VH3,
 murine <tb>
 <tb> anti-Fisher <SEP> 2 <SEP> LPS4 <SEP> 90 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20
 <tb>
   1. The mice were infected under pressure sores by
1 DLIOO (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 g in 0.5 ml of PBS) was administered intraperitoneally before the burn and the infectious injection.



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

  <Desc / Clms Page number 65>

 
 EMI65.1
 



  . Qpg 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
 EMI65.2
 -----) c "UnTihdividu-different was immunized et al.,: tr'45: 309: 1 and served as source of B cells, and the number of CMSPE- was about 2,000 cells per well for The examination was carried out on Fisher immunotypes 1 to 7 pooled together. The cell line finally isolated and the secret monoclonal antibody (isotype IgG) are both designated under the name 14C1 in the text. below.



   In the clinical test, 59/59 (100%) of flagella 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.

  <Desc / Clms Page number 66>

 TABLE VIII
 EMI66.1
 Etüde protection Etud w human 14C1 antiflagella type b the burned mouse model.
 EMI66.2
 
 <tb>
 <tb> of <SEP> thePercentage <SEP> from <SEP> survival <SEP> by <SEP> day2
 <tb> Processing <SEP>
 <tb> 1 <SEP> 2 <SEP> 314Cl,
 <tb> human,
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 90 <SEP> 90 <SEP> 90 <SEP> 90
 <tb> PaF4 <SEP> IVE8,
 murine <tb>
 <tb> antiflagella <SEP> b <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
 <tb> VH3
 <tb> human
 <tb> anti-Fisher <SEP> 2 <SEP> LPS <SEP> 100 <SEP> 40 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> 20
 <tb>
 1.

   The mice were infected with bedsores by
 EMI66.3
 1 DLIOO (90 CFU) of P. aeruginosa F164.



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



  3. The antibody purifies zo in 0.5 ml of PBS) was administered intraperitoneally before the burn and the infectious injection.

  <Desc / Clms Page number 67>

 
 EMI67.1
 



  It emerges from the above that the --de-c ente nvent tttruent-d & s-meanspour fragments thereof which are reactive with FTagellesdeV. crossed against various strains of P. aeruginosa. Surprisingly-deaembr. aeti-fs - = antibody with pitopesdjLjEferents on flagella were isolated. The antibodies of the present invention allow for a more cost-effective way of producing 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 PaF4IVE8 and FA6 IIG5 cited in Examples 2 and 3 were deposited at A. T. C. C. on June 20, 1986 under the references
 EMI67.2
 HB9129 and HB9130, respectively.



  The human lines 20H11 and 2188 cited in Examples 6 and 7 were deposited at A. T. C. C. on December 23, 1986 under the references CRL 9300 and CRL 9301, respectively.



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



   Although the present invention has been described with various details by way of illustration and examples for the purpose of clarity and understanding, it is obvious that it is capable of various variants and modifications without departing from its scope.


    

Claims (1)

  R E V EN D I C A TION S 1. - Composition characterized in that it comprises a monoclonal antibody or a fragment of  EMI68.1  -L i have-tzo-n-deliaison with Les ae .. -2.- mYction characterized in that the antibody or the fragment itself-cl "lnhlbe bacteria. DF it capable of reacting specifically3.-Composition according to any one of claims 1 and 2, characterized in that 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. H89129, 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  <Desc / Clms Page number 69>    EMI69.1  de-Jevendicatjins6RtJ, M. isee - a cell line 9. according to any one of the products of human monoclonal antibodies. 10.-Lineage - in this case designated under the access numbers A.T.C.C. HB9129, HB9130, CRL 9300, CRL 9301, CRI, 9422, CRL 9423 and CRL 9424.    11. - process 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  EMI69.2  uhe and the collection of these antibodies.  12. Pharmaceutical composition useful for the treatment of a human being susceptible to bacteremia and / or sepsis, characterized in that it comprises a prophylactic or therapeutic amount of monoclonal antibodies produced according to claim 11.  EMI69.3   13. Antibody for a binding fragment thereof, characterized in that it is monoclonalreactive 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  <Desc / Clms Page number 70>    EMI70.1  --- a epitope-of-Jragljjes-de-typea-ou b-de-- --- 16.-A composition according to claim 15, --- craractrse-en-qum-secmd-de-ees-antricoTps -monoclonal is able to react with a type of flagella-nonrejacJLfs monocionalr. 17.-CompositiooL claims-15 charactersee -q ge-¯I e- first antibody is a human monoclonal antibody.  18. Composition according to any one of Claims 15 & 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  EMI70.2  -que-ere- c-om qureTle from human blood plasma 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 fraction of gamma globulin 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 fraction of gamma globulin originating from the plasma of human blood is collected from human beings having levels  <Desc / Clms Page number 71>    EMI71.1  raised with Pseudomonas 23.-Pharmaceutical composition characterized in that at least two mono clonal antibodies 9, each of which reacts specifically with a type of reactive immunoglobulin different from the flagellar protein of Pseudomonas aeruginosa and is capable of treating or preventing infections with Pseudomonas aeruginosa. 24. -Compositionpharmaceutiquesuivantla claim 23, characterized in that at least one of the monoclonal antibodies is a human monoclonal antibody.  25.-A pharmaceutical composition according to any one of claims 23 and 24, characterized in that it further comprises at least one antibody  EMI71.2  -monoclonal e act 'human-capable- 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 epitop is present on the flagella of type a or the flagella of type b, but not on both.  28. Composition according to Claim 26, characterized in that the epitope is shown by at least approximately 70% of the flagella of type b.  29.-Composition according to any one of claims 26 and 28, characterized in that 1 kepitop is shown only by flagella type b.  30.- Composition according to claim 29, characterized in that the antibody is pan-reactive.  <Desc / Clms Page number 72>    EMI72.1   31. useful for -F * l feceptH.ible treatment ilntec bacterial, what it - Pharmaceutical compositionprophylactic or therapeutic amount of a monoclonal antibody capable of binding to the flagellum of Pseudomonas aeruginosa, in combination with one or  EMI72.2  piTErsteTfrrjes a therapy of a monoclonal antibody capable of reacting with A of Pseudomonas aeruginosa? -un --anti. econal e-de suitamyT 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.-Monoclonal antibody composition, characterized in that the monoclonal antibody has the same specificity as any of the mono-clonal 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 marker capable of providing a detectable signal.  34. - A monoclonal antibody composition according to claim 33, characterized in that the marker 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 forma-  <Desc / Clms Page number 73>  tion of complexes.  EMI73.1  presence Trousseutiledansaaetectiorf "deTa" terized 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 signal  EMI73.2  - this antibody --u-Jri eacLijEs monoclonal antibody.