DE3745019C2 - Cross-protective human monoclonal antibody compsns. - Google Patents

Abstract

A compsn. useful in the prophylactic and/or therapeutic treatment of bacterial infection comprises human monoclonal antibodies (MAbs) reactive with at least 2 bacterial species of at least 2 genera, at least 1 of the antibodies or binding fragment being capable of specifically reactinq with a non-core carbohydrate epitope shared by serotypes of 2 different species. An immortal cell line secretes a human MAb or binding fragment specifically cross-reactive with an epitope comprising an accessible non-core carbohydrate moiety present on at least 2 serotypes of 2 different species of different bacterial genera.

Description

The invention relates to monoclonal human antibodies, the protect against infections caused by various Bacterial species are caused. The invention According to use antibodies in particular in pharmaceuticals. These funds serve in particular for prophylactic and / or therapeutic treatment of bacterial infections.

Gram-positive and gram-negative bacteria may be present infected patients with life-threatening diseases call. These bacterial infections also lead to serious illness or even death. such Infections occur especially in premature babies Infants, in elderly patients and in patients, whose health is poor, for example reason of burns, surgical interventions, slow healing wounds or malignant tumors. Especially Patients who spend a long time in hospitals are affected by such infections. This includes for example, patients who have been treated surgically Patients with intravascular insult or patients who treated for a long time with immunosuppressants or antibiotics were. Also, newborns are not yet fully developed Immune system extremely susceptible to neonatal sepsis and Meningitis caused by certain gram-negative and gram-positive bacteria.  

Among the most common gram-negative and gram positive organisms include Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Serratia marcescens (S.Marcescens), Enterobacter aerogenes and cloacae (E. Aerogenes / Cloacae), Pseudomonas aeruginosa (P. aeruginosa), Neisseria meningitidis (N. meningitidis), group B Streptococcus and Staphylococcus aureus (S. aureus) (Sonnenwirth, A.C. "The Enteric Bacilli and Similar Gram Negative Bacteria ", pp. 753-790 in Microbiology, 2nd edition, Davis, B.D., Dulbecco R., Eisen H.N., Ginserberg, H.S. Wood, W.B. and McCarty, M., eds. Harper and Row, (1973); McCabe W.R., "Gram-Negative Bacteremia", Adv. Intern. Med., 19; 135-138 (1974); Kreger et al., Gram-Negative Bacteremla III. Reassessment of Etiology, Epidemiology and Ecology in 612 Patients, Am.J.Med.68, 332-343 (1980), Robbins J.B. et al. "Escherichia coli K1 Capsular Polysaccharide Associated With Neonatal Meningitis ", New Engl. J. Med. 290; 1216-1220 (1974); and Hughs, J.M. et al., "Nosocomial Infection Surveillance, 1980-1982) Morb. Mort. Weekly Report, 32; 1SS-16SS (1983). From these infections call at the adult population usually different, however not all, serotypes of certain Gram-negative bacteria, z. E. coli, K. pneumoniae, E. aerogenes / cloacae, P. Aeruginosa and S. Marcescens bacteremia. In the counter This is not fully developed immunologically Premature babies particularly prone to septicemia and Meningitis, which are caused by the encapsulated strains of E. coli, N. meningitidis group B, Hemophilus influenzae type B and the five types of strains the group B Streptococcus. Although other bacteria These infections can become predominant the above bacteria in such infections isolated from the blood.

To control and treat or eliminate such gram-positive and gram-negative infections are so far  mainly antibiotics were used. Because such Infections, however, again and again in severe form occur and there are more and more bacterial strains against anti biotics become resistant and due to some anti biotics inherent toxicity are one such therapy set with antibiotics limits. Therefore, age is native prophylactic and therapeutic treatment possibilities searched.

It is a widely held belief that antibodies, which can react with structures that live on Bacteria are accessible (externally exposed), the Facilitate destruction of the bacterium in various ways can. These include: (1) Direct lysis of the bacteria in Presence of serum complement; (2) bacteriostasis by Blockade of food intake receptors, (3) opsonizing and subsequent phagocytosis of the bacteria in the presence or absence of serum complement, or (4) Ver Preventing the attachment of bacteria to host tissue (Mims, C.A. "Recovery from Infection," in The Pathogenesis of Infections Disease, pp. 198-222, Mims, C.A. Ed., Academic Press (1982)]. For bacteria that are on the surface lying carbohydrate molecules, eg. B. Lipopolysaccharides (LPS) and / or capsules, antibodies appear to be the most effective when they are opsonized [Kaÿser, B., et al, "The Protective Effect Against E.coli of O and K Antibodies of Different Immunoglobulin Classes ", Scand. J. Immunol. 1. 276 (1972)]. Therefore, antibodies that are resistant to this accessible carbohydrate structures are directed, in used effectively to destroy the bacteria become.

Become mammals of disease-causing bacteria infect, they generally produce antibodies, which are specific for LPS or capsules. at these antigens are chemically different  Structures that are repetitive Oligosaccharidmolekülen are composed and their Presence defines the serotype of the bacterial strains. Because it These are often the immunodominant bacteria anti are serotype-specific antibodies (anti- IJPS or capsule body) those who are the most on their potential use as therapeutic antibodies were examined. However, since the cross-reactivity of this Antibody is limited and because the carbohydrate antigens of pathogenic gram-positive and gram-negative bacteria obviously of a very different nature, it would be extremely difficult and costly one to make therapeutic formulation that only sero contains type-specific antibodies (compare Kaÿser B. and Ahlstedt S., "Protective Capacity of Antibodies Against Escherichia coli O and K antigens ", Infect. Immun. 17, 286-292 (1977), and Morrison D.C. and Ryan J.L, "Bacterial Endotoxin and Host Immune Response" Adv. Immunol., 28, 293-450 (1979)]. Nonetheless various reports give rise to speculation according to which Immunotherapeutic ways to treat diseases which can be found by gram-negative bacteria be caused.

It has been found that fractionated human plasma, enriched with immunoglobulins, which are specific and protective against the infecting organisms contain body, has a certain effectiveness against infections, which were caused by P. aeruginosa [Collins, M.S. and Robey, R.E. "Protective Activity of Intravenous Immune Globulin (Human) Enriched in Antibody Against Lipopolysaccharide Antigens of Pseudomonas aeruginosa, "Amer. J. Med., 3, 168-174 (1984)]. However, commercial products are not yet available because these products are subject to certain restrictions,  which their widespread use for treatment life threatening bacterial disease previously prevented.

One reason for the limited accessibility of these immunoglobulin preparations is that it consists of a large pool of Plasma samples are obtained which are on the property a limited number of specific antibodies were preselected. This pool is usually from samples of 1000 donors, some of which pathogenic bacteria can have a low titer. Therefore, the obtained titer of desired antibodies slightly increased at best. Another such Limitation is that the pre-selection process as such a very expensive, continuous screening the donor is required to make a product "same Quality "despite considerable efforts can improve the quality of the product from batch to batch and vary from geographical region to region.

An inherent to the agents based on immunoglobulins Further limitation can be seen in the fact that their Ver use to co-administer large Amounts of foreign proteinaceous substances (eg viruses) leads, which may be contrary biological Can cause effects. Due to the fact that the titers of the desired antibodies are low and that the proportion of foreign substances is large, is thereby the amount administered to the patient specific and thus useful immunoglobulin (s) a suboptimal level limited.

Köhler and Milstein reported in 1975 that certain Cell lines of mice are fused with mouse spleen cells can, where hybridomas arise, the pure monoclonal  To secrete antibodies [Kohler G. and Milstein C., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity ", Nature 256, 495-497 (1975)] Using this technique it is possible to get murine anti body for each particular determinant (or determinants) to produce antigens.

Using this technique, monoclonal Obtaining antibodies from mice derived from mice which with polysaccharide of Neisseria meningitidis group B had been immunized. It was found that these murine monoclonal IgM antibodies to various Bind and anneal K1-positive E. coli strains regardless of their LPS serotypes [Cross, top, Soder Stream, Top and Cross A.S. et al., "The Importance of the K1 Capsule in Invasive Infections Cause by Escherichia coli, J. Inf. Dis., 149, 184-193 (1984)] found that the monoclonal antibodies against a lethal Challenge with E. coli K1 and meningococcal organisms of the Protect group B (Cross, top and Sunderstrom, above). In a further investigation it was found that murine monoclonal antibodies (specific towards Streptococcus type III group B) experimentally generated infections in mice protective effect (Egan M. L. et al, "Protection of Mice from Experimental Infection with Type III Group B Streptococcus Using Monoclonal Antibodies ", J. Exp. Med., 1; 1006-1011 (1983).

Although a mouse derived monoclonal anti body is useful in the treatment of mice, he shows great disadvantages in the treatment of humans. The Immune system of the human being is capable of each one of one Mouse derived monoclonal antibody as foreign to recognize protein. This can be accelerated  Elimination of the antibody lead, so that the pharma ecological effect of this antibody is abolished (Levy, R. and Miller R.A., "Tumor Therapy with Monoclonal Antibodies, Fed. Proc. 42, 2650-2656 (1983).

More serious is that this is due to allergic Reactions, similar to the "blood disease", to one Shock and even death. In clinical Experiment has been found that due to such a reaction (Anti-mouse immunoglobulin) these antibodies only be used in about half of the patients which monoclonal antibodies from mice to Be treatment of various tumors (Sears H.F. et al. "Phase I Clinical Trial of Monoclonal Antibody in Treatment of Gastrointestinal Tumor ", Lancet! 762-764 (1982), and Miller R.A. et al, "Monoclonal Antibody Therapeutic Trials in Seven Patients with T-Cell Lymphoma, Blood 62; 988-995 (1983).

There is therefore a need for monoclonal human antibodies that protect against diseases caused by caused gram-negative and gram-positive bacteria become. Due to the different antigenicity of the Disease-causing gram-positive and gram However, it will hardly be possible for negative bacteria to for each of the many important bacterial pathogens, monoclonal human antibodies of a specific serotype manufacture.

The different antigenicity of gram-negative Bacteria becomes the different regions of the lipo attributed to polysaccharides (LPS). These are around a molecule that is connected to the outer membrane of the gram-negative Organisms is associated. In general, it is assumed that the LPS molecule is composed of three structural regions is set.  

The outer membrane adjacent region is the so-called Lipid A part of LPS. This structurally conserved Region has the endotoxic activity associated with the Gram-negative bacteria-induced disease accompanied. The second structural region, called the core, is often associated with lipid A via a 2-keto-3-deoxy- D-mannooctonate (KDO) and is similar to the lipid A region, usually not accessible to the antibody if the third, the outermost region of the LPS. Although this region is in some gram-negative Bacterial species is partially preserved, many could Deviations regarding the whole core of members of the Enterobacteriaceae family. The outermost region of an LPS molecule is out of itself again composed of oligosaccharide units and is known as the O-specific side chain. The sugar in these oligosaccharide units have molecular units, the one serotype-specific structural antigen-mannig own wrinkles. Thus, the sugars as such their sequence and their bonds with the help of their tertiary Structure the O-side chain antigenicity. It has exposed the antibody for these O groups in the general serotype-specific. Serotypes are usually by their reactivity towards mono specific antisera defined which only one can bind certain antigen determinant (general Explanations can be found in Mayer et al., Meths. Micro biology, 18, 157-201 (1985)).

For the core and lipid A regions of LPS, antisera to protect their protective effect against gram- to show negative infections (Sakulramrung and Domingue, J. Inf. Dis., 151, 995-1104 (1985); McCabe et al., J. Infect. Dis., 1365, 516 (1977), and Mullan et  al. Infect. Immune. 10, 1195-1201 (1974)). In the last Time were monoclonal antibodies of mouse and human which react with the conserved regions can. Although it could be shown that this anti bodies in model systems tailored to them, showed some in vivo efficacy (Teng et al., Proc. Natl. Acad. Sci. USA 82, 1790 (1985), and Bogard and Kung, Patentanm. No. WO 85/01659), were others Laboratories are unable to apply similar effects find (compare Elkins and Metacalf, Infect. Immune. 48, 597 (1985) and Gigliotti and Shenap, J. Inf. Dis. 151, 1005-1011 (1985)). In WO 85/01659 are Antibodies to the lipid A region of LPS and antibodies against the determinants, the nuclear and lipid A region overlap, provided. These antibodies react (usually) do not bind with intact, living, gram-negative bacteria or purified LPS molecules. Because of these results, it is doubtful whether the Core or the lipid A parts of LPS to bacteria in their natural or infectious condition for antibodies too are accessible. It is also generally recognized that anti-nuclear or anti-lipid A antibodies not with gram-positive Bacteria react, since the latter have no LPS. on Because of these results, it is unlikely that monoclonal antibodies against the conserved nucleus or Lipid A regions of LPS in the treatment of Er Illnesses in people can be effective through caused gram-negative or gram-positive bacteria become.

There is thus still a significant need for human monoclonal antibodies in the broad range (Intergenus) are "cross-protective" against negative and gram-positive bacteria Diseases. There is also a need for Ver drive to produce such antibodies.  

According to the invention, new cell lines are provided, which monoclonal antibodies produce in specifically with a variety of bacterial species They can react cross-reacting to an accessible one at least two different bacterial species Handenes, a non-nuclear carbohydrate unit having Bind epitope. Subject of the Invention is also a pharmaceutical agent that several of the monoclonal human antibodies described above contains. This agent preferably has a physiological compatible carrier and can also be one or more of the following ingredients: Further monoclonal Human antibodies that react with other types of bacteria can; a γ-globulin fraction from human blood plasma; a γ-globulin fraction from human blood plasma, at which the plasma has been obtained from a human by whom the mirrors are increased in immunoglobulins, with or without can react to several types of bacteria; and one or several antismicrobial agents.

According to the invention, new cells, which are monoclonal Can produce human antibodies and such anti body-containing means provided. These funds are able to be selective with a variety of bacteria respond to hospital-acquired, neonatal or other infections are responsible. The individual antibodies react in a typical way Non-nuclear carbohydrate epitopes attached to bacteria several genera are present. The cells in question be  sit identifiable chromosomes in which are made from them or a precursor cell-derived germline DNA recombined is so that it encodes an antibody or a binding fragment thereof having a binding site for an antigenic determinant (epitope), which is carbohydrate Molecules common to at least some seros types of 2 or more bacterial species occur. These Monoclonal human antibodies can be used for a variety of Purposes, including diagnosis, Prophylaxis and therapy of bacterial infections.

The cells of the invention are capable of being in one Culture to produce a human antibody on an ongoing basis. These are in particular immortalized Human lymphocytes, the protective monoclonal human anti body for non-nuclear carbohydrate determinants transient molecules containing at least two bacterial species are together, produce. With the Expression "accessible" is expressed that the non-nuclear carbohydrate determinants in the environment for a direct interaction with the monoclonal anti bodies are physically available. The so provided Monoclonal antibodies are for the treatment or the Prophylaxis of severe diseases usable by caused a variety of bacterial infections become. In addition, these non-nuclear coals interact hydrate molecules that are drained into the adjacent environment not directly with the antibody molecules and can be eliminated by the reticuloendothelial system become.

The monoclonal antibodies according to the invention ent Keeping remedies can be used for therapeutic and Prophylactic treatment of nosocomial, neonatal and other infections. nosocomial  Infections are usually caused by the following Bacteria caused: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes / cloacae, Serratia marcescens and Streptococcus agalactiae Group B. According to the invention, such antibodies ent holding means preferred over two, three, four or more of such bacteria. Therefore are Antibodies used to treat neonatal infections, neonatal sepsis and meningitis, for example preferably specific to two or more of following bacterial organisms: Escherichia coli K1, Neisseria meningitidis group B, Streptococcus agalactiae Group B and Hemophilus influenzae type B. Other common infectious bacteria are the following: Staphylococcus aureus, Staphylococcus epidermidis, Streptococ cus peumoniae, Proteus mirabilis, Proteus vulgaris, Bacteroides fragilis, Pseudomonas cepacia, Mycobacterium tuberculosis, Providencia morganii, Salmonella typhi, Pneumocystis carinii, Acinetobacter herellea, pasturella multocida and Klebsiella oxytoca. Regarding further relevant pathogenic bacteria, be the expert are referred to: Hughs J.M. et al, "Noso Comial Infection Surveillance, 1980-1982, "Morb. Mort. Weekly Report 32, 1SS-16SS (1983) and in general Microbiology 3rd Edition, Davis, W.B. and McCarty M., Eds. Harper and Row (1980). On both references is hereby incorporated by reference. The monoclonal antibodies respond with individual members or all members a certain bacterial species in which the mit limbs on the basis of their surface epitopes, in particular LPS or capsule sites, e.g. Serotypes, distinguished can be. It has now surprisingly mono provided with different antibodies Bacterial species can cross-react. To this Bacteria include the clinically important ones listed above Species. This allows a new therapeutic and  Prophylactic treatment. By preselected cross reactive antibodies in combination, one can use a Mixture of less antibodies that produce Be treatment of various species of infectious bacteria can be used.

This will be explained with an example. A mix of two monoclonal antibodies, one of which has at least Two clinically relevant bacteria can cross-react and of which the second with at least two or three can react to different species is for the Be offense against four, five, six or more difference useful species. If you add a third or fourth monoclonal antibody, each of which with at least two clinically important species cross-react can, even one or more of these species the same as the one from the first and / or or second antibody is detected, then you can even five to ten or more species are fought. It can Of course, it will also be required one or more to add monoclonal antibodies, each of which only for a single preselected bacterial species is specific, for example, if monoclonal antibodies that cross with these species can not be available.

There are thus new methods for the treatment of bacterial Allows infections. This is an example he purifies. So you can have a patient from whom you accept or knows he's under a bacterial infection suffers from a particular bacterial species treated by administering an agent, which contains a monoclonal antibody compatible with the Can respond to bacterial species that is believed to that it causes the infection, the monoclonal  Antibody is originally characterized by the fact that he can react with another bacterial species.

You can also treat bacterial infections by that administering agents containing several monoclonal anti contain body, which with a considerable part (i.e., greater than 50%, preferably 60 to 80% or more, most preferably about 90%) of the preselected clinically important bacterial species can react, wherein the number of antibodies is at least two lower as the number of bacterial species. Means, for example If the number "n" is the number of bacterial species, then the agent has about n-2 antibodies, conveniently about n-4 to n-6 or less antibodies for treatment against about 15 to 20 bacterial species. Is it him necessary, a wide range (eg 25 to 50 or more) of bacterial species, then that contains Means typically n-10 to n-20 antibodies or less.

The monoclonal antibodies can be made by immortalizing the expression of nucleic acid sequences encoding antibodies or binding fragments thereof which are specific for a non-nuclear carbohydrate epitope present on multiple bacterial species. The monoclonal antibodies are typically produced by cell-directed Epstein-Barr virus (EBV) transformation of lymphocytes obtained from human donors exposed or exposed to the corresponding Gram-negative bacterium. The antibody-secreting cell lines thus prepared are characterized as continuously growing lymphoblastoid cells which possess a diploid karyotype, are Epstein-Barr nuclear antigen (EBNA) positive and secrete a monoclonal antibody of IgG, IgM, IgA or IgD isotype. As such, the cell-driven transformation process is an invention of Genetic Systems Corporation and is described in detail in U.S. Patent 4,464,465, which is hereby incorporated by reference. The monoclonal antibodies can be used in intact form or as fragments, for example as F _v , Fab, F (ab ') ₂. Normally, however, they are used intact.

However, the cell lines that produce antibodies can also by cell fusion of in ge suitably drug labeled human myeloma, mice myeloma or lymphoblastoid human cells with human B Lymphocytes are produced using hybrid cell lines arise.

The cell lines according to the invention can also be used for others Purpose than for the immediate production of the monoclonal Human antibody find application. The cell lines can with other cells (for example, suitably Drug labeled human myeloma, mouse myeloma, or lympho blastoid human cells) to hybridoma cells become and thus accomplish the transfer of the genes responsible for encode the monoclonal human antibodies. The cell But lines can also serve as a source for the DNA coding for the immunoglobulins, which isolated and after other techniques than the Fusion technology can be transferred into cells. except The genes responsible for the monoclonal antibodies encode, isolate and combine with techniques to used in recombinant DNA is used to ver the specific immunoglobulin at ver to produce different hosts. In particular, one can, by making cDNA libraries from messenger RNA, a single cDNA clone coding for the immunoglobulin is coded and free of introns, isolate and in ge suitable prokaryotic or eukaryotic expressions place vectors and then into a host transform, so the eventuality To enable "mass" production.  

The lymphoblastoid cell lines or the hybrid cell lines can be cloned or screened by conventional techniques be with antibodies that are different in epitopes, in the cell supernatants detected bacterial genera can bind.

The monoclonal antibodies according to the invention can be found in FIG special application as ingredients of pharmaceutical Means which are a therapeutic or prophylactic effective amount of at least one of the invention monoclonal antibody in conjunction with a pharma ceutically acceptable carrier. As a pharma Zeutischer carrier can be any compatible, non-toxic Substance used for the administration of the substance monoclonal antibody to the patient is suitable. Sterile water, alcohols, fats, waxes and inert Solids can be incorporated into the carrier. Pharma ceutically tolerated adjuvants (buffers, dispersants) may also be incorporated into the pharmaceutical agent be. These agents can be a single monoclonal Contain antibodies that are associated with non-nuclear carbohydrate Epitopes can respond which two or more Bacterial species are common, for example nosocomial and neonatal (eg sepsis or meningitis) Cause infections. Alternatively, a Pharmaceutical agent two or more monoclonal anti contain body and thus constitute a "cocktail". So For example, a cocktail that would be monoclonal human contains antibodies, which each before two or more gram-negative, causing infections in humans Bacteria genera protect against the vast majority of common "clinical isolates". desired if one or more monoclonal antibodies which are also used with gram-positive Bacteria can cross-react, causing the pharma  a wider scope of application holds.

Of particular interest are prophylactic and / or therapeutic monoclonal antibody-containing agents, which react with non-nuclear carbohydrate determinants can, which three or more, usually at least five, usually at least ten and up to fifteen or more bacterial serotypes are common, including at least two, usually at least three, usually at least five and suitably less than ten Bacteria genera belong.

Of particular interest are also monoclonal anti body-containing agents containing at least about three preferably at least about five and up to ten and including all the following usual ones nosocomial, infection-causing bacteria react: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes / cloacae, Serratia marcescens and Streptococcus agalactiae group B. It is desirable to treat neonatal infections worth that means with at least two, usually at least three and usually at least four and with up to ten and including all of the following Responding to bacteria causing bacteria: Escherichia coli K1, Neisseria meningitidis group B, Streptococcus agalactiae group B, Hemophilus influenzae Type B, Staphylococcus aureus and Staphylococcus epidermidis.

Each of these agents contains at least two, usually at least three to five and suitably six to ten monoclonal human antibodies, wherein at least one Antibodies with non-nuclear carbohydrate epitopes (eg from the LPS molecules), which are two or more  Bacteria genera are common. This will protect you this mediates. The antibody will usually not bind to all serotypes of any bacteria, but instead can bind to two, three or more serotypes. It is desirable if at least one monoclonal Antibody attached to an accessible non-nuclear carbohydrate Unit of at least two genera of Gram-negative bacteria binds, and at least one monoclonal antibody present is that gram to an accessible carbohydrate unit negative bacteria and a gram-positive bacteria binds.

The molar ratio of the different monoclonal anti Body components usually differ from each other not more than a factor of 10 and expediently around not more than a factor of 5. The molar ratio is conveniently about 1: 1-2 for each of the other anti body components.

The monoclonal human antibodies can also be "single" be applied, especially if the disease Exciter has been identified or a close Range of pathogens within the binding spectrum belongs to the special antibody.

The monoclonal human antibodies according to the invention can also in combination with other monoclonal antibodies, compare z. For example, the application entitled "Mono clonal Antibodies Cross-Reactive and Protective Against P. aeruginosa serotype "with USSN 807 394, incorporated is 10.12.1985, which is hereby incorporated by reference) as well as with existing blood plasma products, as in Commercially available γ-globulin and immunoglobulin products, the application find in the prophylactic or therapeutic treatment of bacterial diseases, be used in humans. For immunoglobulins is  the plasma preferably obtained from human donors, the have elevated levels of immunoglobulins, which with different infectious bacteria genera can react.

Reference is made to the compendium "Intravenous Immune Globulin and the Compromised Host", Amer. J. Med., 76 (3a), 30.03.1984, p. 1-21. Thereon is hereby incorporated by reference.

The monoclonal antibodies according to the invention can be used in Form of separately administered agents together with antibiotics or antimicrobial agents. To The antimicrobial agents include pencicillins and Cephalosporins (for example, carbenicillin, penicillin G and the like) in conjunction with an aminoglycoside (for example, gentamicin, tobramycin, etc.). It can also various other active ingredients (for example Cephalosporins, sulfa preparations, etc.), which are those skilled in the art are well known to be used.

The monoclonal human antibodies of the invention and the these pharmaceutical compositions containing this invention In particular, agents may be administered orally or parenterally be enough. The pharmaceutical agents are preferred wise parenteral, i.e. subcutaneously, intramuscularly or administered intravenously. Thus, according to the invention Prepared for parenteral administration, the a solution of the monoclonal human antibody or a Cocktail thereof, dissolved in a compatible carrier, before preferably an aqueous carrier. A variety aqueous carrier may find application, for example Water, buffered water, 0.4% saline, 0.3% glycine solution and the like. These solutions are sterile and generally free of particles. These funds can be done by standard, well-known sterilization techniques be sterilized. Thus the "conditions" of these means  the physiological conditions as far as possible ent If necessary, they can pharmaceutically speak Contain compatible excipients. These include means for Adjusting the pH, buffer, means for adjusting the Toxicity and the like. As examples you can call: Sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The concentration of the anti Body in these formulations can be over a wide Range varies, i.e. less than about 0.5%, more commonly 1% or at least about 1% up to 15 or 20% by weight. When choosing the concentration, consider before Weighing the volume of liquid, the viscosity, etc. in Agreement with the chosen mode of administration.

So can a typical pharmaceutical agent for a intramuscular injection 1 ml of sterile, buffered water and 50 mg monoclonal antibodies. A typical one Means for intravenous infusion may be 250 ml sterile Ringer's solution and 150 mg of monoclonal antibody ent hold. The usual method of preparation parenteral administered agents are well known in the art and are still detailed, for example, described in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980). To this Publication is hereby incorporated by reference.

To store the monoclonal antibodies of the invention, they can be lyophilized. You can then contact closing shortly before use in a suitable Carriers are reconstituted. It is known that these Operation with conventional immunoglobulins successfully can be set. According to the invention, known Lyophilization and Reconstitution Techniques Application Find. The skilled person knows, however, that lyophilization and  Reconstitution to a differently strong anti loss of body activity leads (as example example in conventional immunoglobulins the IgM antibodies a greater loss of activity than the IgG antibodies). Care must therefore be taken to compensate for this wear.

The monoclonal human antibodies or a cocktail thereof containing agents can be used for prophylactic and / or therapeutic treatment of bacterial infections be used. In the therapeutic application the remedy to an already infected patient in administered in such an amount that the patient healed is, or in such an amount, that the infection and whose symptoms are at least partially eliminated.

Such a dose is considered to be a therapeutically effective Dose. How big the effective amount has to be depends on the severity of the infection and the general Condition of the patient's immune system. In general however, will be an amount of from 1 to about 200 mg of antibody per kg / body weight in doses of 5 to 25 mg per kg administered. It must not be forgotten that the In general, according to the invention provided substances extremely severe disease states are administered, for example, in life-threatening or, where appropriate life threatening situations, especially at Bacteremia and endotoxemia. As a result of the invention according to the monoclonal human antibodies in these cases none foreign substances are present and no rejection reaction of "foreign substances" takes place, it is possible and may be desirable by the attending physician see, significantly larger amounts of these antibodies to process.  

The antibody of the invention or a cocktail thereof containing agent is administered for prophylactic Applications to a patient who has not been through the corresponding bacterium has been infected to the Resistance of the patient against such a possible Strengthen infection. Such a quantity is called prophylactically effective dose. At this The exact quantities depend on the health condition of the patient and the general immunity level off. In general, however, an amount of 0.1 to 25 mg per kg, in particular 0.5 to 2.5 mg per kg, administered.

The remedies may take the form of a gift or in the form of several Doses administered with the dose and the is selected by the attending physician. On In any case, the pharmaceutical formulations should have a Provide amount of antibody (s) according to the invention, sufficient for effective treatment of the patient is.

The monoclonal antibodies according to the invention can various ways in vitro are used. The monoclonal antibodies can be used, for example, for Bacterial typing, for isolation of specific Bacterial strains or fragments thereof, for vaccine production etc. are used.

For diagnostic purposes, the monoclonal anti be marked body or not. At diagnosis assays is generally the formation of a complex by the binding of the monoclonal antibody to the LPS of the organism detected. Are the antibodies without Label, then found in agglutination assays Application. In addition, unlabeled antibodies  together with other labeled (second antibodies) be set, the latter with the monoclonal anti Body can react, being for human immuno globulin-specific antibodies. In alternative The monoclonal antibodies can be labeled directly be. You can ver a great variety of markers apply, including radionuclides, fluorescence causing sub punching, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. count. There are several types of immunoassays for Ver addition. For example, the following may be mentioned, the are 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, incorporated herein by reference.

The monoclonal antibodies of the invention may also be used in enzyme immunoassays, wherein the in question antibodies or second antibodies of of a different species are conjugated to an enzyme. If a sample, such as human blood or lysate one or more bacteria of a certain kind or contains a serotype, it will be with the in question Antibodies combined. The binding takes place between the Antibodies and such molecules as those selected Have epitopes. These cells then can not bound reagents are separated. Subsequently, will a second antibody labeled with an enzyme together. After that, the amount of in a specific way to the cells bound antibody-enzyme conjugate be Right. There may also be others well known to those skilled in the art Working methods are applied.

Kits may also be provided in which the in question antibodies for the detection of bacterial infection or to detect a  certain antigen are used. The invention Monoclonal antibody-containing agents are becoming more common wise in lyophilized form in a container bereitge represents and is either alone or in connection with other specific for other gram-negative bacteria Antibodies. The antibodies with a marker may be equipped or no such mark are in the kits together with buffers, for example tris, phosphate, carbonate, etc .; Stabilizers, biocides; inert proteins, e.g. Bovine serum albumin; or like that. In general, these are Materials in an amount of less than about 5% by weight present, based on the amount of the effective antibody. They are generally present in an amount that ge is about 0.001 wt .-%, based again on the Antibody concentration.

It is also often desirable to reduce the effective loading An inert diluent or a Incorporate inert excipients. The excipient can be about 1 to 99 wt .-% of the total make up. Becomes a second antibody used at the monoclonal It can usually bind to antibodies, then it usually lies in one separate ampoule in front. The second antibody is on conventional manner conjugated to a label and formulated in the same way as the above written antibody formulations.

The following examples serve to illustrate the invention.

Example I

In this example, procedures for producing the described monoclonal antibody, the intergenus Cross-reactivity to members of the following species  shows: Escherichia coli (E. coli), Serratia marcescens (S. marcescens), Klebsiella pneumoniae (K.pneumoniae), and Enterobacter aerogenes (E. aerogenes). In this Example is also the in vivo protective activity of Antibody to a lethal challenge homologous (cross-reactive) E. coli and E. aerogenes serotypes described.

A. Obtaining suitable human cells

Suitable human B cells (lymphocytes) were removed from the peripheral blood of an individual obtained from a cystic fibrosis disease. mononuclear Cells became normal from the peripheral blood Centrifugation techniques to Ficoll-Paque (Boyum A., Isolation of Mononuclear Cells and Granulocytes From Human Blood. "Scand. J. Clin. Lab. Invest., 21, Suppl. 97, 77-89 (1968) and twice in calcium-magnesium free, Washed phosphate-buffered saline (PBS) and then in 1 ml of 90% crude fetal bovine serum (FBS) and 10% dimethylsulfoxide and at -196 ° C frozen in liquid nitrogen.

To transform the mononuclear cells, an ampoule containing 5 × 10⁷ cells was rapidly thawed to 37 ° C. The cell suspension was added to 10 ml of Iscove's medium containing 15% FBS and centrifuged for 10 min at room temperature and at 250 g. The mononuclear cells were freed from the T cells (lymphocytes) by means of a modified E-rosetting procedure. The cells were resuspended to a concentration of 1 x 10⁷ cells / ml in PBS with 20% FBS at 4 ° C. One ml of this suspension was placed in a 17 × 100 mm round bottom polystyrene tube containing 1 × 10⁹ red sheep blood cells treated with 2-aminoethylisothiouronium bromide (AET) from a 10% (v / v) solution in Iscove's. Medium (Madsen M. and Johnson HE, "A Methodological Study of E-rosette Formation Using AET Treated Sheep Red Blood Cells", J. Immun. Methods, 27, 61-74 (1979)). The suspension was mixed vigorously for 5 to 10 min at 4 ° C. The cells bound by the E-rosette method were removed by centrifugation through Ficoll-Paque for 8 min at 2500 g and at 4 ° C. The E-rosette test-negative peripheral blood-derived mononuclear cells (E⁻PMBC), which bind to the interface, were washed once in and in Iscove's medium (containing 15% w / v FBS (g / ml ), L-glutamine (2 mM / L), sodium pyruvate (1 mM / L), penicillin (100 IU / ml), streptomycin (100 μg / ml), hypoxanthine (1 × 10 ^-4 M) and thymidine (1, 6 x 10 ^-S M)). This medium is hereinafter referred to as HAT medium.

B. Cell-guided transformation of peripheral blood derived mononuclear cells

He became the cell-directed transformation of E⁻PBMC ranges by transforming the E⁻PBMC Cell line were cultured. The transforming Cell line was an EBNA-positive, lymphoblastoid human Cell line derived from ethylmethanesulfonate mutagenesis GM 1500 lymphoblastoid cell line. The Selection in the presence of 30 μg / ml 6-thioguanine changed the cells to give them hypoxanthine-guanine Phosphoribosyltransferase was missing and therefore HAT were sensitive.  

The cell line was designated as 1A2 cell line and in the American Type Culture Collection (A.T.C.C.) on March 29, 1982 under the A.T.C.C. Number CRL 8119 behind sets. 1A2 cells in the logarithmic growth phase were suspended in HAT medium and incubated with E⁻PBMC in a ratio of 30 1A2 cells per E⁻PBMC combined. The cell mixture was in 10 with 96 wells with Round bottom equipped microtiter plates in a concentration of 62,000 cells / well in a volume of 200 μl / Ver given deepening. The culture was incubated at 37 ° C in a be Moistened atmosphere with a content of 6% CO₂ in cubed. 5 days after the transformation, the Cultures "fed" in which half of the supernatant replaced with fresh HAT medium. The wells were on an inverted microscope every day Signs of cell proliferation studied. 10 days after "Plating" the cell mixture and after the 1A2 cells died due to the HAT selection became one new media formulation given in the wells, the identical to the HAT medium but no aminopterin component contained. 15 days after the "plating" was observed that 100% of the wells proliferate Cells and that in most of the cells Wells of sufficient "density" were present to to remove them and the supernatants on anti-E. coli or anti-S. to test marcenscens antibodies.

C. Detection of specific antibody secreting cells

The supernatants were assayed for the presence of anti-E. coli or anti-S. marcenscens antibodies with the help of a Enzyme-labeled immunoadsorbing assays (ELISA) (Engvall E., "Quantitative Enzyme Immunoassay (ELISA) in Microbiology ", Med. Biol. 55, 193-200 (1977)). The antigen plates consisted of a series of flat  bottomed, 96-well Immunlon 2 micro titer plates. The wells contained a mixture from either live E. coli or S. marcescens sero types attached to the surfaces of the poly-L-well depressions. lysine (PLL) were fixed. 50 μl PLL (1 μg / ml) in PBS were added to each well and added there for 30 min Leave room temperature (RT). The plates were washed 3 times PBS washed. In each well was either PBS or 50 μl of a mixed bacterial suspension with O.D.₆₆₀ = 0.2 given. The plates were incubated at 37 ° C for 60 min. And 3x with saline / 0.02% Tween 20 (common salt / T) wash to remove unbound bacteria. It The following different antigen plates were screened used: 1) A mixture of E. coli serotypes 01 (A.T.C.C. No. 23499) and 04 (A.T.C.C. No. 12791); 2) one Mixture of S. Marcescens serotypes 07, 015, 016 and 018 (All of the typifying strains mentioned were obtained from Communicable Disease Center (CDC) Atlanta, GA); and 3) a microtiter plate without bacteria.

For the ELISA assay, the wells were first washed with Blocks 200 μl of a mixture containing 5% w / v dry lean milk, 0.0001% foam A (Foam A) and 0.01% w / v thimerosal in 500 ml of PBS contained a non-specific protein prevent binding. After 1 hour incubation at RT plates were washed 3x (200 μl / well / wash medium from common salt / T). In each well was 50 μl a mixture containing 0.1% Tween 20 and 0.2% Bovine serum albumin in PBS (PTB). The supernatants from the wells of the culture plate were replica plated (replica plated) into corresponding wells the antigen and control plates (50 μl / well). The Plates were incubated at RT for 30 min. The supernatants were then removed and the plates were 5x with cook salt / T washed. 50 μl biotinylated anti-human immuno globulin from goat (Ig) (TAGO # 9303040) diluted to  1: 250 in PTB) were added to each well. To Incubation at RT for 30 min., Which became biotinylated Reagent removed. The wells were 5x with cook salt solution / T washed. Then 50 μl of one were prepared before provided avidin: biotinylated horseradish peroxidase Complex (Vectastain ABC Kit, Vector Laboratories) in given every well. After 30 min. At RT was the Vectastain ABC reagent removed. The wells were Washed 5x with brine / T and 100 μl of the substrate (0.8 mg / ml ortho-phenylenediamine dihydrochloride in 100 mM Citrate buffer, pH 5.0 plus 0.03% H₂O in desalted H₂O mixed in equal volume just before plating) (Plating = filling in the wells of the plate) were added to each well. After 30 minutes Incubation in the dark, 50 ul of 3N H₂SO₄ in each Ver given depth to terminate the reaction. The Culture supernatants containing antibodies compatible with the Bacteria can react with which the plates are coated were detected by measuring absorbance 490 nm using a Dynatech MR 580 MicroELISA reader.

The culture supernatants from six transformations were on analyzed the manner described above. It was a Well (7D7) identifies which activity on the E. Coli and S. Marcescens serotype plates, but not on the control plates. In subsequent ELISA's with individual E. coli serotypes it was determined that this well contained an antibody that was at least with E. coli serotypes: 08, (A.T.C.C. # 23504), and 075 (A.T.C.C. No. 12798), but not 04, 06: K2, 08: K8, 09: K9 or 022: K13 (A.T.C.C. Nos. 12791, 19138, 23501, 23505 and 23517) could react. In addition ent This depression held one with S.Marcescens serotypes 012, 013 and 015, but with no other of the 20 be S. Marcescens LPS serotypes knew reactive anti body.  

D. Cloning of Specific Antibody-Producing cell

The cells in well 7D7 became different Male (4) clones until all clonal supernatants, the with the aid of the ELISA assay described above were seeking a positive response to E. coli sero Types 08 and 075 and S. Marcescens serotypes 012, 013 and 015 showed. None of the clonal supernatants showed an unusual reactivity template. This suggests that the cultural over 7D7 was a real intergenerational Cross-reactivity for the E. coli described above. and S. Marcescens serotypes and only one Cell line (each showed individual serotype reactivity) contained. The cells were cloned by limiting the Dilution in 96-well plates with round Soil in the absence of feeder cells. The media consisted of Isocove's medium with one Content of 15% v / v FBS, L-glutamine (2 mM / L), sodium pyruvate (1 mM / L), penicillin (100 IU / ml) and Streptomycin (100 μg / ml). The cultures were all 3 days "fed" by half the supernatant replaced by fresh medium. The cells pointed generally between the second and the third Weeks after plating a sufficient lympo blastoid cell density for analysis on anti-E. coli and S. Marcescens serotype specificity.

Thus, in this experiment, a cloned obtained transformed human cell line, the continuous (immortal) and a monoclonal human anti body for a determinant on the surface of the E. coli and S. marcescens serotypes described above secreted.  

The continuous transformed identified as 7D7 Humanzellinie was before the priority date in the American Type Culture Collection, Rockville, MD A.T.C.C. No. CRL 9009.

E. Further characterization of the intergenus cross Reactivity

The antibody from the cloned 7D7 cell line was examined for further intergenus cross reactivity by a modification of the usual immunoblot technique. In particular, the cross-reactivity to the bacteria K. pneumonia E. aerogenes and E. cloacae was examined by placing "spots" of bacteria on a rastered nitrocellulose paper disc which reacted the disc containing the bacteria with the antibody and the antibody reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system. For this purpose, 1.0 .mu.l of the bacteria (OE₆₆₀ = 0.4) per grid section as a spot on a nitrocellulose paper disc (Schleicher and Schuell, 37 mm nitrocellulose disc, screened 0.45 microns). Each slice can easily contain 60 different bacteria samples. The spiked slices were air dried, fixed in 25% v / v isopropanol for 30 min, and blocked for 10 min in the dry skim milk reagent described in the ELISA assay described above. The blocked slices were washed 3 × 5 min each in PBS / Tween 20 and transferred to the lids of 35x10 mm tissue culture dishes. The antibody-containing supernatant (1.0 ml) was added to the lid and incubated at RT for 60 min. After washing 5 times in PBS / Tween 20 for 5 min, 1: 1000 (PBS) alkaline phosphatase-conjugated goat anti-human immunoglobulin (TAGO, Burlingame, CA) was added and left at room temperature for 60 min. The disks were washed as described above and immersed in 1 to 2 ml of a fresh substrate prepared as follows:
16.5 mg Bromchlorindolylphosphat and 8.5 mg Nitroblue tetrazolium were dissolved in 50 ml of alkaline phosphatase buffer (0.1 M Tris-HCl, pH 9.5 with 0.1 M NaCl and 5 mM MgCl₂) ge. The solution was stored in the dark and filtered immediately before use. After a satisfactory color development (10 to 15 min.), The reaction was quenched by washing the disk in distilled water which was changed several times. The developed discs can be stored after drying.

The discs contained 50 K. pneumoniae capsule-typed Reference strains obtained from Dr. med. George Kenney, University Washington, Department of Pathobiology, Seattle, WA and the American Type Culture Collection; 4 E. Aerogenes clinical blood isolates; and 6 E. Cloacae clinical blood isolates (blood isolates released from Harborview Hospital, Seattle, WA). The Enterobacter isolates were isolated (speciation) using the API 20 E system of 23 standardized biochemical tests (API-Analytab Product, Plainview, NY). In this process, the Genus and the species of gram-negative Bacteria but not the serotype identified. Therefore enterobacteria, but not E. coli, S. Marcescens and K. Pneumoniae, not concerning the Serotypes, but only in terms of the genus and the Identified species.

These experiments revealed that the 7D7 antibody was more Possesses intergenus cross-reactivity. This antibody reacts with the following serotypes.

From the monoclonal human antibody 7D7 could thus festge he states that he has intergenus cross-reactivity for Bacteria belonging to the species E. Coli, S. Marcescens, K. pneumoniae and E. aerogenes, but not E. cloacae, belong.

F. Characterization of monoclonal antibodies

The finding that the monoclonal antibody ver different types of bacteria (Genera) Cross-reacted, led to the conclusion that the anti body against a common protein or carbohydrate was directed. Of these two molecular species could be shown to be suitable for intragus cruciate Responsible reactions (Mutharia L. and Hancock R.E.W. "Characterization of Two Surface-Localized Antigenic Sites on Porin Protein F of Pseudomonas aeruginosa, "Canadian J. of Microbiol., 31, 381-386 (1985) and Orskov F. and Orskov I., "Serotyping of Escherichia coli ", in Methods in Microbiology, Vol. 14, Bergan T .; Ed. Academic Press, Orlando, FL, 43-112 (1984)).

A biochemical characterization of the molecular Species recognized by the 7D7 antibody was accomplished by immunoblot analysis. Washed bacteria were transferred from 20 ml of one over Night-grown culture (for E. coli, S.Marcescens and E. aerogenes serotypes) or over Overnight grown plates (K. pneumoniae) in 1.0 ml extracted a solution containing 64 ml of 50 mM Tris pH 7.6, 530 ml glycerol, 0.3 g deoxycholate (sodium salt),  0.14 ml of β-mercaptoethanol and 6 ml of deionized water (Schechter I and Block K. "Solubilization and Purification of trans-formesyl pyrophosphate-squalene synthetase ", J. Biol. Chem. 246, 7690-7696 (1971)). After 18- hourly incubation at 4 ° C, the suspension was added Centrifuge 10 g for 10 min. The supernatant was removed and the protein was quantitated with. Help of the Bio-Rad Protein Assays (Bio-Rad Laboratories, Richmond, CA). Between 100 and 1000 ng of the protein (varies for each bacterium extract) from each bacterium became one Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis Kusecek B. et al. "Lipopolysaccharide, Capsule, and Fimbriae as Virulence Factors Among 01, 07, 016, 018 or 075 and K1, K5 or K100 Escherichia coli ", Infection and Immunity, 43, 368-379 (1984)). Separated molecular species were transferred from the gel to a nitrocellulose membrane (NCM), as for example in the Literature is described, see: Towbin H. et al, "Electrophoretic Transfer of Proteins From Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications, Proc Natl Acad Sci., 76, 4350-4354 (1979). The NCM blot was blocked for 1 h in PBS-Tween (Batteiger B. et al. The Use of Tween 20 as a Blocking Agent in the Immunoglogical Detection of Proteins Nitrocellulose Membranes, J. Immunol., Meth., 55, 297-307 (1982)). The blot was incubated for 1 h at room temperature in 10 ml of the spent culture supernatant from the 7D7 line incubated. After rinsing in PBS-Tween for 5 minutes, the Blot incubated in goat anti-human Ig, with alkaline phosphatase conjugated and as in this together hang with the bacteria-nitrocellulose slice assay ent wraps. In all tracks, the deoxycholate extracts of contained positive bacteria Reactions are detected. In these tracks seems the 7D7 antibody is a series of regularly spaced ones  To recognize molecular units, which become a ladder-like Pattern on the immunoblot. This profile is correct completely the same as that in the polyacrylamide Gel electrophoresis analysis of LPS in the presence of SDS ge had been found. There it could be shown that the heterogeneous size profile indicated by the bands is based on a population of LPS molecules that are characterized by Weight increments differ by the number of per Molecule present O-antigen oligosaccharide side chains units is equivalent (Pavla E.T. and Makela P.H., "Lipopolysaccharide heterogeneity in Salmonella thypimurium Analyzed by Sodium Dodecyl Sulfate / Polyacrylamide Gel Electrophoresis, Eur. J. Biochem., 107, 137-143 (1980) and Goldman R.C. and Leive L., "Heterogeneity of Antigenic Side-Chain Length in Lipopolysaccharides from Escherichia coli 0111 and Salmonella typhimurium LT2 ", Eur. J. Biochem. 143-154, (1980)). These data show that the monoclonal Antibody 7D7 is directed against an antigenic determinant, which is common to the LPS molecules, which on some Serotypes of E. coli, S. marcescens, K. pneumoniae and E. Aerogenes can be found.

To further define the molecular nature of the antigen Deoxycholate extracts were used before electrophoresis the proteolytic enzyme proteinase K (Eberling W. et al "Proteinase K from Tritirachium album Limber", Eur. J. Biochem. 47, 91-97 (1974)). For the production of Sample, 10 μg of proteinase K became 50 μg of the protein sample given. The mixture was heated to 65 ° C for 60 min. The Samples were electrophoresed as previously described and subjected to an immunoblot. The after treatment observed with proteinase K were immunoblot patterns identical to those observed without treatment had been respected. This indicates that with the 7D7 Antibody-reactive antigen is not a protein by nature is.  

To specifically find out if 7D7 is using a coal hydratepitop reacted, the electrotransferierte became Deoxycholate sample of mild oxidation with periodate below Throw before the nitrocellulose paper with the antibody was reacted. From this reaction could be shown that they are the carbohydrate determinants and thus their subsequent reactivity with antibodies zer interferes without altering protein or lipid epitopes (Woodward M. P. et al., Detection of Monoclonal Antibodies Specific for Carbohydrate Epitopes Using Periodate Oxidation, J. of Immunol., Methods, 78, 143-153 (1985)). For this purpose, after the electroblot the nitrocellulose paper, containing the electrophoresed sample with PBS-Tween blocked as described here. The paper was with Rinsed with 50 mM acetic acid-sodium acetate pH 4.5 buffer. The Nitrocellulose paper was dissolved in 50 mM periodic acid in the acetic acid buffer was added, in the dark for 60 min Room temperature incubated. The treated paper was Rinsed 3X in PBS-Tween and treated as described previously with the Antibody reacted. The way this way treated electroblotted deoxycholate extracts no longer with the 7D7 monoclonal antibody.

These data clearly show that this is due to this Antibody recognized epitope is a carbohydrate that the LPS molecule of the bacteria described herein is.

The isotype of the 7D7 monoclonal antibody was determined using an ELISA method similar to that described above Specificity test is similar, but biotinylated Goat anti-human IgG (γ-chain specific, TAGO) or biotinylated goat anti-human IgM (Mu chains specifically, TAGO) as a second stage reagent of the "broader" reactive biotinylated anti-human Ig Goat was used. Both reagents were in a 1: 500 dilution and the antigen plate  contained "pools" of PLL-immobilized E. coli-08 and 075 strains. A positive reaction of the 7D7 monoclonal Antibody with E. coli strains was only immunized with the anti- IgM reagent observed what an IgM isotype for the mono indicating clonal antibodies. A specialist on the here in The area in question knows that if the above Ver drive would repeat several times and the isotypes the intergenus thus obtained cross-reactive monoclonal Antibody would determine, you further z. B. IgM and IgG isotypes would find (Frosch M. et al "NZB Mouse System For Production of Monoclonal Antibodies to Weak Bacterial Antigens: Isolation of an IgG Antibody to the Polysaccharide Capsules of Escherichia coli K1 and Grop Meningocci ", Proc Natl Acad Sci., 82, 1194-1198 (1985)).

G. In vitro activity

The in vitro functional activity of 7D7 monoclonal Antibody was detected in an in vitro opsonophagocytosis Assay examined. This is the bactericidal activity of the antibody in the presence and absence both of human neutrophils as well as of human complement ver equalized.

Bacteria were prepared by adding either 10 ml tryptic soy broth (TSB) with 50 μl of an overnight Broth culture (for E. coli, S. Marcescens and E. Aerogenes) or for K. pneumoniae a Worfel-Fergusan agar streaked Petri dish has been. For broth cultures, the tubes were allowed to stand for 3 hours a shaker at 37 ° C incubated. After that 1.5 ml of the culture was centrifuged for 1 min at 10,000 g. The spent culture media were discarded and the Pellet was dissolved in 3.5 ml Hank's balanced salt solution containing 0.1% gelatin and 5 mM HEPES (HBSS / gel) contained. In case of ge on the agar plate The bacteria were growing  Colonies scraped off the plate and placed in sterile HBSS / gel given. The bacterial concentrations in the case of bacteria that had grown under both conditions, were adjusted to 3 x 10³ bacteria / ml by the O.D.₆₆₀ was measured and ver was diluted (in about 1: 50 000). Human neutrophils were isolated according to van Furth and Van Zwet ("in Vitro Determination of Phagocytosis and Introcellular Killing by Polymorphonuclear and Mononuclear Phagocytes ", in Handbook of Experimental Immunology, Vol. 2, D.M. Weir, Ed. 2nd ed., Blackwell Scientific Publications, Oxford, 36.1-36.24 (1973)), with various modifications were made. The buffy coat of 10 ml heparinized blood was underlaid with Ficoll-Paque and centrifuged. The pellet of red blood cells (RBC) was washed once with RPMI 1640 medium and incubated in RPMI 1640 medium resuspended in an equal volume of PBS (37 ° C). 3 ml of this suspension were added to 6 ml of 2% dextran in PBS given by 37 ° C. The content became cautious, however thoroughly mixed. To 20 minutes incubation at 37 ° C, the red blood Let the body settle, the supernatant (contains Neutrophils), 2X in PBS of 4 ° C and 1X in HBSS / gel washed and in it up to 5 × 10⁷ neutrophils / ml suspended. For the complement source with E. coli and S. Marcescens was used, human serum was 2 × with living bacterial pools (Bjornson A.B. and Michael J.G. Factors in Human Serum Promoting Phagocytosis of Pseudomonas aeruginosa I. Interaction of Opsonins with the Bacterium, J. of Inf. Dis., 130 Suppl. 5119-5126 (1974)) according to those used in this assay Organisms adsorbed. The serum was continued with boiling zymosan (Bjornson, supra) adsorbed to to remove the serum component propperidin. It deals this is a molecule responsible for the activation of the Mutual complement path is required. For  opsonophagocytic assays using K. Pneumoniae and E. aerogenes did not become complement source adsorbed normal human serum with a final concentration used by 1%.

The plates for quantifying the number of surviving / Destroyed bacteria were prepared by first 24-well plates heated to 37 ° C for 3 to 5 hours were. A 0.4% solution of agarose in TSB was prepared by treating the mixture in an autoclave for 5 min and then cooled to 50 ° C in a water bath was let. About 15 minutes before the end of the last Incubation period in the Opsonophagocytoseassay was a 24-well plate from the incubator (37 ° C) removed and placed on a 42 ° C hot plate. Then 0.4 ml of TSB / agarose was added to each well. The Plate was returned immediately to the incubator (37 ° C) so that the agarose is never at a temperature was cooled below 37 ° C.

For the assay, 25 μl of the 7D7 culture supernatant and 25 μl of a suitable bacterial strain in each case 2 × in 96-well microtiter plates with round bottom and incubated at RT for 30 min. Subsequently were 15 μl of the human complement, 15 μl human neutrophils (5 x 10⁶ / ml) and 70 ul HBSS / gel. The whole Surface of the plate was covered with a sterile cotton bausch wiped off; a plastic adhesive for sealing the Plate was placed on the entire plate and on the between applied to the wells zones and the plate was rotated at 37 ° C for 1 h. After incubation, the plate became Centrifuged at 100 g for 5 min. The mass for sealing the plate was carefully removed and the plate top The area was covered with a sterile dipped in 70% ethanol Dry cotton cloth. 50 microliters were removed from each  Microtiter well removed and into individual Ver depressions of plates for quantification purposes 24 wells given ge already 0.4 m / well molten (38 to 40 ° C) contained 0.4% TSB / agarose. These plates were placed on a flat bed shaker and shaken for 1 min. at 150 rpm. The agarose could Cure for 15 minutes at room temperature. In the end, each one was Overcoating with 0.4 ml TSB / agarose. Subsequently was allowed to cure for 15 min. at 4 ° C before the plates were incubated overnight at 37 ° C. After 18 h, the Colonies were counted and the data were collected for each tion as Colony Forming Units (colony forms units (CFU)).

The bacterial serotypes used here were up on K. pneumoniae only in the presence of the monoclonal anti body 7D7, an active source of complement and human neutrophils (Table 1) inactivated. Was this Experiment with various non-responsive with 7D7 bacterial serotypes repeated, then no cerium Disturbance of bacteria observed (data are not up leads). This demonstrates the functional specificity monoclonal antibody 7D7 and its capacity, To opsonize bacteria and their phagocytosis to below support. The common effects of opsonins (specific anti bodies) and polymorphonuclear leukocytes (neutrophils) the primary cause of immunity for these bacterial strains. Thus, these data suggest that the 7D7 antibody may be used as appropriate Administration protection against lethal challenges with those described here Would grant bacterial serotypes.  

Table 1

H. In vivo activity

To verify the hypothesis set out above Investigations carried out to determine how far animals were protected. These investigations were with 7D7 antibody and at least one organism the E. coli and E. aerogenes described herein. Species performed. 7D7 antibodies and negative Control antibody (6F11, monoclonal IgM human antibodies, specific for Pseudomonas aeruginosa Fisher immunotype 2) were first consumed from the Culture supernatants by precipitation with solid ammonium concentrated sulfate (final concentration 50%) (Good A.J. et al, "Purification of Immunoglobulins and Their Fragments, in Selected Methods in Cellular Immunology, Mishell B.B. and Shiigi S.M., Hersg. W.H. Freeman and Company, San Francisco, CA (1980) 279-286). The precipitated material was in sterile Water reconstituted and extensively against PBS dialyzed.

The IgM antibody in the ammonium sulfate salt precipitate was determined by affinity chromatography on a Column with a murine monoclonal anti-human IgM Antibodies purified. For the preparation of this column 1 g of dehydrated cyanogen bromide was activated Sepharose 4B (Pharmacia) with 15 ml of ice-cold 1 mM HCl mixed in distilled water. The hydrated Gel was dissolved in 30 ml of coupling buffer (0.1 M carbonate (NaHCO₃) in 0.5 M NaCl, pH 8.2), washed under Formation of a wet cake drained and with the in 1 to 3 ml of the coupling buffer dissolved ammonium salt precipitate combined. The gel suspension was thorough 2 h at room temperature ver mixed and then centrifuged for 5 min. At 200 g.  

For blocking still available reactive sites, the supernatant was removed. 10 ml 1 M ethanolamine was added to the gel and it became further mixed as described above. The suspension was Centrifuged at 200 g for 5 min and the supernatant was ver worfen. To prepare an application-ready gel was it 1x in 0.1 M acetate / saline buffer (6.8 g sodium acetate trihydrate and 14.6 g NaCl were distilled in 500 ml dissolved water containing 2.9 ml of glacial acetic acid, pH 4.0), washed 2x in coupling buffer and 2x PBS. The Gel was poured on a Pharmacia C10 / 10 column and until stored for use at 4 ° C.

For purification of the immunoglobulin, 0.5 ml of the Salt fractionated material to 2.0 ml diluted in PBS and added to the affinity column. To the sample was loaded with PBS, pH 8.0, washed until the absorbance monitor indicated that no further protein is present in the flow agent is. The bound antibody was treated with 2 M MgCl in PBS eluted. The protein concentration was for each fraction determined at O.D.₂₈₀. The peak fractions were united. The fraction containing the antibody was desalted with a G-25 Sephadex column and if necessary by centrifuging (microconcentration) (Centricon 30, Amicon Corp., Danver, MA) to 1-2 mg / ml concentrated. The purity of the final preparation was by SDS-polyacrylamide gel electrophoresis and by subsequent staining of the proteins with silver nitrate (Morrissey J. H. "Silver Stain for Proteins in Polyacryl Amide Gels: A Modified Procedure with Enhanced Uniform Sensitivity "; Anal. Biochem. (1981) 177, 307-310).

For the investigation of the antibody activity a used above described ELISA assay.  

For each bacterial challenge, females became non-breeding related Swiss Webster mice weighing 20 to 22 g used. These mice were in three groups with each divided 10 mice. A representative Experiment was carried out as follows:

Each group containing the antibody became intravenous 200 μl PBS (sterile, contains 25 μg of purified anti body). Two hours later, all the animals received intraperitoneally a challenge of 300 μl live bacteria. The challenge contained three times the lethal dose (3x LD₅₀) of the corresponding bacterial strain. The bacteria suspension was produced from a broth culture in the logarithmic growth phase, from which the bacteria were washed 2 × in PBS and to a suitable concentration in PBS had been resuspended. The animals were killed during one Observed over a period of 5 days. 24 to 48 hours after the Challenge were all animals in group B (irrelevant Antibodies) and group C (irrelevant organism) dead. In contrast, all animals were the 7D7 Antibodies (Group A) had been kept alive and free of symptoms.

This animal protection model was used to study the therapeutic effect of the 7D7 antibody against bacterial Challenges with organisms, which added to the three above led genera to be used. A summary the data is given in Table 2.  

Table 2

Table 2a

These data demonstrate that the monoclonal human Antibodies 7D7 mice against lethal challenges with bacteria which can lead to three different gram- belong to negative bacterial species. Because 7D7 with a Carbohydrate epitope that is present at LPS and LPS molecules are less accessible to K. pneumoniae (Orskov I. and Orskov F. "Serotyping of  Klebsiella ", in Methods in Microbiology, Vol. Bergan T., ed. Academic Press, Orlando, FL (1984) 143-146), protection by the 7D7 antibody was present K. pneumoniae infections not apparent (the data are not listed). Nevertheless, the intergenus cross Reactive human monoclonal antibody 7D7 able to Administration of 25 μg of the purified antibody, To confer protection against infections of organisms which to the gram-negative bacterial species E. coli and E. Aerogenes belong.

Example II

In this example II are methods of production and Selection of a monoclonal human antibody be described the intergenus cross-reactivity against mit members of the species Serratia marcescens, Klebsiella pneumoniae and Enterobacter aerogenes. Farther is the in vitro opsonizing activity of this anti body against homologues S. Marcescens, K. Pneumoniae and E. Aerogenes serotypes explained. The method described in Example 1 (explained there essentially in parts A to G) for the preparation of a monoclonal human antibody repeated. This antibody is cross-protective (cross-protective = cross-protective) Infections caused by the bacteria described here caused. In this example, however, it was him necessary to make specific modifications to to characterize the antibody described herein and to investigate. Below are the changes in the Assay method and with the mono described here clonal body results obtained.

• 1. The culture supernatants of 6 transformations were analyzed by the method described above. This led to the identification of a well (4F10) which showed activity on the S. Marcescens serotype plate, but not on the E. coli or control plate. In subsequent ELISA studies with individual S. Marcescens serotypes, it was found that this well contained an antibody which was compatible with S. Marcescens serotypes 015 and 018, but not with any of the other known S. Marcescens LPS. Serotypes responded.
  Prior to the priority date of the present invention, the continuous transformed human cell line identified as 4F10 was deposited with the American Type Culture Collection of Rockville, MD as ATCC No. CRL 9007.
• 2. The antibody obtained from the cloned 4F10 cell line was examined for further intergenus cross-reactivity by a modification of the standard immunoblotting technique. In particular, the cross-reactivity to bacteria K. pneumoniae, E. aerogenes and E. cloacae was investigated by placing "spots" of bacteria on a rastered nitrocellulose paper disc and reacting the disc containing the bacteria with said antibody and the antibody reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system.
  From these experiments it could be deduced that the 4F10 antibody has further intergenus cross reactivity. This antibody reacted with the following serotypes: The monoclonal human antibody 4F10 thus had intergenus cross reactivity for bacteria belonging to the species S. Marcescens, K. pneumoniae and E. aerogenes.
• 3. Using the immunoblot technique, positive responses were found in all tracks containing deoxycycerate extracts of the bacteria described herein. In these tracks, the antibody 4F10 appears to recognize either a broad band of components or a series of regularly spaced molecular units resulting in a ladder-like pattern. This profile was fully consistent with that found in electrophoretic polyacrylamide gel analyzes of carbohydrate moieties having either extensive molecular heterogeneity due to the oft-repeating specific sugar sequence or LPS molecules which differed by weight increments, that of number the O-antigenic oligosaccharide side-chain units are equivalent per molecule (Vimr ER et al, "Use of Procaryotic-Derived Probes to Identify Poly (Sialic Acid) in Neonatal Neuronal Membranes", Proc. Natl. Acad. Sci., (1983) , 81, 1971-1975 and Holden KG et al., "Gel Electrophoresis of Mucous Glycoproteins, I. Effect of Gel Porosity", Bio chemistry (1971) 10, 3105-3109). These data indicate that the monoclonal antibody 4F10 is directed against an antigenic determinant shared by or shared by carbohydrate molecules found on some serotypes of S. Marcescens K. pneumoniae and E. aerogenes.
  The immunoblot patterns observed after treatment with proteinase K were identical to those obtained without treatment. This shows that the antigen reacting with the antibody 4F10 is not a protein.
  To determine if 4F10 reacted with the carbohydrate epitope, the electro-transferred deoxycholate sample was subjected to mild periodate oxidation prior to reaction of the nitrocellulose paper with the antibody. The electroblotted deoxycholate extracts thus treated no longer reacted with the monoclonal antibody 4F10. These data strongly suggest that the epitope recognized by this antibody represents a carbohydrate unit present on molecules comprising the bacteria described herein.
• 4. The isotype of the monoclonal antibody 4F10 was with The help of an ELISA method, which is similar to the one in Bei play I described specificity test is similar. However, the antigen plate contained a pool of PLL-immobilized S. Marcescens 015 and 018 serotypes, A positive reaction of the monoclonal antibody 4F10 with S. Marcescens serotypes was found only with the Anti-IgM reagent observed. This shows that the mono clonal antibody is an IgM isotype. The skilled person is clear that if the method of this example repeated several times and the isotypes of thus obtained intergenus cross-reactive monoclonal Antibodies would be determined, one more isotypes, z. As IgM and IgG isotypes, would find.
• 5. The in vitro functional activity of the monoclonal antibody 4F10 was examined in an opsonophagocytic assay comparing the bactericidal activity of the antibody in the presence and absence of human neutrophils as well as the human complement.
  The bacterial serotypes used here were inactivated only in the presence of the monoclonal antibody 4F10, an active complement source and of human neutrophils (Table 3). After repeating the experiment with various bacterial serotypes that do not react with 4F10, no destruction of the bacteria was observed (the data are not listed). This demonstrates the functional specificity of the monoclonal antibody 4F10 and its capacity to opsonize bacteria and promote their phagocytosis.

Table 3

Example III

In this Example III are methods of preparation a monoclonal human antibody described so probably with Escherichia coli capsule type K1 as well with Neisseria meningitidis (N. meningitidis) group B poly saccharide can react. Furthermore, the protective activity this antibody in vivo against a lethal challenge homologous E. coli and N. meningitidis bacterial species explained. That in Example I (there essentially in the Parts A to G) was repeated, to produce a monoclonal human antibody, the cross-protects against infections, which by the here be caused bacteria are caused. It was, however necessary to carry out specific modifications, to that described in the present example To characterize and study antibodies. by standing are the changes made in the assay procedure and those with the monoclonal described herein Antibody obtained results described.

• 1. Culture supernatants from 5 transformations were analyzed by the method described above. This resulted in the identification of 4 wells (5D4, 2C10, 9B10 and 8A8) that contained anti-E.Coli specificity on the E. coli serotype plate but not on the S. Marcescens or control plate. In subsequent ELISA assays performed as described above, it was determined on individual E. coli serotypes that these wells contained an antibody capable of reacting with at least the following E. coli serotypes: 01 (ATCC 23499), 07 K1 (ATCC 12792), 016: K1 (ATCC 23511) and 050 (CDC 1113-83) However, this antibody did not react with: 04 (ATCC 12792), 06: K2 (ATCC 19138), 08: K8 (ATCC 23501). , 09: K9 (ATCC 23505) or 022: K13 (ATCC 23517). Monoclonal antibody 9B10 was chosen for further analysis because of its more advantageous cloning behavior and because of the increased antibody production.
  The continuous, transformed human cell line, designated here as 9B10, was deposited with the American Type Culture Collection of Rockville, MD as ATCC No. CRL 9006 prior to the priority date of the present application.
• 2. The finding that the monoclonal antibody from each of the clones reacted with the identical group of E. coli O antigen serotypes indicated that these antibodies are directed against a bacterial surface structure common to these serotypes. Various approaches have been tried to define the common surface structure of these E. coli serotypes. As shown below, two (07: K1 and 016: K1) of the four E. coli serotypes identified by antibody 9B10 possessed capsule serotype K1, while the other two (01 and 050) did not have their K antigen sero type have been typed. It was therefore considered possible that the antibody 9B10 contained reactivity for the K1 antigen and that the E. coli strains having the O antigen serotypes 01 and 050 also had the K1 capsule serotype.
  The temperature sensitivity of the K1 capsule has already been exploited in order to prove its presence. By heating K1-positive E. coli serotypes for 60 min in a 100 ° C boiling water bath, the subsequent ability of these strains to react with anti-K1 sera is removed and their ability to react with anti-O Antigen sera, (Orskov F. and Orskov I., "Serotyping of Escherichia coli", in Methods in Microbiology, Vol. 14, T. Bergan, ed., Academic Press, London (1984) p. 44-105). The reciprocal effects on heating are primarily due to the capsule removal and the increased accessibility of the antibody to lipopolysaccharide (LPS) molecules. E. Coli K1 positive serotypes (07 and 016) and non-K1 typed serotypes (01 and 050) were heated as described below and probed with antibody 9B10 and LPS serotype specific heterologous sera (Difco Bacto-E. coli typing reagents) in the ELISA procedure. The heat-treated organisms lost all of their reactivity to antibody 9B10 and showed increased reactivity towards their homologous LPS serotype-specific sera. In contrast, untreated organisms (control) continued to react strongly with the culture supernatants of 9B10 and reacted only slightly with their corresponding LPS serotype specific antisera.
  The polysaccharide (carbohydrate) of Neisseria meningitidis group B bacteria (a homopolymer of sialic acid, alpha 2, 8-linked poly-N-acetylneuraminic acid) is known to be chemically and antigenically homogeneous with the E. coli KI polysaccharide (Grados O. and Ewing WH "Antigenic Relationship between Escherichia coli and Neisseria meningitidis", J. Immunol. (1973), 110, 262-268). These data suggest that if the 9B10 monoclonal antibody has specificity for E.coli K1 capsules, this antibody would also need to contain specificity for N. Meningitidis group B polysaccharide. Further, it can be seen that monoclonal antibodies containing specificity for the polysaccharide Group B of N. meningitidis would also have to be reactive to E.coli strains containing the K1 capsule. Two experiments were performed to firstly test the ability of antibody 9B10 to react with N. meningitidis and secondly to test the ability of an antibody to N. meningitidis group B polysaccharide with four E. coli 9B10-reactive ones Serotypes to respond.
  Highly purified polysaccharide Group B (Connaught Laboratories, Toronto, Canada) and live N. Meningitidis Group B bacteria were reacted with antibody 9B10 in an ELISA as described above. The monoclonal antibody 9B10 reacted strongly with both antigen preparations. To prove the reverse specificity, a commercially available N. Menin gitidis Group B Meningitis Test Kit (Directagen Direct Antigen Detection System, Hynson, Westcott and Dunning, Baltimore, MD) was used. This kit uses latex particles coated with a murine monoclonal antibody for the Group B polysaccharide. In agglutination assays using the 9B10 48888 00070 552 001000280000000200012000285914877700040 0002003745019 00004 48769 positive E. coli serotypes, all four serotypes showed strong reactivity with the antibody coated particles. E. Coli serotypes known to be negative for K1 antigen were also negative in this assay. These data show overall that the monoclonal antibody 9B10 can react with the E.coli K1 capsule and the type-specific carbohydrate on N. meningitidis group B. Since many of these assays have been performed on intact, live bacteria, it can be deduced that monoclonal antibody 9B10 is specific for part of an externally exposed region of the poly-sialic acid molecule.
• 3. The isotype of the monoclonal antibody 9B10 was determined by an ELISA method similar to that described in the play I described in the specificity tests. However, the antigen plate contained a pool of PLL immobilized E. coli K1-positive serotypes. A positive reaction of the monoclonal antibody 9B10 with the K1-positive E. coli serotypes was only with the anti-IgM reagent. This shows one IgM type for the monoclonal antibody. The expert know that if you follow the procedure of this example repeated several times and the isotypes of him K1-specific monoclonal antibody be true, one more isotypes, z. IgM and IgG isotypes would find.
• 4. The in vitro functional activity of monoclonal antibody 9B10 was examined in an opsonophagocytosis assay comparing the bactericidal activity of the antibody in the presence and absence of human neutrophils and human complement.
  K1-positive E. coli serotypes were inactivated only in the presence of monoclonal antibody 9B10, an active complement source, and human neutrophils (Table 4). After repeating this experiment with various K1-negative E. coli serotypes, no bacterial destruction was observed (the data are not listed). This demonstrates the K1 specificity of monoclonal antibody 9B10 and its capacity to opsonize bacteria and promote their phagocytosis. Since the combined effect of opsonins (specific antibodies) and polymorphonuclear leukocytes (neutrophils) appeared to be the primary mechanism of immunity to K1-positive E. coli serotypes, these data indicate that the antibody 9B10, after appropriate administration, protects against would confer lethal challenge with each of the E. coli K1 encapsulated serotypes regardless of its O antigen serotype.

  Table 4

• 5. To test the above hypothesis, animal protection studies were carried out with the antibody 9B10 and various K1-positive and K1-negative E. coli serotypes and with a N. meningitidis group B serotype (strain H313, obtained from Dr. Carl Frasch, Laboratory of Bacterial Polysaccharides, Office of Biologics, Food and Drug Administration, Bethesda, MD).
  For each bacterial challenge, non-pedigree Swiss Webster mice weighing 20 to 22 g were used. The mice were divided into three groups of 10 mice each. A representative experiment was performed as follows: Each group containing the antibody was injected intravenously with 200 μl of PBS (sterile containing 25 μg of purified antibody). Two hours later, all animals were challenged with 300 μl of live bacteria (containing 3 times the LD₅₀ amount of the corresponding bacteria) intraperitoneally. The bacterial suspension was prepared from a broth culture in the logarithmic growth phase. The bacteria were centrifuged out, washed twice in PBS and resuspended in PBS to a suitable concentration. The animals were observed for 5 days. By 24 to 48 h after challenge, all animals in group B (irrelevant antibody) and group C (irrelevant organism) were dead. In contrast, all animals that had received antibody 9B10 (group A) were still alive alive and free of symptoms.
  This animal protection model was chosen to demonstrate the therapeutic effect of antibody 9B10 on bacterial challenge, which challenge is caused by organisms belonging to the two species described herein. A summary is given in Table 5.

  Table 5

  These data demonstrate that the monoclonal human antibody 9B10 is capable of protecting mice from a lethal challenge with bacteria belonging to two different Gram-negative bacterial species. The intergenus cross-protective antibody was able to prophylactically protect against infection caused by organisms belonging to the Gram-negative bacterial species E. coli and N. meningitidis group B.

Example IV

In this Example IV are methods of making a monoclonal human antibody, which explains a Intergenus cross reactivity against members of the species Escherichia coli (E.coli), Enterobacter cloacae (E.loclacae) and Streptococcus group B possesses. In this example is continues to explain an antibody that interacts with species can cross-react, leading to two major bacterial groups  count, namely gram-negative (E. coli and E. gloacae) and gram-positive (Streptococcus group B). Also is the in vivo protective activity of this antibody before lethal challenge with homologous E. coli and Streptococcus Group B serotypes explained. That in the example I (there in the essential Ver described in Parts A to G) Ver driving was repeated to obtain a monoclonal human To produce antibodies that is cross-protective Infections caused by the bacteria described here caused. However, it was required to be specific Perform modifications to the in this example to characterize and under-described antibodies search. Below are the ones in the assay procedure changes made and those described here Monoclonal antibodies obtained results closer he purifies.

• 1. Supernatants were screened for the presence of anti-Group B Streptococcus antibody by the ELISA technique as described in Example I. The antigen plates consisted of a series of Immunolon 2 96-well round bottom microtiter plates whose wells contained mixtures of Group B Streptococci capsule types which were fixed to the surfaces of the well with poly-L-lysine (PLL). Various antigen plates were used in the screen: (1) a mixture of Group B Streptococcus types Ia (ATCC No. 12400), Ib (ATCC No. 12401) and Ic (ATCC No. 27591); (2) a mixture of types II (ATCC No. 12973) and III (clinical isolate, obtained from Dr. C. Wilson, Children's Orthopedic Hospital, Dept. Infectious Disease, Seattle, WA); and (3) a microtiter plate without bacteria.
  The culture supernatants from two transformations were analyzed according to the method described above. This resulted in the identification of a well (4B9) which had activity on both group B Streptococcus typing plates but not on the control plates. In subsequent ELISA studies with individual group B Streptococcus types, it was found that this well contained an antibody that reacted with all five typing reference strains.
  Thus, in this experiment, a cloned transformed human cell line could be provided which is continuous (immortal) and secretes a monoclonal human antibody for a determinant on the surface of the group B Streptococcus types described.
  Prior to the priority date of the present application, the continuous human cell transformed line designated 4B9 has been deposited with the American Type Culture Collection of Rockville, MD under the designation ATCC No. CRL 9008.
• 2. The antibody from cloned cell line 4B9 was assayed for cross-reactivity for gram-negative and gram-positive bacteria using a modification of the standard immunoblot technique. In particular, the cross-reactivity for the following bacteria was investigated: E. coli, K. pneumoniae, S. marcescens, E. aerogenes, E. cloacae, hemophilus influenzae, and staphylococcus aureus. For this purpose, "spots" of the bacteria were placed on a screened nitrocellulose paper disc. The disc containing the bacteria was reacted with the antibody. The antibody reactions were developed with an alkaline phosphatase / nitro blue tetrazolium enzyme system as described in Example I.
  These experiments revealed that antibody 4B9 has cross-reactivity with certain Gram-negative species. This antibody reacted with E. coli LPS serotypes 04, 07, 018, and 025, as well as with the E. cloacae clinical isolates. The monoclonal human antibody 4B9 thus has intergenus cross-reactivity between the Gram-negative and Gram-positive bacteria belonging to the species E. coli, E. cloacae and Streptococcus group B.
• 3. The finding that the monoclonal antibody cross-reacted with various different bacterial genera belonging to both the Gram-positive and the Gram-negative bacterial group showed that the antibody was directed against a common protein or carbohydrate. The biochemical characterization of the molecular species recognized by antibody 4B9 was performed by immunoblot analysis. For analysis of the gram-negative genera, washed bacteria were extracted into deoxycholate as described in Example I. In the case of Gram-positive bacteria, 1.0 L of bacteria was added to modified Todd-Hewitt broth (Difco, Todd-Hewitt broth, containing 2.8 g / L of anhydrous sodium phosphate, pH 7.8) for 6 h at 37 ° C, recovered by centrifugation and washed 3 × in PBS. The bacteria were resuspended in 85 ml of protoplast medium (40% sucrose w / v in 0.03 M potassium phosphate buffer, pH 6.9, containing 10 mM MgCl₂). The suspension was heated to 37 ° C for 10 min. In about 3000 units of mutanolysin (Sigma) were added. The mixture was shaken at 37 ° C for 90 minutes or until the OD₆₆₀ of the suspension had been reduced by more than 90%. The digested material was centrifuged for 15 min. At room temperature and at 2000 g. The supernatant was dialysed for 48 h against PBS (Young MK and Mattingly SJ, "Biosynthesis of Cell Wall Peptidoglycan and Polysaccharide Antigens by Protoplasts of Type III Group B Streptococcus", J. Bact., (1983) 154, 211-220). The dialysate was concentrated 10-fold by positive pressure dialysis through a PM-10 filter (Amicon Corp., Danvers, MA).
  Carbohydrates that bind to wheat germ agglutinin were purified by affinity chromatography on a wheat germ lectin-pharose 6MB column (Sigma). The bound digestion described herein was eluted from the column with 10 ml of 0.1 M N-acetylglucosamine. The eluate was dialyzed against distilled water at 4 ° C. The purified affinity eluate was dried by lyophilization. The dry weight of the resulting material was determined (Gray BM et al "Interaction of Group B Streptococcal Type-Specific Polysaccharides with Wheat Germ Agglutinin and Other Lectins", J. Or Immunol. Meth., (1984), 72, 269-277). In all tracks containing deoxycholate extracts of the bacteria described here, positive reactions were detected. In those tracks containing extracts of Gram-negative bacteria, the antibody 4B9 appeared to recognize a series of regularly spaced molecular units, resulting in a ladder-like pattern on the immunoblot. This profile was fully consistent with that observed in the electrophoretic polyacrylamide gel analysis of LPS in the presence of SDS. There it could be shown that the heterogeneous size profile indicated by the bands is based on a population of LPS molecules which differ by weight increments equivalent to the number of O-antigenic oligosaccharide side-chain units present per molecule (Pavla ET and Makela PH, see above and Goldman RD and Leive L., supra). In those tracks containing extracts of Group B Streptococcus types, the antibody 4B9 appeared to know components that were present over a broad range. This profile was consistent with that found in polyacrylamide gel electrophoresis analyzes of carbohydrate moieties having extensive molecular weight heterogeneity with a frequently repeating specific sugar sequence (Vmir ER et al, supra, and Holden KG, supra ). These data demonstrate that monoclonal antibody 4B9 is directed against an antigenic determinant common to molecules present on some serotypes of E. coli, E. cloacae and Streptococcus group B.
  To further define the molecular nature of the antigen, deoxycholate extracts were treated with the proteolytic enzyme proteinase K before being electrophoresed (Eberling, W., supra). Immunoblot patterns observed after treatment with proteinase K were identical to those observed without treatment. This indicates that the antigen reacting with antibody 4B9 is not a protein by nature.
  To accurately determine whether the antibody 4B9 reacted with a carbohydrate epitope, the electro-transferred deoxycholate and wheat germ agglutination-affinity purified sample were subjected to mild periodate oxidation before reacting with the nitrocellulose paper containing the antibody was (see Example I). The electroblotted deoxycholate extracts thus treated did not react with the monoclonal antibody 4B9. These data strongly suggest that the epitope known by this antibody is a carbohydrate moiety present in molecules having both the gram-negative and gram-positive bacteria described herein.
• 4. The isotype of the monoclonal antibody 4B9 was with Help of an ELISA determined that described above Specificity test is similar. However, the included Antigen plate immobilized a pool of PLL Group B Streptococcus types II and III. A positive Reaction of Monoclonal Antibody 4B9 with the Group B Streptococcus strains were treated only with the observed an IgM isotype for IgM reagent the monoclonal antibody is displayed.
• 5. The in vitro functional activity of monoclonal antibody 4B9 was examined in an opsonophagocytosis assay by comparing the bactericidal activity of the antibody in the absence and presence of both human neutrophils and human complement.
  The bacterial strains used here were inactivated only in the presence of the monoclonal antibody 4B9, an active complement source and human neutrophils (Table 6). After repeating this experiment with various 4B9 non-responsive bacterial serotypes, no bacterial destruction was observed (data not shown). This demonstrates the functional specificity of monoclonal antibody 4B9 and its capacity to opsonize bacteria and promote their phagocytosis. Since the combined effects of opsonins (specific antibodies) and polymorphonuclear leukocytes (neutrophils) appear to be the primary mechanism of immunity for these bacterial strains, these data indicate that antibody 4B9, after appropriate administration, protects against lethal challenges with those described herein Bacteria granted.

  Table 6

• 6. To test the above hypothesis, animal protection experiments were carried out with the antibody 4B9 and at least one organism of each genus described here.
  For each Gram-negative bacterial challenge, female, non-cognate Swiss-Webster mice weighing 20 to 22 g were used. The mice were divided into three groups of 10 mice each. A representative experiment was conducted as follows: Each group receiving the antibody was injected intravenously with 200 μl of PBS (sterile, containing 25 μg of purified antibody). Two hours later, all animals were challenged with 300 μl of live bacteria (containing three times the LD₅₀ amount of the corresponding bacterial strain) intraperitoneally. The bacterial suspension had been prepared from a log culture in the logarithmic growth phase. Out there, the bacteria have been spun down. They were washed 2X in PBS and resuspended to a suitable concentration in PBS. The animals were observed for a period of 5 days. Twenty-four to 48 hours after challenge, all animals of group B (irrelevant antibody) and group C (irrelevant organism) were dead. In contrast, the animals receiving antibody 4B9 (group A) were all alive and free of symptoms.
  To investigate the protection against Streptococcus group B a neonatal rat model was used. Non-pediatric young Sprague-Dawley rats (kept with their mothers) less than 48 hours old received the antibody and bacteria in substantially the same manner as in the mouse protection studies. The primary differences were as follows: (1) both the antibody and bacterial challenges were injected intraperitoneally and (2) the inoculum size was reduced to 20 μl.
  These animal protection models were used to demonstrate the therapeutic effect of antibody 4B9 on bacterial challenges with organisms belonging to the three species described herein. A summary of this data is given in Table 7 wiederge.

  Table 7

  These data show that human monoclonal antibody 4B9 is able to protect mice and rats from lethal challenges with bacterial genera belonging to both the Gram-negative and the Gram-positive bacterial group. Intergenus cross-reactive human monoclonal antibody 4B9 was able to mediate protection after infection after administration of an amount of 25 μg (purified antibody) caused by organisms belonging to the Gram-negative bacterial genera E. coli and the gram-positive bacteria of the group B Streptococcus.

Example V

In this example V are methods of manufacture and Selection of a human monoclonal antibody described of an intergenus cross-reactivity against members of the Generic Serratia marcescens, Klebsiella pneumoniae and Possesses Enterobacter aerogenes. Furthermore, in this Example the in vitro opsonizing activity of this anti body against homologues S. Marcescens, K. Pneumoniae and E. aerogenes serotypes. That in Example I (there essentially in sections A to G) be was repeated, but it was necessary was to perform specific modifications to the Characterize and describe antibodies described herein investigate. Below are the in the assay procedure changes made and those described here Monoclonal antibodies obtained results closer he explained:

• 1. Culture supernatants from four transformations were analyzed by the method described above. This resulted in the identification of a well (7E10) that had binding activity on at least one of the four K. pneumoniae serotype plates containing the capsular serotypes; 1, 2, 3, 4, 6, 8, 9, 19, 20, 21, 24, 27, 31, 43, 44 and 55, but not on the control plate (no bacteria). The antibody from the 7E10 cell line was screened for further intergenus cross reactivity by a modification of the standard immunoblot technique. In particular, the cross-reactivity for the following bacteria was investigated: K. pneumoniae, E. aerogenes, S. marcescens, E. coli and P. aeruginosa. For this, spots of bacteria were placed on a screened nitrocellulose paper disc. The disc containing the bacteria was reacted with the antibody and the antibody reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system.
  From these experiments it could be deduced that the antibody 7E10 sits further intergenus cross-reactivity be. This antibody reacted with the following species and serotypes: The monoclonal human antibody 7E10 thus had intergenus cross-reactivity for bacteria belonging to the genera K. pneumoniae, S. Marcescens and E. aerogenes.
• 2. The isotype of the monoclonal antibody 7E10 was with Help determined by an ELISA, the be in Example I be but with specificity tests the antigen plate a pool of PLL (poly-L-lysine) immobilized K. pneumoniae K8 and K11 serotypes ent held. A positive reaction of the monoclonal antibody 7E10 with K. pneumoniae serotypes was only with the Anti-IgM reagent observed. This shows an IgM isotype for the monoclonal antibody. The expert on the It is clear in this area that, if one the method described in this example several  Times would repeat and the isotypes of it so keep intergenus cross-reactive monoclonal anti body would determine other isotypes, eg. B. IgM and IgG isotypes.
• 3. The in vitro functional activity of the monoclonal antibody 7E10 was examined in an opsonophagocytosis assay, which compared the bactericidal activity of the antibody in the presence and absence of both human neutrophils and human complement. The bacterial serotypes tested here were inactivated only in the presence of monoclonal antibody 7E10, an active complement source, and human neutrophils (Table 8). If this experiment was repeated with serotypes that did not react with the antibody 7E10, then no destruction of the bacteria could be observed (the data are not listed). These experiments illustrate the functional specificity of the monoclonal antibody 7E10 as well as its capacity to opsonize bacteria and to support their phagocytosis.

  Table 8

Example VI

In this example VI are methods of manufacture and Selection of a monoclonal antibody described, the Intergenus cross-reactivity against members of the genera Serratia marcescens and Klebsiella pneumoniae. In this example is also the opsonizing in vitro Activity of this antibody against homologous S. Marcescens and K. pneumoniae serotypes. That in Example I described method (there substantially in the Ab cut A to G described) was repeated, where it however, it required specific modifications to administer the antibody described here characterize and investigate. Below are the made changes in the assay method and the results obtained with this monoclonal antibody listed.

• 1. Culture supernatants from four transformations were analyzed by the method described above. This resulted in the identification of one well (8C9) that had binding activity on at least one of the four K. pneumoniae serotype plates containing the following capsule serotypes: 1, 2, 3, 4, 6, 8, 9, 19 , 20, 21, 24, 27, 31, 43, 44 and 55; However, not on the control plate (no bacteria) possessed. The antibody from cell line 8C9 was assayed for further intergenus cross reactivity using a modified standard immunoblot technique. In particular, the cross reactivity for the following bacteria: K. pneumoniae, E. aerogenes, S. marcescens, E. coli and P. aeruginosa was investigated. For this purpose, spots of the bacteria were placed on a screened nitrocellulose paper disc.
  This bacteria-containing disc was reacted with the antibody. The antibody reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system.
  From these experiments it could be deduced that the antibody 8C9 has further intergenus cross-reactivity. This antibody reacted with the following species and serotypes: K. pneumoniae S. Marcescens K5, 6, 7, 14, 27, 36, 55, 64 03 The monoclonal human antibody 8C9 thus had intergenus cross reactivity for bacteria belonging to the genera K. pneumoniae and S. marcescens.
• 2. The isotype of the monoclonal antibody 8C9 was with Help determined by an ELISA, the be in Example I be described specificity tests, but the Antigen plate immobilized a pool of PLL K. pneumoniae contained K14 and K27 serotypes. A positive reaction of the monoclonal antibody 8C9 with K. pneumoniae serotypes were identified only with the anti-IgM reagent. This demonstrates one IgM isotype for the monoclonal antibody. It is for the skilled person that, if the Ver driving this example would repeat several times and the isotypes of the intergenus cross thus obtained would investigate reactive monoclonal antibody, other isotypes, eg. For example, find IgM and IgG isotypes would.
• 3. The in vitro functional activity of monoclonal antibody 8C9 was examined in an opsonophagocytosis assay, comparing the bactericidal activity of the antibody in the presence and absence of both human neutrophils and human complement. The bacterial serotypes used here were only inactivated in the presence of monoclonal antibody 8C9, an active complement source, and human neutrophils (Table 9). When this experiment was repeated with serotypes that did not react with the 8C9 antibody, no destruction of the bacteria could be observed (the corresponding data are not listed). These experiments illustrate the functional specificity of the monoclonal antibody 8C9 and its capacity to opsonize bacteria and promote their phagocytosis.

  Table 9

Example VII

In this example VII are methods of production and Selection of a human monoclonal antibody described Intergenus cross reactivity against members of the genera  Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes and Enterobacter cloacae. In this case play is still the in vitro opsonic activity of this Antibody to homologous S. Marcescens, K. pneumoniae, E. aerogenes and E. cloacae serotypes. The im Example I method (essentially the in the parts A to G) was repeated, however, it required specific modifications to use the antibody described here characterize and investigate. Below are the Changes made in the assay procedure and those with the monoclonal antibody described herein obtained results explained in more detail.

• 1. Culture supernatants from four transformations were analyzed by the method described above. This resulted in the identification of one well (1E4), the binding activity on at least one of the four K. pneumoniae serotype plates containing the following capsule serotypes: 1, 2, 3, 4, 6, 8, 9, 19, 20, 21, 24, 27, 31, 43, 44 and 55 but not on the control plate (no bacteria). The antibody of the cell line 1E4 was examined for further intergenus cross-reactivity by means of a modified standard immunoblot technique. In particular, the cross-reactivity for the following bacteria was investigated: K. pneumoniae, E. aerogenes, S. Marcescens, E. coli, E. cloacae and P. aeruginosa. For this purpose, spots of bacteria were placed on a screened nitrocellulose paper disc. This disk containing the bacteria was reacted with the antibody. The antibody reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system.
  From these experiments it was found that the antibody 1E4 possesses intergenus cross reactivity. This antibody reacted with the following species and serotypes: The human monoclonal antibody 1E4 thus had intergeneric cross reactivity for bacteria belonging to the species K. pneumoniae, S. Marcescens, E. cloacae and E. aerogenes.
• 2. The isotype of monoclonal antibody 1E4 was determined by an ELISA similar to the specificity test described in Example I except that the antigen plate contained a pool of PLL immobilized K. pneumoniae K3 and K8 serotypes. A positive reaction of the monoclonal antibody 1E4 with the K. pneumoniae serotypes was observed only with the anti-IgM reagent.
  This shows an IgM isotype for the monoclonal antibody. It will be apparent to one of ordinary skill in the art that, if one were to repeat the procedure of this example several times and determine the isotypes of the intergenus cross-reactive monoclonal antibody thus obtained, one would find additional isotypes, e.g. B. IgM and IgG isotypes.
• 3. The in vitro functional activity of the monoclonal antibody 1E4 was examined by means of an opsonophagocytosis assay in which the bactericidal activity of the antibody was compared in the presence and in the absence of both human neutrophils and human complement. The bacterial serotypes used here were only inactivated in the presence of the monoclonal antibody 1E4, an active complement source and human Neutrophil (Table 10). If this experiment was repeated with serotypes that did not react with the antibody 1E4, then no destruction of the bacteria could be observed (the corresponding data are not listed). These experiments illustrate the functional specificity of the monoclonal antibody 1E4 and its capacity to opsonize bacteria and promote their phagocytosis.

  Table 10

Example VIII

In this example VIII are methods of production and Selection of a human monoclonal antibody described Intergenus cross-reactivity against members of the possesses the following genera: Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes and Pseudomonas aeruginosa. In this example is also the in vitro Opsonizing activity of this antibody against homologous S. Marcescens, K. Pneumoniae, E. Aerogenes and P. Aeruginosa Serotypes described. The Ver described in Example I. drive (there essentially in sections A to G) was repeated, but it was necessary to perform specific modifications to the be to characterize and underwrite written antibody search. Below are the changes in the assay procedure as well as the monoclonal ones described herein Antibody results obtained explained.

• 1. Culture supernatants from four transformations were analyzed by the method described above. This resulted in the identification of one well (9D1) that had binding activity on at least one of the four K. pneumoniae serotype plates containing the following capsule serotypes: 1, 2, 3, 4, 6, 8, 9, 19, 20, 24, 27, 31, 43, 44 and 55 but not on the control plate (no bacteria). The antibody from cell line 9D1 was assayed for further intergenus cross-reactivity using a modified standard immunoblot technique. In particular, the cross reactivity for the bacteria K. pneumoniae E. aerogenes, S. Marcescens, E. coli and P. aeruginosa was examined. Spots of bacteria were placed on a screened nitrocellulose paper disc. This disk containing the bacteria was reacted with the antibody. The anti-body reactions were developed with an alkaline phosphatase / nitroblue tetrazolium enzyme system.
  These experiments demonstrated that antibody 9D1 has further intergenus cross reactivity. This antibody reacted with the following species and serotypes: The monoclonal human antibody 9D1 thus had intergenus cross reactivity for bacteria belonging to the genera K. pneumoniae, S. Marcescens, E. aerogenes and P. aeruginosa.
• 2. The isotype of the monoclonal antibody 9D1 was in a ELISA determined that described in Example I. Specificity tests are similar, but with the antigen plate a pool of PLL immobilized K. pneumoniae K13 serotypes contained. A positive reaction of the monoclonal Antibody 9D1 with the K. pneumoniae serotypes was observed only with the anti-IgM reagent. This will be a IgM isotype for the monoclonal antibody shown. It is the expert in the field in question here Clear, that one, if one described in this example Repeat procedure several times and the isotypes the thus obtained intergenus cross-reactive monoclonal Antibody would determine to find other isotypes would, for. For example, IgM and IgG isotypes.
• 3. The in vitro functional activity of the monoclonal antibody 9D1 was examined in an opsonophagocytosis assay, which compared the bactericidal activity of the antibody in the presence and absence of both human neutrophils and human complement. The bacterial serotypes used herein were inactivated only in the presence of monoclonal antibody 9D1, an active complement source, and human neutrophils (Table 11). If this experiment was repeated with serotypes that did not react with the antibody 9D1, then no destruction of the bacteria could be observed (the corresponding data are not listed). These experiments illustrate the functional specificity of the monoclonal antibody 9D1 as well as its capacity to opsonize bacteria and promote their phagocytosis.

  Table 11

Example IX

In this example IX are methods of production of a monoclonal human antibody, the intergene Cross reactivity against members of the following species has Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Serratia marcescens Marcescens). Further in this example is the in vivo Protective activity of this antibody against a lethal Challenge homologous P. aeruginosa, E. coli and S. marcescens serotypes described. The Ver described in Example I. go (there essentially in sections A to G) described) was repeated to obtain a monoclonal human antibody to produce the cross-protective against Infections that are caused by bacteria to which he binds. Here in this example are specific modifications of that described in Example I. Procedure to characterize the antibody and to investigate. The ones made in the assay procedure Changes as well as those with the monoclonal antibody he The results obtained are explained below.

• 1. The supernatants were in the presence of anti-P. Aeruginosa antibodies screened using the ELISA technique described in Example I. The antigen plate consisted of a 96-well flat bottom immunolone 2 microtiter plate (Dynatech, Alexandria, VA). The wells of this microtiter plate contained a mixture of poly-L-lysine PLL-immobilized bacteria belonging to seven P. aeruginosa Fisher reference strains (Fisher MW et al., J. of Bacteriology (1969), 98, 835-836, ATCC Nos. 27312-27318).
  Culture supernatants from transformation were analyzed by the method described above and resulted in the identification of a well having activity on the P. aeruginosa plate but not on the PLLL control plate. In subsequent ELISA studies with 17 individual P. aeruginosa serotypes belonging to the International Antigenic Typing Scheme (IATS, ATCC Nos. 33348-33364), it was determined that a "master" well contained 9C3 antibodies binding to IATS Serotype type 1 (Liu PV, Int., J. Syst. Bacteriol. (1983) 33, 256-264, incorporated herein by reference).
  This experiment thus resulted in a cloned transformed human cell line that is continuous (immortal) and secretes a single monoclonal human antibody that binds to a determinant on the surface of P. aeruginosa IATS type 1.
  The continuous transformed human cell line designated 9C3 was deposited with the American Type Culture Collection of Rockville, MD prior to the priority date of the present application under the number ATCC CRL 92.39.
• 2. The antibody from the cloned cell line 9C3 was also tested for its cross-reactivity to Gram-negative and Gram-positive bacteria. This was done by a modified standard immunoblot technique. In particular, the cross-reactivity for the following bacteria was investigated: E. Coli, K. pneumoniae, S. Marcescens, E. aerogenes, E. cloacae, Haemophilus influenzae and Staphylococcus aureus. Spots of bacteria were placed on a screened nitrocellulose paper disc. The disc containing this bacteria was reacted with antibody 9C3. The anti-body reactions were visualized with an alkaline phosphatase / nitro blue tetrazolium enzyme system (described in Example I).
  These experiments show that antibody 9C3 binds to E. coli serotype 06 and S. Marcescens serotypes 012 and 14. The monoclonal human antibody 9C3 thus has intergenus cross reactivity between the Gram-negative bacteria belonging to specific serotypes of the species E. Coli, S. Marcescens and P. aeruginosa.
• 3. The finding that the monoclonal antibody cross reacted with various different bacterial genera shows that this antibody can bind to a common antigenic determinant. The biochemical characterization of the molecular species recognized by antibody 9C3 was performed by immunoblot analysis (described in Example I). Reactions were found in deoxycholate extracts of the reactive serotypes of P. aeruginosa and E. coli, but not by S. Marcescens. Although it is not clear why the antibody 9C3 did not react with the S. Marcescens preparation, it is possible that the antibody recognizes a conformational epitope that has been destroyed by the methods of preparation. For the E. coli and P. aeruginosa bacterial extracts, the antibody 9C3 appears to recognize a series of regularly spaced molecular entities which result in a ladder-like pattern on the immunoblot. This profile was fully consistent with that observed in polyacrylamide gel electrophoresis analyzes of LPS in the presence of SDS. There it could be shown that the heterogeneous size profile indicated by the bands is due to a population of LPS molecules differing in weight increments, which is equivalent to the number of O-antigenic oligosaccharide side-chain units present per molecule ( Pavla ET and Makela PH, supra, and Goldman RD and Leive L., supra).
  To further define the molecular nature of the antigen, deoxycholate extracts were treated with proteolytic enzyme, proteinase K, before being subjected to electrophoresis (Eberling W., supra). The immunoblot patterns observed after proteinase K treatment were identical to those observed without treatment. This shows that the antigen reacting with antibody 9C3 was not a protein.
• 4. The isotype of the monoclonal antibody 9C3 was with an ELISA method, the above be described specificity tests, but the Antigen plate PLL immobilized P. aeruginosa Fisher serotype Contained 4 bacteria. A positive reaction of monoclonal antibody 9C3 with P. aeruginosa Strain was observed only with the anti-IGM reagent. This shows an IgM isotype for the monoclonal anti body.
• 5. The in vitro functional activity of monoclonal antibody 9C3 was examined in an opsonophagocytosis assay comparing the bactericidal activity of the antibody in the absence and presence of both human neutrophils and human complement.
  The bacterial strains used here were killed only in the presence of the monoclonal antibody 9C3, an active complement source and human neutrophils (Table 12). When this experiment was repeated with various bacterial serotypes unresponsive to the antibody 9C3, no bacterial destruction was observed. This illustrated the functional specificity of monoclonal antibody 9C3 and its capacity to opsonize bacteria and promote their phagocytosis. Since the combined action of opsonins (specific antibodies) and polymorphonuclear leukocytes (neutrophils) appears to be the primary cause of immunity for these bacterial strains, these data indicate that the antibody 9C3, after appropriate administration, protects against lethal challenges with those described herein Can mediate bacterial strains.

  Table 12

• 6. To study the protective properties of antibody 9C3, protection experiments were performed on animals with at least one organism from each genus described here.
  The model in which mice are burned (modeled mouse) was used for the protection experiments with P. aeruginosa Fisher 4 and S. Marcescens 014. For each bacterial challenge, non-cognate female Swiss Webster mice weighing 22 to 25 g were used. The mice were divided into three groups of 7 or 8 mice each. An exemplary experiment was performed as follows: The day before the experiment, each mouse was shaved and treated with a depilatory to remove all the fur on the back (burn site). On the day of the experiment, each animal received 0.1 ml of anesthetic saline containing 0.7 ml of 0.85% NaCl, 0.2 ml of xylazine (20 mg / ml), 0.1 ml of ketamine (100 mg / ml ). The dosage was such that 20 mg / kg of cylazine and 180 mg / kg of ketamine per mouse were administered. In anesthetized mice, 10% of total body area (full thickness) was burned with a gas flame (3rd degree). Immediately after the wounds had been added to the mice, they were injected under the fire scab with 0.5 ml of the spent culture fluid (4 ° C, premixed with 5 to 10 LD doses of the bacteria) containing the antibody.
  The bacterial suspension had been recovered from a log culture log culture from which the bacteria had been centrifuged. The bacteria were washed twice in PBS and resuspended in PBS to a suitable concentration. The animals were observed for a period of 10 days. Three to five days after challenge, all animals in group B (irrelevant antibody) and group C (irrelevant organism) were dead. In contrast, all animals that received antibody 9C3 (group A) were alive and free symptoms (see Table 13).
  For the protection studies with E. Coli 06, healthy Swiss Webster mice (20-22 g) were divided into three groups of 10 mice each. Each group receiving the antibody was injected intravenously with 20 μl of PBS (sterile, containing 25 μg of purified antibody). Two hours later, all animals were challenged intraperitoneally with 300 μl of live bacteria (containing 3 LD₅₀ doses of the corresponding bacterial strain). The results are summarized in Table 13.

  Table 13

These data show that the monoclonal human antibody 9C3 is able to challenge mice with lethal challenges To protect bacteria, which causes three gram-negative Genera belong. The Intergenus Cross-reactive Monoclonal Human antibody 9C3 was able to mediate protection with a culture supernatant containing antibody or a purified antibody against infection, the caused by organisms which are among the most gram- negative genera E. coli, S. marcescens and P. aeruginosa belong.

The above statements show that the cell of the invention Lines Compositions of monoclonal human anti provide bodies and fragments thereof, which ver different bacterial species, both gram-negative and also gram-positive, can cross-react and before to cross-protect this. This allows for deployment prophylactic and therapeutic agent that is effective are against by most, if not all, bacteria Genera caused nosocomial and neonatal infections By combining the antibodies it is possible to have a broad protection against a large part (usually however not especially) of clinically significant bacteria to achieve. In addition, the cell lines provide antibodies ready to use in immunoassays and other well-known Ver drive find application.

Claims (19)

A human monoclonal antibody characterized in that it is capable of reacting with at least two bacterial species of at least two genera, the antibody or a binding fragment thereof being capable of specifically reacting with a non-nuclear carbohydrate epitope, the serotype of two different species is common. 2. Monoclonal human antibody according to claim 1, characterized in that the antibody or a binding fragment thereof with Sero types and species of at least three bacteria species and genera responds. 3. Monoclonal human antibody according to claim 1, characterized in that at least one with him responsive bacterial species gram-positive and a second species gram-negative is. 4. Monoclonal human antibody according to claim 1, characterized in that it Protects infections caused by bacteria which are called to at least two belong to different genera.   5. Monoclonal human antibody according to one of Claims 1 to 4, characterized in a specific way with a non-nuclear carbohydrate epitope responds, the sero types of two or more species of bacteria under different genera and a plurality of Serotypes of at least one species is common, these are the species Escherichia coli, Neisseria meningitidis, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Pseudomonas aeruginosa and Streptococcus agalactiae group B is. 6. Monoclonal human antibody according to claim 5, characterized in that it comprises at least one of the following combinations of bacteria species: (a) E. coli, S. marcescens and E. aerogenes; (b) E. coli and N. meningitidis; (c) E.coli, E. cloacae and Streptococcus agalactiae group B; (d) K. pneumoniae and S. marcescens and E. aerogenes; (e) K. pneumoniae and S. marcescens; (f) K. pneumoniae, S. marcescens, E. aerogenes and E. cloacae; (g) K. pneumoniae, S. marcescens, E. aerogenes and P. aeruginosa or (h) E. coli, S. marcescens and P. aeruginosa.   7. Monoclonal human antibody according to one of Claims 1 to 6 for the treatment of neonatal Sepsis or meningitis, characterized that he is in a specific way with a Non-nuclear carbohydrate epitope responds to the serotypes two or more bacterial species below different genera is common, it being these species are Escherichia coli Ki, Neisseria meningitidis group B, Hemophilus Influenzae type B and Streptococcus agalactiae Group B acts. 8. Monoclonal human antibody according to claim 1, characterized in that he or a binding it's capable of fragmenting it with a total of four bacterial species too react. 9. Immortal cell line, the monoclonal human antibody or binding fragments thereof according to one of claims 1 to 8 secreted in specifically with at least two Serotypes of different species different Bacteria genera existing epitope with a accessible non-nuclear carbohydrate unit can cross-react. 10. cell line according to claim 9, characterized records that it is the bacterial species  around at least two of the following species Escherichia coli, Serratia marcescens, Neisseria meningitidis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Pseudomonas aeruginosa and Streptococcus agalactiae group B. 11. Cell line according to claim 9 with the ATCC accession numbers: CRL 9006, CRL 9007, CRL 9008, CRL 9009 and CRL 9239. 12. Monoclonal human antibody or binding Fragment of it that is capable to bind to a carbohydrate epitope which with a monoclonal antibody can react from a cell line Claim 11 is produced. 13. Use of a cell line according to one of Claims 9, 10 or 11 for the preparation of a monoclonal human antibody specific cross with a non-nuclear carbohydrate epitope It can react to at least two serotypes of at least two different species different bacterial genera is present.   14. Method for producing monoclonal human antibodies according to one of claims 1 to 8, characterized that isolates the B cells of an individual, these B cells to form a variety of antibodies immortalizing producing clones, these clones on the Production of antibodies that are specific for a screent Non-nuclear carbohydrate epitope of at least two different bacterial species are, and the cultivated selected clones and produced thereby Monoclonal antibody wins. 15. The method according to claim 14, characterized in that the B cells are immortalized by EBV transformation. 16. A human monoclonal antibody according to any one of claims 1 to 8, characterized in that it is a monoclonal Antibody or a binding fragment thereof is, the or that with E. coli and Streptococcus group B serotypes Intergenus cross-reacts. 17. Monoclonal human antibody according to any one of claims 1 to 8, characterized in that it is a monoclonal Antibody or a binding fragment thereof is, the or that with E. coli capsule type K1 and N. meningitidis group B Polysaccharide reacts. 18. Pharmaceutical agent containing a therapeutic or prophylactically effective amount of a monoclonal Human antibody according to any one of claims 1 to 8, 12 or 16 and 17 in combination with a pharmaceutical compatible carrier. 19. Pharmaceutical agent according to claim 18, in addition containing an adjuvant.