WO1990013660A2 - Human monoclonal antibodies to sero-specific determinants of gram-negative bacteria - Google Patents

Abstract

Prophylactic or therapeutic compositions are described that are useful for treating bacteremic infections consisting of human monoclonal antibodies that bind to Pseudomonas aeruginosa, Klebsiella, and E. coli alone or in combination. Also described are methods for using the composition to treat patients suffering from bacteremic infections.

Description

HUMAN MONOCLONAL ANTIBODIES

TO SERO-SPECIFIC DETERMINANTS OF

GRAM-NEGATIVE BACTERIA

This invention is in the area of immunology, and particularly concerns the generation of human monoclonal antibodies that are useful to treat gram-negative bacterial infections. More specifically, it concerns the generation of human monoclonal antibodies to the three most prevalent bacteria responsible for nosocomial infections, Pseudomonas aeruginosa, Klebsiella, and Escherichia coli.

Antibiotic therapy is the primary recourse to treat nosocomial acquired gram- negative bacterial infections. Unfortunately, antibiotic therapy has limited effectiveness as nearly a third of the patients that become infected eventually die. For example, in the United States alone nosocomial bacteremia develops in about 194,000 patients, and of these about 75,000 die. Maki, D.G., 1981, Nosocomial Infections, (Dickson, R.E., Ed.) page 183, Yrke Medical Books, U.S.A. It has been shown that six major gram- negative bacilli are responsible for most of these deaths, and these are Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella, Enterobacter and Serratia.

Because of the limited effectiveness of antibiotics in the treatment of gram- negative bacteremia recent developments have focused on passive immunization as an alternative, or supplement to antibiotic therapy. This approach is based on the observation that immunization of man or experimental animals with bacterial vaccines, or the bacterial endotoxin, lipopolysaccharide (LPS) elicits an immune response directed mainly against LPS, and that such antibodies are protective against subsequent bacterial challenge. LPS is one of the major components of the outer cell membrane of most gram-negative bacteria, as well as being a primary constituent of bacterial endotoxins. Thus, a good deal of work has been expended towards developing antibodies that bind to and neutralize the effects of gram-negative bacteria, with particular attention being devoted to lipopolysaccharides, and more specifically to the lipid A moiety of LPS.

Lipopolysaccharides consist of at least three significant antigenic regions, and these are lipid A, core polysaccharide, and O-specific polysaccharide. The latter is also referred to as O-specifϊc chain or simply O-antigen. The O-specific chain region is a long-chain polysaccharide built up from repeating polysaccharide units. The number of polysaccharide units differs among different bacterial species and may vary from one to as many as six or seven monosaccharide units. While the O-specific chain varies among different gram-negative bacteria, the lipid A and core polysaccharides are similar if not identical. Because passive transfer of anti- LPS antibodies has been shown to be associated with survival in patients suffering from bacteremia, most of the effort towards generating passive immunization modalities have focused on generating antibodies to the three antigenic regions of LPS. The immunotherapeutic use of type- specific anti-LPS antibodies has perhaps been most extensively studied in the treatment of bacteremia due to Pseudomonas aeruginosa. It has been shown that such antibodies are protective in a variety of animal models. Pseudomonas aeruginosa infections, in Bacterial Vaccines, page 317, Academic Press, 1984. Also see Pollack, M., Antibody- Mediated Immunity in Pseudomonas Disease and Its Clinical Application, in

Im munoglobulins: Characteristics and Uses of Intravenous Preparations, Study With Immuno globulins & Preparations. Alving, B.M. and Finlayson, J.S., Eds., page 273, U.S. Department of Health and Human Services, (1979).

Other studies have shown the importance of LPS in E. coli induced bacteremia. For instance, Ziegler, el al., 1982, New England J. of Medicine. 307:1225 have shown the preparation of human antisera to the core region of E. coli LPS. The study consisted of vaccinating healthy patients with a mutant strain of E. coli that lacks the O- specific polysaccharide, thus exposing the core-antigen and permitting more efficient antibody production against it. For example, in the E. coli J5 strain, the O-specific polysaccharide is deleted via a mutation that effectively causes a loss of the enzymes involved in the synthesis of the O-antigen, or the enzyme required to attach the O- antigen to the core. In any event, antisera generated in this fashion against the O- antigen was used to treat bacteremia and reported to be biologically effective. Indeed, when the antisera was administered to near terminally ill bacteremic patients, the recovery rate nearly doubled.

In addition to passive immunization of patients with antibodies generated against LPS, considerable effort has been expended towards realizing passive immunization schemes based on the generation of antibodies against bacterial capsular antigens. A good deal of this work has been done with Klebsiella wherein the protective effect of sero type-specific capsular polysaccharide antibody preparations has been documented. Cooper, J.M, and Rowley, D., 1982, Journal of Experimental Biology Medicine & Science, 60:629; Cryz, S.J. et al., 1984, Infectious Immunology, 45:139, and Cryz, S.J. et al., 1984, J. of Infectious Disease, 150:817. In addition to having a plasma membrane and cell wall, Klebsiella may be capsulated, which capsules consist of negatively charged acidic polysaccharides. A number of the antigens have been identified and have molecular weights under 50,000. Indeed, about 77 such antigens have been identified, and are classified based on their different serological properties. Because of the inherent advantages associated with monoclonal antibodies, the favored approach towards establishing effective passive immunization schemes against gram-negative bacterial infections is the generation of monoclonal antibodies that recognize LPS, capsular antigens, or other bacterial antigens that are involved in the production of bacteremia. As described below, several investigators have generated either murine or human antibodies.

European Patent Application, Publication No. 256,713, describes human lymphoblastoid cell lines and hybridomas that produce antibodies reactive with LPS present on Pseudomonas aeruginosa. The antibodies are immunospecific for one or more of the Fischer immunotypes of Pseudomonas aeruginosa.

European Patent Application, Publication No. 163,493, also shows human lymphocyte cell lines which secrete human monoclonal antibodies to serotypic determinants on LPS. Human monoclonal antibodies specifically reactive with Pseudomonas aeruginosa are shown.

European Patent Application, Publication No. 101,039, describes murine monoclonal antibodies that recognize Pseudomonas aeruginosa. Mice were immunized with formalin fixed bacteria.

PCT Patent Application, International Application No. PCT/US 84/02022, shows murine monoclonal and polyclononal antibodies that recognize LPS. Host animals were also immunized with fixed bacteria.

European Patent Application, Publication No. 174,204, shows human monoclonal antibodies that react with the lipid A antigenic determinant of LPS.

Specifically shown are human IgM antibodies that protect animals from gram-negative bacterial endotoxin.

PCT Patent Application, International Application No. PCT/US 86/01204, shows cell lines that secrete human monoclonal antibodies capable of binding to and neutralizing Pseudomonas aeruginosa exotoxin A. The virulence associated with Pseudomonas aeruginosa is thought to result from several bacterial products including LPS. One such product is the enzyme exotoxin A. It is an extra cellular enzyme and has been demonstrated to inhibit eukaryotic protein synthesis. Iglewski, B. and Kabat, D., 1975, Proc. Nat'l. Acad. Sci. USA, 72:2284.

UK Patent Application No. 2192185 shows human monoclonal antibodies that bind to Pseudomonas aeruginosa flagella. Some of the antibodies protect against lethal challenge of Pseudomonas aeruginosa.

PCT Patent Application, International Application No. PCT/US84/01643, shows both human and murine monoclonal antibodies that bind to LPS, and methods of using the same for the treatment or prevention of gram-negative bacteremia. U.S. Patent No. 4,771,127, shows Pseudomonas aeruginosa vaccines that protect against the endotoxic effects of LPS. The vaccines consist of LPS covalently coupled to either tetanus toxoid or Pseudomonas aeruginosa toxin A. Such conjugates have a molecular weight greater than 350,000, are non-toxic and non-pyrogenic, and upon immunization of a host animal induce protective anti-LPS antibody, as well as antibody which neutralizes the effects of tetanus toxin or toxin A.

Finally, U.S. Patent No. 4,755,381, shows vaccines to Klebsiella consisting of capsular polysaccharide antigens.

Despite the existence of numerous monoclonal or polyclonal antibody preparations to some of the most prevalent bacteremic gram-negative bacteria these antibodies have limited medical applications because of their restricted cross-reactivity, immunogenicity in the case of murine antibodies, or poor binding properties. The use of polyclonal antibodies is similarly limited, and suffers from the further drawback that polyclonal antibodies are time consuming and expensive to prepare. Because bacteremia can be a fatal disease that generally kills a patient within 48 hours, there is little time to determine the type of bacteria responsible for the disease in order to subsequently administer the appropriate antibody. Thus, it will be appreciated that what is needed for effective treatment are antibodies that cross-react with various bacteremic strains, that may be used alone or in combination.

Regarding the need for highly cross-reactive antibody it has been stated that of the 77 known capsular serotypes of Klebsiella about 25 are responsible for 70% of the reported Klebsiella bacteremic isolets. The situation is not as complicated for

Pseudomonas aeruginosa. The majority of infections are attributable to 5 of the 17 known serotypes. Thus, an effective pass immunization scheme for treating a wide spectrum of gram-negative bacterial infections, would preferably consist of a few monoclonal antibodies that are highly cross-reactive with different virulent gramnegative bacteria including Pseudomonas aeruginosa, Klebsiella, and E. coli. or a "cocktail" of antibodies that has the same binding specificity.

The invention described herein relates to the generation of antibody secreting hybrid cell lines that secrete antibodies that bind to and neutralize the bacteremic effects of gram-negative bacteria. The antibodies may be used alone, or in combination.

A second object of the invention is the description of cell lines that secrete human monoclonal antibodies that bind to antigenic determinants that effectively neutralize the bacteremic effects of Pseudomonas aeruginosa, Klebsiella, or E. coli.

A third object of the invention is the description of a "cocktail" of human monoclonal antibodies to Pseudomonas aeruginosa, Klebsiella and E. coli. A fourth object of the invention is the description of highly cross-reactive monoclonal antibodies to Klebsiella wherein eight monoclonal antibodies bind to, and effectively neutralize the bacteremic effects of 22 Klebsiella serotypes.

A fifth object of the invention is the presentation of methods for producing human monoclonal antibodies against bacteremic strains of Pseudomonas aeruginosa consisting of immunizing human volunteers with polysaccharide-toxin A conjugate vaccines and isolating lymphocytes from the volunteers. The latter are used to produce stable hybrid cell lines that secrete the antibodies of interest.

A sixth object of the invention is the presentation of methods whereby a vaccine to Klebsiella capsular polysaccharide antigens is produced and used to immunize human volunteers thereby producing antibody secreting cells that can be employed to ultimately yield stable antibody secreting hybrid cell lines.

A further object of the invention is a description of methods of using the above described antibody compositions to treat patients suffering from bacteremia.

Table 1 shows binding of antibodies to lipopolysaccharide purified from various strains of Pseudomonas aeruginosa. Similar results were obtained when whole bacteria representative of the various serotypes were used.

Table 2 shows the properties of various monoclonal antibodies generated to Klebsiella capsular polysaccharides.

Table 3 presents the properties of two monoclonal antibodies to E. coli O- polysaccharides.

Table 4 shows the properties of monoclonal antibodies that recognize

Pseudomonas aeruginosa serotypes.

Table 5 illustrates the in vivo protective capacity of monoclonal antibodies that recognize Pseudomonas aeruginosa serotypes, IT-1 through IT -7, and IATS-3 and IATS-4.

The invention described herein relates to the production of human monoclonal antibodies reactive with various gram-negative bacteria that are highly virulent and responsible for the majority of hospital required bacteremic infections. Monoclonal antibodies are described that are reactive with lipopolysaccharide associated with E. coli or Pseudomonas aeruginosa, as well as monoclonal antibodies that bind to capsular antigens of Klebsiella. The invention was realized in several steps and each will be described separately below. These are the preparation of immunogen(s) suitable for either in vivo or in vitro immunization, use of the immunogen to formulate a vaccine to sensitize human lymphocytes, immortalization of the lymphocytes by either

transformation or cell fusion techniques thereby resulting in transformed lymphocytes or hybrid cell lines, and the identification of those transformed cell lines or hybridomas that secrete the appropriate monoclonal antibodies, and characterization of the monoclonal antibodies with regard to their gram-negative binding properties and anti- bacteremic effects.

Mentioned below are several prior art patents and scientific publications. These references are relevant to the instant invention and are all hereby incorporated by reference.

Preparation of Immunogen

Pseudomonas aeruginosa

The preferred immunogen is lipopolysaccharide, and more preferred is the O- polysaccharide determinant associated therewith. LPS can be isolated from various Pseudomonas aeruginosa strains using techniques well known to those skilled in the art, particularly the hot phenol water method described by Westphal et al., Ueber Die Extraktion Von Bakterien MitPhenol-Wasser. Z. Naturfosch, 2:148. Additionally, other methods may be utilized including that described by Darveau and Hancock, 1983, J. of Bact., 155:831. LPS can be subsequently purified by treatment with RNAse, DNAse and pronase as described by Cryz, S .J. et al., 1984, Infect. Immun., 44:508. Purified LPS may be used as immunogen, or alternatively the O-polysaccharide determinant can be isolated and used directly as immunogen. If O-polysaccharide is used directly it is preferably coupled to an appropriate carrier to ensure that it will elicit an immune response. The latter procedure is desirable as O-polysaccharide alone is not readily immunogenic. Pier, U.S. et al., 1978, Infectious. Immun., 22:919 and Chester, I.L. et al., 1973, J. of General Microbiology, 78:305. The preferred carriers and methods for coupling them to O-polysaccharide are described in U.S. Patent No. 4,771,127. Klebsiella

The preferred Klebsiella antigen consist of repeating units of monosaccharides which comprise high molecular weight polymers. These polysaccharides are found predominantly in the capsule surrounding Klebsiella, and as such are termed capsular polysaccharide antigens. They are responsible for ctetemώiing the antigenic specificity of various bacterial species, including Klebsiella. The Klebsiella polysaccharide capsular antigens are referred to as "K" antigens, the "K" stands for kapsel which is German for capsule. There are about 77 known Klebsiella capsular serotypes.

Orskov, I. and Fife-Asbury, M.A., 1977, Int. J. Syst. Bacteriol, 27:386. Capsular polysaccharides can be isolated using a number of techniques, and the isolated product used as immunogen. In many instances, the antigens have been shown to be protective against fatal Klebsiella infection, particularly Klebsiella pneumonia. Capsular polysaccharide antigen can be obtained from culture supernatants of Klebsiella grown in a culture medium specifically designed to support capsule production. The capsular polysaccharide antigens can be purified as described by Cryz, S.J. et al., 1985, Infect. Immun., 5 0:225; and Cryz, S.J. et al., 1985, J. of Infect. Diseases. 151(4). Alternative methods are known for isolating and formulating capsular antigens in highly immunogenic form and these are described in Infect.

Immun., 1984, 45:139. Also, U.S. Patent No. 4,755,381 describes a procedure for producing capsular antigen preparations that lack pyrogenic activity.

E. coli

A number of procedures are known for isolating E. coli lipid polysaccharide in immunogenic form. See for example, European Patent Application, Publication No. 174,204. Indeed, most of the procedures described above relating to LPS isolation and purification from Pseudomonas aeruginosa are similarly applicable here.

Immunization

The above described immunogens may be used to generate an immune response either in vivo or in vitro, and sensitized lymphocytes resulting therefrom can be used to prepare hybrid cell lines that secrete the appropriate monoclonal antibody. The preferred immunization procedure involves sensitizing lymphocytes to the antigen of choice by immunizing human volunteers, and isolating therefrom the antibody secreting cell fraction and immortalizing the cells therein by one of several procedures.

The preferred procedures whereby individuals are immunized with

Pseudomonas aeruginosa or Klebsiella are described in U.S. Patent Nos. 4,771,127 and 4,755,381, respectively. Similar procedures may also be followed to immunize individuals with LPS or O-polysaccharide of E. coli. It will be apparent to those skilled in the art, that in lieu of immunizing individuals, lymphocytes may be isolated from individuals that are experiencing, or have experienced a bacteremic attack, and used to produce permanent antibody secreting hybrid cell lines. For example,

immunocompromised human patients are generally susceptible to bacterial infections, particularly those suffering from various malignancies, extensive burns, etc., and lymphocytes isolated therefrom may be a source of antibody secreting cells.

Human lymphocytes can also be sensitized in vitro following one or more procedures generally known in the art. In vitro techniques are generally described by Luben, R. and Mohler, M., 1980, Molecular Immunology, 17:635, Reading, C.

Methods in Enzymology, 121 (Part One):18, or Voss, B., 1986, Methods in

Enzymology, 121:27. Moreover, a number of in vitro immunization systems have been shown to be effective for sensitizing human B-cells. Reading, C, 1982, J. of Immun. Methods, 53:261.

Lymphocytes sensitized either in vivo or in vitro can be converted into immortalized antibody-producing hybrid cell lines thereby making available a permanent source of the desired antibody. One procedure for performing this step is immortalization by viral transformation. The preferred viral transformation technique involves the use of Epstein-barr virus. The virus is capable of transforming human B- cells, and has been used to generate human monoclonal antibodies. Crawford, D. et al., 1983, J. of General Virology, 64:697; Kozbor, V. and Roder, J., 1983, L

Immun. Today, 4:72.

In lieu of producing permanent antibody producing hybrid cell lines by viral transformation, antibody producing cells can be immortalized employing suitable cell fusion techniques which realize hybrid cells that produce the antibodies of interest. The hybrid cell lines may be formed by fusing antibody producing cells with an appropriate immortalizing cell line. The latter is preferably of lymphoid origin and may be a lymphoblastoid cell or a plasmacytoma cell.

A third procedure whereby sensitized lymphocytes may be immortalized consist of a combination of the above two techniques, that is viral transformation and cell fusion. The preferred combination consist of transforming antibody secreting cells with Epstein-barr virus, and subsequently fusing the transformed cells to a suitable fusion partner. The fusion partner may be a mouse myeloma line, a heteromyeloma line, or a human myeloma, or other immortalized line. PCT Patent Application No. 81/00957; Schlom et al., 1980, PNAS USA, 77:6841; Croce et al., 1980, Nature. 288:488. The preferred fusion partner is a mouse-human hetero hybrid, and most preferred is the cell line designated F3B6. This cell line is on deposit with the

American Type Culture Collection, Accession No. HB8785. It was deposited April 18, 1985. The procedures for generating F3B6 are described in European Patent Application, Publication No. 174,204.

Techniques applicable to the use of Epstein-Barr virus transformation and the production of immortal antibody secreting cell lines are presented by Roder, J. gi &L, 1986, Methods in Enzymology, 121:140. Basically, the procedure consist of isolating Epstein-Barr virus from a suitable source, generally an infected cell line, and exposing the target antibody secreting cells to supernatants containing the virus. The cells are washed, and cultured in an appropriate cell culture medium. Subsequently, virally transformed cells present in the cell culture can be identified by the presence of the Epstein-Barr viral nuclear antigen, and transformed antibody secreting cells can be identified using standard methods known in the art.

The techniques for cell fusion as applied to the production of immortalized antibody secreting cell lines are generally described by Kohler, G. and Milstein, C, 1975, Nature. 256:495. These initial studies involved fusing murine lymphocytes and plasmacytomas to produce hybridomas. Subsequently, the technique has been applied to produce hybrid cell lines that secrete human monoclonal antibodies. The latter procedures are generally described in Abrams, P., 1986, Methods in Enzymologv. 121: 107, but other modifications are known to those skilled in the art.

Regardless of which procedure is used, the antibody secreting cells are combined with the fusion partner and the cells fused with a suitable fusing agent, preferably polyethylene glycol, and more preferably polyethylene glycol 1000. The latter is added to a cell pellet containing the antibody secreting cells and the fusion partner in small amounts over a short period of time accompanied with gentle agitation. After the addition of the fusing agent, the cell mixture is washed to remove the fusing agent and any cellular debris, and the cell mixture consisting of fused and unfused cells seeded into appropriate cell culture chambers containing selective growth media. After a period of several weeks, hybrid cells are apparent, and may be identified as to antibody production and subcloned to ensure the availability of a stable hybrid cell line.

Identification/Characterization of Monoclonal Antibodies Hybrid cells produced by fusing antibody secreting cells and an appropriate fusion partner, or Epstein-Barr virus transformed cells that produce the desired antibody can be identified using convenient immunochemical screening techniques. The hybrid cells may be grown in appropriate cell culture media, and the supernatant screened for the presence of monoclonal antibody using, for example, an enzyme linked immunosorbent assay (ELISA). The latter is described by Engvall, E., 1977, Med. Biol., 55:193. Basically, the procedure consist of coating flat-bottom 96 well microtitre plates with the appropriate antigen, reacting the antigen with cell culture supernatant, removing the supernatant and then revealing the presence of antibody using a suitable second antibody that has been labelled with a reporter group. It is well known in the art that the reporter group is preferably a radioactive tracer or a fluorescent molecule.

Antibody specificity can be tested directly against different strains of bacteria using ELISA assays, or alternatively, purified antigen may be tested, such as lipopolysaccharide, or O-polysaccharide derived therefrom. The conditions for binding either intact bacteria, lipopolysaccharide, or O-polysaccharide to plastic surfaces to run the ELISA are known to those skilled in the art. European Patent Application No. 163,493; and European Patent Application No. 174,204.

Additionally, antibody specificity can be further characterized by immunoblot analysis and immunofluorescence assay. Immunoblot analysis is particularly useful to determine the specificity of the antibodies to various lipopolysaccharides. The procedure can be carried out as described by Trobwin, H. et al., 1979, PNAS USA. 76:4350 and Burnette, W., 1981, Analytical Biochem., 112:195. The methods for performing immunofluorescence are also known in the art. The isotype of the monoclonal antibodies can be determined using standard immunochemical techniques.

Finally, the ability of the monoclonal antibodies, either alone, or in combination to protect against bacteremia can be determined using one of several sepsis animal model systems. These are described by Cryz, S.J., et al. 19 83, Infectious Immun.. 39:1072 and Strieritz, D.D., 1975, J. of Infect. Disease, 131:688.

It will be apparent to those skilled in the art that while the preferred embodiment of the instant invention is intact monoclonal antibodies, either one or more, alone or in combination with other monoclonal antibodies having various specificities for different bacteria involved in the production of bacteremia, that the antibodies may be altered and still maintain their biological activity. Thus, encompassed within the scope of the invention are antibodies modified by reduction to various size fragments, such as F(ab')₂. Fab, Fv, or the like. Also, the hybrid cell lines may be considered to be a source of the DNA that encodes the desired antibody, which may be isolated and transferred to cells by known genetic techniques. An example of the latter would be the production of single chain antibody having the antibody combining site of the hybridomas described herein. Single chain antibodies are described in U.S. Patent No. 4,704,692.

It will be appreciated by those skilled in the art that a key feature of applicants' invention is the realization that human monoclonal antibodies having different specificities for bacteria involved in producing bacteremia may be combined in pharmaceutical compositions to form a "cocktail". Thus, a preferred embodiment of applicants' invention is a "cocktail" consisting of monoclonal antibodies that recognize Pseudomonas aeruginosa, Klebsiella, and E. coli. More preferred is a cocktail consisting of monoclonal antibodies that recognize various Pseudomonas aeruginosa strains (Table 1), various strains of Klebsiella (Table 2), and various serotypes of E. coli (Table 3).

It will further be appreciated by those skilled in the art that the human monoclonal antibodies described herein can be administered either alone, or as a "cocktail" in combination with various pharmaceutically acceptable diluents or carriers. Such are widely known to those skilled in the art and are formulated according to standard pharmaceutical practices.

It will also be appreciated by those skilled in the art that the scope of the invention includes uses of the subject antibodies both in therapeutic and prophylactic regimes in combination with antimicrobial agents, preferably antibiotics. The dosage of the monoclonal antibodies in combination with the dosage of antibiotics can be determined by a prescribing physician as is normal medical practice. It is expected that the dosage will vary according to the age, weight, and response of the recipient patient Having generally described what applicants believe their invention to be, illustrative of the invention are examples presented below. It is intended that the examples be considered in an exemplary spirit only, and not restricted of the scope of the invention.

Example I

Isolation of Pseudomonas Aeruginosa Lipopolysaccharide

O-polysaccharide Determinant

LPS was isolated from the appropriate Pseudomonas aeruginosa strains by the hot phenol water method of Westphal et al., described above, and further purified by treatment with RNAse, DNAse, and pronase as described by Cryz, S J., et al., 1984, Infect. Immun., 44:508. Typically, LPS prepared in this way contains less than 1% (wt/wt) protein and less than 1% (wt/wt) nucleic acids. The 0-polysaccharides containing serospecifϊc antigenic determinants was derived from LPS by mild acid hydrolysis. Cryz, S.J., et al., 1986, Infect. Immun., 52:161. Briefly, this consisted of suspending LPS in 1% (vol/vol) acetic acid and heating the suspension to 100°C for 90 minutes. This treatment effectively causes the lipid A component of LPS to precipitate which can be removed by centrifugation at 5,000 x g for 15 minutes. In order to remove residual nonhydrolyzed LPS, the mixture was chromatographed over a AcA34 column (LBK Produkter-, Promma, Sweden), gel bed 5 x 45 cm equilibrated with water. The O-polysaccharide obtained in this manner was iyophilized and analyzed for pyrogenic activity. Only pyrogen free O-polysaccharide was used to produce a conjugate vaccine which was used to immunize human volunteers from which antibody producing cells were obtained and subsequently used to generate the sought after hybrid cell lines.

In addition to the above, further details regarding the generation of O- polysaccharides from Pseudomonas aeruginosa strains are described in U.S. Patent No. 4,771,127. Example II

Isolation of Klebsiella Capsular Polysaccharide Antigens The capsular polysaccharides are obtained from strains of Klebsiella cultivated on a medium designed to promote capsular production. The procedure is generally described by Cryz, S J. et al., 1985, Infect. Immun., 50:225 and Cryz, S.J. et al., 1985, J. of Infect. Diseases. 151:4. Further, additional procedures are shown by Cryz, S J. et al., 1984, J. of Infect. Disease. 151:817 and by Cryz, S J. et al., 1984, Infect. Immun., 45:139. Briefly, Klebsiella cultures were inoculated onto agar plates and the cultures grown for 18-24 hours at 37° C. The cultures were grown up by inoculation into 30 ml of HYBM broth (2% (wt/vol) Hycase-SF(Hunko Sheffield,

Memphis, Tennessee, USA)), 0.3% (wt/vol) yeast extract (Difco Laboratories, Detroit, Michigan, USA)), and 2% (wt/vol) maltose added as a 10% sterile stock solution in a 125 ml flask. Cultures were grown at 37°C while rotating at about 100 revolutions per minute, for 8 hours. Next, 1 ml of this culture was used to inoculate 500 ml of HYEM broth in a 2 litre baffled Erlenmeyer flask, and grown at 37°C for 16 hours with agitation (100 revolutions per minute). At the end of the culture period, the microbial purity of each flask was confirmed by gram-stain.

Subsequently, the strains of Klebsiella were removed from the growth flask by centrifugation, and the capsular polysaccharides isolated from cell-free culture supematants with 0.5% (wt/vol) cetavlon (M-cetyl-N, N, N-trimethyl, ammonium bromide, E. Merke & Company). This material was detoxified of trace amounts of lipid polysaccharide by deacylation. The cetavlon isolation procedure, subsequent purification steps, and detoxification procedures are described in detail in U.S. Patent No. 4,755,381.

Prior to formulating the capsular polysaccharides for immunization, the polysaccharides were subjected to NaOH-treatment, also described in U.S. Patent No. 4,755,381. This treatment results in large molecular weight polysaccharide antigenic fractions which are nonpyrogenic.

Example III

Isolation of E. coli LipopoIysaccharide/O-polysaccharide

The procedures described in Example I were similarly employed here to obtain O-polysaccharide from E. c_Qli lipopolysaccharide. LPS was isolated from serotypes, 01,02, 04, 06, 07, 08, 012, 015, 016, 018, 025, and 075. Example IV

Preparation of Pseudomonas Aeruginosa Immunogens O-polysaccharide Pseudomonas aeruginosa/toxin A immunogenic conjugates were prepared wherein the O-polysaccharide moiety was obtained, as described above, from different strains of Pseudomonas aeruginosa. These strains were Fisher type 2, 5, and 7 available from M. Fisher, Parke Davis & Company, Detroit, Michigan, and Fisher type 3 and 4 and 6, available from Walter Reed Army Institute of Research, Washington D.C. Additionally, Fisher type 1 is available from B. Wretlind, Danderyd Hospital, Danderyd, Sweden; andHabs serotypes 3 and 4 are available from I.L. Pitt, Central Public Health Laboratory Service, London, England

The various O-polysaccharides were covalently linked to toxin A via adipic acid dihydrazide. It is noteworthy that coupling of toxin A to polysaccharide by this reaction results in the detoxification of toxin A. Such conjugates are generally about 350,000 in molecular weight, nontoxic and nonpyrogenic. Upon subsequent immunization with the conjugate, antibody production is elicited both to the

polysaccharide as well as toxin A.

More specifically, O-polysaccharide was oxidized with NaIθ4 by dissolving the polysaccharide in distilled water to a final concentration of 5 mg/ml with a final concentration of 0.1 M NalO₄. The oxidation was allowed to proceed for 2 hours at 22° C and was protected from light. At the end of this time, the reaction was stopped by the addition of 0.53 ml of ethylene glycol. The material was extensively dialyzed against distilled water and subsequently lyophilized.

Next, toxin A was prepared as described by Cryz, S .J., et al., 1984, Infect. Immun., 43:795. Toxin A was derived from a spontaneously isolated hyperproducer of the molecule, Pseudomonas aeruginosa strain PA103 (available from Dr. B.

Wretlind, Karolinska Institute, Stockholm, Sweden) termed PA103-FeR. Generally, such preparations consist of greater than 95% toxin A protein.

Oxidized polysaccharide was coupled to toxin A using adipic acid dihydrazide. Briefly, the procedure consisted of initially reacting adipic acid dihydrazide with toxin A in solution consisting of 5 mg/ml of 0.05 M NaPO₄, pH 7.2 with 10 mg/ml of both adipic acid dihydrazide (available from Huka A.G., Buchs, Switzerland) and 1-ethyl-3 (-3-dimethylaminopropyl (carbodϋmide). This reaction was allowed to proceed for 4 hours, followed by subsequent dialysis against 0.05 M NaPO₄, pH 8 buffer, for 72 hours at 40°C. A second dialysis was performed consisting of 0.5 M NaPO₄, pH 8 buffer for an additional 4 hours at 22°C. Insoluble material was apparent at the end of the dialysis period, and was removed by centrifugation. The toxin A protein concentration was adjusted to 5 mg/ml and this material was covalently coupled to the oxidized polysaccharide.

Toxin A derivatized with adipic acid dihydrizide in 0.5 M NaPO₄, pH 8 buffer (5 mg/ml) was added to an equal amount of oxidized polysaccharide (5 mg/ml). The mixture was stirred for 6 hours at 22ºC after which 3.1 ml of 0.25 M NaCNBH₃ was added. The mixture was stirred for 5 days at 22ºC and dialyzed for 24 hours against phosphate buffer saline, pH 7.4 containing 0.02% merthiolate. Insoluble material was removed by centrifugation, and the mixture chromatographed over a column of AcA34 (LKB-Produkter, Promma, Sweden). The conjugate was eluted in the void volume of the column, indicating that it had a molecular weight greater than 350,000.

Using the above procedures, O-polysaccharide toxin A conjugates were made of the Fisher serotypes 1, 2, 3, 4, 5, 6, 7, and Habs 3 and 4. These conjugates were then combined to comprise a nonavalent vaccine wherein a single dose of the vaccine contained 25 μg of O-polysaccharide of each serotype.

Four healthy adult volunteers were immunized twice intramuscularly at day 0 and day 28 with the vaccine. Peripheral blood lymphocytes were then isolated 7, 35 and 125 days after the first immunization, and subsequently used to produce permanent antibody secreting cell lines as described below.

Example V

Preparation of Klebsiella Immunogens

A vaccine was made from NaOH treated capsular polysaccharide material from Klebsiella serotypes K2, K3, K5, K9, K10, K15, K16, K17, K18, K21, K22, K25, K28, K30, K35, K43, K52, K53, K55, K60, K61, K62, K63, and K64. Capsule polysaccharide material from each of the bacterial strains possess a high molecular weight (Kd less than or equal to about 0.1 on Sepharose CL-4B) with the majority of carbohydrate eluting before a K_d = 0.5 have been reached.

A 24 valent Klebsiella capsular polysaccharide vaccine was formulated by reconstituting the capsular polysaccharides from the various Klebsiella serotypes in sterile distilled water such that one human dose is equal to about 300 μg of total antigen. Three healthy adult volunteers were immunized subcutaneously in the deltoid region, and peripheral blood lymphocytes isolated from the patients between 28 and 35 days post-vaccination. The peripheral blood lymphocytes were used as described below to construct permanent hybrid cell lines that secrete the monoclonal antibodies of interest.

Further details regarding the Klebsiella capsular polysaccharide vaccine are described in U.S. Patent No. 4,755,381. Example VI

Preparation of E.coli Immunogens

E.coli O-polysaccharides, isolated from the serotypes described in Example II, were coupled to toxin A as described in Example III using adipic acid dihydrazide. One healthy adult volunteer was immunized twice intramuscularly at day 0 and 28, and peripheral blood lymphocytes were isolated at day 35 and used to generate permanent hybrid cell lines as described below.

Example V II

Pseudomonas Aeruginosa/Klebsiella/E.coli Antibody

Secreting Hybrid Cell Lines

Peripheral blood lymphocytes were isolated from individuals immunized as described above with either the Pseudomonas aeruginosa, nonavalent vaccine, the polyvalent Klebsiella vaccine, or with one of the E. coli immunogen conjugates. These were then infected with Epstein-barr virus, and the resultant transformed lymphocytes fused to a fusion partner cell line, and the hybrid cell lines so generated isolated and characterized as to antibody production.

More specifically, mononuclear cells were separated on Ficoll-hypaque

(Pharmacia), and monocytes depleted from the mixture by adherence to plastic.

Standard laboratory techniques were utilized to effect these procedures.

Next, nonadherent cells were enriched for antibody producers by antigen- specific panning. Panning is a technique generally known in the art, and involves incubation of a population of antibody secreting cells on a plastic surface coated with the appropriate antigen. Those cells that express antibody on their surface bind antigen, and consequently adhere to the plastic surface, whereas those do not, do not adhere and can be removed by washing. Thus, specific antibody secreting cells are enriched for by this technique.

More specifically, 6- well plates (Costar) were coated with antigen, either LPS, O-polysaccharide, or Klebsiella capsular polysaccharide whereby 150 μg of antigen was coated per well in 0.05 M HCO₃ buffer, (pH 9.6) at 4ºC overnight. The wells were blocked after the overnight incubation period with phosphate buffered saline containing 1% bovine serum albumin for at least 1 hour at 4ºC, and subsequently washed with phosphate buffered saline/BS A. Next, 10⁷ lymphocytes in 1 ml of PBS/BS A were added to each well of the six well plates. The lymphocytes were allowed to incubate on the plates for 70 minutes, after which any nonadherent cells were removed by aspiration. The adherent cells were incubated with cell culture medium (IMDM, Sigma Chemical Co., St. Louis, Missouri) containing 10% fetal calf serum.

The adherent cells were subjected to Epstein-Barr virus transformation by adding an equal amount of culture media obtained from growing the Epstein-Barr virus infected marmoset cell line, B95-8, and thus containing the virus, to media bathing the adherent cells. The cells were cultured in this environment at 37'C for 3 hours, and in this way the lymphocytes in the adherent cell population were subjected to Epstein-Barr infection. Following the infection period, the cells were washed and plated onto 96 well microtitre plates at a density of about 10⁴ - 10⁵ cells/well in IMDM medium, plus 10% fetal calf serum, 30% conditioned medium, the latter derived from a

lymphoblastoid cell line, JW5. The medium also contained 5 x 10^-5 M 2- mercaptoethanol, 50 μg/ml gentamycin sulfate (Sigma), and 600 ng/ml cyclosporine A (Sandimmun, Sandoz, Basel, Switzerland).

After about 14 to 21 days of incubation, cell culture supematants were screened by ELISA for antibody binding activity against various strains of whole Pseudomonas aeruginosa or Klebsiella bacteria, purified LPS , or purified capsular polysaccharide. Cells which exhibited antibody were expanded, retested by ELISA, subcultured at low density, and grown up and fused to the cell line F3B6 using polyethylene glycol and the plate fusion technique known in the art and described by Larrick. Larrick, J.W., 1985, Human Hvbridomas and Monoclonal Antibodies. E.G. Engleman, S.K.H.

Foung, J.W., Larrick, and A.A. Raubitschek, Editors, Plentm Press, New York, page 446. F3B6 is a heteromyeloma cell line that is sensitive to growth in media containing 100 μM hypoxanthine, 5 μg/ml azaserine and 5 μM ouabain. Thus, hybrids were selected in drug supplemented culture media, screened by ELISA and cloned by limiting dilution.

ELTSAs were performed by methods generally known in the art, and more specifically as described by Cryz, S J., et al. 1987, J. of Clin. Investigation, 80:51 with the following minor modifications. Pseudomonas aeruginosa lipopolysaccharide stock solutions of Fisher IT1, IT2, IT4 and IT5 were made up at a concentration of 5 mg/ml in 36 mM triethylamine. Microtiter plates (Dynatech Lab., Alexander, Virginia) were coated at a concentration of 1 μg/ml in NaHCO₃-coating buffer, pH 9.6. Stock solutions of Fisher IT3, 6 and 7, as well as Habs 3 and 4 at 2 mg/ml were made in distilled water, and coated at a concentration of 10 μg/ml.

Klebsiella capsular polysaccharides were coated onto microtiter plates using standard procedures but with varying concentrations depending on the serotype. K10, K16, and K30 were coated at a concentration of 0.2 ug/ml; K61 at 0.5 ug/ml; K55 and K62 at 1.0 ug/ml; K3 at 2.0 ug/ml; K5, K9 and K64 at 5.0 ug/ml; and K17 at 10 ug/ml.

E. coli lipopolysaccharide stock solutions were made up at a concentration of 5 mg/ml in 36 mM triethylamine and microtiter plates were coated at a concentration of 10 ug/ml in PBS-NaN₃ buffer, pH 7.2.

ELISA assay on whole bacteria was also conducted as is generally known in the art and consisted of growing up the appropriate bacterial strains overnight in TSB- medium supplemented with 1% glucose, or in medium favorable for capsule induction. The cells were pelleted by centrifugation at 10,000g for 10 minutes at 4ºC, washed twice in phosphate buffered saline containing 0.02% NaN₃ and diluted to 1 x 10⁸ cells/ml in PBS-NaN₃. 0.2 ml of the cell suspension was added to microtitre wells (Immunolon, Dynatech) and allowed to incubate for 3 hours at 37°C. Next, the bacteria coated plates were washed four times with phosphate buffered saline and incubated with hybrid cell culture supernatant sought to be tested for the presence of antibody. The incubation period was for 90 minutes at 37ºC after which the supernatant was removed, and the bacteria washed three times with phosphate buffered saline containing 0.02% Tween 20. To reveal the presence of antibody in the culture supernatant, horseradish peroxidase conjugated goat anti-human IgG, IgA, IgM

(available from Kirkegaard & Perry Labs., Gaitagersburg, Maryland) were added in PBS-Tween. The plates were incubated for 2 hours at room temperature, washed three times with PBS-Tween, and the appropriate horseradish peroxidase substrate, ABTS, added and the absorbance read at 405 nm in a Titertk-ELIS A-reader (Flow Labs, Mclean, Virginia).

Using the above described procedures, several monoclonal antibodies were generated that recognize Pseudomonas aeruginosa serotypes, Klebsiella capsular polysaccharides, and E. coli 0-polysaccharides. Table 1 shows monoclonal antibodies and their serotype specificity to Pseudomonas aeruginosa. Table 2 shows monoclonal antibodies that bind to capsular polysaccharides of Klebsiella. In addition, Table 2 also shows the isotype of the monoclonal antibodies, and the results of immunofluorescent assays using intact bacteria. Table 3 shows the properties of two antibodies that bind to E. coli o-polysaccharides. Antibodies that recognize the various Pseudomonas aeruginosa serotypes were also characterized with respect to isotype, binding specificity, as determined by immunoblotting and immunofluorescence of intact bacteria, and agglutination activity. These results are presented in Table 4. Also shown in this table is the International Antigenic Typing System specificity of the monoclonal antibodies. Isotype determination of the monoclonal antibodies was conducted using standard techniques known in the art. Table 4 shows that all of the antibodies that recognize Pseudomonas aeruginosa are of the IgM class, and have the kappa light chain.

The antigenic specificity of the various monoclonal antibodies was characterized by immunoblot analysis using the procedures described by Towbin, et al. above, and Bumette, above. Briefly, 3 μg of lipopolysaccharide isolated from the various bacterial strains was electrophoresed using SDS-PAGE employing a 7.5 - 20% linear gradient of acrylamide. The SDS-PAGE procedures are described by Laemmli, U.K., 1979, Nature, 227:680, and the gels were silver stained as described by Tsai, 1982,

Analytical Biochemistry, 119:115. The electrophoretically separated

lipopolysaccharides were transferred onto nitrocellulose membranes for screening the various hybridoma culture supematants. The membranes were blocked, and then treated with culture supematants to test for antibody, followed by revealing antibody presence in the supematants with horseradish peroxidase conjugated goat anti-human IgG, IgA, and IgM. Immunoreactive bands were visualized with 4-chloro-1-naphtol and hydrogen peroxide.

The immunoblot results revealed that all the monoclonal antibodies that recognize Pseudomonas aeruginosa react with the latterlike O-side chain of US isolated from the various strains of Pseudomonas aeruginosa, and, more over, failed to react with the core-lipid A region of LPS.

More specifically, purified LPS from nine different Pseudomonas aeruginosa serotypes, IT1 through 7 and Habs 3 and 4, were subjected to SDS-PAGE gel electrophoresis which produces a slow migrating latterlike pattern of bands

corresponding to O-side chains, and faster migrating bands corresponding to the corelipid A complex. After electrophoresis, the electrophoretically separated material was transferred onto nitrocellulose membranes, and the membranes subjected to treatment with an appropriate monoclonal antibody. Reaction with a horseradish peroxidase conjugated goat anti-human second antibody demonstrated that all the monoclonal antibodies react with the O-side chain determinants of LPS, but do not react with the core-lipid A region. Excluding 2-8AH79, all the monoclonal antibodies reacted with a single isotype. 2-8AH79 reacted with the O-polysaccharides of Pseudomonas aeruginosa isotypes 1, 3, 4, and 6. It is further worth noting that the monolconal 4- 8KH15, CF6-0169 and A17H reacted with only the highest molecular weight fractions of the O-antigen, whereas 1-8KH53, M410 and 1RR2OH13 in addition to binding to the highest molecular weight fractions, also recognized faster migrating bands. The hybridoma M410 has previously been described by Larrick et al.., as LTR 228. Larrick et al., In Vitro Expansion of Human B Cells for Production of Human Monoclonal Antibodies. In E. Engleman, S. Foung, J. W. Larrick, and A. A.

Raubitschek (eds). Human Hybridomas and Monoclonal Antibodies. Plenum Press, New York (1985).

In addition to immunoblotting, the specificity of the various antibodies was assessed by indirect immunofluorescence on fixed bacteria. Briefly, this consisted of placing a drop of the appropriate bacterial suspension onto a cover slip and fixing the bacterial with methanol for 5 minutes at 4°C. The fixed bacteria were incubated with undiluted hybridoma supematants for 15 minutes after which the slides were washed and further incubated with a second antibody labelled with fluorescein isothiocyanate. The second antibody was goat anti-human IgG, IgA, or IgM. The slides were mounted with Mowiol (Hoechst A.G., Frankvert, FRG).

The immunofluorescent results were scored as to strong immunofluorescence (+++) and weak immunofluorescence (+).

The ability of anti-Pseudomonas aeruginosa monoclonal antibodies to agglutinate bacteria was tested. The techniques employed were standard, and it was observed that certain of the antibodies strongly agglutinated bacteria of homologous immunotypes. 2-8AH79, however, exhibited no agglutination.

Finally, the capacity of several anti-Klebsiella monoclonal antibodies to opsonize the appropriate bacteria strains was tested using standard techniques. These experiments showed that MT74H31 and MT71H105 have good opsonic activity.

Example VIII

In Vivo Protective Effects

The ability of various monoclonal antibodies to protect against bacterial challenge was tested using the murine bum sepsis model. Details regarding this model system are described by Cryz, S.J., 1983, Infectious Immun., 39:1072, and by Stieritz, D.D., and Holder, LA., 1975, J. of Infectious Disease, 131:688. Briefly, 18- 20 gram female mice were anesthetized in an atmosphere of 2-chloro-1, 1, 2- trifluoraethyl-difluormethylether (Ethran; Abbott Labs., North Chicago, Illinois). The animals were then burned over a 2 cm² area of the back, and immediately challenged with the appropriate strain of bacteria in 0.5 ml phosphate buffered saline. The bacteria were injected subcutaneously. Twenty hours prior to burning, the experimental mice received the appropriate antibody intravenously.

Antibody doses between 0.4 μg and 5 μg/mouse were highly protective as shown by a great increase in the LD50 of the bacterial strain used to challenge the animals. The results are shown in Table 5. It is apparent that the monoclonal antibodies directed against Pseudomonas aeruginosa serotypes exhibit protection ranging from 10 to 10,000 fold.

Example IX

Antibody Dependent In Vitro Killing

An experiment was conducted to determine the in vitro killing properties of two anti-Klebsiella monoclonal antibodies. The experiment was performed using standard microbiological techniques, and with MT74 H31 and MT71H105 which recognize Klebsiella serotypes K16 and K55, respectively. Antibody present in cell culture supernatant was tested, and cell culture media from the cell line F3B6 was used as a control. The results, presented below, clearly show that these two monoclonal antibodies are highly effective at killing bacteria that display the appropriate serotype. MT74H31 andMT71H105 exhibited 85% and 95% killing, respectively.

Antibodv NHS % Killing Supernatant Dilution

MT74H31 5 85 1:10

F3B6 5 0 1:10

MT71H105 5 95 1:80

F3B6 5 0 1:10

The following cell lines have been deposited with the American Type Culture Collection, and the deposit satisfies the requirements of the Budapest Treaty:

1-8KH53

4-8KH15

4-10KH139

MT74H31

MT71H105

Table 1

ELISA binding of HmAb to purified LPS and whole

bacteria from P. aeruginosa serotypes

The experiments were done with undiluted hybridoma supernatant (100 μl/well), the antibody concentration was in the range of 10-40 μg/ml. The microtiter plates were coated with 10 μg/ml of purified LPS or whole bacteria.

The binding activity of MAb is expressed as optical densities at 405 nm. The data shown are for binding with purified LPS which gave comparable results with whole bacteria.

1 IT1 to 7: Fisher immunotypes 1 to 7 which correspond to the International Antigenic Typing System (IATS) 6, 11, 2, 1, 10, 7 and 16,

respectively.

H3 and H4: Habs immunotypes 2 and 4 which correspond to IATS 3 and 4, respectively.

1) The serospecificity of the HmAb was determined by ELISA and immunofluorescence (IF) analysis. ELISA plates were coated with intact bacteria of 77 different aerotypes. The serespecificity of antibody binding in ELISA was confirmed by an immunofluorescence assay using intact bacteria. (+++), strong aurface staining of intact bacteria.

2) The binding activity of antibodies from undiluted hybridoma subernatant to purified CPS is expressed as optical densities at

405 nm. TABLE 3

Specificity of human monoclonal antibodies to E. coli o-polysaccharides.

1) (+++); strong surface staining of intact bacteria using immunofluorescence (IF) microscopy analysis.

2 ) The binding activity o f HmAb from undiluted hybridome supernatant to purified LPS of E. coli serotypes 04 and 06 is expressed as optical densities at 405 nm.

1Challenge was performed with a strain of P. aeruginosa expressing a homologous serotype.

2Control mice received 0.2 ml of F3B6 culture supernatant intravenously 20 hrs prior to challenge.

3Treated mice received approximately 1-4 ug of HmAb in 0.2 ml of hybridoma culture supernatant 20 hrs prior to challenge.

4Determined by dividing the LD₅₀ value of treated mice by that of control mice.

Claims

WE CLAIM:

1. A composition comprising human monoclonal antibodies that bind to Pseudomonas aeruginosa serotypes IT 1 through 7 and IATS 3 and 4.

2. Hybrid cell line A17H193.

3. Hybrid cell line 4-10KH139.

4. Hybrid cell line A306H.

5. Hybrid cell line 1RR20H13.

6. Hybrid cell line 1-8KH53.

7. Hybrid cell line 4-8KH15.

8. Human monoclonal antibody secreted by hybrid cell line A17H193.

9. Human monoclonal antibody secreted by hybrid cell line 4-10KH139.

10. Human monoclonal antibody secreted by hybrid cell line A306H.

11. Human monoclonal antibody secreted by hybrid cell line 1RR20H13.

12. Human monoclonal antibody secreted by hybrid cell line 1-8KH53.

13. Human monoclonal antibody secreted by hybrid cell line 4-8KH15.

14. A composition comprising monoclonal antibodies that bind to

Pseudomonas aeruginosa serotypes that are also bound by human monoclonal antibodies secreted by hybrid cell Unes A17H193, 4-10KH139, A306H, 1RR20H13, 1-8KH53 and 4-8KH15.

15. A composition comprising human monoclonal antibodies that bind to one or more Klebsiella capsular polysaccharide antigens.

16. Hybrid cell line MT74H31.

17. Hybrid cell line MT133H26.

18. Hybrid cell line MT71H105.

19. Hybrid cell line MT72H99.

20. Hybrid cell line MT168H11.

21. Hybrid cell line S2-AH92.

22. Hybrid cell line S 3-4H68.

23. Hybrid cell line KJ6- 1H74.

24. Human monoclonal antibodies secreted by hybrid cell line MT74H31.

25. Human monoclonal antibodies secreted by hybrid cell line MT133H26.

26. Human monoclonal antibodies secreted by hybrid cell line MT71H105.

27. Human monoclonal antibodies secreted by hybrid cell line MT72H99.

28. Human monoclonal antibodies secreted by hybrid cell line MT168H11.

29. Human monoclonal antibodies secreted by hybrid cell line S2-AH92.

30. Human monoclonal antibodies secreted by hybrid cell line S3-4H68.

31. Human monoclonal antibodies secreted by hybrid cell line KJ6-1H74.

32. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line MT74H31.

33. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line MT133H26.

34. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigen that are also bound by a human monoclonal antibody secreted by hybrid cell line MT71H105.

35. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line MT72H99.

36. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line MT168H11.

37. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line S2-AH92.

38. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line S3-4H68.

39. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by a human monoclonal antibody secreted by hybrid cell line KJ6-1H74.

40. A composition comprising human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by human monoclonal antibodies secreted by hybrid cell lines MT74H31, MT133H26, MT71H105,

MT72H99, MT168H11, S2-AH92, S3-4H68 and KJ6-1H74.

41. A composition comprising:

a) human monoclonal antibodies that bind to one or more

Pseudomonas aeruginosa serotypes, IT1 through 7 and IATS-3 and IATS-4; and

b) human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens K3, K5, K7, K9, K10, K16, K17, K20, K26, K30, K46, K49, K55, K61, K62, K64, K68, K69, K74, K79, K81 and K82.

42. A composition as described in claim 41 further comprising a human monoclonal antibody to E. coli serotype 04.

43. A composition as described in claim 42 further comprising a human monoclonal antibody to E. coli serotype 06.

44. A composition comprising:

a) human monoclonal antibodies that bind to Pseudomonas

aeruginosa serotypes that are alsobound by human monoclonal antibodies secreted by hybrid cell lines A17H193, 4-10KH139, A306H, 1RR20H13, 1-8KH53 and 4-8KH15; and

b) human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are alsobound by human

monoclonal antibodies secreted by hybrid cell lines MT74H31, MT133H26, MT71H105, MT72H99, MT168H11, S2-AH92, S3-4H68 and KJ6-1H74.

45. A composition as described in claim 44 further comprising a human monoclonal antibody to E. coli serotype 04.

46. A composition as described in claim 45 further comprising a human monoclonal antibody to E. coli serotype 06.

47. A method of treating or preventing bacteremia comprising administering to a patient a composition comprising human monoclonal antibodies that bind to

Pseudomonas aeruginosa serotypes that are also bound by human monoclonal antibodies secreted by hybrid cell lines A17H193, 4-10KH139, A306H, 1RR20H13, 108KH53 and 4-8KH15, and human monoclonal antibodies that bind to Klebsiella capsular polysaccharide antigens that are also bound by human monoclonal antibodies secreted by hybrid cell lines MT74H31, MT133H26, MT71H105, MT72H99, MT168H11, S2-AH92, S3-4H68 and KJ6-lH74.

48. A method as described in claim 47 further comprising human monoclonal antibodies that bind to E. coli serotype 04.

49. A method as described in claim 47 further comprising human monoclonal antibodies that bind to E. coli serotype 06.

50. A pharmaceutical composition for treating or preventing bacteremic infections comprising the composition of claim 44, and an effective amount of an antibiotic.

57. Human polyclonal antibodies that bind to Pseudomonas aeruginosa serotypes IT-1 through IT-7, and IATS-3 and IATS-4.

58. Human polyclonal antibodies that bind to Klebsiella capsular polysaccharide antigens K3, K5, K7, K9, K10, K16, K17, K20, K26, K30, K46, K49, K55, K61, K62, K64, K68, K69, K74, K79, K81 and K82.