Compositions of human monoclonal antibodies with multiple protection.

29-07-1988 дата публикации
Номер:
OA0000008477A
Принадлежит: Genetic Systems Corp
Контакты:
Номер заявки: 59-00-1987063
Дата заявки: 06-02-1987

[1]

The present invention concerns the use of immunological techniques for the preparation of novel products useful in the treatment and diagnosis of bacterial infections and, more particularly, has the preparation and application of human monoclonal antibodies capable, protect against infection caused by different genera. bactériesc of

[2]

The bactéries'bactéries' gram-positive and gram-negative could cause diseases that threaten the life of patients who are infected by it.•these bacterial infections often causes morbidity and mortality importantesc found the occurrence of increasingly frequent these - infection in infants born avantavant.terme, elderly patients and•having a state and suffering from serious medical - by ' example of burns, a traumastism surgical, wound-healing and long. of malignant neoplasm. These infections are typically original nosocomi 'en (it is to say acquired in hospitals), particularly the etet.elles strike patients who underwent prolonged hospitalization associated with surgery, infection intrasvasçulaire or long-term therapy with immunosuppressive agents or d-'antibiotiques. Means further, neonates whose immune system is immature fruit.. are apparently highly likely to be exposed to.. a neonatal sepsis. and. meningitis- caused by bacteria -.

[3]

•gram-negative and gram-specific positive.

[4]

Among the - organisms that encountered most frequently in - the 'diseases gram-negative and gram-positiveson include: Escherichiacoli' (Ξ, Escherichia coli), Klebsiella (pneumoniae are K), Serratia (3. marcescens to), the Enterobacter clocae acrogenes and (I. aerogenes was/clocae), or Pseudomonas aeruginosa (P-. pseudomonas aeruginosa), îîeisseria meningitids (n-. further antigen), group b streptococcus and Staphylococcus aureus

[5]

(Staphylococcus aureus) [Sonnenwirth UI, "tea Fermentation bacilli bacteria NDA riken gram negatively Bacteria Limited", ρρ. 753 - 790, in microbiology- ,, 2ème editing, the Davis B.D., Eagle R, versterking Η Ν.., Ginserberg HS, the Wood W. B. and McCarty Μ ., EDS, Harper for NDA (1973) ; rowwise. McCabe and W.R., "gram at a negative Bacteremia", VDA. Labeling. 19 Med., 135 - 138 (1974); Kreger et, " gram at a negative Bacteremia lll. Yonggang d'Etiology, Johns, and Food in-612", with am. The MED J. 68, 332 - 343 (1980); Required from J.B. and al, "Escherichiacoli Κ 1 Capsular polysaccharide which Epitopes associated with neonatal nursing meningitides", JAMA nu. J Med., 290, 1216 - 1220 (1974); and Hughs J.M. and al, "Nosocomial infection in monitoring, 1980 - 1982", MORB-.

[6]

Death. Playspaces, 32, (1983) 1ss - 16ss]. Among these infections, several serotypes but less than all, of certain gram-negative bacteria, for example E. coli, K. pneumoniae K, E aerogenes was/clocae, P-. ^ aeruginosa and marcescens was, from causing bacteremia in the adult population. In contrast to adult, the neonate an immature immunologically is particularly susceptible to sepsis and meningitis encapsulated strains caused by e. coli, N. meningitidis of group b, haemophilus influenzae type b and the five types of strain of Streptococcus group b. although other bacteria can also cause these infections, the bacteria mentioned above are the predominant isolates blood infections mentioned above.

[7]

_ antibiotics have represented a long time the primary therapeutic tool control and eradication infections gram-positive and gram-negative. However, the permanence of the occurrence et~ and severity of these infections, the permanence of the emergence of new antibiotic-resistant strains and L, has inherent toxicity of some antibiotics, show jetproof within a therapeutic by antibiotics. These observations have brought to the practice of finding a novel therapeutic dRapproches and prophylaxis.

[8]

It is believed very generally that antibodies reactive with structures accessible (exposed to outside) of live bacteriathe R can facilitate destruction of these bacteria by a mechanism which concha, " mechanisms which include:

[9]

(1) direct lysis of the bacteria in the presence of a serum complement, (2) a bacteriostasis by receptor blockade of nutrient absorption,

[10]

(3) fixing of opsonin and subsequent phagocytosis of bacteria in the presence or in the absence of serum complement, or

[11]

(4) obstruction sputtering. I ' adhesion of bacteria to the tissues of their. hôhô.te [referred to, .

[12]

AC, "sets a from-infecting", in tea d'hiv pathogenesis.; Of Infectious RJ, pages. 198 and 222, Mins, ç.a., Ed., (1982) 3. Academy press assembly, for bacteria that have surface molecules .'d ' carbohydrate of.

[13]

carbon, such as for example lipopolysaccharides (LPS-) and/could; dice capsule, it appears that an antibody is most effective by attachment mechanisms of opsonin [Kaijser B et, "tea circuit protective action of antioxidant E.coli d'O-NDA K. Mitochondrial Antibodies but of Immunoglobulin class", J. Assoc. J Immunol. 1, 276 (1972)]. Therefore, antibodies directed to carbohydrate structures may provide accessible means effective to remove bacteria.

[14]

In general, mammals which are exposed to dice bacteria causing diseases produce antibodies, or specific LPS. a capsule. AAR antigens have different, chemical structures composed of oligosaccharide molecules are frequently performed and whose presence determines tee serotype bacterial strains. Because; often represent immunodominant bacterial antigens, the serotype-specific antibody (anti-LPS factor or anti-cap) have been most studied among lengths of a therapeutic antibody having a potentiality.

[15]

However, due to die the reactivity multi, limited portions thereof, and the nature. apparent extremely diverse ^ carbohydrate antigens on pathogenic bacteria existing carbon gram-positive and gram-negative, it would be extremely difficult and çoOteux die performing forming therapeutic containing only one serotype specific antibodies [see e.g. Kaijser B and Ahlstedt sec., "circuit protective demand of antibodies against foot Escherichiacoli Κ antigenically NDA 0", disgusting. Immune, 17, 286 - 292 (1977) ;. and Morrison and EMI and Ryan "KL.," BacterialEndotoxins NDA the host an immune imidazoquinoline. ", VDA. Immunol. 28, 293 - 450 (1979) Nevertheless, various references have given suggests that one can find immunotherapeutic approaches to find diseases caused by gram-negative bacteria.

[16]

Human plasma fractionated, immunoglobulin-rich containing specific antibodies and protective against organisms causing infections, has shown some efficacy against infections caused by P. aeruginosa [the Collins M.S. and Robey I.R., "pliers where frequent salt of an Intravenous an immune globulin (specific performance) Enriched in-mediated cytotoxic antibodies against antigenically or Pseudomonas aeruginosa lipopolysaccharide from OC", bitter. THE J.

[17]

Med., 3, 168 - 174 (1984)]. However, not available yet easily products on the market because of certain inherent limitations which have prevented from spreading their use in the treatment of bacterial diseases that could threaten the life.

[18]

One of these limitations associated with the composition of the immunoglobulins is that they are gathered from large sets of plasma samples for the presence of a preselected limited number of antibodies particuliers.-to-.Typiquement, the assemblies consist of samples from one thousand donors which may only presents faiblçs titles in a few pathogenic bacteria. Thus, is found at more than a modest increase in the antibody titer should ultimately obtained.

[19]

Another of these limitations is that the preselecting process requires by itself a permanent and very coQteux screening the population of donors to ensure permanence of product quality. Despite considerable efforts, the products can still vary considerably a batch and geographic region to the other.

[20]

•another of these inherent limitations of the composition of the immunoglobulins is still that their use leads to simultaneous administration of large amounts of substances protêinées exogenous (e.g. viruses), representing potential causes of adverse biological effects. The combination low titers of antibodies. and should the high content of exogenous substances limit, often, àà .des lower levels than optimally, the amount " of (e) specific immunoglbbulinès (e) and (e) thus beneficial that can be administered to a patient.

[21]

In 1975, Kohler and Milstein have indicated that certain cell lines could be fused with mouse spleen cells of mouse to generate hybridomas capable of secreting. "monoclonal" antibody pure [Kohler illumination G. and Milstein C, "continu crops increased ability to price differentially and tailor products", in nature, 256, 495 - 497 (1975)]. Vavènement. with this technology, is arranged to make a. mouse antibody directed against any determinant or qualifiers (e) existing on particular antigens.

[22]

With this technology, is-made mouse monoclonal antibodies from mouse ' immunized using a polysaccharide from Neisseria meningitidis group b. it has been observed that these mouse IgM monoclonal antibodies bind and ran on several strains e. coli ml-positive whatever their serotypes LPS-[with cross, super, Soderstrom, super, and cross A.S. and neck! ., "tea significance of imprinted ml cap in-invasively infested because bypass Escherichiacoli", J IFN. Dis., 149, 184 - 193 (1984)]. Further, the monoclonal antibodies were, in mouse, protecting against lethal attacks carried by the E.coli kl and organisms vaccines group b (with cross, super, and Sunderstrom, above). In another example, it was indicated that mouse monoclonal antibodies specific group B streptococcus III were protective against an infection model experimental mouse [in Egan M.L. and coil polymers ., "protective d'AMPA des recordings on infecting avec la III serogroup B the Streptococcus estimation using a monoclonal Mitochondrial Antibodies", J with exp. Mëd ., 1, 1006 - 1011. (1983)]..

[23]

While useful for the treatment of mouse, a mouse monoclonal antibody has major drawbacks when using in humans. The human immune system is capable of recognizing any mouse monoclonal antibody as a foreign protein. This can result in accelerated removal of the antibody and thus completely eliminating its pharmacological effect [commented R and I the Miller CED,, "Tumor therapy increases the monoclonal avec la Mitochondrial Antibodies", FEDs. Proc., 42, 2650 - 2656 (1983)]. More seriously, designed and that this should lead to a shock and even death to caused allergic reactions analogous to that tone calls "the serum sickness". The clinical experience has shown that the responses to the anti-mouse had limited the utility of these antibodies expensive approximately half of the patients receiving mouse monoclonal antibodies for the treatment of various tumors [low Sears RF et, "I phase of newly monoclonal antibody mediated cytotoxic in-her dimerization inhibitor OC a gastrointestinal Tumor", the Lancet, 1, 762 - 764 (1982); and Miller R. and al, "monoclonal antibody mediated cytotoxic improved therapeutic apparatus-course chemotherapy in the Seven avec la T Cel1Lymphoma", Β 1ood, 62, 988 - 995 (1983).].

[24]

It is therefore a-needless to human monoclonal antibodies that are protective against bacterial diseases gram positive and gram-negative. However, the diversity of properties antigens pathogenic bacteria gram-positive and gram-negative suggests strongly that the preparation of human monoclonal antibodies serotype-specific to each of the many important pathogenic bacteria would be impracticable.

[25]

The diversity of properties antigens of gram-negative bacteria is assigned to variable regions of lipopolysaccharide (LPS-), molecules associated with the outer membrane, gram-negative organisms. It is considered in general that the LPS molecule is composed of three structural regions. The region closest to the outer membrane is the so-called lipid a portion of LPS. This conserved region structurally has the endotoxic activity associated with diseases gram-negative. The second structural region that tone calls the core is bonded to the lipid a, often by way of a remainder 2 a-céto~3-deoxy d-mannooctonate (cks) and, as well as for the region of lipid a, it is usually not a-accessible to the antibody when the third outermost region of the LPS is present. Although this region is partial -, telling a conserved within a few species of gram-negative bacteria, we found many deviations in the complete core among the members of the family enterobacteriaceae. The outermost region of a LPS molecule consists of repeating oligosaccharide units and is known as the O-specific side chain. The sugars of these patterns oligosaccharides comprise molecular entities with a variety structural antigen serotype specific. Thus, sugars themselves, their sequence and their linkages nerit the determinants. antigenicity of O-chain side by their tertiary structure. It was generally found that antibodies to these-sets 0 were serotype-specific. The serotypes are defined typically by their reactivity with monospecific antisera which does have a binding activity for only one particular antigen determinant. See generally Mayer and coil polymers ., Meths. Microbiology, 18, 157 - 201 (1985).

[26]

Fusion proteins were prepared. isérums directed against the core and the regions of lipid a of LPS for best efforts demonstrate protection against gram-negative infection. Sakulramrung and Dominguez J IFN. SID, 151, 955 - 1104 (1985); McCabe and al, J disgusting. SID. 1365, 516 (1977); and ' Mullan et, disgusting. Immune ., 10, 1195 - 1201 (1974). Most recently, we préparé_des mouse monoclonal antibodies reactive with the manhole and conserved regions. Although these antibodies have sometimes presented a partial efficiency in vivo in model systems specifically tailored (Teng et, process. Proc. CDAA. Itself. Revenue 82, 1790 (1985); and Bogard and fu, applying 5,411,607 n° w085/01659), other labs have not been able for the purpose of demonstrating a similar effect. See Elkins and Metcalf configuration, disgusting. Immune. 48, 597 (1985); and Gigliotti. and Shenap, J IFN. SID. 151, 1005 - 1011 (1985). Further, the antibodies do not generally react with (or bind to nene.se) gram-negative bacteria viable intact or purified LPS molecules. These findings suggest that it is doubtful that the core or the portions of lipid a of LPS may be accessible to an antibody on bacteria in their natural state or an infectious state. It is also generally1 accepted that the antibodies antinoyau or antilipide has not reacted with gram-positive bacteria because they do not possess LPS. Because of these findings, it is unlikely that monoclonal antibodies directed against the conserved regions of the core or of lipid a of LPS can be effective in the treatment of bacterial diseases human gram-negative or, in this regard, gram-positive.

[27]

The II therefore there is a great need of human monoclonal antibodies that are widely (Coning) reciprocally protectants against diseases caused by gram-positive and gram-negative and the need exists for methods of preparing and using practices of these antibodies. The present invention satisfies these needs.

[28]

Prepared cell lines that provide human monoclonal antibodies capable of specific multiple reactions

[29]

•with a variety of bacterial species by binding an epitope accessible comprising a radical carbohydrate not part of the core present on at least two different bacterial species. Further comprising providing methods of prophylactically treating a human patient subject confectioneries bacterial infection and therapeutic treatment of a patient suffering from such an infection by administering an effective amount of a composition containing a plurality of human monoclonal antibodies, wherein at least one of said antibodies is capable of reacting with an epitope-based carbohydrate, not part of the. core, shared by two or more bacterial species. Preferably the composition comprises a physiologically acceptable carrier and may also contain one or more of the following: additional human monoclonal antibodies capable of reacting with other bacterial genera; a gamma globulin fraction from human blood plasma; a gamma globulin fraction from human blood plasma in which the plasma is obtained from a human having elevated levels. of immunoglobulins reactive with one or more antimicrobial agents.

[30]

The present invention therefore provides new cells capable of producing human monoclonal antibodies and compositions comprising the antibodies, the compositions being capable of selectively react with a number of genera of bacteria that cause infections nosocomiennes, neonatal and other, in - which the individual antibodies typically react with epitopes of carbohydrate is not part of the core present on multiple genera of bacteria. The cells of the invention have identifiable chromosomes in which the true that your line sprout from them themselves or a precursor cell has been rearranged to encode an antibody or binding fragment thereof having a binding site for an antigenic determinant (epitope) shared by molecules of carbohydrate found on at least some serotypes of two or more bacterial genera. These human monoclonal antibodies can be used in any sort of way, including for. the diagnosis, prophylaxis and the therapy of bacterial diseases.

[31]

Typically, the cells of the invention the present invention will be cells capable of stably producing a human antibody by culturing, and especially lymphocytes immortalized human which give rise to the formation of human monoclonal antibodies protective against carbohydrate determinants that are not in the existing core molecules accessible shared by more than one bacterial species. By "reachable", it denotes that the carbohydrate determinants that are not in the core are physically available in the environment to be used direct interaction with the monoclonal antibodies. The monoclonal antibodies so produced are useful in the treatment or prophylaxis-of serious diseases caused by any a series of bacterial infections. Further, these molecules of carbohydrate is not part of the core and which are released in 1' immediate environment, are also free to interact directly with the antibody molecules and likely to be eliminated via the reticuloendothelial system.

[32]

The compositions containing the monoclonal antibodies of the present invention are typically useful in the therapeutic and prophylactic treatment of nosocomial infections, neonatal or other infections. nosocomiennes infections typically being from infections caused by the following bacteria: the Escherichia coli, or Pseudomonas aeruginosa, Klebsiella, Enterobacter species aerogenes was/clocae, a Serratia and Streptococcus agalactiaeand of group b, we should prefer antibody compositions which are protective against two, three, four or more of these bacteria. Similarly, neonatal for use, for example in sepsis and neonatal meningitis, it is desirable that the antibodies are specific for two or more of the following bacterial organisms: Escherichia coil ml, of Neisseria meningitidis group b, group b Streptococcus agalactiaeand and Hemophilus influenzae type b. other bacterial common infectious comprise the following: the Staphylococcus aureus, Staphylococcus epidermis ·, Streptococcus pneumonia, mirabilis, Proteus vulgaris is, Bacteriodes fragilis could, cepacia, M. tuberculosis, morganii Providencia species, S. typhi, P. carinii, âcinetobacterherellea, Pasturella multocida antigens, the Klebsiella oxytoca. In respect of other pathogenic bacteria concerned known specialists, see Hughs J.

[33]

et, "Nosocomial infection in monitoring, 1980 - 1982", MORB-. Death.

[34]

Playspaces, 32, (1983) 1ss - 16ss, and, generally, Microbiology, 3ème editing, the Davis B.D., Eagle R, versterking H.N., Ginserberg HS, the Wood W. B. and McCarty Μ ., SDR. (1980) NDA rowwise Stephen Harper, both being incorporated herein as a reference. The monoclonal antibodies will react with individual or with all members of a particular bacterial species, these members which are distinguishable by their surface epitopes, including a history of LPS or capsules, for example their serotypes.

[35]

The unexpected determination of reactivity multiple of monoclonal antibodies against several bacterial species, including species clinically important listed herein, provides novel means of therapeutic and prophylactic treatments. Using antibodies to preselected multi reactivity in combination, can be prepared a mixture of antibodies for some! a processing control a number of different species of infectious bacteria.

[36]

By way of non-limiting example, a mixture of two monoclonal antibodies, V a; of them to multiply reactivity against at least two bacterial species of clinical significance and. the second multi-reactivity against at least two or three different species will be useful in treatment of fighting four, five, six or more different species. Adding a third or of one-fourth monoclonal antibody, each of which has a multi-reactivity against at least two species of clinically significant - even if one or more than one of these species is the same as that recognized by the first and/or second antibody - will increase its usefulness in treating fighting five to ten or more than ten species. Naturally, it may be necessary to add yet one or more monoclonal antibodies, each specific being only one individual bacterial species preselected, for example when multi-reactive monoclonal antibodies against of this species are not available.

[37]

Similarly, the present invention provides also for new methods for treating bacterial infections. Again, by way of non-limiting example, a novel method comprises treating a patient suspected to be the object or be susceptible to bacterial infection caused by a bacterial species selected. The treatment comprises the administration of a composition based on a monoclonal antibody reactive to the bacterial species suspected of causing the infection, the monoclonal antibody is originally characterized by reactivity with a different bacterial species.

[38]

As, another example, include a method of treating a bacterial infection by administering compositions which comprise a number of monoclonal antibodies reactive with a substantial proportion (cjcj.es T-i.e. greater than 50%, preferably 60 to 80% or more, particularly about 90%) of clinically significant bacterial species preselected, the number of antibody is at least less than two to the number of bacterial species. Typically, if "the n" represents the number of bacterial species, the composition will include about n - 2 antibody, more typically about n 4 or n-8 or less antibody for treatment to about 15 to 20 against bacterial species.

[39]

In situations Ou treatment ' for combating a broad spectrum (e.g. 25 to 50 or more) of bacterial species is desired;

[40]

the composition will comprise typically n 10 to n - 20 antibody or less.

[41]

. The preparation of monoclonal antibodies can be effected in an immortalizing the expression of nucleic acid sequences that encode for antibodies or binding fragments thereof that are specific for an epitope of hydrate-of carbon is not part of the core on several bacterial species. Typically, the monoclonal antibodies can be prepared by cell transformation caused by the virus of epstein-to-8arr (e8v). lymphocytes obtained from human donors that are or that have been exposed to gram-negative bacteria respective. Cell lines secreting antibody thus prepared are characterized in that they give rise permanently to lymphoblastoid cells having a diploid karyotype ', in that they' are positive with respect to the nuclear antigen of epstein-Barr virus (EBNA) and that they secrete a monoclonal antibody type either IgGs, the IgM, IgA or DDI. The cellular transformation process in itself is an invention of Genetic Systems Corp. and is described in detail in the Patent of the United States of America no. 4,464 ' 465 incorporated herein as a reference. The monoclonal antibodies may be used intact or as fragments, for example Fv-> Fabs, FCAb ' the j ^, but are traditionally used intact.

[42]

Or alternatively, could be cell lines producing antibodies by cell fusion between human myeloma, myeloma cells of mouse or human lymphoblastoTdes suitably tagged drug with human b cells for performing hybrid cell lines.

[43]

The cell lines according to the presented invention can find another use than the direct preparation of human monoclonal antibodies. The cell lines may be fused with other cells (e.g. human myeloma, myeloma or mouse cells lymphoblastoTdes human suitably tagged drug) to make hybridomas and thereby perform the transfer 1 genes encoding human monoclonal antibodies. Or, the cell lines can be used as a source of DNA-encoding the inrninoglobulines that of isolating and transfer to cells using different techniques of fusion. Further, may be insulated genes encoding monoclonal antibodies and the TES using recombinant techniques for the preparation of the DNA-specific immunoglobulins in a variety of hosts. In particular, by preparing libraries of a known of the messenger, may be isolated single clone encoding the immunoglobulin and free of introns, and installed in a prokaryotic or eukaryotic expression vectors suitable and then transported in a host for a final mass production.

[44]

lymphoblastoTdes cell lines or hybrids may be cloned and screened in accordance with conventional techniques through the antibodies that are capable of binding to the epitopes of the different genera of bacteria detected in the supernatants of cells.

[45]

The monoclonal antibodies of the present invention have particular utility as ingredients in pharmaceutical compositions containing a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of the present invention in combination with a pharmaceutically effective carrier. A pharmaceutical carrier can be any compatible non-toxic substances suitable for administration of the monoclonal antibodies to patients. May be included in the carrier sterile water, alcohol, fats, waxes and inert solids. May also incorporate pharmaceutically accepted adjuvants (buffering agents, dispersing agents) in these pharmaceutical compositions. These compositions may comprise a single antibody-monoclonal antibody reactivity to multiply against carbohydrate epitopes. carbon not part of the core shared by two or more bacterial species that cause e.g. nosocomiennes and neonatal infections (e.g. sepsis or meningitis). Or alternatively, a pharmaceutical composition may contain two or more monoclonal antibodies to form a "coktail". For example, a coktail containing human monoclonal antibodies, each being protective against two or more genera of gram-negative bacteria responsible for human infections, would have activity against the vast majority of common clinical isolates. If desired, one can select one or more multi-reactive monoclonal antibodies against bacteria, gram-positive, showing possible wider product applications.

[46]

Are interesting the prophylactic and/or therapeutic monoclonal antibodies capable of reacting with carbohydrate determinants that are not in the core shared by three or more, usually at least five and more, usually at least ten and up to fifteen or more bacterial serotypes which ' comprise at least one, two, usually at least three, more usually at least five - and usually less than ten bacterial genera.

[47]

The compositions of particular interest are monoclonal antibodies that react with at least about three, preferably at ' least about five and up to and including all common bacteria. nosocomiennes. cause infections following: the Escherichia coli, or Pseudomonas aeruginosa, Klebsiella, Enterobacter species aerogenes was/clocae, a Serratia.=affinity antagonists of Streptococcus group B. agalactfae pourpour.le ections of treatment, it is desirable that the compositions react with at least two, usually at least three, and more usually at least four and up to and including all of the following types of bacteria that cause infections: Escherichiacoli ml, of Neisseria meningitidis group b, group b Streptococcus agalactiaeand, haemophilus influenzae type-b, aureus and Staphylococcus epidermidis.

[48]

Each of compositions will comprise at least two, usually

[49]

at least three to five, and more usually from six to ten human monoclonal antibodies, one at least of these antibodies reactive with epitopes of carbohydrate does. not part of the core (e.g. epitopes of LPS molecules) shared by two or more- two bacterial genera and provides protection. Typically, the. antibody does not will bind to all serotypes of each bacterial but can bind to two, three or more serotypes. It is desirable that there is at least one monoclonal antibody which sese.lie. to a radical carbohydrate not part of the accessible core of at least two genera of gram-negative bacteria and at least one monoclonal antibody that binds to a radical carbohydrate accessible from a gram-negative bacterium and a gram-positive bacterium..

[50]

The molar ratios of various components of a monoclonal antibody does not usually differ from each other. further, 'd ' a 10 factor;

[51]

normally not more than a factor of 5, and will exist usually in molar ratios of about 1/1 to 2 with respect to each of the other components. 1' antibody.

[52]

The human monoclonal antibodies also may find their application individually, especially, one in which the pathogen has been identified, or when it is restricted to narrow of pathogens within the spectrum of antibody binding particular.

[53]

The human monoclonal antibodies according to the present invention may

[54]

- also be used in combination with other monoclonal antibodies (see requesting the same applicant entitled "monoclonal antibody Mitochondrial Antibodies with cross-reactive protein NDA circuit protective antioxidant Ρ. aeruginosa-Car" deposited under number U.S.S.N. 807,394 deposited 10 December 1985, incorporated herein for reference) also bb.ien than with products. existing blood plasma, e.g. gammaglobulins and immunoglobulins commercially available for use in the prophylactic or therapeutic treatment of bacterial diseases of-humans. Preferably, in respect of the immunoglobulins, the plasma will be obtained from human donors having high levels, of immunoglobulins reactive with various infectious bacterial genera. See generally the compendial " Intravenous immune stimulation. The host DNA GlobulinCompromised ", bitter. The MED J.

[55]

76 (3a), 30 March 1984, page 1 to 231, incorporated herein as a reference.

[56]

The monoclonal antibodies of the present invention can be used as separately administered compositions of agents in combination with antibiotics or antimicrobials. Typically, the antimicrobial agents may include a penicillin or a cephalosporin (e.g. carbenicillin, penicillin G, and so on) in conjunction with an aminoglycoside (e.g. qentamjcine^ ^ to verbsanwcinjE ^ FETsc. the L ^, ' but can be used for many other agents (e.g. cephalosporins, sulfonamides, and so on) well known to those skilled.

[57]

The human monoclonal antibodies and pharmaceutical compositions based on the antibodies according to the present invention are particularly useful for oral or parenteral administration. Can be preferably administered pharmaceutical compositions administered parenterally, it is i.e. subcutaneously, intramuscularly or intravenously. The present invention thus provides compositions for parenteral administration which comprise a solution of the human monoclonal antibody or a coktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. May be used a wide array of carriers' aqueous, e.g. water, buffered water, 0.4% saline, 0.3% glycine solution, like these solutions are sterile and generally free of particulate matter. These compositions may be sterilized

[58]

ii

[59]

by1 sterilization techniques conventional and well, known. These Compohandler -

[60]

85 80 may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, for example adjusting agents and pH-buffering, modifiers of the toxicity, like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, so on the antibody concentration such formulations can vary widely, for example less than about 0.5%, usually 1% or at least about 20% or up to 15 by weight and is selected primarily based on fluid volumes of. viscosities, and so on in accordance with the particular mode of administration chosen.

[61]

Thus, a pharmaceutical composition may be prepared is typical for intramuscular injection which contains up to 1 ml sterile buffered water and 50 mg of monoclonal antibody. Preparing a composition could be typical for intravenous injection which contains up to 250 ml of sterile solution Ringers and 150 mg of monoclonal antibody. The actual methods for preparing compositions likely abuts parenteral administration are well-known or obvious for the skilled and are described in greater detail for example in Remington-Peppers pharmaceutique forensic science, 15ème editing, by Mack Publishing, Easton in, of Pennsylvania (1980), incorporated herein as a reference.

[62]

monocTonaux the antibodies according to the present invention may be lyophilized for storage and be reconstituted in an appropriate vehicle prior to use. It has been found that this technique was effective with conventional immunoglobulins and discovered that employ

[63]

1 reconstitution and freeze-drying techniques known in the technology. The skilled worker will appreciate that lyophilization and reconstitution can lead to varying degrees of loss of activity of the antibody (e.g. immunoglobulins or conventional, the IgM antibodies tend to lose more activity that the igg antibodies) and that it may be necessary to adjust the levels of use for for compensation.

[64]

The compositions containing the human monoclonal antibodies according to the present invention or a coktail of these antibodies can be administered to prevent and/or therapeutically treat bacterial infections. In the therapeutic application, these compositions are administered to a patient who is already infected in an amount sufficient to cure or at least partially arrest the infection and these complications. The amount adequate to accomplish these goals is defined as "therapeutically effective dose". Amounts effective for this use depend on the severity of the infection and the general status of the patient's own immune system but are more typically ranging from about 1 to about 200 mg of antibody per kilogram of body weight, doses between 5 and 25 mg per kg being those that tone used most commonly. Bearing in mind that the products according to the present invention may generally be used in the case of serious diseases, it is to say those which threaten life or are susceptible to the threaten, especially in case of bacteremia or endotoxemia. In those cases, owing to the absence of exogenous substances and the lack of rejection of "foreign substance" obtained grSce to the human monoclonal antibodies according to the present invention, it is possible and may feel that it is desirable for the treating physician to deliver significant excess of these antibodies.

[65]

In prophylactic applications ^ compositions comprising the antibodies of the present invention or a coktail of these antibodies are administered àà.un patient who is not yet infected with the corresponding bacteria to enhance the patient's resistance to this potential infection. This amount is defined to be a "prophylactically effective amount"..' in this use, the precise amounts again depending on the health status of the patient and general level of immunity, but they are generally between 0.1 and 25 mg per kg, especially 0.5 and 2.5 mg per kg.

[66]

May be performed with a single or more administrations compositions and dosage levels according to a program to be selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of antibodies according to the present invention sufficient to effectively treat the patient.

[67]

The monoclonal antibodies of the present invention can further find a wide range of use in vitro. For example, monoclonal antibodies can be used for typing bacteria, for the separation of specific bacterial strains or dede.leurs moieties, for the preparation of the vaccines, andc.

[68]

For diagnosis, the monoclonal antibodies may either be labeled, either unlabeled. Typically, the diagnostic tests involve the detection of the formation of a complex by the binding of the monoclonal antibody with the LPS of the organism. When unlabeled, the hill icorps find use in agglutination assays. Further, the antibodies can be used unlabeled in combination with other labeled antibodies (secondary antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for human immunoglobulin. Or alternatively, the monoclonal antibodies can be labeled directly. One can employ a variety of markers, such as radiopaque elements, fluorescent elements, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, binders (particularly haptens), like many types of immuno-assays are available and, as an example, some of these assays are described in the patents of the United States of America no. 3,817 827, 3,850 752, 3,901 654, 3,935 074, 3,984 533, 3,996 345, 4,034 074 4,098 876 and, all of which are incorporated herein as a reference.

[69]

Used-to-commonly the monoclonal antibodies according to the present invention in immunoassays in-enzyme wherein the antibodies according to the present invention, or the secondary antibody from a different species, are associated with an enzyme. ^ whem ' a sample, e.g. human blood or a lysate of human blood, containing one or more bacteria of a certain " genus or serotype, is associated with the antibody of the invention, binding occurs between the antibodies and these molecules which present the selected epitopes. These cells may then be separated from the unbound reagent can be added and a secondary antibody (labeled with an enzyme). After that, the presence of the conjugate specifically binding to the anticorpsenzyme cells is determined. Onon.peut also utilize other conventional techniques well known to skilled artisans.

[70]

May also provide kits for use with s to the antibodies of the invention for detection of bacterial infection or the detection of the presence of a chosen antigen. Thus, the monoclonal antibody composition of the present invention may be provided, usually in a lyophilized form in a container, either alone, or in combination with other antibodies specific other gram-negative bacteria. The antibodies, which may be conjugated or non-conjugated to a label, are included in the kits along with buffers such as tris, phosphate, carbonate, and the like, stabilizers, biocides, inert proteins, e.g. bovine serum albumin, and the like generally, these products will be present in less than about 5% by weight relative to the amount of active antibodies and usually present in total amount of at least

[71]

; 0,001 ¾ weight again to the concentration of the antibody. It will be frequently desirable to include an inert extender or excipient to dilute the active ingredients, the carrier can then be present in an amount between about 1 and 99% by weight relative to the total weight of the composition. When employing a secondary antibody capable of binding to the monoclonal antibody, it will usually be present in a separate vial. Typically, the secondary antibody is conjugated to a label and it enters the formulation analogously to antibody formulations described above.

[72]

The experimental data and the information that follow are exemplary non-. limiting.

[73]

SUCH AS I

[74]

This example demonstrates methods for preparing a human monoclonal antibody having reactivity intergenre multi against members of the genera: the Escherichia coli (e coli), Serratia (marcescens was), K. pneumoniae (K.. k. pneumoniae) and Enterobacter aerogenes (I. aerogenes was). This example provides further demonstration of the protective activity in vivo antibody against a lethal challenge with homologous serotypes (multi-reactivity) e. coli and e. aerogenes was.

HAS. Obtaining suitable human cells

[75]

Human B cells are obtained from peripheral blood suitable an individual known to have been attacked by cystic fibrosis. Are separated from peripheral blood mononuclear cells by conventional centrifugation on ficall-a passover [Boyum A, "insulation d'Mononuclear cells involved NDA granulocyte des Human blood and", J. Assoc.

[76]

0. Clinch. LABs. Increases be ., 21', ACS. 87, 77 - 89 (1968)] and washed twice in 'a phosphate buffered saline free calcium-magnesium (PBSs)' before suspending in 1 ml 90% to Fetal bovine serum (FBS for) and dimethyl sulfoxide to 10% and -196 °c to freezing in liquid nitrogen.

[77]

In process for transforming the mononuclear cells, thawed rapidly at 37 °c an ampule containing 5 x 10 ^ cells. Is- adds the suspension of cells to 10 ml of medium containing 15% Iscôve fBS and centrifugation is performed at room temperature for 10 minutes at 250 x g for. The cellules_mononucléaires are rid of their T cell (lymphocyte) using a modified operating mode rosette-th. In short, the releases cells in suspension at a concentration of 1 x 10^cells/ml in PBS containing 20% fBS to 4 °c. 1 Ml of the suspension is placed in a tube round bottomed polystyrene 17 x 100 mm, to which was added

[78]

1 x 10 sheep red blood cells treated by bromide (PES) 2 a-aminoethylisothiouronium from solution to 10% (v/v) in the middle of Iscôve [m and each district H.E Johnson's "has now subordinated Study both e-rosette forming ion exchange EAT treated fairly characterized herein Per R7 cells involved", J immune.

[79]

Methods 27, 61 - 74 (1979)]. Mixed vigorously the suspension for 5 to 10 minutes at 4 °c and the cells rosette-e are removed through centrifugation by ficall-a passover during 8 minutes at 2500 x g to 4 °c. Peripheral blood mononuclear cells negative for the rosette-th (th " PBMC) - is gathering at the interface were washed once in the middle of iscôve-and is the. again suspended in the same medium containing 15% p/v of fés (grams/ml.), L-glutamine (2 mm/1), sodium pyruvate (1 mm/1), penicillin (100 UI/ml.), streptomycin (100 pg/ml), of T' said hypoxanthine (1 x 10- The m ^) and thymidine (1, 6 x 10" ^ the m). This medium is designated CIRs called hat in medium.

[80]

B cell transformation périphé blood mononuclear cellsdecoding apparatus '

[81]

Cell transformation of e " PBMCs is effected by coculture of e~PBMCs in the presence of a cell line transformation. Transforming the cell line is a cell line from human lymphoblastoTdesEBNA positive by mutagenesis at éthyméthane sulfonate of the cell line lymphoblastoTdes gm to 1500. A selection in the presence of 30 mg/ml of 6 a-thioguanin renders the cells deficient hypoxanthineguanine phosphoribosyl transferase and thus sensitive auau.hat. The cell line is a cell line termed 1a2 and it has been deposited to 1' American Type culture (line A.T.C.C.) 29 March 1982 under the CRLs n° line A.T.C.C. 8119. 1A2 cells in logarithmic growth phase are suspended in the HAT medium combined with the E~PBMCs at ratio of 30 cells 1a2 by e " PBMCs. The cell mixture is placed on 10 plates microtîtrage to 96 well round bottomed, a concentration of cells/62,000 wells at a 200 ml volume/well, and incubated at 37 °c culture in a humidified atmosphere containing 6 ¾ of COGs. Cultures are maintained five days after transformation by replacing half the supernatant by the medium hat in nine. Observing the well every other day at reversing microscope for observing the signs of cell proliferation. Ten days after inoculation the mixture of cells and 1a2 after the cells are dead due to the selection by the hat in, the supply wells is done using a new medium formulation identical HAT except that it lacks the component aminopterin. Fifteen days after seeding, it is observed that 100% of the wells contain proliferating cells and that, in most of the wells, the cells have a sufficient density polishing removal and control in the supernageants anti-E. coli or anti e. marcescens was.

[82]

C detecting cells secreting an antibody specific

[83]

The supernageants screened for detecting the presence of anti-E. coli or anti e. marcescens was using a technique of an immunosorbent assay test enzyme-linked (eiïisa) [Engvall E, "a quantitative enzyme-Immunoassay kits (ELISAs) in-Microbiology", the MED. Biol., 55, 193 - 200 (1977)]. The plaquesd ' antigens are a series of microtiter plates Immunlon 2 to flat-bottom 96 well, whose wells contain a mixture of one or the other serotypes viable e. coli or S. marcescens was attached confectioneries and the surface of the wells by poly-L-lysine-(PLLs). Briefly, 50 ml is added PLL (1 mg/ml.) in PBS to each well, during 30 min, at room temperature (TR). Plates are washed three times with PBS and, either PBS, 50 ml of a slurry is mixed bacteria to oo.d.ggg=0.2 are added to each well. Plates are incubated at 37 °c for 60 minutes and washed three times with saline to 0.02% tween 20 (salt/T-) unattached to eliminate bacteria. The various plates of antigens used in screening include:

[84]

1) a mixture of e. coli serotypes 01 (line A.T.C.C. no. 23499), and

[85]

04 (Line A.T.C.C. no. 12791) ;.

[86]

2) a mixture of serotypes 07, 015, 016 and 018 of S. marcescens (all strains of tipage reference are obtained of Communicable Disease DCML (CDCs), Atlanta Georgia, a Ga); and

[87]

3) a microtiter plate bacteria free.

[88]

For the modus operandi ELISAs, test site are first blocked with 200 ml. a mixture containing 5% p/v of milk solids non fat, cover with 0.0001%, and 0.01% p/v of azide in 500 ml PBS for preventing the binding of a non-specific protein. After incubation for 1 hour at room temperature, washed the plates three times with 200 ml/well/wash/T-saline solution. For each well, is added 50 ml of a mixture containing 0.1% tween 20 and 0.2% bovine serum albumin in PBS (Ptp). The supernatants of the culture wells of the plate are replicas in the corresponding wells seeded plates of antigens and control plates (50 ml/well) and the plates are incubated at room temperature for 30 s at minute. The supernatants are then removed, washed the plates five times with saline/T and V is added to each well 50 ml immunoglobulin (lg) goat anti-treated human bio-tin (tag0 ≠ 9303040 to - dilution of 1/250 in the Ptp). After a ' incubation of 30 minutes at room temperature, removing the reactive bio-tin, wells are washed five times with saline/T and adding to each well 50 ml of a preformed complex/avidin peroxidase réfort treated bio-tin (Vectastain ABC-a kit, innovation vector Laboratories Ltd). After 30 - minutes at room temperature, the ABC Vectastain reagent is removed, wells are washed five times with saline/T and adding to each well 100 ml of substrate (0.8 mg/ml of octenidine phenylendiamine in 100 mm citrate buffer, pH 5.0 than 0.03% H ^ in hr ^ O-demineralized, mixed with an equal volume just before seeding). After 30 minute incubation in the dark, adding to each well 50 ml H ^ ^ OS 3n to terminate the reaction.

[89]

The culture supernatants containing an antibody reactive with the plates coated with bacteria are detected by measuring absorbance at 490 nm index on a player micro ELISAs Dynatech the MR 580.

[90]

The culture supernatants from six transformations are analyzed by the above method, leading to the identification of a well (707)' that has activity on the plates of serotypes e. coli and S. marcescens was not on the control plates. It is determined in an ELISA test later, using individual e. coli serotypes, that this well contains an antibody reactive with at least serotypes e. coli:

[91]

08 (Line A.T.C.C. no. 23504), and 075 (line A.T.C.C. no. 12798), but not 04, 06: Κ 2, 08: Κ 8, 09: Κ 9 or 022: Κ 13 (line A.T.C.C. no. 12791, 19138, 23501, 23505 and 23517 respectively). This well further contains an antibody reactive with the serotypes of S. marcescens 012, 013 and 015 but not with any other of the twenty LPS serotypes of S. marcescens known.

[92]

0. Cloning of specific antibody-producing cells

[93]

Cells in the well 7d7 are subjected to several rounds of cloning (four) until all clonal supernatants tested by the procedure above provide an ELISA-positive feedback with the serotypes 08 075 and e. coli and with the serotypes 012, 013 and 015 of

[94]

j. marcescens was. There is never a example where none supernatant demonstrates segregating in the clonal does, its pattern of reactivity, this suggests that the culture supernatant from the well 707 has a true multi intergenre reactivity against e. coli serotypes. and

[95]

Marcescens was exposed and that it does not contain more than one cell line (each demonstrating reactivity for an individual serotype). The cells are cloned by limiting dilution in 96 well plates to rounded bottom, in the absence of feeder cells. The media consisting of Iscove medium containing 15ï v/v fBS, L-glutamine (2 mm/1), sodium pyruvate (1 mm/1), penicillin (100 UI/ml.) and streptomycin (100 mg/MV). The cultures are fed every three days by replacing half the supernatant by fresh media. In general, the wells has a cell density between two lymphoblastoTdes sufficient and three weeks after seeding for that tone can make the analysis of the specificity against serotype of the anti one-th. coli and of S. marcescens was.

[96]

Thus, in this experiment, there is obtained a cloned transformed human cell line which is continuous (immortal) .and secret an antibody, directed against a human monoclonal determining superficial > serotypes of E. colicoli.et marcescens was.

[97]

Before the application of the present application, the transformed human cell line identified as continuous 7d7 has been deposited at the American 11432, of Rockville, MD1 under the n° aa.t.c.c.

[98]

CRLS 9009.

[99]

E further characterization of the reactivity multi intergenre

[100]

Attempting an antibody from cell line 707 cloned for testing its additional intergenreréactivitémultiple by modifying the conventional immunotSche technique. Specifically, studied the reactivity multi against bacteria pneumoniae are K, E aerogenes and e-cloacae by depositing bacteria on a nitrocellulose paper disk grid, by reacting the disk containing the bacteria, with the antibody and developing the reactions of antibodies using an enzyme system/AI câlinetatrézoliumnitroblue-phosphatase. In.;

[101]

brief, is deposited 1.0 PI of bacterium (oo.è .ggg=0.4) by gate section of a paper disk nitrocellulose (Schleicher and Schuell, disk nitrocellulose of 37 mm, array of 0.45 MW). Each disk conveniently hold 60 different samples of bacteria. The seeded disks are air dried, fastening them in 1' isopropanol at 25 ¾ v/v during 30 minutes and are locked during 10 minutes in the reagent to the non-fat dry milk, as described for the ELISA. The disks are washed troistrois.fois blocked during 5 min each in PBS/Tween 80 20 and is transferred to the can lids 35 x 10 mm tissue culture die. The supernatant containing t'a antibody (.1, 0 ml) is introduced on the cover and is incubated at room temperature for 60 min. Following three washes of 5 minutes in PBS/Tween 80 20, added during 60 minutes at ambient temperature the thence 2 ml goat-to-antihuman immunoglobulin (TAG0, Burlingame, AC) conjugated with alkaline phosphatase (PBSs) diluted to 1/1000. The disks are washed as above and are submerged in 1 à - 2 ml fresh substrate prepared as follows: 16.5 mg is dissolved bromo-chlorindolylphosphatenitroblue tetrazolium and 8.5 mg in 50 ml de buffer alkaline phosphatase (0.1 m in TrisHCl, pH of 9.5 with NaCl 0, 1m and MgCl^5 mm), the solution being kept in the dark and is filtered immediately before use. After a suitable colored development (10 to 15 min), is. stops the ' by rinsing of the disk in more loads of distilled water. May store the disk developed■after drying.

[102]

The disks contain: 50 reference strains of K. pneumoniae are capsule type, obtained in the ooctor DiGeorge Kenny, University of Washington State, Pathobiology, in Seattle, wa and of VAmerican 11432, 4 iSoiats clinical blood E. aerogenes and clinical blood isolates. 6. of e-cloacae (isolates blood obtained. the Harborview Hospital in, in Seattle, UVA). The Enterobacter isolates are typed (speciation) in. using API 20th of 23 standardized biochemical tests (APIs Analytab Agri, Plainview, ny is). This method identifies the genus and species of gram-negative bacteria but not their serotype. 0th thereby, unlike the e coli, marcescens was and

[103]

K pneumoniae proteins, it does not identify the serotype of Enterobacters, but only their genus and their species.

[104]

From these experiments, it is observed that the antibody has a reactivity 7d7 intergenre multi additional. This antibody reacts with the following serotypes:

[105]

K pneumoniae are E.coli marcescens was E aerogenes was .

[106]

I-kl4, 47.60 08.75 012, 13, 15. clinical isolates

[107]

Therefore observed that 1' human monoclonal antibody 7d7 intergenre multi has a reactivity against bacteria belonging to the species Escherichia coli, marcescens was, K pneumoniae are, E aerogenes was but not e-cloacae.

[108]

, F.. Characterization of monoclonal antibodies

[109]

The finding that the monoclonal antibody reacted with various genera of bacteria suggests that the antibody is raised against a protein or a carbohydrate shared between it. It has been demonstrated that these two molecular species were responsible for multiple reaction intragenresOiutharia L and Hancock R.E.W., "of covalent deux area-LocalizedAntigenic sites are Porin protein adduct F. d' Pseudomonasaeruginosa", the Canadian J d'Oceanogr ., 31, 381 - 386 (1985) and Orskov F. and Orskov I, "Serotyping d'ofEscherichia CoTi", in-University of Arizona Microbiology, volume of 14, Bergan Τ ., DE. When Academic press assembly, to Orlando, fl., 43 - 112 (1984)

[110]

A biochemical characterization of molecular species recognized by antibody 7d7 is carried out by analyzing to 1' immunotSche. Briefly, bacteria washed from 20 ml of culture broth grown during the duration of an overnight (for serotypes e. coli, marcescens was and e. aerogenes was) or plates grown during the duration of an overnight (K pneumoniae antigens) are extracted in 1.0 ml of a solution suitable. 64 ml 50 mm tris at pH 7.6, 30 ml of glycerol, 0.3 g of deoxycholate (sodium salt), 0.14 ml of beta-mercaptoethanol and 6 ml of deionized water [Schechter I and Block Κ ., "appropriate solubilization NDA purifying d' trans Formesyl pyrophosphate squalene synthetase inhibitors", J. Agric. Chem., 256, 7690 - 7696 (1971)]. After 18 hours of incubation at 4 °c, is passed through the suspension to the centrifuge at 10,000 g for 10 mins. Removing the supernatant and effect a quantitative analysis of the protein by using the bio-protein assay (bio-research Laboratories, in Richmond, AC). Between 100 and 1000 ng of protein (varying amounts depending on each bacterial extract) from each bacterium are subjected to a dielectrophoresis in the presence of sodium dodecylsulfate (of SOS) - polyacrylamide gel [Kusecek B et, "lipopolysaccharides, cap, NDA pili aces for virulence factors on the unadjusted 01, 07, 016, 018 and 075 and fourth KL, k5 or k100, the Escherichia coli", and infection than ISIS, 43, 368 - 379 (1984)]. The molecular species separated are transferred from the gel to a nitrocellulose membrane (Ncm can) as described elsewhere [Towbin et H, " des protins Elèctrophoretic incomes of the polyacrylamide gel, nitrocellulose-to-can often hear:

[111]

Procedure to char and some application ", process. Proc. CDAA. IBS ., 76, 4350 - 4354 (1979)], - and the tSche of is blocked for 1 hour Ncm can in PBS-to-Tvveen [Batteiger B et, . "Tea utilisation d' Tvveen 20 AS agent is an agent in the fixed Immunological detection de protins on nitrocellulose membrane or pro", J Immunol. Metha. 55,.297 - 307 - (1982)]. Is incubated during 1 hour tSche at room temperature in 10 ml " supernatant liquor from the washing line 707. After four washes 5 minutes in PBS-to-Tvveen, incubated the tSche in Ig-goat-anti human conjugated to alkaline phosphatase and developed as described herein. 1 polishing ' test bacterium on nitrocellulose disk. Noted positive reactions on all tracks which. upon deoxycholate extracts contain bacteria described herein. In these- ' tracks, it is listed tt.que the antibody 7d7 recognizes a series of regularly spaced molecular entities giving rise to an effect on scale 1' iirenunotâche. This profile is entirely consistent with that found in the analysis by polyacrylamide gel electrophoresis of the LPS in the presence of SDS for which. it has been found that the profile dimension heterogeneous presented by the stripes was OD 3 a population of LPS molecules differing by weight increments equivalent to the number of units of oligosaccharide side chain 0-antigenic present per molecule [Pavla SE and Makela w.w., "lipo Heterogeneity in discussion or Northwest Territories boundary bypass the sodium sulfate-polyacrylamide gels/FACE", €. J Biochem., 107, 137 - 143 (1980) and Goldman R.C. and Leive L, " Heterogeneity d'Antigenic-a-car chain gun Longueur in-from-lipopolysaccharide from Escherichia coli strain 0111 DNA from Salmonella typhimurium lt2, €. J Pharmacol, 107, 143 - 154 (1980)]. These data indicate that the monoclonal antibody is directed against a 7d7 antigenic determinant shared by the LPS molecules found on some serotypes e. coli, marcescens was, K pneumoniae and

[112]

E aerogenes was.

[113]

To further define the molecular nature of the antigen, deoxycholate extracts are treated prior to electrophoresis with a proteolytic enzyme, proteinase K [Eberling W et, "recombinant proteinase k des Tritirachium photo album Limber", €. J Pharmacol. 47, 91 - 97 (1974)] ., . To prepare the sample, 10 mg of proteinase K are added to 50 mg of the protein sample and the ' mixture, is heated to 65 °c during 60 min. Passed the samples in electrophoresis and immunotSche as here. The schemas

[114]

•immunotSche observed after treatment of proteinase K are identical to the patterns observed without treatment and thus suggest that the antigen reactive with the antibody 707 is not the nature of a protein.

[115]

To determine specifically whether the 707 reacts with a carbohydrate epitope, the sample is deoxycholate electro -

[116]

, transferred to moderate oxidation periodate having reacting the nitrocellulose paper with the antibody. It has been shown that this reaction destroyed the determinants to carbohydrate-based and thus their reactivity dêtruisait later with 1' antibody without altering the epitopes of protein or lipid Woodward's M.P. and al, "detection de a monoclonal Mitochondrial Antibodies arrest specific zu in carbohydrate epitope of periodate oxidation of ion exchange", J d'Immunol. In wafer processing, 78, 143 - 153 (1985)]. In short, after being blocked the nitrocellulose paper labeled électrotSche containing the sample electrophoresed with PBS-to-Tvveen as described herein, rinsed tee. paper using a buffer at pH 4.5 acetic acid confectioneries tatetate.de CEA, na! 50 mm apart. Incubated for the paper. nitrocellulose in periodic acid 50 mm dissolved in the acetic acid buffer, during 60 min, -.; <ians. the dark,, confectioneries, ambient temperature. The treated paper is rinsed three times in PBS-to-Tvieên and reacted with the antibody as described herein. The extracts of deoxycholate transferred by électrotSche treated thereby born are more reactive with the monoclonal antibody 707. These data suggest strongly that the epitope recognized by this antibody is a carbohydrate present on the LPS molecule bacteria described herein.

[117]

The isotype of the monoclonal antibody 7d7 is determined in a procedure similar to the tests of specificity of ELISA described above except that use is made of an anti-human IgG chève treated bio-tin (specific gamma chain, TAGO) or IgM goat anti-treated human bio-tin (specific mu chain, TAGO) as a second stage reagent instead Ig goat anti-treated human bio-tin more broadly reactive. The reagents are used both at a dilution of 1/500 plate and the antigen contains sets of e. coli strains 08 075 and immobilized to the PLL. There is a positive feedback of the monoclonal antibody 7d7 with e. coli strains only in-presence of IgM reagent, demonstrating an IgM isotype for the monoclonal antibody. It will be appreciated by skilled artisans that is repeated several times the above process and if it is determined the isotypes of intergenre multi-reactive monoclonal antibodies so obtained, we would find other additional isotype, for example IgM and IgG [Frosch m and al, "NZB foam system for facilitating production section of the monoclonal antibodies to the BacterialWeak antigenically: insulation polar organic salt of an igg fusion mediated cytotoxic the polysaccharide capsule d'Escherichiacoli Κ 1 NDA GropMeningocci", process. Proc. CDAA. IBS ., 82, 119.4 - 1148 (1985)].

[118]

GM. In vitro activity

[119]

Examined and 1 'activité functional in vitro. 1' 707 monoclonal antibody in an assay of opsono-to-in vitro phagocytosis, which compares the bacteriocidal activity of the antibody in the presence and in the absence to the fois.de human neutrophils and de - human complement.

[120]

Are prepared bactériesbactéries.soft-by inoculation of tryptic soy broth (CST) to 1. '50 using UV-of one bouillon: culture grown during the duration of an overnight (for the e coli * marcescens was and e. aerogenes was), either, for the' the K. pneumoniae are, by seeding a petri dish containing agar agar of Worfel-to-Férguson. For the a-broth cultures, incubated at 37 °c on the tubes in a shaker during 3 hours, after which 1.5 ml of the centrifugal OH-culture for 1 minute to 10 OOfr x g for, final disposal of the spent culture medium and further processing the pelletized suspended in 3.5 ml of a balanced salt solution containing 0.1% gelatin Hank and 5 mm HEPES (HBSS/freezing). For bacteria cultured ' on plate of agar agar, is scratching the colony for removal from the plate run them in

[121]

gel HBSS sterile. Bacterial concentrations of bacteria

[122]

3

[123]

grown under the two conditions are adjusted to 3 x 10 bacteria/ml in measuring the oo.d.ggg and by suitable dilutions (approximately 1/50 000). Human neutrophils are separated according to a van Furth and Van Zwet ["vitro Determination phagocyto sis foll NDA Introcellular commuting Mononuclear phagocytic Polymorphonuclear NDA bypass", in Handbook of recordings when looking for, in volume 2, D. M. the Weir, Ed., 2ème editing, Blackwall Scientific is editions, the Oxford, 36, 1 - 36, 24 (1973)] with several modifications. A supernatant layer from de .10 ml heparinized blood is mixed with- due '. medium ff.ico.ll-a passover and moves on to the centrifuge. The pellet of blood cells (RBCs) roiige is washed once with RPMI 1640 and resuspended in an equal volume of PBS 37 °c. 3 Ml of the suspension is added to 6 ml 2% dextran (in PBS at 37 °c) and blending of the contained moderately but bottom-end-to-end-over IP. Incubation at 37 °c de.20 minutes to settle to leave! the RBCs, removing the supernatant (containing neutrophils), washed twice in PBS at 4 °c, once in gel HBSS and is suspended in the even at a concentration of 5 X-. 10 ^ neutrophils/milliliter.

[124]

For the source of complement used with the e. coli and S. marcescens to, adsorbed twice human serum in the presence of collections of live bacteria [Bjôrnson etet.mi.chael AB J.G., " the human sera success of competition-promoting phagocyto sis foll d'ofPseudomonas aénjginosaT. Collapsed and Opsonins. useful bacterium with the use ", . J OC. IFN. Oisois.j. 130 . 57.5.., . (1974) S.i19-to-s126] corresponding to the organisms used in the test. This serum is again adsorbed with zymosan boùi: Y: IF.. (.. canola:, the supra.) for. remove a component of ' serum, . the; ^. ^ born ropérWjipoVê cu! ^ e - néèessait. ". to 1! activation of

[125]

T-another path ' complement, PPs ^ IBLs E bpsonophagqcytiqûes. for tests. using the K. priéümpniae. and the. I ^: ' aerogenes was,: 1a source is human serum complement normâl-to-honhon.adsorbé . used at a final concentration.

[126]

•1%. - - '■.. ;••

[127]

The plates used to quantify the ratio of bacteria

[128]

Surviving•/ destroyed are prepared at the beginning by heating 24 well plates at 37 °c for 3 to 5 hours. A solution to 0.4% agarose in the Tsb is prepared by passing to V. the mixture for 5 minutes in an autoclave and allowing it to cool water bath at 50 °c sputtering. Approximately ' 15 minutes before the end of the incubation period final in the test panel arrays, by removing a plate 24 to 37 °c incubator-well, it is placed on a hot plate to 42 °c and adding to each well mixing CST/0.4 ml of agarose. The plate is returned immediately in the incubator at 37 °c such that 1' in agarose cools only never below die 37 °c.

[129]

For the test, 25 ml of the supernatant culture 707 and 25 ml of a bacterial strain suitable are added in duplicate to microtiter plates to 96 well round bottomed and incubated at room temperature for 30 min -. Followed by the addition of 15 ml of this human complement, 15 ml of human neutrophils (5 x 10 ^ / ml.) and 70 ml HBSS gel. Wiped from the entire surface of the plate with a cotton pad and applying a sterile plastic plate adhesive so as to cover the entire wafer and the stlre spaces. existing between the shaft and the rotation of the plate 1 to 37 °c during time. After incubation, the plate is passed to the centrifuge for 5 minutes to 100 x g for, gently removed the cover from the plate and dried the plate surface with sterile cotton immersed in 70% ethanol. 50 Ml is taken of each microtiter well and added to individual wells of plates of quantitative immunoassay to 24 wells already containing

[130]

0.4 w/mixing well to 0 * 4% of CST/agarose column (38 to 40 °c). Plates are placed over a shaker with flat bottom during 1 minute to 150 rpm/min and allowed 1! in agarose cured for 15 minutes at ambient temperature the I. Finally, it adds. to each well an overcoat for mixing CST/0.4 ml of agarose, that followed by a period of 15 minutes to 4 °c curing before the incubating. plates to 37 °c for the duration of a night.

[131]

After 18 hours,, is enumerated the-columns and over to the data.

[132]

as colony forming units (CFUs) for each condition.

[133]

The bacterial serotypes used herein, except the K. pneumoniae antigens, are only inactivated in the presence of monoclonal antibody 707, a source of active complement and human neutrophils (table 1). When repeats this experience with more. bacterial serotypes unconsumed 707, is observed, not of killing bacteria (data not shown), demonstrating the specificity of the antibody. functional monoclonal antibody. 707 and its ability to bind L-.' opsohine on bacteria-and promote phagocytosis. Since the combined actions of opsonins (spécifiquésj polymorphonuclear leukocytes and antibody (neutrophil) appear be le: primary mechanism. immunity of these bacterial strains, these data suggest that, after appropriate administration, the antibody 707. would provide protection against fatal threats dice carried by serotypes die bacteria, described herein.

[134]

TABLE 1

[135]

THE I! Bacteria! Neutrophil antibody complement I-bacterium introduced! I% for destroying!

[136]

! !E coli 08 and075! !*! !7D7! the I -hasthe I!0•! !
I!T-b.1the I!
!E coli 08 and0751+!6F11! +!0!
;1![!1
the IE coli 08 and075the I-17D7+!0T-
the Ithe L!;1!
!E 08 075 transporter comprising stored characteristics!+17D7! + '!85V!
"!1T-
Smarceacena sec.012 and 015the I4 *17D7-!0the I
!1.!!J
marceacena sec.012 and 015!+!6F11+!0!
!!-1 -"
marceacena sec.012 and 0151-17D7+!0"
!1I!
the Imarceecens sec.012. 015 andT-+F.7D7+T-85V1
111!the I!
E-to-'àerogenes isolates (2)!+ 'F.7D7-the I0!
F.!"F.J
1(2) Isolates the E. aerogenes was1.+■!6F11+!01
1T-!T-!
"(2) Isolates the E. aerogenes was!-17D7+!0!
1T-!1!
1(2) The E. aerogenes was Isolata1+!7D7+J85V1
"11!!

[137]

! ! THE I! ! ! ! i-•!

[138]

(-)" heat inactivated human complement (56I C. for 30 timed).

[139]

(6F11) - culture supernatant containing monoclonal IgM human

[140]

directed against Pseudomonas aeruginosa Fischer-type 2.

[141]

HR. In vivo activity

[142]

To test T-assumption above, carried out animal protection studies using the antibody 707 and at least one organism species from e. coli and E. aerogenes was described herein. Antibodies of contrSle 707 and negative (6f11, monoclonal antibody human IgM specific of Pseudomonas aeruginosa Fisher immunotypic 2) are first concentrated from culture supernatants worn by precipitation with solid ammonium sulfate (50% final concentration) (Good's A.D. and al, "purifying d': immunoglobulin, NDA their naturally moieties", in-selected on the University of Arizona shirt when looking for, MishelT' bb.b; and Shiigi ss.m,, EDS, W. H. the Freeman and Company study, Francisco, AC (1980) 279 - 286). The precipitated material is reconstituted in sterile water and subjected to an extensive dialysis in the presence PBS.

[143]

The IgM antibodies in the precipitated salt of ammonium sulfate is purified by affinity chromatography on a column of d! - tolerances and procedures and nor-tee packed with IgM antibody mouse monoclonal. To prepare the column, one gram Sepharose 4b activated by cyanogen bromide (Pharmacia is) dehydrated is mixed with 15 ml of HCl 1 NIMs ice-cooled in distilled water. The hydrated gel is washed in .30 ml of coupling buffer (carbonate is 0, 1m (NaHCOgrams ) naCI in 0, 5 Μ, at pH 8.2), is drained to prepare a wet cake and combining the precipitate of ammonium salt dissolved in 1 to 3 ml of coupling buffer. The suspension gel is mixed end-to-end during 2 hours at room temperature and is then passed to. the centrifuge at 200 g for 5. minutes. For blocking the reactive sites still available, removing the supernatant, 10 ml is added to the gel ethanol friend does Im and mixing is continued as above. - In going from the suspension to the centrifuge, at 200 g for 5 minutes and tone tosses the supernatant. The gel is prepared for use with a washing in acetate buffer 0, the LM/saline solution (6.8 g of sodium acetate trihydrate and i4, 6 g of NaCl are dissolved in 500 ml of distilled water containing 2.9 ml of glacial acetic acid, pH of 4.0), two washes in buffer coupling and two washes in PBS. The gel is poured into a column Pharmacia is c10/10 and stored to 4 °c until use.

[144]

Purify the immunoglobulin, .0, 5 ml of fractionated material at salt is' diluted to 2.0 ml dahs-to-.du PBS and tone adds to the affinity column. After the sample loading, the column is washed with PBS, pH of 8.0 until the monitoring device for the absorption coefficient indicates more than another protein in what flows. The bound antibody is eluted with MgCl 2m in PBS, brr VBE1 - determines the protein concentration for each fraction to ôô.d.2g0 and tone gathers the fractions of peaks. The fraction containing the antibody is desalted on a Sephadex-G 25 and, if necessary, concentrated: to using centrifugation of microconcentration (Centricon 30, Amicon TX, Danver, am) to 1 to

[145]

2 mg/ml to. Tested the purity of the final preparation by electrophoresis on SDS-polyacrylamide gel that tone forwards of a revealing proteins at silver nitrate (j-Η Morrissey.., "Rolls-polyacrylamide gel Stain zu protins thein: A alaising transforaminal Precipitation with Enhanced thermometrically SSE", Anal.. Toxicol. (1981) ., 117, 307 - 310) .et the activity of the antibody by ELISA as indicated above.

[146]

For each attack by bacteria, mice female type the decisions to Webster weighing between 20 and 22 g are divided into three groups of ten mice each. A representative experiment is carried out as follows:

[147]

following:
GroupBacteriumAntibody
WITHE coli 087D7
BE coli 086F11
C014 Marcescens was7D7

[148]

Each group receiving an antibody receives an intravenous injection of 200 ml sterile PBS containing 25 mg of purified antibody. Two hours later, all animals are treated intraperitoneally by 300 ml of live bacteria containing 3 VLD ^ Q of their bacterial strain respectively. The bacterial suspensions were prepared from a culture broth in a logarithmic growth phase from which the bacteria were centrifuged, washed twice were found in PBS and the resuspension at the appropriate concentration in PBS. Animals is observed for a period of 5 days. 24 To 48 hours after the attack, all the animals of group b (irrelevant antibody) and group c (organism irrelevant) died. On the contrary, the animals which received the antibody 7d7 (group a) are all living and systemic.

[149]

This model die for animal protection is used to demonstrate the therapeutic effect of the antibody 707 against bacterial attack by organisms belonging to the three genera exposed above. A summary of these data is given in table 2.

[150]

Bacterium of- Antibody! Survival/% survival
TH.escherichia coli-a 087D7! 10/10 VBE1100
TH.escherichia coli 086Fllhas0/10 1!.0
E.014 * marcescens was3707the I/O-loto
!

[151]

TABLE 2

[152]

The antibody 6f11 is specific of Pseudomonas aeruginosa Fisher method irranunotype 2 and serves as an antibody negative control.

[153]

The S. marcescens to^014 is not reactive with the antibody 7d7 and it serves as a nonspecific control organisms.

[154]

j is 1, 1.% survivalC.
I-j-antibody.Day n°
i-J.0i-I-the I; 2;3i-J.4
The I|6f1112i-I-J.2i-I-I-|21i-I-I-1
707 the I!12i-J.10i-I-I-j-64l-I-J.4

[155]

Table 2a

[156]

C. Studies of LD5 q and protection are implemented using mouse that rendered neutropenic injection of cyclophosphamide as follows: day 0:150 mg/kg body weight, day 2:50 mg/kg body weight. A TES 4 day mouse receive the antibody and bacteria, as described above.

[157]

These data demonstrate that•1 ' human monoclonal antibody - 707 is able to protect mouse lethal attacks by bacteria of three different species of gram-negative bacteria. Since the 707 is reactive with a carbohydrate epitope present on LPS, but that molecules of LPS K. pneumoniae are less accessible (Orskov I and Orskov sec., "Serotyping d' Klebsella", in-University of Arizona Microbiology, volume of 14, Bergan T, Ed., Academy press assembly, to Orlando, fl. (1984) 143 - 146), the protection by antibody 7d7 against infections caused by the K. pneumoniae infection is not evident (data not shown). However, the human monoclonal antibody reactivity to multi 7d7 intergenre is able to provide shielding using 25 µg of purified antibody against infections caused by organisms belonging to. species of gram-negative bacteria E.coli and e. aerogenes was.

[158]

SUCH AS II

[159]

The example II gives the demonstration of methods for the preparation and selection of a human monoclonal antibody having reactivity intergenre multi facing members of Serratia species, Klebsiella pneumoniae and Enterobacter aerogenes. Further, this example gives the demonstration of the binding activity in vitro opsonin " antibody against homologous serotypes of S. marcescens, K pneumoniae antigens and e. aerogenes was. Repeated mode of operation such as I (essentially. as described in parts a through g) to prepare a human monoclonal antibody which gives a multiple protection against infections caused by bacteria described above except that it is necessary to make specific modifications to characterize and. testing the antibody described in this example. The following represents changes in test operating modes and the results obtained with the monoclonal antibody described herein.

[160]

1. The culture supernatants from six transformations are analyzed by the above method and lead to the identification of a well (4f10) that has activity on the serotype of plate

[161]

Marcescens was but not on the E.coli or on the control plate. It is determined in a subsequent ELISA using individual serotype of S. marcescens that this well contains an antibody reactive with the serotypes of S. marcescens 015 and 018 but with any other twenty known serotypes of LPS of S. marcescens.

[162]

Before depositing the application priority, the human cell line transformed continuous (immortal) identified under the n° 4f10 was deposited to VAmerican 11432, of Rockville, MD1 n° under the line A.T.C.C. CRLs 9007.

[163]

2. Attempting an antibody from cell line 4f10 cloned for multi intergenre reactivity testing in additional to using a modified die technology standard imnunotSche. Specifically, studied the reactivity multi against bacteria pneumoniae are K, E aerogenes and e-cloacae by depositing bacteria on a nitrocellulose paper disk grid, by reacting the disk containing the bacteria with the antibody and developing the reactions of antibodies using an enzyme system nitroblue tetrazolium/AI câline phosphatase inhibitors.

[164]

From these experiments, it is observed that the antibody has a reactivity 4f10 intergenre multi additional. This antibody reacts with the following serotypes:

[165]

K pneumoniae are marcescens was E aerogenes was K3, 12, 29, 31, 68, 72 015.18 clinical isolates the human monoclonal antibody reactivity 4f10 intergenre thus has a multi-.l from bacteria belonging to the species marcescens was j-K pneumoniae are, E aerogenes was.

[166]

3. By using the technique of immunotS ' goosefoot, noted positive reactions in all tracks containing extracts of deoxycholate bacteria described above. It appears in these tracks that the antibody 4f10 recognizes either a broadband component, or a certain series of regularly spaced molecular entities giving rise to a course to scale. This profile is entirely consistent with that encountered, assays of polyacrylamide gel electrophoresis radicals carbohydrate that demonstrate a significant heterogeneity of molecular weight due to sequence-specific sugar repeating often or LPS molecules differing by increments of weight equivalent to the number of units of an oligosaccharide side chain per molecule 0 antigenic (transplantation establishments E.R. et, "utilisation d' prokaryotic-a-derived hypothetical probes objectives to a poly (Sialic acid is also) for neonatal in-neural membrane", process. Proc. CDAA. IBS ., (1983), 81, 1971 - 1975; and to Holden K.G. et, "freeze FACE d' MALT Glycoproteins, I OC action of the gel Porosity",

[167]

For Biochemistry (1971) 10, 3105 - 3109). These data indicate plays the antibody

[168]

i-to. '; the V " ^•4f10 monoclonal antibody is directed against an antigenic determinants shared by

[169]

! ' i- the L,M

[170]

carbohydrate molecules. of. the carbon dating. snowboard ^ some serotypes of S. marcescens, K, K. pneumoniae and e. aerogenes was.

[171]

The schemas of d * immunotSche observed after treatment with proteinase K are identical to the patterns observed without treatment and thus suggest that the antigen reactive with the antibody 4f10 does not have the nature of a protein.

[172]

To verify specifically if the 4fl0 reacts with a carbohydrate epitope, .. the sample is subjected to an oxidation deoxycholate électrotransféré moderate the ati periodate oxidation prior to reacting the nitrocellulose paper with an antibody. The extracts of deoxycholate electrodeposited treated in this way are not reactive with the monoclonal antibody 4f10. These data indicate intensely that the epitope recognized by this antibody is a radical carbohydrate present on molecules forming part bacteria described above.

[173]

4. Determined the isotype of the monoclonal antibody 4f10 by a procedure similar to the tests.de ELISAs specificity described in the example-I, except that the plate antigêne contains a set of serotypes of S. marcescens 015-and 018 immobilized by a PLL. To observe a positive reaction of the monoclonal antibody to-4f10 with the serotypes of S. marcescens only with reagents IgM, demonstrating an IgM isotype in the monoclonal antibody. It will be appreciated by skilled artisans that is repeated several times the process of this example and if it is determined the isotypes of monoclonal antibody. to reactivity multi intergenre thus obtained, there would .'d ' other isotype, for example IgM and IgG.

[174]

5. Examined in vitro functional activity of monoclonal antibody in an assay 4f10 opsonophagocytic which compares the activity of bacteriocidal. 1' antibody in the presence and in the absence of both human neutrophils and of human complement.

[175]

The bacterial serotypes used herein. are only inactivated in the presence of the monoclonal antibody 4f10, a source of active complement and human neutrophils (table 3). When repeats this experience with several bacterial serotypes not reactive with the 4f10, not observed bacterial killing (data not shown), demonstrating the functional specificity of monoclonal antibody 4f10 and its ability to bind the opsonin on bacteria and promote their phagocytosis.

[176]

TABLE 3

[177]

I

[178]

1

[179]

!

[180]

; i-

[181]

J-j-bacteria neutrophils

[182]

+!7D714 -
7D7;-!
+the I•6 ' hysteresisT-+!
-;7D7Y+!
+2 j.7D74 -;;
7D7!
+•6F11+5
-2. 7D7+•!
4 -.17D7•+Y

[183]

t-T-

[184]

t-T-

[185]

Marcescens was 018 and!" 015

[186]

;

[187]

! Sec." arcescens 018 015 and

[188]

2

[189]

! O, and 015 018 raarcescens

[190]

Antibody

[191]

7D7

[192]

6Fllb. .

[193]

7D7

[194]

! !

[195]

",,! % destruction of! the OMP centeredly; gramsthe ACT ethe R GBPI introduced! ! !

[196]

1 +

[197]

+

[198]

0

[199]

0

[200]

018 015 And marcescens was!

[201]

!

[202]

! K pneumoniae are k3 k12 and

[203]

!

[204]

! K pneumoniae are k3 k12 and

[205]

the I

[206]

! K pneumoniae are k3 k12 and

[207]

Y

[208]

1 K pneumoniae are k3 k12 and

[209]

the I

[210]

! - (2) Isolates the E. aerogenes was!

[211]

! !

[212]

! (2) Isolates the E. aerogenes was!

[213]

! !

[214]

! (2) Isolates the E. aerogenes was!

[215]

THE I -!

[216]

t-E(2) isolates serogenes!

[217]

! !

[218]

!

[219]

85%

[220]

O

[221]

O

[222]

0

[223]

60%

[224]

O

[225]

0

[226]

O

[227]

85%

[228]

! ! i-I to 1! ! ! ! ! the I

[229]

has

[230]

(-)"=human complement heat inactivated (56 °c during 30 min).

[231]

(6F11)" culture supernatant containing monoclonal IgM human

[232]

directed against Pseudomonas aeruginosa Fischer-type 2.

[233]

SUCH AS III

[234]

The example III gives the demonstration of methods of preparing a human antibody which is reactive to both polysaccharide 1' coli capsular kind kl and Neisseria meningitidis

[235]

(N. meningitides) group b and gives further demonstration of the protective activity of the antibodies in vivo against a lethal challenge of bacterial species homologs E.coli and N. meningitides. The process is repeated in the example-I(essentially described in parts a through g) to prepare a human monoclonal antibody which gives a multiple protection against infections caused by bacteria described above, except that it is necessary to make specific modifications to characterize and assay the antibody described in this example. The following represents the changes in the test operating modes and in the results obtained with the monoclonal antibody described herein.

[236]

1. Analyzing culture supernatants from five, transforms by the above method, which leads to the identification of four wells (5d4, 2c10, and 8a8 9b10) that contain anti-E-specificity. coli on the plaque.de e. coli serotype but not on the S. marcescens was or the control plates. It is determined in a subsequent ELISA conducted as indicated thereto above on individual serotypes e. coli that the wells contain at least an antibody reactive with the serotypes e. coli: 01 (A.T.C.C. 23499); 07: kl (A.T.C.C. 12792); 016: Κ (A.T.C.C. 23511) and 1 050 (CDCs 1113-83), but not the 04 (A.T.C.C. 12791); 0 δ: Κ 2 (A.T.C.C. 19138);

[237]

08: Κ 8 (A.T.C.C. 23501); 09: Κ 9 (A.T.C.C. 23505) or 022: Κ 13 (A.T.C.C. 23517). Because of these improved performance during cloning and production increased antibody, the monoclonal antibody 9b10 is selected for for further analysis.

[238]

Before depositing the priority application, the transformed human cell line (immortalized) continued herein identified n° 9b10 has been deposited at 1' American Type culture, of Rockville, MD1 under the 9006 n° line A.T.C.C. CRLs.

[239]

2. Noting that the monoclonal antibodies from each of the clones that have been reacted with the identical group of serovars of antigen 0 e. coli indicate that these antibodies are directed against a surface structure common to these serotypes of bacteria. Several approaches are used to define the surface structure common to these serotypes e. coli. As stated above, two of the four serotypes e. coli (07: kl and 016: kl) identified by antibody 9b10, have the capsular serotype ml while the other two (01 and 050) have not been typed for detector their serotype antigen K 5 hence the possibility remains that the antibody 9b10 contains a reactivity with the antigen souches.de kl and that the E coli serotypes having antigen-0 01 and 050 also possess the capsular serotype fourth KL.

[240]

Other researchers have availed of the lability of the capsule ml to establish its presence. The heating of serotypes e. coli positive at KL in a bath of boiling water to 100 °c during 60 min eliminates the subsequent ability of these strains to react with sera anti kl and improves their ability to react with sera antigens anti 0 (Orskov F. and Orskov I, "Serotyping d'Escherichiacoli" inches. University of Arizona Microbiology, volume of 14, T Bergan, ed., Academy Prfss, the London (1984) PPs. 44 - 105). It is likely that the reciprocal effects of boiling are due to elimination of the capsule and to increased accessibility of antibodies to; lipopolysaccharide (LPS-) molecules, a TES serotypes e. coli positive fourth KL (07 and 016) and untyped serotypes for KL (01 and 050) are heated as hereinbefore and reacted with the antibody 9b10 and sera specific heterologous serotype of LPS (Agar Bactô e coli-herd TSARs.) according to the procedure of ELISA. The organisms heat treated to lose their reactivity with the antibody 9b10 and they perceive increase their reactivity with serum from their homologous serotype LPS-while the organisms (untreated control) remain highly reactive with culture supernatants of 9b10 and low-reactive anti-sera with their respective specific serotype of LPS. VBE1

[241]

It has been found that the polysaccharide (carbohydrate) from bacteria Neisseria meningitidis of group b.. (a homopolymer of sialic acid, poly-n-acetyl-neuraminic acid bound in the alpha position 2.8) was of a chemically and antigenic homogeneous with the polysaccharide of the E. colicoli.kl. (Grados 0. and Ewing W. H., "Antigenic relationship between humans Escherichia coli and Neisseria meningitidis are", 0. Immunol. (1973) 110, 262 - 268). These data suggest that, if the monoclonal antibody 9 Β 10 contains specificity with respect to the capsule e. coli ml, then the antibody should also. contain a specificity against the polysaccharide of N. meningitidis of group b and, further, that the monoclonal antibodies containing specificity against the polysaccharide of the

[242]

Group b of the N. meningitides, should also demonstrate reactivity against strains of e. coli having the capsule fourth KL. Two experimental protocols are used to test

[243]

(1) the ability of 1' 9b10 antibody reacted with the N. meningitides; and (2) the ability of an antibody directed against a polysaccharide

[244]

N. meningitidis of group b to react with four serotypes reagents at 9b10 e-coli.

[245]

A polysaccharide is reacted extremely purified from group b (Conaught Laboratories Ltd, in Toronto, Canada is) and viable bacteria of

[246]

N meningitids of group b with the antibody 9b10 in one operational mode ELISAs as stated above. The monoclonal antibody 9b10 react strongly against both antigen preparations. To prove the specificity reciprocal, used a test kit of meningitis caused by N. meningitidis of group b commercially available ("directagene" system for directly detecting an antigen, Hinson, Westcott, and Dunning, in Baltimore, MD1) that uses latex spheres coated with a mouse monoclonal antibody directed against the polysaccharide of the group b. in agglutination assays using e. coli serotypes 9b10 positive, four serotypes all demonstrate a strong reactivity with the beads coated with antibodies. E. coli serotypes are known to be negative to the antigen KL are also negative in this test system. Collectively, these data indicate that the monoclonal antibody is reactive with the capsule 9b10 KL e. coli and with the carbohydrate type specific existing on the N. meningitidis of group b. further, since these tests are implemented withthe I intact bacteria and viable, this suggests that T-antibody-monoclonal antibody is specific for a few 9b10 parts of a region exposed to the outside of the molecule of polysialic acid.

[247]

i-a is *. determines V-isotype of the monoclonal antibody 9b10 in a procedure similar ELISAs. specificity tests described in the example I-except that the plate antigen contains an assembly 'de serotypes e. coli positive fourth KL immobilized by the PLL. We observed a positive reaction of monoclonal antibody with the serotypes 9b10 e. coli positive fourth KL only with the reagent IgM, demonstrating for3 "* the monoclonal antibody an IgM isotype. It will be appreciated by those skilled neural ' cautiousO if tone repeats the process of this example several times and that tone determines the isotype of monoclonal antibodies specific to the ml thus obtained, we would find other isotype, for example IgM and IgG.

[248]

4. Examined in vitro functional activity of monoclonal antibody in an assay 10 9 Β opsonophagocytic which compares 11 bacteriocidal activity of the antibody in the presence stored characteristics in the absence of both human neutrophils and of human complement.

[249]

E. coli serotypes positive fourth KL does are inactivated presence ^ ' 9b10 monoclonal antibody, a source of active complement and human neutrophils (table 4). When repeats this experience with several serotypes e. coli negative at fourth KL, not observed bacterial destruction, demonstrating specificity at KL monoclonal antibody 9b10et its ability to fix the opsonin on bacteria and promote their phagocytosis. Since the combined action of opsonins (specific antibodies) and polymorphonuclear leukocytes (neutrophils) appears to be the, primary mechanism of immunity in the serotypes e. coli positive fourth KL, these data suggest that the antibody 9b10 could provide, after a suitable administration ., protection against lethal. attacked by any of the serotypes: e. coli encapsulated ml whatever its serotype antigen-O bond.

[250]

... TABLE. 4.

[251]

.!. !. !

[252]

Bacteria•j of neutrophilsT- . Antibody!•!. " t-telling Bacterium introduced•.!. ! X destruction of

[253]

! " -!f...•
J .11.
I!. "Escherichia coli 01:1 018 and ' Κ stcï/"4 - **1.•9È10, '1has. !0
9.ï *.1'As TR -•" -
! E coli 01: kl and 0.1btki>;••. /.!.1; 6FU.(1+10
E•.. .. / Λ. *•ii th, Escherichia coli 01: kl and/OlBîikl ' -;;:;" VBE1 " * the I '■•' -1.% B10. -.! '0 -
! E coli-to-QlîKl. et * 018: mlT- "! *•** · ; / ν•!.RAS' the O ': . + - '* - - - I-:•99%■
! Having N. meningitidis group b!.. +! the I9 Β · 10.. 1. T-THE O.
The n -!, ! I-I-meningococcus group b+i-I-.6F11 - 't-I-+I-•1 -O
! Having N. meningitidis group b!-!9B10the I+!0the L
! !J1
! Having N. meningitidis group b•ii+!9B10the I+199%!
! !1the IF.1

[254]

the Ba (-) ** human complement heat inactivated (56". C. during 30 min). (6F11) ** culture supernatant containing monoclonal IgM human

[255]

directed against Pseudomonas aeruginosa Fischer-type 2.

[256]

5. To test the above hypothesis, studies conducted protective animal with the antibody 9b10 and several serotypes e. coli kl and positive as well as negative at fourth KL with serotype of

[257]

Ν. meningitidis of group b (strain h313, resulting in the Dr. curry IMPERIAL OIL, laboratory bacterial polysaccharide, Office follower, US FDA administration, the Bethesda, MD1);

[258]

For each bacterial attack, females Swiss Webster mice weighing between 20 and 22 g are divided into three groups of ten mice each. A representative experiment is carried out as follows:

[259]

- Group-BacteriumAntibody
WITHE coli ml '9B10
BE coli ml6F11
10CE coli k29B10

[260]

Each group receiving an antibody, is injected intravenously 200 ml sterile PBS containing 25 mg of purified antibody. Two hours later, injected to all animals intraperitoneally 300. ml of live bacteria containing 3^VLD their bacterial strain respectively. The bacterial suspension was-prepared from a culture broth in a logarithmic growth phase from which the bacteria were centrifuged, washed twice were found in PBS and the resuspension at the appropriate concentration in PBS. Animals is observed for a period of 5 days. 24 To 48 hours after the attack, - all animals of group b (irrelevant antibody) and group c (organism irrelevant) died. On the contrary, animals that have sated L'abticorps9bî0 (group a) are all living free symptSmés.

[261]

This animal protection model is used to demonstrate the therapeutic effect of 11 9b10 antibodies against bacterial attack by organisms belonging to the two species indicated above. Shown in the table 5 a summary of these data.

[262]

i-I-! Bacterium driver! the I. !Antibody! Survival /! ! [% survival!
j-e coli ml! 1! i-I-9B10 -i-I-I-lo/îo1 1 I-100 i-
! E coli ml!6Fllhasthe I: the O/lothe Io-I-
ii E.coli k2b. 19B10the I/O-lo 1i-I-I-0 y
! Having N. meningitidis group b!9B10i-I-5/5the IO-O
! Having N. meningitidis group b! I-I-6F11! 0/5 i-i-J.0!
the j k2 E.coli! i-I-9B10i-0/5 1i-I-o-I-

[263]

TABLE 5

[264]

The antibody 6f11 is specific of Pseudomonas aeruginosa Fisher method 2 immunotypic etet.il serves as a negative control antibody.

[265]

The ^ e coli k2 is not reactive with the anticojrps 9b10 and serves as an nonspecific control organisms.

[266]

These data demonstrate that the human monoclonal antibody 9b10 is able to protect mice from killing by bacteria belonging to two different gram-negative bacterial species. The antibody to multiple protection intergenre is capable of protecting against infection by passively organisms belonging to the gram-negative bacterial species E.coli and N. meningitides group b.

[267]

EXAMPLE VI

[268]

The example IV gives a demonstration of methods of preparing a human monoclonal antibody having reactivity intergenre multi facing members of Escherichia coli (e coli), the Enterobacter cloacae (I. clocae) and streptococcus group b. this example gives further demonstration of antibody reactivity against multi

[269]

i species belonging to two main divisions bacterial.: the gram-negative (e. coli and e-cloacae) and gram-negative (group b streptococcus). And this example gives even still further demonstration of the protective activity in vivo antibody against a lethal challenge of homologous serotypes e. coli and Streptococcus Group B is repeated the such as I (essentially described in parts a through g) to prepare a human monoclonal antibody that is capable of multiple protection against infections caused by bacteria described above except that it is necessary to make specific modifications to characterize and test the antibody described in this example. Thereby suitindic changes in test procedures and the results obtained with the monoclonal antibody described herein.

[270]

1. In the supernatants tested antibodies for group b streptococcus test using an enzyme-linked immunosorbent assay of (ELISAs) as in example i. the plates of antigen are a series of microtiter plates Immunolon 2 to flat-bottom 96 well, which the wells contain mixtures of capsule types GBS 8 fixed to the surfaces of the wells by poly-L-lysine-(PLLs). The various plates of antigen used in the screenable include :.

[271]

(1) type mixing the group b streptococcus (aa.t.c.c.

[272]

no. 12400); lb (line A.T.C.C. no. 12401); the (line A.T.C.C. no. 27591);

[273]

(2) a mixture of type II isolates (line A.T.C.C. no. 12973) and III (clinical isolate obtained from the Dr. to C. Wilson's, Children the Patent Hospital in, Dept; of Infectious disease but, in Seattle, WA to); and

[274]

(3) a plate microtiter not withstanding bacteria.

[275]

The culture supernatants from two transformations are analyzed by the above method, leading to the identification of a well (4b9) that has activity on the two sheets of typing group b streptococcus but not on the control plates. It is determined in an ELISA later in the presence of individual types of group b streptococcus that this well contains an antibody reactive household clothes each of five strains reference typing.

[276]

In this way, in this experiment, a human cell line transformed cloned which is continuous (immortal) and secretes a human monoclonal antibody directed against a determinant existing on the surface of kinds of group b streptococcus exposed above.

[277]

Before depositing the traffic request, from the human transformed continuous identified under the n° 4b9 has been deposited at 1' American Type culture, of Rockville, MD1, under the n° aa.t.c.c.

[278]

CRLS 9008.

[279]

2. Attempting an antibody from cell line 4b9 cloned in to determine the reactivity multi against gram-positive and gram-negative by a change in the standard immunotSche technique. Specifically, studied the reactivity multi against E. coli bacteria, K pneumoniae are, marcescens was, E aerogenes was, e-cloacae, influenzae and Staphylococcus aureus, by depositing bacteria on a grid of nitrocellulose paper disk, by reacting the disk containing the bacteria with the antibody and developing antibody reactions-phosphatase enzymatic/AI câlinenitrobluen tetrazolium (as described in the example-I).

[280]

From these experiments, it is observed that the antibody 4b9 multi has a reactivity to a particular species of gram-negative bacteria. This antibody reacts with the serotypes 04, 07, 018 and 025 of the LPS of e. coli and with the isolates CF iniqtfes of e-cloacae. Thus, 1' antibody " monoclonal antibody reactivity 4b9 thus has a multi intergenre between the gram-negative bacteria and gram-positive species E.coli, E cloacae and group b streptococcus.

[281]

3. The finding that the monoclonal antibody reactivity more multiple different genera of bacteria belonging to both bacterial division gram-positive and gram-negative, suggests that this antibody is directed against a protein or carbohydrate. carbon shared between it. The biochemical characterization of the molecular species recognized by the antibodies is performed by analysis by 4b9 immunotâche. For the analysis of gram-negative genera, is extracted from the washed bacteria in deoxycholate as described in Example I for gram-positive bacteria, 1.0 1 bacteria grown during 6 hours in the broth of Todd-to-solid phase modified (Agar, broth Todd-to-solid phase containing 2.8 g/l of anhydrous phosphate, at pH 7.8) to 37 °c, are collected by centrifugation and washed three times in PBS. They are then hung in these bacteria. in 85 ml of medium SiON is protoplast (sucrose 40% p/v in a potassium phosphate buffer 0, 3 Μ, at pH 6.8, containing a-MGCs^10 mm) and the slurry is heated to 37 °c during 10 min. Added approximately 3,000 units mutanolysin (delta) and the mixture is shaken at 37 °c; during 90 minutes or until the 00^of the suspension has been reduced>90%. Digested material is passed to the centrifuge to 2,000 g for 15 minutes at room temperature and dialyzing the supernatant in the presence PBS during 48 hours (Young's modulus M.K. and fewer technical ARCHAMBAULT, "Biosynthesis the Innovation in Wall peptidoglycan which NDA polysacharide antigenically bypass Protoplasts d'III measure groups Β of Streptococcus", J Bact ., (1983) 154, 211 - 220). Concentrated ten times the dialysate by dialysis to positive pressure through a PM and 10 (Amicon TX Danvers, am).

[282]

The carbohydrate-binding agglutinin wheat are purified by affinity chromatography on Sepharose chromatography column packed with 6 mb lectin wheat (delta). The product digested bound, described above, is eluted from the column to 1' aide.de 10 ml of n-acetylglucosamine 0, 1m and dialyzing the eluate in the presence of distilled water at 4 °c. The eluate affinity purified is freeze-dried and used to the dry weight of the resulting material (the Gray B.M. and al, "collapsed and serogroup B Streptococcal-type growth arrest specific polysaccharide avec la Wooster tubelike AgglutininLectins poetry", J gold Immunol. Metha ., (1984) 72, 269 - 277). Noted positive reactions in all tracks which comprise the extracts at deoxycholate bacteria described above. In the tracks containing extracts of gram-negative bacteria, it appears that the antibody 4b9 recognizes a variety of regularly spaced molecular entities, resulting in a ladder-shaped on t'a immunotâche. This profile is entirely consistent with that found in the analysis by gel electrophoresis in the presence of LPS polyamide - SOS, and where it has been demonstrated that the profile dimension heterogeneous presented by the stripes was dq-a population of molecules of LPS differing by increments of weight equivalent to the number of units of oligosaccharide side chain antigen-0 present per molecule (Pavla SE and Makela w.w., super and Goldman, R.D. and Leive L, above). In these tracks containing tests of types of group b streptococcus, it appears that the antibody 4b9 recognizes components present over a wide band. This profile is consistent with that tone encounter in assays of gel electrophoresis of polyamide of carbohydrate radicals which showed significant heterogeneity of molecular weight and a sequence of sugar,, specific frequently recurring (toto vmir E.R. et, above and to Holden K.G., above). These data indicate that the monoclonal antibody is directed against a 4b9 antigenic determinant shared by molecules encountered on some serotypes e. coli, E cloacae and group b streptococcus.

[283]

To further define the molecular nature of the antigen, are treated at dioxycholate extracts with a proteolytic enzyme, proteinase K, prior to electrophoresis (Eberling W, above). The schemas immunotSche observed after treatment with proteinase K are identical to the patterns observed without treatment and thus suggest that the antigen reactive with the antibody 4b9 does not have the nature of a protein.

[284]

To check specifically whether the 4b9 reacts with a carbohydrate epitope, are subjected samples deoxycholate electroblotted and agglutination of affinity-purified wheat germ àà .une moderate periodate oxidation before reacting on nitrocellulose paper with the antibody (see example-I). The extracts at deoxycholate electrodeposited treated in this way are not reactive with the monoclonal antibody 4b9. These data suggest a-.fortement that this epitope recognized by this antibody is a radical carbohydrate present in molecules belonging to both gram-negative and gram-positive bacteria described above.

[285]

4. Determined Visotype 4b9 of monoclonal antibody by a process similar to the tests of specificity of ELISA described above, except that the plate antigen contains a set of type II and type III group b streptococcus iimnobilise by a PLL. To observe a positive reaction of monoclonal antibody 4b9 with the strains of Streptococcus group b with the reagent that IgM, which indicates the presence of a IgM on the monoclonal antibody.

[286]

5. Examined in vitro functional activity of monoclonal antibody 4b9 in an assay that compares the opsonophagocytic activity of the bacteriocidal antibodies in the presence and in the absence of both human neutrophils and of human complement.

[287]

The bacteria strains used herein are only inactivated in the presence of monoclonal antibody 4b9, a source of complement and human neutrophils (table 6). When repeats this experience with several bacterial serotypes unreactive at 4b9, not observed of killing bacteria (given not shown), demonstrating the functional specificity of monoclonal antibody 489 and its ability to bind the opsonin on bacteria and promote their phagocytosis. Since the combined actions of opsonins (specific antibodies) and " polymorphonuclear leukocytes (neutrophils) appear to represent the primary mechanism of immunity against these bacterial strains, these data suggest that the antibody 4b9 could, after appropriate administration, provide protection against lethal attacks carried by the strains of bacteria described herein.

[288]

TABLE 6

[289]

Bacteria

[290]

Neutrophils

[291]

Antibody

[292]

Complement

[293]

% for destroying! Bacterium introduced! !

[294]

1!
TH.escherichia coli 018 and 025•+ '! 4B9-20
TH.escherichia coli 018 and 025! +* to GOL 6f114 *0
I ':escherichia coli 018 and 025!! 4B9T-+0
TH.escherichia coli 018 and 025! +! 4B9T-+85
TH.clocae isolates! +•4B9the I-0
TH.clocae isolatesthe I +! 6F11the I+0
TH.clocae isolatesthe I! 489 j.! the IT-the I0
TH.clocae isolates! + the L "! I-j-4b9! f.!4 *F. the I F.85

[295]

1!!

[296]

Bacteria! * I-neutrophilsAntibodyr! ! J-j-I-|complement. C/o destroying bacterium introduced
Group b streptococcus.the I! +4B9•! ! the T -!0
Kinds Ta and TTI 'i-J.! !
Group b streptococcusTHE J +6F11! +!0
Kinds the and IIIof ii-F.
Group b streptococcus!. -4B9! +!0
Kinds the and III-;t-J.
Group b streptococcusthe I +4B9! +. '!85
Kinds the and IIIthe I "1.1, 1 t-T-F.*!

[297]

* - (-)=•■human complement heat inactivated (56 °c during 30 min)..

[298]

(6F11) - culture supernatant containing monoclonal IgM human

[299]

directed against Pseudomonas aeruginosa Fischer-type 2.

[300]

the I 6. To test the above hypothesis, is performed animal protection studies using the antibody 489 and of at least one microorganism proyenant each kind. described herein.

[301]

For each attack bacteria granvnégatives, of. mouse females Swiss Webster weighing between 20 and 22 g are divided into three. groups of ten-mouse each. Carried out an experiment, representative as follows:

[302]

Group bacterium. Q...Antibodies'

[303]

Has e " çoli 018 -... / 4b9

[304]

B..U.S.•, Ecoli Glâ ^ Receive&wire " "'

[305]

C. sec. marceseens 01:4 'the R - 4B9

[306]

Each group receiving T-antibody, OH-injeoinjeo.te ': by - intravei pathway. 200 ml sterile PBS containing neuse 25 µg of purified antibody. Two hours later, injected to all animals intraperitoneally 300 PI of live bacteria containing 3 VLD ^ Q of their bacterial strain respectively. The bacterial suspension was prepared. from broth culture phase ' logarithmic growth wherein the bacteria is centrifuged, washed twice were found in PBS and the resuspension at the appropriate concentration in the PBS. Animals is observed for a period of 5 days. 24 To 48 hours after the attack, all the animals of group b (irrelevant antibody) and group c (organism irrelevant) died. On the contrary, the animals which received the antibody 4b9 (group a) are all living and systemic.

[307]

In studies of Streptococcus group protecting counteracted ll.è ' b., a model of newborn rats. Sprague-Dawley rats juveniles (housed with their mothers), Sgés of less 48 hours, receive the antibody and bacteria essentially as described by in studies of mouse protection. The main differences are the following:

[308]

1) the antibodies and bacterial attack-are both carried intraperitoneally ., and

[309]

2) the size of the inoeiilum is reduced to 20' PI.

[310]

These models of animal protection are useful for demonstrating the therapeutic effect of the antibody 4b9 against attacks carried by bacterial organisms belonging to the three, species here exposed. A summary of this data is set forth in the. Table 7.

[311]

J.•: 1:*.. / **•'•" *1
Bacterium ofi-I-Antibody.J. 1Survival/Attàque! % j-survival
025 E.colii-I-j-j is4B91' J IS 110/10O-O-H - - -••
025 E.coli6F11310/10the I-O
014 Marcescens wasb.i-I-I-J.4B91! i-J.0/10i-I-O-
The Streptococcus group 84B910/10o-O
the and IIIi-I-J.the I 1
Group b streptococcus!6F110/10the I-O
the and IIIi-j-j-ii iiTHE I...|J IS 1
014 Marcescens was9B10j-j-j is0/10! 0 i-I-

[312]

;. TABLE 7.: the R, ;

[313]

9 The antibody 6f11 is specific of Pseudomonas aeruginosa Fisher method immunotypic 2 and serves as an antibody negative control.

[314]

b. The S. marcescens was 0i4 is not reactive with the antibody 7d7 and serves as an organisms of controlled non-specific.

[315]

These data demonstrate that the human monoclonal antibody is able to protect 4b9 mouse and rats against fatal attacks carried by genera of bacteria belonging to both divisions of gram-negative bacteria and bacteria gràm-positive. The human monoclonal antibody reactivity to multi 4b9 intergenre is able to provide shielding Vg 25 using purified antibody against infections made by organisms belonging to the genera of gram-negative bacteria e. coli and to gram-positive bacteria belonging to group b streptococcus..

[316]

EXAMPLE V.

[317]

The example V gives the demonstration of methods for the preparation and selection of a human monoclonal antibody having reactivity multi intergenre against members of the genera Serratia, Klebsiella pneumoniae and Enterobacter aerogenes. Further, this example demonstrates the in vitro activity of fixing dede.cet opsonic antibodies against homologous serotypes of S. marcescens, K pneumoniae antigens and e. aerogenes was. The process is repeated in the example-I(essentially as described in parts a to g) except that it is necessary to make specific modifications to characterize and test the antibody described in this example. The following nts sente .les changes to the test procedures and to the results obtained with the monoclonal antibody described herein.

[318]

1. The supernatants are analyzed from four transforms by the method. above which lead to the identification of a well (7e10) having binding activity on at least one of the four, of serotypes of K. pneumoniae are plates containing serotypes' capsule:

[319]

1, 2, 3, 4, 6, 8, 9, 19, 20, 21, 24, 27, 31, 43, 44 and 55, but not on the control plate (without, bacterium). One attempts to the antibody from the cell line to determine reactivity 7e10 multi additional intergenre with alterations in the standard immunotSche technique. Specifically studied multi reactivity against bacteria pneumoniae are K, E aerogenes was, marcescens was, e coli and P. aeruginosa by depositing-bacteria on a nitrocellulose paper disk grid, by reacting the disk containing the bacteria with the antibody and developing the reactions of antibodies using an enzyme system nitroblue tetrazolium/alkaline phosphatase.

[320]

From these experiments, it is observed that the antibody has a reactivity 7e10 intergenre multi additional. This antibody reacts with the following species and serotypes:

[321]

The human monoclonal antibody reactivity 7e10 thus has a multi intergenre against bacteria belonging to the genera

[322]

K pneumoniae are, marcescens was and e. aerogenes was.

[323]

2. Determined the isotype of the monoclonal antibody ELISAs 7e10 by a procedure similar to the tests described in the specificity exempie I-except that the plate antigen contains a set of serotypes of K. pneumoniae are k8. Kll and immobilized to the PLL (poly-L lysine). To observe a positive reaction of monoclonal antibody with the serotypes 7e10 of K. pneumoniae are that with the IgM reagent, demonstrating an IgM isotype the presence on the monoclonal antibody. The skilled worker will appreciate that if tone repeated several times the process of this example and that one determines the isotype of multi cross-reactive monoclonal antibodies so obtained genus, we would find other additional isotype, for example IgM and IgG.

[324]

3. Examined in vitro functional activity of monoclonal antibody 7e10 in an assay that compares the opsonophagocytic activity of the bacteriocidal antibodies in the presence and in the absence of both human neutrophils and of human complement. The sërotypés bacterial used herein are inactivated ERT has AEG presence of monoclonal antibody 7e10, a source of active complement and human neutrophils (table 8). When tone repeats this experience with serotypes does not react with the antibody JEIO, only destruction of bacteria die not observed (data not shown). AAR experiments demonstrate the functional specificity of monoclonal antibody ' 7e10 and its capacity S fix the opsonin on bacteria, and to promote their phagocytosis.

[325]

1J IS 1
T-the If. F.% destruction of
BacteriaNeutrophils!Antibody( ComplementBacterium introduced
!J! !
1I! !
e.marcescens to 012I+F.7E10-!3 !0
F.b.! !
e.marcescens to 012+f. F. F.!; F.6F11! +!0
e.marcescens to 012!-7E10! +!0
e.marcescens to 012" f.+7E10! +! f86%

[326]

TABLE 8

[327]

K pneumoniae are marcescens was E aerogenes was

[328]

K2, 8, 11, 12, 13, 21, 26 04.12 clinical isolates

[329]

29, 30, 33, 42, 68, 69

[330]

Bacteria

[331]

. Neutrophils

[332]

Antibody

[333]

Complement

[334]

% destruction of bacterium introduced

[335]

J. K. pneumoniae are ** k8 and Kll! the I+ii the I! 7E30 *-1, 1 f.the O -
K pneumoniae are * k8 and Kll! the I+i-I-6f11HAS -10
K pneumoniae are k6 and Kll!-! 7E10+T-0
K pneumoniae are k8 and Kll!+! 7EIO+!94%
E aerogenes was Isolac.!+■! 7EIO-F.O
E aeroganes isolate!+! 6F11+THE O.
E aerogènea isolate 1 T.-! 7E10+T-O
I aetogenes isolate *! T-! 7E10•+1!60%
VBE11

[336]

has. b. -•

[337]

*=■and. see. in the footnotes to the ' table 3.

[338]

EXAMPLE VI.

[339]

The example IV the deircpnstration-of jnéthodes℮ CENTS preparation and df selection of a human monoclonal antibody that has ^ reactivity-multiple-intergenre against members of the genera. Serratia marcescens. marcescens to and Klebsiella pneumoniae. This example further demonstrates activity in vitro attachment opsonic antibody against homologous serotypes of S. marcescens and K. pneumoniae antigens. The process is repeated from the example I-(substantially as described in the; parts a to g), except that it is necessary to make specific modifications to characterize and test the antibody described in this example. The following represents changes in the test procedures and the results obtained with the monoclonal antibody described herein.

[340]

.1. Onanalyse the culture supernatants from four transforms by method - above leading to the identification of a well (8c9) which has a binding activity on at least one, of the four plates of serotypes of K. pneumoniae proteins containing capsule serotypes: 1, 2, 3 ., 4, 6, 8, 9, 19, 20, 21, 24, 27, 31, 43, 44 and 55, but not on there control plate (without bacterium). One attempts to the antibody.

[341]

from the cell line 8c9 to determine yet the reactivity multi intergenre by changingthe immunotSche technique:

[342]

the standard. Specifically studied multi reactivity against bacteria pneumoniae are K, E aerogenes was, . Marcescens was, e coli and P. aerugî-to-5 nosa by depositing bacteria on a paper disk of nitrocellulose. grid, by reacting the - disk comprising the bacteria with the antibody and developing reactions in antibody Taided ' an enzyme system nitroblue tetrazolium/alkaline phosphatase.

[343]

From these experiments, it is observed.. that the antibody has a reactivity 8c9 intergenre multi additional. This antibody reacts with the following species and serotypes. :, •'

[344]

K pneumoniae antigens ... Marcescens was K5, 6, 7, 14, 27, 36, 55, 64. ' -03

[345]

Thus, the human monoclonal antibody has a reactivity 8c9 intergenre multiply. against bacteria. the genera K pneumoniae and marcescens was.

[346]

2. Determined the isotype of L.' ànticprps 8c9 monoclonal antibody by a process similar to the tests of ELISAs, specificity described in the example. I-except that the plate antigen contains a set of serotypes of K. pneumoniae are immobilized k27 k14 and even, the PLL. N is' observes a positive reaction of monoclonal antibody of K. pneumoniae are 8c9.avec serotypes with the reagent that IgM, demonstrating the existence of a IgM 'on the monoclonal antibody .'. The skilled worker will appreciate that if tone repeated several times the process of this example and if it is determined the isotype antibodies monoclo penal. to reactivity and multi intergenre

[347]

; obtained, we would find other additional isotype, e.g.

[348]

•IgM and IgG.

[349]

3. Examined the functional activity in vivo. vitro the monoclonal antibody in an assay 7e10 opsonophagocytic-which compares the activity of the bacteriocidal antibodies in the presence and in the absence of both human neutrophils and of human complement. The bacterial serotypes used herein are inactivated that in the presence of d-ànticorps 8c9 a monoclonal, a source of active complement and human neutrophils.

[350]

(Table 9). When tone repeats this experience with non-reactive with the antibody serotypes 8c9, not observed destructiorfde bacteria (data not shown). These experiments demonstrated the functional specificity of monoclonal antibody 8c9 as well as its ability to bind the opsonin on bacteria and promote their phagocytosis.

[351]

TABLE 9

[352]

Bacteria

[353]

Neutrophils

[354]

Anticoms

[355]

Y. hasT-
03 Marcescens was1! 8C9T-0
03 Marcescens was, ~+b.!OF FU&+the I0
03 Marcescens wasVBE1-! 8C9+T-0
03 Marcescens was1+! 8C9+Y70%
K pneumoniae are k14 k27 and1+! ' 8C9-the I0
K pneumoniae are k34 k27 and;+! 6F11+the I0
K pneumoniae are k3a etK27i-J.-! 8C9. the I+Y0
K., K. pneumoniae k1a k27 andi-I-+! T-8c9■+!93%
.the IT-J

[356]

Complement

[357]

% destruction of bacterium introduced

[358]

has' b.

[359]

and=see in the footnotes to table 3.

Example of vIL '

[360]

The example VII provides the demonstration of methods for the preparation and selection of a human monoclonal antibody having reactivity multi intergenre directed against members of the genera Serratia Klebsiella, Enterobacter species aerogenes and the Enterobacter cloacae. This example provides further demonstration of in vitro activity of opsonin binding of this antibody against homologous serotypes of S. marcescens, K pneumoniae are, E aerogenes and e-cloacae. The process is repeated in the example-I(essentially as described in parts a through g), except that it is necessary to make specific modifications to characterize and test the antibody described in this example. The following represents the changes in the test operating modes and in the results obtained with the monoclonal antibody described herein.

[361]

1. The culture supernatants of four transformations are ' analyzed by the above method which lead to the identification of a well (1e4) having binding activity on at least to one of between the four serotypes of K. pneumoniae are die plates containing serotypes capsule:

[362]

The I, 2, 3, 4, 6, 8, 9, 19 ., 20, .21, 24, 27, 31, -43, 44 and 55, but not on the control plate (without bacterium). In attempting the antibody of Ε 1 cell line 4 to determine reactivity intergenre multiply by modifying the standard immunotâche technique. Specifically, studied the reactivity against bacteria multiply. K pneumoniae are, E aerogenes was, marcescens was, e. coli and P. aeruginosa by depositing bacteria on a nitrocellulose paper disk grid, by, reacting the disk containing the bacteria with the antibody and developing jmticorps reactions using an enzyme system nitroblue tetrazolium/AI câline phosphatase inhibitors.

[363]

From these experiments, it is observed that the antibody has a reactivity 1e4 intergenre multi additional. This antibody reacts with the following species and serotypes:

[364]

K pneumoniae are marcescens was E aerogenes was e-cloacae. Ml, 3, 8, 9, 13, 15, 29, . 015 Clinical clinical isolates 31, 33, 36, 68, 69 isolates the human monoclonal antibody reactivity 1e4 thus has a multi intergenre against bacteria belonging to the genera K pneumoniae are, marcescens was ., e-cloacae and e. aerogenes was. '.

[365]

2. Determined the isotype of the monoclonal antibody 1e4 in a procedure similar to the tests of specificity of ELISA described in the example - I-except Que plate antigen contains a set of serotypes of K. pneumoniae antigens immobilized on etet.k8 k3.] has a PLL. . Is n; 'observes a positive reaction of. 1' 1e4 monoclonal antibody with the serotypes of K. pneumoniae are that in the presence of the reactive IgM, which indicates the presence of a IgM on the monoclonal antibody. The skilled worker will appreciate that if tone repeated several times the process of this example and if tone determines the isotype of multi-reactive monoclonal antibodies so obtained intergenre, we would find other additional isotype, for example IgM and IgG.

[366]

3. Examined in vitro functional activity of monoclonal antibody in an assay 1e4 opsonophagocytic bacteriocidal activity which compares the antibodies in the presence and in the absence of both human neutrophils and of human complement. The bacterial serotypes used herein are inactivated that in the presence of the monoclonal antibody 1e4, a source of active complement and human neutrophils (table 10). When tone repeats this experience with non-reactive with the antibody serotypes 1e4, not observed of killing bacteria (data not shown). These experiments demonstrated the functional specificity of monoclonal antibody 1e4 as well as its ability to bind the opsonin on bacteria and promote their phagocytosis.

[367]

1 * T-bacteriaNeutrophils1, 1 * I-antibodyI-j-supplement% destruction of bacterium introduced
! ! 015 Marcescens was!!+the I " 1e4the I _has Y0
015 Marcescens was!!+1 6f11 b.the I +O
015 Marcescens was!!-! 1E4 '+!0
015 Marcescens was!!+! 1E4+!86%
! K pneumoniae are k3 k29 and!+! 1E4!0
! K pneumoniae antigens K3 and k29! i-I-+! 6F11+!O
! K pneumoniae are k3 k29 and!-1 1E4+ ' i-0
! K pneumoniae are k3 k29 and!+! 1E4 '+!80%
! (2) Isolate the E. aerogenes was!+! 1E4!0
! (2) Isolate the E. aerogenes was!* +! 6F11+ Y0
! (2) Isolate the E. aerogenes was.!! 1E4+ Y0
! (2) Isolate the E. aerogenes was!! 1E4.+!80%
! E-cloacae isolate ii'" +! 1E4•!0
! E-cloacae isolate!+! 6F11 -+ 10
! E-cloacae isolate!. .. the R 'the I 1- - Εψ ψ) ^ -•. : -..' of fibre.
! E-cloacae isolate!1e4 9.ï.' +. !.•; '80%■'

[368]

TABLE 10

[369]

and=see in the footnotes to table 3.

[370]

EXAMPLE VIII.

[371]

.. The L'example VIII gives the demonstration and methods of preparation

[372]

selection of a human monoclonal antibody having reactivity multi intergenre directed against members of the genera Sérratia marcescens was, Klebsiella, or Pseudomonas aeruginosa the Enterobacter aerogenes and. Further, this example demonstrates the in vitro activity of securing opsonin of this antibody against ' homologous serotypes of S. marcescens,

[373]

Κ. K. pneumoniae, P. aerogenes and E. The process is repeated in the example-I(essentially as described in parts a through g), except that it is necessary to make specific modifications to characterize and test the antibody described in this example. The following represents the changes in the test operating modes and in the results obtained with the monoclonal antibody described herein.

[374]

1. Culture supernatants are analyzed four transformations per the above method which lead to the identification of a well (901) having binding activity on at least one of the four plates of serotypes of K. pneumoniae proteins containing serotypes capsule:

[375]

1, 2, 3, 4, 6, 8, 9, 19, 20, 21, 24, 27, 31, 43, 44 and 55, but not on the control plate (without bacterium). In attempting the antibody of cell line 901 to determine additional intergenre multi reactivity with alterations in the standard immunotSche technique.

[376]

Specifically, studied the reactivity against bacteria multiply pnuemoniae K, E aerogenes was, marcescens was, e. coli and P. aeruginosa by depositing bacteria on a nitrocellulose paper disk grid, by reacting the disk containing the bacteria with the antibody and developing the reactions of antibodies using an enzyme system nitroblue tetrazolium/alkaline phosphatase.

[377]

From these experiments, it is observed that the antibody has a reactivity 9d1 intergenre multi additional. This antibody reacts with species and serotypes following :.

[378]

K pneumoniae are marcescens was E aerogenes was P. E9, 13, 15, 29, 33 03, 9, 15, 18 clinical isolates f6 the human monoclonal antibody reactivity 901 thus has a multi intergenre directed counteracted bacteria belonging to K pneumoniae are, marcescens was, E aerogenes and P..

[379]

2. Determined the isotype of the monoclonal antibody. ELISAs 9d1 in a procedure similar to the tests described in the example 1 specificity except that the plate antigen contains a set of serotypes of K. pneumoniae are k13 immobilized to the PLL. To observe a positive reaction of monoclonal antibody 9d1 with the serotypes of K. pneumoniae are that in the presence of the reactive IgM, which indicates the presence of a IgM on the monoclonal antibody. The skilled worker will appreciate that if tone repeated several times the process of this example and if tone determines the isotype of multi-reactive monoclonal antibodies so obtained intergenre, we would find other additional isotype, for example IgM and IgG.

[380]

3. Examined in vitro functional activity of monoclonal antibody in an assay 9d1 opsonophagocytic bacteriocidal activity which compares the antibodies in the presence and in the absence to the ' times of human neutrophils and of human complement -. The bacterial serotypes used herein are inactivated that in the presence of the monoclonal antibody 901, a source of active complement stored characteristics of human neutrophils (table 11). When repeats this experience with non-reactive with the antibody serotypes 901, not observed of killing bacteria (data not shown). These experiments demonstrated the functional specificity of monoclonal antibody 9d1-to-âinsi that its ability to bind the opsonin on bacteria and promote their phagocytosis.

[381]

TABLE 11

[382]

!

[383]

' Bacteria i neutrophil antibody complement % destruction of bacterium introduced

[384]

03 Marcescens wasOf i+1, 19D1hasi-I-0
03 * Marcescens was1Ib. 6f11+!0
03 Marcescens wasF.-!901+!0
03 Marcescens wasT-+ 'J901187%
K - pnuemoniae k13+19D1-!0
K. pneumoniae are k13 *1+ "6F11+10
K pneumoniae are k13J-19D1+0
K pneumoniae are k131 f.+I9D1+J50%.
E aerogenes was isolate(2)!+!9D1-10
E aerogenes was isolate(2)!+16FI1+1 ii F.0
E aerogenes was isolate(2)!+Y9010
E aerogenes was isolate1 (2)!+J IS 1901+Î !70%
P. f6!. +!9DI-T-0
P. f6!!6F11+10
P. f61, 1-! I90110
P. f6J IS 1+ -the I9D1+the T 175% -

[385]

and=see in the footnotes to table 3.

[386]

EXAMPLE IX.

[387]

The example IX. gives the demonstration of methods for the preparation and selection of a human monoclonal antibody having reactivity multi intergenre directed against members of the genera Pseudomonas aeroginosa (P.), the Escherichia coli (I. coli cells), and Serratia marcescens (marcescens was). Further, this example demonstrates the in vitro activity of securing of opsonin ., this antibody against homologous serotypes of P. aeruginosa, e coli and P. marcescens was. The process is repeated in the example 1 (essentially as described in parts a through g), except that it is necessary to make specific modifications to characterize and test the antibody described in this example. The following represents the changes in the test operating modes and in the results obtained with the monoclonal antibody described herein.

[388]

1. Supernatants examined in the presence of anti -

[389]

P. aeruginosa by an ELISA technique as described in the example 1. The plate antigen consists of a microtitration plate ' titration Immunolon 2 to 96 well flat bottom (Dynatech, ADOH, will), whose wells contain a mixture of bacteria immobilized poly-L lysine (PLLs) belonging, to the seven reference strains of P. aeruginosa Fisher method (Fischer M.W. and al, J (1969) 98 OC AB, 835 - 836,

[390]

27312 - 27318 n° line A.T.C.C.).

[391]

The culture supernatants from a transformation are analyzed by the above method which leads to the identification of a well having an activity on the plate of P. aeruginosa, but not on the control plate to the PLL. It is determined in a subsequent ELISA, in the presence of ten seven serotypes of P. aeruginosa individual belonging to multinational antigen typing scheme (IATS, aa.t.c.c.

[392]

no. 33348 - 33364), that a well master 9c3 contains antibodies that bind to serotype-type 1 (the Liu G.P., Int.. J press machine. Bactertol. (1983) 33, 256 - 264, which is incorporated herein for reference).

[393]

As such, in this experiment, a human cell line transformed which is continuous (immortal) and secretes a human monoclonal antibody single liantcà determinant existing on the surface of P. aeruginosa IATS-type 1.

[394]

Before depositing the request 'priority, the human cell line transformed continuous identified under the no." 9c3 has been deposited at 1' American Type culture, of Rockville, MB, under nthe R AA.T.C.C.

[395]

9239 CRLS.

[396]

2. It also tries to the antibody from the cell line 9c3 cloned to determine responsiveness in multi facing of gram-positive and gram-negative by a modification of the technique of d1 conventional immunotâche. Specifically studied multi reactivity against E. coli bacteria, K pneumoniae are, marcescens was, E aerogenes was, e-cloacae, Haemophilus influenza and Staphylococcus aureus by depositing the bacteria on a grid of nitrocellulose paper disk, by reacting the disk containing the bacteria with the antibody 9c3 and by viewing the reactions of the antibody with an enzyme system nitroblue tetrazolium/alkaline phosphatase (as described in the example-I).

[397]

From these experiments, it is observed that the antibody binds to serotype 06 9c3 e-coli serotypes and 012 and of S. marcescens to 014.

[398]

The human monoclonal antibody reactivity 9c3 thus has a multi intergenre against gram-negative bacteria belonging to the species-specific serotypes E parcel, marcescens was and P. aeruginosa.

[399]

3. The finding that the monoclonal antibody has multiple feedback with several different bacterial genera suggests that the antibody can bind to an antigenic determinant shared between these genera. The biochemical characterization of the molecular species recognized by the antibodies is performed by analysis by 9c3 immunotâche as in example I the effects in the extracts at negative from deoxycholate serotypes of P. aeruginosa and E. coli but not of S. marcescens was. Although the reason for which the antibody is reactive with the preparation 9c3 of S. marcescens is not clear, it is possible that the antibody recognizes. a conformational epitope which has been described by the preparation processing. For the e. coli bacterial extracts and P. aeruginosa, it appears that the antibody 9c3 recognizes a variety of regularly spaced molecular entities, resulting in a scheme to run on scale 1' immunotâche. This profile is entirely consistent with that found in the analysis by gel electrophoresis of polyamide of LPS in the presence of SDS for which it has been shown that the profile dimension heterogeneous' is presented dq-a population of molecules differing by LPS weight increments equivalent to the number of units of oligosaccharide side chain antigen-0 present per molecule. (Pavla Ε. Τ .and Makela Ρ. Η ., super, and Goldman R.D. and Leive L, above).

[400]

To define in greater detail the molecular nature of the antigen, are treated at deoxycholate extracts using a proteolytic enzyme, proteinase K, with their electrophoresis (Eberling W, above). The schemas immunotâche observed after treatment with proteinase K are identical to the patterns observed without treatment and thus suggest that the antigen reactive with the antibody 9c3 does not have the nature of a protein.

[401]

4. Determined Tisotype 9c3 of monoclonal antibody in a procedure similar to the tests of specificity of ELISA described above except that the plate antigen contains four bacteria of serotypes of P. aeruginosa Fisher method immobilized to the PLL. To observe a positive reaction of monoclonal antibody 9c3 with the strain of P. aeruginosa than with the IgM reagent, demonstrating an IgM isotype the presence on the monoclonal antibody.

[402]

5. Examined in vitro functional activity of the antibody

[403]

! the monoclonal 9c3 in an assay that compares the opsonophagocytic bactëriocide activity of the antibody in the presence and absence both of human neutrophils and of human complement.

[404]

The bacterial strains used herein are killed in the presence of the monoclonal antibody 9c3, a source of active complement and

[405]

the R . human neutrophils (table 12). When tone repeats this experience with several bacterial serotypes unreactive at 9c3, not observed for destroying bacteria, demonstrating the functional specificity of monoclonal antibody 9c3 and its ability to bind the opsonin on bacteria and promote their phagocytosis. Since the combined action of opsonins (specific antibodies) and polymorphonuclear leukocytes (neutrophils) appears to be the primary mechanism of immunity against these bacterial strains, these data indicate that the antibody 9c3 could, after appropriate administration, provide protection against fatal attacks carried by bacterial strains are described below:

[406]

6. For testing the protection characteristics of the antibody 9c3, protection studies performed on animals with at least one organism from each genus herein described.

[407]

!1T-F.
! TH.06 transporter comprising!+! 9C3.>>!0!
! (b.)1the I
! F...a Colt 06!+! 6F11+ 102
!F.1the IY
! TH.06 transporter comprising!-! 9C3+!0!
1 TH.ecoli 06!+! 9C3+ the I98%!
1|1!
THE S!.014 marceseens!+! 9C3!0!
!(111
b.
THE S!.014 marceseens!+! 6F11the I +0!
THE S!.014 marceseens!-l-9c3+!0F.
THE S!.014 marceseens!+! 9C3+!94%"
! W,aeruginosa-Fisher method 4!! 9C3!0F.
! W.aeruginosa-Fisher method 4!+! 6F11the I +02
F.JYT-
! W.aeruginosa-Fisher method 4!-! 9C3the I +0!
!F.!the I!
! W.aeruginosa-Fisher method 4!+! 9C3+!79%F.
!1JT-!
JF.1!F.

[408]

1 ABLEAU 12

[409]

! ! Bacteria antibody Coraplément! % dCs destruction of! ! Bacterium introduced!

[410]

! the I! E + 06 transporter comprising!! 1! 9c3.the T 0 F.>>!!

[411]

** 006f11 (has) culture supernatant containing monoclonal IgM human human complement heat inactivated (56 °c during 30 min).

[412]

directs against Pseudomonas aeruginosa Fischer-type 2.

[413]

For the experiments protection, there is used a mouse model

[414]

processed by of P. aeruginosa Fisher method 4 and S. marceseens 014. For each bacterial attack, mice females Swiss-Mebster weighing 22 to 25 g are divided into three: groups of 7 to OTf 8 each mouse. An experiment example takes place as follows:

[415]

GroupBacteriumAntibody
' HASP. f49C3
BP. f46F11
_ C.P. f29C3

[416]

The day before the experiment, each mouse is shaved and treated with a depilatory to remove all back (brQlé side). The day of the experiment, each animal receives in each thigh 0.1 ml saline anesthetic containing 0.7 ml 0.85% NaCl, 0.2 ml xylazine (20 mg/ml.) and 0.1 ml of (100 mg/ml.) cetamine, such that the dose is 20 mg/mouse of 180 mg/kg xylazine and kilograms. cetamine.

[417]

The anesthetized mouse receive on 10% of the total surface of the body to a burn the gas flame at 3ème degree, full thickness. Immediately after being inflicted the wound, is injected to the mice in wounds, 0.5 ml of antibody at 4 °c spent culture fluid containing 5 to 10 premixed with VLD ^ Q of bacteria. The bacterial suspension is prepared from a culture broth in a logarithmic growth phase from which the bacteria were centrifuged, washed twice were found in PBS, were found to be resuspended at the appropriate concentration in PBS. Animals is observed for a period of 10 days.

[418]

Three to five days after this action, all animals died in the group b (irrelevant antibody) ^ and the group c (unmodified ^ relevant). On the contrary, the animals which received the antibody 9c3 (group a) sontsont.tous live and systemic (see table 13).

[419]

In studies of protecting against e-coli 06, Swiss-Webster mouse healthy (20 to 22 grams) are divided into three groups of 10 each mouse. Each receiving group of the antibody, is transdermal " ^ PI-PBS sterile intravenous 200 containing 25 mg of purified antibody.

[420]

Two hours after, to all animals are injected intraperitoneally with 300 ml of live bacteria containing 3 VLD5Q their bacterial strain respectively (the results are shown table 13).

[421]

Expér.-! Bacterium driver! [j.Antibody! Survival/I-the I! the I % survival
1i-P! 1.pseudomonas aeruginosai-f4!1 9C3! I6/7! 86 i-
ponse. Ipseudomonas aeruginosaI-I-f46Fll(has) !|0/7i-I-O-
i-I-Ppseudomonas aeruginosaF2(b.) i-I-9C3! the I0/7i-I-O-
2i-I-eecoli 06|9C3! the I6/10the j. 60, 1
i-I-eecoli 061|6F11 ii VBE10/10i-I-O-
3! 1 SEC.marcescens was014!|9C3! the I8/8the I 100!
! I-sec.marcescens was014 the I 16F11!0/8i-I-O-

[422]

TABLE 13

[423]

has The antibody 6f11 is specific of Pseudomonas aeruginosa Fisher method immunotypic 2 and serves as an antibody negative control -.

[424]

^ P. aeruginosa f2 is not reactive with the antibody 9c3 and it serves as a nonspecific control organisms.

[425]

The data demonstrate that the monoclonal Tanticors 'human 9c3 is able to protect mouse fatal attacks carried by bacteria belonging to three genera gram negative.'the antibody human monoclonal reactivity 9c3 multi intergenre is capable of providing protection by means of a culture supernatant containing the antibody or purified antibody against infection by the organisms belonging to the genera willwill grara-negative E.coli, marcescens was and P. aeruginosa. -

[426]

Based on the foregoing, it can be appreciated that the cell lines of the present invention provides compositions of human monoclonal antibodies and fragments thereof to reactivity is multiple and multi-reactivity against various bacterial species both gram negative and gram positive. This allows for more easily at the point are prophylactic and therapeutic compositions that can be effective against infections nosocomiennes and from neonatal to most, if not all genera of bacteria. By combining the antibody, it is possible to obtain broad protection against a great part normally not yet clinically significant antibodies. Further, the cell lines provide antibodies that find their applications in immunoassays in other well-known procedures.

[427]

Although the present invention has been described with some detail by way of example and by way of example for purposes of clarity and understanding, it is evident that tone may make some changes and amendments while remaining within the scope of the attached claims.



[428]

Cell lines have been produced that secrete human monoclonal antibodies capable of binding to molecules of different bacterial species. These antibodies have been found to be protective against lethal challenges of various bacterial genera. Pharmaceutical compositions containing these antibodies, which can be in combination with other monoclonal antibodies, blood plasma fractions and antimicrobial agents, and the prophylactic and therapeutic use of such compositions in the management of infections are included. Prior to filing of this patent application the continuous transformed human cell lines 9B10, 4F10, 4B9, 7D7, and 9C3, described herein, were deposited in the American Type Culture Collection and given the designations CRL 9006, CRL 9007, CRL 9008, CRL 9009, and CRL 9239, respectively.



1, - A composition useful in the therapeutic or prophylactic treatment and/of. bacterial infections, said composition comprising an effective prophylactic or therapeutic amount of a number of human monoclonal antibodies - and being reactive with at least two bacterial species .'d ' at least two genera, at least one of the antibodies or binding fragment. the antibodies capable of reacting specifically with a carbohydrate epitope is not part of the shared core by ' serotypes are two different species.

2. - A composition according to claim 1, wherein at least one of the antibodies or one of its fragments binding reacts with serotypes for species ofthe R at least three species and bacterial genera.

3. - A composition according to claim 1, wherein at least one of these bacterial species is a gram-positive and the second is gram-negative.

4. - A- the composition of claim 1 -, wherein each

; at least two of these antibodies is protective against infections caused: by bacteria of at least two kinds.

5, - A composition useful in the prophylaxis and/or therapy of bacterial infections, which composition comprises an amount of at least one therapeutic or proprylactic two human monoclonal antibodies or binding fragments thereof in which each of these ' antibody reacts specifically with a carbohydrate epitope is not part of the shared core by serotypes of two or more than two bacterial species of various types and a plurality of serotypes in at least one species, wherein these species include:

The Escherichia coli, N. meningitidis, Serratia, Klebsiella, Enterobacter species aerogenes was, the Enterobacter cloacae, Pseudomonas aeruginosa and Streptococcus agalactiaeand of group b.

6, - A composition according to claim 1, having at least one human monoclonal antibody which -. is reacted with at least one of the combinations of the following bacterial species:

(has) E.coli, marcescens was and e. aerogenes was;

(d) e. coli and N. meningitides;

(d)" th. coli strain, E cloacae and. the Streptococcus, Streptococcus agalactiae group b;

(d.) K pneumoniae are, marcescens was and e. aerogenes was.;

(i) K pneumoniae and marcescens was;

(f.) Κ. pnuemoniae, marcescens was, Ε. aerogenes and Ε. clocae; ·

(gm). X pnuemoniae ., marcescens was, Ε. aerogenes and Ρ Pseudomonasaeruginosa.; or

(hr). Ε Escherichia coli, Pseudomonas aeruginosa and Ρ marcescens was.;

7. - A pharmaceutical composition comprising a composition according to any one of claims 1, 5 6 and a physiologically acceptable carrier.

8. - An immortal cell line which secretes a human monoclonal antibody or a binding fragment thereof having reactivity with a specific multi-epitope comprising a carbohydrate radical is accessible not part of the core present on at least two serotypes or two different species of different genera of bacteria.

9. - A cell line according to claim 8 wherein these bacterial species represent at least two of the Escherichia coli, Serratia, Neisseria, K. pneumoniae, the Enterobacter cloacae, the Enterobacter aerogenes was, Pseudomonas aeruginosa and Streptococcus agalactiaeand of group b.

; 10, - A cell line according to claim 8 referred n° line A.T.C.C. n° CRLs 9006, 9007 CRLs, CRLs 9008, 9009 and CRLs 9239 CRLs.

11. - A human monoclonal antibody or a binding fragment thereof capable of binding àà.un carbohydrate epitope reactive with a monoclonal antibody produced by a cell line according to claim 10.

12. - A kit for use in detecting the presence of at least two members of species of different bacterial genera, the kit comprising a monoclonal antibody composition containing at least one monoclonal antibody, wherein the antibody reacts; with a carbohydrate determinant is not part of the core shared by at least two members of different bacterial species and a label providing a detectable signal covalently bound to said antibody or linked to a second antibody reactive with the monoclonal antibody.

13. - A composition useful in the prophylactic and/or therapeutic neonatal sepsis or meningitis, the rim composition comprising a prophylactic or therapeutic amount of at least two human monoclonal antibodies or the binding fragment, wherein each antibody reacts specifically with a carbohydrate epitope is not part of the shared core by serotypes of two or more than two bacterial species of different genres, wherein these species include: Escherichiacoli ml, of Neisseria meningitidis group G, influenzae type R and the group b Streptococcus agalactiaeand.

14. - A composition useful in the prophylactic and/or therapeutic effect of a certain preselected number of bacterial species, cettecomposition comprising an amount effective prophylactic or therapeutic of two or more human monoclonal antibodies or binding fragments thereof, these antibodies being capable of ' react with a total of at least four bacterial species, and a compatible carrier.

15. - A method of preparing a pharmaceutical composition useful in the prevention or treatment of bacterial infections, which method comprises:

- combine a number of human monoclonal antibodies wherein the composition is reacted with at least two, bacterial species of at least two genera, wherein at least one of these monoclonal antibodies and binding fragment thereof is capable of reacting specifically with a carbohydrate epitope is not part of the shared core by serotypes are two different species.

16. - A method of preparing a pharmaceutical compositien. useful for the prophylactic treatment of OA and/therapeutic sepsis or neonatal meningitis wherein this method involves combining a

•prophylactic or therapeutic amount of at least two human monoclonal antibodies or binding fragments thereof die, wherein each antibody specifically reacts' with a carbohydrate epitope is not part of the shared core by serotypes of two or more different bacterial genera - species in which these species, include: e-to-cot'cot' i ml, of Neisseria meningitidis, haemophilus influenzae and Streptococcus agalactiaeand of group b '.

17. - A method for producing ^ ' human monoclonal antibodies with reactivity-multiple-specific with an epitope comprising a carbohydrate radical not part of the core or present on at least two serotypes moimoi.ns two different species of different genera of bacteria, characterized in that it comprises culturing a cell lines such as defined, in any one of claims 8, 9 or 10 and recovering said antibody.

18. - A method of producing human monoclonal antibodies capable of specifically binding to an epitope consisting of a carbohydrate is not part of the core, shared by serotypes are two different genera OD bacteria, in method comprising:

isolating the cells - b of an individual,

- to immortalize these b cell to form a plurality of antibody-producing clone,

- to select these clones for the production of antibodies specific for an epitope consisting of a carbohydrate is not part of the core, shared by serotypes are two different genera of bacteria,

- and culturing these clones grown and then collecting the monoclonal antibodies so produced.

19. A method according to claim 18, characterized in that said B cells are immortalized by EBV-transformed.