CA1340018C - Monoclonal antibodies reactive with cachetin - Google Patents

Abstract

Cell lines are provided that produce improved neutralizing monoclonal antibodies reactive with human cachectin. The antibodies have various therapeutic and diagnostic uses.

Description

1340~ L~

IMPROVED MONOCLONAL ANTIBODIES REACTIVE WITH CACHECTIN

FIELD OF THE INVENTION

The present invention relates to the application of immunological techniques to provide novel materials useful in diagnosing and treating, inter alia, bacterial infection, and, more particularly, to the production and application of improved monoclonal antibodies that are capable of specifically reacting with human cachectin.

BACKGROUND OF THE INVENTION

The introduction of invasive stimuli, such as parasitic, bacterial or viral infections, or neoplastic cells, induce significant metabolic changes in the susceptible host. If serious, the changes may eventually disrupt normal homeostatic mechanisms, both locally and systemically, leading to the depletion of host energy stores advancing to wasting (Cachexia), tissue damage, multiple organ system failure, shock and death.
Until recently, clinicians believe that the systematic patterns were primarily due to actions of the invasive agents themselves. However, it is now known that the invasive stimuli caused the host to generate various cytokines, the combined actions of which cause most of the undesirable biological responses. These host-derived inflammatory mediators present new opportunities for developing treatment regimens against a wide variety of inflammatory disease states.
One of the most potent cytokines is cachectin, which is primarily released by macrophages after appropriate stimulation. Cachectin, also known as Tumor Necrosis Factor, is a protein having 157 amino acids *

normally found in vivo as a dimer or other multimer. The calcula-ted molecular weight of human TNF monomer is about 17,000 daltons.
Cachectin acts to suppress biosynthesis of several adi-pocyte-specific proteins, such as lipoprotein lipase. It also acts to induce the biosynthesis or release of numerous other pro-teins, including C'Lass I major histocompatibility antigen, granu-locyte-macrophage-colony stimul'~ting factor, and interleukin 1.
(See, generally, Beutler and Cerami, New ~. J. of Med., 316:379-385 (1987).
The recoqnition of cachectin's broad influence on various disease states has led to attempts in controlling its actions. For example, experiments have shown that antibodies specifically reactive with cachectin may be therapeutically useful in controlling the immunomodulatory responses now known to be associated with cachectin (see, U. S. Patent Nos. 4,603,106 and 4,684,623. In particular, neutralizing antibodies capable of binding various epitopes on hum~n cachectin at high affinity have significant potential for medi~ting the toxic effects of excess cachectin levels.
Thus" there exists a need for improved antibodies capable of neutralizing the toxic effects of cachectin ln vivo.
The present invention fulfills these needs.
SUMMARY OF THE INVENTION
Novel ce'Ll lines are provided which produce monoclonal antibodies capable of binding to human cachectin epitopes with enhanced neutralization capabi'Lity ln vivo. Additionally, methods are provided for treating a hum~n susceptible to bacteremia or sepsis or already infected with an endotoxin-bearing bacteria by administering a prophylactic or therapeutic amount of a composi-tion comprising at least one monoclonal antibody or binding - 1340~ i ~

f ragment thereof ~apable of react ing wlth human cachect l.n and exhibiting neutra:..izing activlt y in an Lq2'~ cell cytolytic assay at less than about 400ng, typl cally between about 50 to 200 ng or more, t}-le composit ion preferab~Ly further including a physiologically a(: ceptable carl-ier . The composit ion may also contain any one ol more of the following ~dditional monoclonal ant ibodies capabl~? of react inq with bacteri.a:l endotoxlns or exotoxins; monock)nal antlbod:~.es capable of reacting with serotype determinants Ot-l p~lrticul.ar ba( t erial stralns bearing endotoxins; a 10 gamma globulln fraction frorn hurnan blood p]asma; a gamrna globulln f ract lon f rom hurncln blood plasrna, where the plasma may be obtalned frorn a hurnan exhit)iting elevated levels of irnmunoglobullns reactive with bacl erial endotoxins; and one or more antlmicrobial agents. Further, clinical usec; of the monoclonal antibodies are provldecl, includirlg the produ( t ion cf diagnostic klts .
In part :~ cular, the presellt lnvent ion provides a compositlon comprising a pharmaceutlcally acceptable carrier or excipient, in admï.xture with a rnonoclonal ant ibody produced by the cell line designat ed A .I'.C.C. Accession Number HB9736 or A.T.C.C
20 Accession Nurnber HB9737, or b.inding fragmer,ts thereof.
The present invent i(-:)n also provides a composit ion cornprising a pharlllaceut ical ly acceptable carrier or excipient, ln admixture with a monoclonal ant ibody produced by the cell line designated A.T.C.~ . Accession Nurnber HB9736, or a binding fragment thereof and a rnonc:~clonal ant ibc)dy produced by the ce].l line designated A.T.C.C. Accession Nurnber H~9737, or a binding fragment t he reof .

3a 134 0018 The present invent if ~ll furt her prc~vides a pharmaceut ical composition usefuL for t:reatin(3 or prevent~ng an infection, said compos lt lon compr i s ing a monoc 'lonal ant ibody produced by the cel L
1ine designated A T.C.C. Accession Nutnber E~Bg736 or A.T.C.C.
Accesslon Number IJiBg737, c,r bindlng fragments thereof, an ant imicrobial agellt, a galrla g Lobul ir, f ract } on f rom hurnan blood plasma and a phys iologlcally acceptable car rler.
The present inve~ntion yet also pl-ovides a cell line des ignat ed A . T . C . ~ . Access ion Number HB~ 7 3~' . The present 10 lnvention furtheL provicles a cell line deslgnated A.T.C.C.
Accesslon Number HB9737 . The invent lon al~o includes the monoclonal ant ibodies pr oduce-l by these ce 1 1 llnes .
The pre ,ent invent iOIl also provides a kit for use ln detect ing the E~resence of endo',-oxin-bearing bacterla infection ln a host, said kit compric;lrl,g a monoclonal ant ibody composit ion containing monoclonal antibodies produced k~y a cell line designated A.T.C.~'. Accession Number HBg73~ or A.T.C.C. Accession Number HB9737 , or binding fra(~lnents ti-lereof, and labels providin~
for a detectable ',ignal cc,valelltly bonded t-o said antibody or 20 bonded to second ~lnt ibocli~s react ive with each of said monoclonal ant ibodies .
~ES~'RIPTICIN OF Ii'EIE SPECIFIC EMBODIMENTS
In acco~-dance with 1 he present irvention, novel cells capable of produc ing neutraliz lng monoclon~l ant ibodies of desired affinities and compi~is1tions colnprising SlJC~ antibodies are provided, such compositiorls be1ng capable c,f selectively recognizing epitopes present OIl hulnan cachect in . The sub.~ect cells have identii-iable chromosomes in which the germ-line DNA

., .

3~ 134~01~
from them or a precllrsor cell has rearranged to encode an antibody having a binding site for a de~ired epitope on hurnan cachectin.
These monoclonal antiboclies carl be used in a wide variety of ways, including diagnos~s and therap~.
The rnonl~clona]. antibodles so provi(ied are particularly useful, when col-npared to ~rior art antibodies, in the treatment or prophylaxis of se!~-ious disease;" such as ~acteraemia, sepsis, cachex:ia, or other (iisease stat-es associated wlth elevated levels of cachectin. Thus, the antibodies will typically have neutralizing a(-t- i~tity at ''C' ..~ .

13~0018 less than 400 ng, preferably less than about 300 ng, most prefer-ably about 50 to 2()0 ng, in the L929 cell cytolytic assay. This higher level of activity permits utilizing significantly lower dosage levels for treatments.
The preparation of monoclonal antibodies can be accom-plished by immortaLizing a celL line capable of expressing nucleic acid sequences that code for antibodies specific for an appropri-ate epitope on human cachectin. The immortalized cell line may be a mammalian cell line that has been transformed through oncogene-sis, by transfection, mutation, or the like. Such cells includemyeloma lines, lymphoma lines, or other cell line capable of sup-porting the expression and secretion of the immunoglobulin, or binding fragment thereof, in vitro. The immunoglobulin or frag-ment may be a naturally-occurring immunoglobulin of a mammal other than the preferred mouse or human sources, produced by transforma-tion of a lymphocyte, particularly a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, e.g., a myeloma, to produce a hybrid cell line. Typically, the spleno-cyte will be obtained from an allima1 immunized against cachectin-related antigens or fragments thereof containing an epitopic site.Immunization protocols are well known and can vary considerably yet remain effective. (See, Goc3ing, Monoclonal Antibodies:
Principles and Practice, 2d Ed.f Academic Press, N.Y. [1986].
The hybrid cell lines may be cloned and screened in accordance with conventional techniques, and antibodies in the cell supernatants cletected that are capable of binding to the desired human cachectin determinants with appropriate affinities.
The appropriate hybrid cell lines may then be grown in large-scale culture or injected into the peritoneal cavity of an appropriate host for production of antibodies ascites fluid.
By virtue of having t:he antibodies of the present inven-tion, which are known to be specific for the human k~
. ~ .

cachectin protein, in some cases the supernatants of subsequent experiments may be screened in a competition assay with the subject monoclonal antibodies as a means to identify additional examples of the desired anti-human cachectin monoclonal antibodies (so-called "blocking antibodies"). Thus, hybrid cell lines can be readily produced from a variety of sources based on the availability of present antibodies specific for the particular cachectic determinants at the appropriate affinity levels.
Alternatively, where hybrid cell lines are available that produce antibodies specific for the subject epitopic sites, these hybrid cell lines may be fused with other neoplastic B-cells, where such other B-cells may serve as recipients for genomic DNA coding for the antibodies. These antibodies may be functional at the cell surface and not, for example, as receptors. While rodent, particularly murine, neoplastic B-cells are most commonly utilized, other mammalian species may be employed, such as lagomorpha, bovine, ovine, equine, porcine, avian or the like.
The monoclonal antibodies may be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgE, or subclasses of IgG known for each species of animal. Generally, the monoclonal antibodies may be used intact, or as binding fragments, such as Fv, Fab, F(ab') 2' but usually intact.
The cell lines of the present invention may find use other than for the direct production of the monoclonal antibodies. The cell lines may be fused with other cells (such as suitably drug-marked human myeloma, mouse myelomas or human lymphoblastoid cells) to produce hybridomas or triomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies. Alternatively, the cell lines may be used as a source of the chromosomes encoding the immunoglobulins, which may be isolated and transferred to cells by techniques other than fusion. In - 6 - 134 001 g addition, the genes encoding the monoclonal antibodies may be isolated and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin in a variety of hosts. Particularly, by preparing cDNA libraries from messenger RNA, a single cDNA clone, coding for the immunoglobulin and free of introns, may be isolated and placed into suitable prokaryotic or eukaryotic expression vectors and subseq-lently transformed into a host for ultimate bulk production. (See, generally, U. S. Nos.
4,172,124; 4,350,683; 4,363,799; 4,381,292; and 4,423,147. See a_ , Kennett, et al., Monoclonal Antibodies, Plenum, New York [1980], and references cited therein.
More specifically, in accordance with hybrid DNA tech-nology, the immunoglobulins or fragments of the present invention may be produced in bacteria or yeast. (See, Boss, et al., Nucl.
Acid. Res., 12:3791 and Wood, et al., Nature 314:446. For example, the messenger RNA transcribed from the genes coding for the light and heavy chains of the monoclonal antibodies produced by a cell line of the present invention may be isolated by differential cDNA hybridization employing cDNA from BALB/c lymphocytes other than the subject clone. The mRNA that does not hybridize will be rich for the message coding for the desired immunoglobulin chains. As necessary, this process can be repeated to further enhance the desired mRNA levels. The subtracted mRNA
composition may then be reverse-transcribed to provide for a cDNA
mixture enriched for the desired sequences. The RNA may be hydrolyzed with an appropriate RNAse and the ssDNA made double-stranded with DNA polymerase I and random primers, e.g., randomly fragmented calf thymus DNA. The resulting dsDNA may then be cloned by insertion into an appropriate ~ector, e.g., virus vectors, such as lambda vectors or plasmid vectors (such as pBR322, pACYCI84, etc.). By developing probes based on known sequences for the constant regions of the light and heavy chains, those cDNA clones having the gene coding for the desired light and heavy chains can be identified by hybridization. Thereafter, the genes may be excised from the plasmids, ~' 13~0018 manipulated to remove superfluous DNA upstream from the initiation codon or constant region DNA, and then introduced in an appropriate vector for transformation of a host and ultimate expression of the gene.
Conveniently, mammalian hosts (e.g, mouse cells) may be employed to process the chain (e.g., join the heavy and light chains) to produce an intact immunoglobulin, and furthermore, secrete the immunoglobulin free of the leader sequence, if desir-ed. Alternatively, one may use unicellular microorganisms for producing the two chains, where further manipulation may be re-quired to remove the DNA sequences coding for the secretory leader and processing signals, while providing for an initiation codon at the 5' terminus of the sequence coding for t:he heavy chain. In this manner, the immunoglobulins can be prepared and processed so as to be assembled and glycosylated in cells other than mammalian cells. If desired, each of the chains may be truncated so as to retain at least the variable region, which regions may then be manipulated to provide for other immunoglobulins or fragment spe-cific for the cachectin epitopes (see, e.g., European patent application publication numbers 0239400 published September 30, 1987, and 0125023 publishecl November 14, 1984.
The monoclonal antibodies of the present invention are particularly useful because of their high affinity for epitopes of human cachectin. Also, some of the monoclonal antibodies are protective _ vivo, permitting incorporation into pharmaceutical products, such as antibo(1y combinations for bacterial infections.
Monoclonal antibodies of the present invention can also find a wide variety of utilities in vitro. By way of example, the monoclonal antibodies can be utilized for purifying native or recombinant human cachectin, for selectively removing human cachectin in a heterogeneous mixture of proteins, or the like.
For diagnostic purposes, the monoclonal antibodies may either be labeled or unlabeled. Typically, the diagnostic assays entail detecting the formation of a complex through the binding of the monoclonal antibody to the protein. When unlabeled, the anti-bodies find use i~ agglutination assays. In addition, unlabeled antibodies can be used in combination with other labeled anti-bodies (second antibodies~ that are reactive with the monoclonal antibody, such as antibodies specific for immunoglobulin. Alter-natively, the mon~clona:L antibodies can be directly labeled. A
wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Numerous types of immunoassays are available, and by way of example, some include those described i~ U. S. E'atent Nos. 3,817,827; 3,850,752;
3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.
Commonly, the monocLonal antibodies of the present invention are utilized in enzyme immunoassays, where the subject antibodies, or second antibodies from a different species, are conjugated to an enzyme. When a sample containing human cachec-tin, such as human blood or lysate thereof, is combined with the subject antibodies, bindirlg occurs between the antibodies and those molecules exhibiting the desired epitope. Such complexes may then be separated from the unbound reagents, and a second antibody (labeled with an enzyme) added. Thereafter, the presence of the antibody-enzyme conjugate specifically bound to the cells is determined. Other conventional techniques well known to those skilled in the art may also be utilized.
Kits can also be supplied for use with the subject antibodies for detecting human cachectin in solutions or the presence of human cachectin epitopes in 13~001~

recombinant fractions. Thus, the subject monoclonal antibody composition of the present invention may be provided, usually in a lyophilized form, either alone or in conjunction with antibodies specific for endotoxins, exotoxins or gram-negative bacteria. The antibodies, which may be conjugated to a label or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., bovine serum albumin, or the like. Generally, these material will be present in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt. of the total composition. Where a second antibody capable of binding to the monoclonal antibody is employed, this will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above.
The monoclonal antibodies of this invention can also be incorporated as components of pharmaceutical compositions containing a therapeutic or prophylactic amount of at least one, but commonly a mixture comprising two or more, of the monoclonal antibodies of this invention with a pharmaceutically effective carrier. A
pharmaceutical carrier should be any compatible, non-toxic substance suitable to deliver the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically accepted adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Such compositions can contain monoclonal antibodies specific only for human cachectin.
Alternatively, a pharmaceutical composition can contain monoclonal antibodies reactive directly with bacteria can 13~0018 be utilized to form a "cocktail." For example, a cocktail con-taining monoclonal antibodies against human cachectin epitopes and against groups of the various bacterial strains (e.g., different serotypes) causing sepsis would be a universal product with acti-vity against the great majority of the clinical isolates respons-ible for the disease.
The mole ratio of the various monoclonal antibody compo-nents will usually not differ by more than a factor of 10, more usually by not more than a factor of 5, and will usually be in a mole ratio of about 1:1-2 to each of the other antibody compo-nents. When used in combination, the monocLonal antibodies of the present invention will be generally used in equal molor ratios.
The monoclonal antibodies of the present invention may also be used in combination with existing b]ood plasma products, such as commercially available gamma globulin and immune globulin products used in prophylactic or therapeutic treatment of bacter-ial sepsis in humans. Preferably, for immune globulins the plasma will be obtained from human donors exhibiting elevated levels of immunoglobulins reactive with endotoxin bear-ing bacteria. (See, generally, the compendium "Intravenous Immune Globulin and the Compromised Host," Amer. J. Med., 76(3a), March 30, 1984, pp.l-231.
The monoclonal antibodies can also be used as separately administered compositions qiven in conjunction with antibiotics or antimicrobial agents. Typically, the antimicrobial agents may include a penicillin in conjunction with an aminoglycoside (e.g., gentamicin, tobramycin, etc.), but numerous additional agents (e.g., cephalosporins) well-known to those skilled in the art may also be utilized.
The monoclonal antibodies and phar-maceutical composi-tions thereof of this in~ention are particu]arly useful for oral or parenteral administra~ion. Preferably, the pharmaceutical compositions may be administered parenterally, i.e., subcutaneous-ly, intramuscularly, intraarterially or intravenously. Thus, this -- 1 :L

invention provides compositions for parenteral administration which comprise a solution of the monoclonal antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4~ saline, 0.3% glyçine and the like. These solutions are sterile and generally free of particulate matter.
These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptahle auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acet:ate, sodium chloride, potassium chloride, calcium chloride, sodium lactal:e, etc. The concentration of antibody in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., preferably for the particular mode of administration selected.
Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 500 mg of monoclonal antibody. A typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of monoclonal antibody. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15 Ed., Mack Publishing Company, Easton, Pennsylvania (1980).
The monoclonal antibodies of this invention can be lyophilized for storage and reconstituted in a suitable ~ ~' carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g., with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.
The compositions containing the present monoclonal antibodies or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatment of any of a variety of bacterial infections. In therapeutic application, compositions are administered to a patient already infected with one or more bacterial strains in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this will depend upon the severity of the infection and the general state of the patient's own immune system, but generally range from about 1 to about 200 mg of antibody per kilogram of body weight with dosages of from 5 to 25 mg per kilogram being more commonly used. It must be kept in mind that the materials of this invention may generally be employed in serious disease states, that is, life-threatening or potentially life-threatening situations, especially bacteremia and endotoxemia.
In prophylactic applications, compositions containing the present antibody or a cocktail thereof are administered to a patient not already infected to enhance the patient's resistance to potential infections. Such an amount is defined to be a "prophylactically effective does." In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.1 to 25 mg per kilogram, especially 0.5 to 2.5 mg per kilogram.

Single or multip]e administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formula-tions should provide a quarltity of the antibody(ies) of this in-vention sufficient to efEectively treat or prophylax the patient.
EXPERIMENTAL
Production and Purification of Cachectin Recombinant human cachectin (Pennica, et al., Nature, 312:724-729 (1984) and Shirai, et al., Nature, 313:803-806 (1985), was expressed as an intracellular protein in yeast from a synthet-ic gene encoding an initiat:ion methionine and the sequence of mature cachectin, with codons chosen to reflect that of highly expressed yeast genes in accordance with standard techniques. The synthetic sequences were placed downstream from a hybrid alcohol dehydrogenase/glyceraldehycle-3-phosphate dehydrogenase (ADH2/GAPDH) promoter. Cachectin synthesis was induced in trans-formed cultures of Saccharomyces cerevisiae by glucose depriva-tion. About 5-10% of tota] yeast protein was cachectin.
The cachectin was purified from crude yeast extracts by (a) Ammonium sulfate fractionation, (b) Q-Sepharose* Fast Flow Column Chromatography, (c) S-Sepharose* Fast Flow Column Chromato-graphy, and (d) Sepharcryl S-200* (HR) Column Chromatography.
This procedure gave about 80% recovery of cachectin activity as assayed with actinomycin D-treated L929 cells (Ruff and Gifford, (1981) Tumor Necrosis Factor, Lymphokines, 2:235-272). The specific activity of purified cachectin was 2-4 x 108 units/mg, and the purity greater than 98% as judged by SDS
polyacrylamide gel electrophoresis.
Production of Monoclonal Antibodies to Cachectin BALB/c mice were immunized intraperitoneally with 5 to 25 ~g Freund's complete i~d~uvant, and boosted twice with the same dose in Freund's incomplete adjuvant at 3 weekly intervals. A
final intraperitoneal boost: of the same dose in aqueous solution was given, and the fusion performed 3 to 4 days later.
*Trade-mark .~

1340ol8 Spleen cells (about 108 cells) from immunized mice were fused with about 2 x 107 myeloma cells (P3-X63-Ag8.653) (Kearney et al., J. Immunol. (197'~) 123:1548-1550) using polyethylene gly-col according to Kohler ancl Milstein (Nature 256:495 (1979)). The cells were plated in microt:iter plates, and the hybrids were selected by hypoxanthine aminopterin thymidine (HAT) medium.
Five fusions were performed with spleen cells from five mice immunized with cachect:in. By using the solid phase enzyme-linked immunosorbent assays, 60 positive clones were obtained producing monoclonal antibodies to cachectin.
The hybrids from positive wells were cloned 3 times by limit dilutions to make certain that each was a true clone. Four-teen hybridomas positive for cachectin were cloned and character-ized.
Cachectin/TNF Bioassay: L-929 Cell Cytotoxicity TNF activity was measured using a cytolytic assay with actinomycin D-treated L929 cells as described by Ruff and Gifford in Lymphokines 2:235, (198]).
L929 cells (CCLl, American Type Culture Collection, Rockville, MD) are maintained in RPMI 1640 supplemented with lOmM
Hepes and 10% fetal bovine serum (or DME + 10% FBS). Confluent cultures (3-4x107 cells/75 cm flask) are released by brief trypsin treatment (rinsing with O.C)5% trypsin) in physiologic salt solu-tion containing 5 mM EDTA and 10 mM Hepes, pH 7.4 and resuspended in fresh medium containing actinomycin D (1 ~g/ml). The cells are then plated in 96 well microtiter dishes (5-7x104 cells/well).
After 2 hours in culture, serially diluted samples are added to wells, (less than 10-20% serum), and plates incubated overnigbt (5% C02, 37~C). Samples are assayed in quadruplicate.
The next day, following microscopic evaluation, tbe medium is decanted, and the wells fi]led witb a solution of 0.2~ crystal violet, 10% formalin and 0.01 phosphate pH 7-7.5 for 5 min., wash-ed thoroughly with water and dried. The degree of lysis is quantitated spectrophotometrically (550-570 nM) on a microtiter r~
~.~

- 15 - 134001&

plate reader. Assay result:s are expressed as U/ml, with one unit (U) defined as the amount of cachectin resulting in lysis of 50%
of the cells.
In Vitro Neutralization Assay Recombinant human cachectin (20 ng/ml in 0.02 M Tris NCl, pH 8.0, 0.15 M NaCl~ ] mg/ml BSA) is mixed with equal volumes of diluted antibody and incubated at 37~C for 60 minutes. The mixture is diluted 1:10 with fresh medium containing actinomycin D
(~g/ml); and 0.1 ml of serially diluted (two-fold) samples in quadruplicate is added to microtiter dish wells. The residual cytolytic activity is determined using the L929-cell cytotoxicity assay.
Characterization of Monoclonal Antibodies Fourteen hybridomas positive for cachectin were identi-fied and subcloned. The antibodies produced by these hybridomas were characterized for their ability to compete with monoclonal antibody 18-1-1. (Tracey, et al., Nature 3 :662-664 (1987) for cachectin binding in an ELISA. As shown in Table 1, the monoclo-nal antibodies fall into three classes; those that do not compete with 18-1-1, those that do, and those that show intermediate competition. To verify t:hat those monoclonal antibodies which did not compete with ~ '~
~..'' 18-1-1 were specific for cachectin, a cachectin capture assay was performed. On a solid phase were bound the monoclonal antibodies to capture cachectin, and monoclonal antibody 18-1-1 was used as the labeled antibody. All the monoclonal antibodies were specific for cachectin by this assay.
Experiments with conditioned media from hybridoma cells were also performed to determine the extent the Mabs neutralized the cell killing activity of cachectin. All of the 14 monoclonal antibodies were produced in mouse ascites fluid for further characterization. The efficacy of the purified monoclonal antibodies in protecting L929 cells against killing by cachectin was determined. The neutralizing activity of MAb 1-2-4Bl had been confirmed with the purified material. Incubation of MAb 2-2-3E3 (50 ng/ml) with an equal volume of human recombinant cachectin (20 ng/ml) at 37- for 60 min., showed that 50% of the cytolytic activity of cachectin was neutralized as determined by the L929 cell killing assay. As shown in Table 1, MAb 2-2-3E3, which recognizes the same epitope as MAb 18-1-1, is required in 1/8 the amount of 18-1-1 for cell protection. Also shown in Table 1, is that most of the antibodies of the same specificity as 18-1-1 neutralized cachectin (at 100%) in the L929 cell killing assay. Other antibodies, such as 1-2-4B1, which have different epitope specificities from that of 18-1-l, also possessed significant neutralizing activity.

Table 1 Profile of Monoclonal Antibodies Reactive with Human Cachectin Hybridoma MAb, typea Competition assay Neutralization Assay with 18-1-lb Medium Purified Ig, ng 18-1-1 IgG + + 400 1-2-4D4 IgG + +
1-2-4B2 IgM - -1-2-4Bl IgG - + 400 l-l-lF3 IgG - +
2-2-2FS IgG + + >800 1-1-4C8 IgG - +
2-2-3E3C IgG + + 50 1-2-2F8 IgG + +
1-1-2E3 IgG + + 200 1-2-2Al IgG +
2-3-lA8 IgG + +
1-2-4C6 IgG +

a) Determined by SDS-PAGE of MAb partially purified from ascites fluid by precipitation with ammonium sulfate.

b) Determined .in a Solid Phase Competition ELISA as follows. Conditioned medium from 24 hr hybridoma cultures (50~1) and HRP-labelled 18-1-1 (25~1, 0.2 ~g/ml in PBS + 10% goat serum) were added to each well.

c) The cell llnes 1-2-4Bl and 2-2-3E3 were deposited on June 8, 1988 wlth the A.T.C.C. and deslgnated Accession Numbers HB9737 and ~9736, respectlvely.
Efflcacy of post-treatment wlth antl-TNF ~onoclonal anti~odY
fraqment ln preventlnq the deleterlous effects of sePsls ln the baboon.
~ aboons were lntravenously admlnistered a two hour LDloo lnfuslon of Escherlchla coll (3. coll). Anlmal~ were monltored for 10 hours and observed untll death, or for a maxlmum of 7 days. The amlnoglycoslde antlblotlc, gentamicln, was admlnlstered at deslgnated times followlng a two hour lnfuslon of E. Coll. A bonus ln~ectlon of antl-TNF monoclonal antlbody 2-2-3E3 F(abl~2 fragment (10 mg~kg) was admlnlstered to four baboons at Tl30 mlnutes (one-quarter of the way through the bacterlal lnfuslon, l.e., "post-treatment"). One addltlonal baboon was glven the same dose of ~. coll plus gentamlcln and treated wlth an lsotype control ~f an lrrelevant F(a~l)2 fragment.
~1) Survival Data DATE EXPERIMENT ~OON * 3AB00N WT. WSE 3. COLI SURVIVAL ~E
(KG + SEX~
2/25/88 + Antibody 1 5.8 M 3.7 x 101~ 17 hours 3/1/88 + Antibody 2 14.3 M 4.6 x 101~ 24 hours 3~17/88 + .~ntibody 3 7.3 M 6.2 x 101~ 7+ days 4/5/88 + Antibody 4 6.6 FM 4.4 x 101~ 7+ days 4/6/88 Control 5 6.4 ~M 6.5 x lolO 21 hours ( Carrler 77040-~

13~0018 18a ~2) Post-mortem observatlons Summary of post-mortem examlnatlons conducted on baboons #1,2,5 (post-mortems not lncluded on survlvlng baboons #3,4 because prlor work wlth survlvlng anlmals showed no adverse hlstological effects). The gross post mortem flndlngs on anlmals #1,2,5 were very slmllar to each other and are as follows:

19 13400~8 Lungs: Hemorrhagic (3 Kidneys: Congested, necrotic (3+++) Adrenals: Hemorrhagic, necrotic (4 Liver: Congested (2 ) Spleen: Engorged and congested Intestines: Normal in appearance but intussusceptions in small intestine were observed in animals #1,2.
Heart, pancreas, stomach, colon: Normal in appearance.

(3) Physiological and other parameters monitored for ten hours (a) Parameters associated with survival benefits in baboons #3,4 and different from nonsurviving baboons (#1,2,5) were: tMean systemic arterial pressure (MSAP), tpH, llactate, lBUN, lcreatinine, lSGPT, lcortisol, tglucose, lFDP and tfibrinogen.
(b) Parameters no different between surviving (#3,4) and nonsurviving (#1,2,5) baboons were: Heart and respiration rates, WBS, platelets, pCO2, PO2, CPK, hematocrit, colony (E. coli) blood concentrations, body temperature.
When the studies were repeated with whole antibody, no significant differences were noted. Thus, both binding fragments and intact antibodies may be utilized in accordance with the present invention.
From the foregoing, it will be appreciated that the cell lines of the present invention provide means for producing monoclonal antibodies and fragments thereof reactive with human cachectin at low neutralizing amounts.
This allows prophylactic and therapeutic compositions to be more economically developed and safely administered to be effective against infections due to most endotoxin bearing bacterial strains. In addition, the antibodies will find uses in various diagnostic and other therapeutic procedures.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (14)

1. A composition comprising a pharmaceutically acceptable carrier or excipient, in admixture with a monoclonal antibody produced by the cell line designated A.T.C.C. Accession Number HB9736 or A.T.C.C. Accession Number HB9737, or binding fragments thereof.

2. A composition comprising a pharmaceutically acceptable carrier or excipient, in admixture with a monoclonal antibody produced by the cell line designated A.T.C.C. Accession Number HB9736, or a binding fragment thereof and a monoclonal antibody produced by the cell line designated A.T.C.C. Accession Number HB9737, or a binding fragment thereof.

3. A composition according to Claim 1 or Claim 2, further comprising an antibiotic agent and/or a gamma globulin fraction from human blood plasma.

4. A composition according to Claim 3, wherein the gamma globulin fraction from human blood plasma is obtained from humans exhibiting elevated levels of immunoglobulins reactive with endotoxin-bearing bacteria.

5. A pharmaceutical composition useful for treating or preventing an infection, said composition comprising a monoclonal antibody produced by the cell line designated A.T.C.C. Accession Number HB9736 or A.T.C.C. Accession Number HB9737, or binding fragments thereof, an antimicrobial agent, a gamma globulin fraction from human blood plasma and a physiologically acceptable carrier.

6. A composition according to Claim 5, wherein the gamma globulin fraction from human blood plasma is obtained from humans exhibiting elevated levels of immunoglobulins reactive with endotoxin-bearing bacteria.

7. A cell line designated A.T.C.C. Accession Number HB9736.

8. A cell line designated A.T.C.C. Accession Number HB9737.

9. A method of producing monoclonal antibodies that can immunologically bind endotoxin-bearing bacteria comprising:
cultivating the cell line of Claim 7 or Claim 8 and recovering said antibodies.

10. A monoclonal antibody, or binding fragment thereof, produced by the cell line of claim 7.

11. A monoclonal antibody, or binding fragment thereof, produced by the cell line of claim 8.

12. A monoclonal antibody according to claim 10 or claim 11, conjugated to a label capable of providing a detectable signal.

13. A monoclonal antibody according to claim 12, wherein the label is a fluorescer or an enzyme.

14. A kit for use in detecting the presence of endotoxin-bearing bacteria infection in a host, said kit comprising a monoclonal antibody composition containing monoclonal antibodies produced by a cell line designated A.T.C.C. Accession Number HB9736 or A.T.C.C. Accession Number HB9737, or binding fragments thereof, and labels providing for a detectable signal covalently bonded to said antibody or bonded to second antibodies reactive with each of said monoclonal antibodies.