CA2071872A1 - Cytokine antibody for the treatment of sepsis - Google Patents

Abstract

Compositions and methods for prophylactically or therapeutically treating sepsis consisting of antibody to IL-6 and/or M-CSF
wherein the antibodies are administered alone or in combination.

Description

U ~
WO 91/08774 PCl/llS9~/074~1 CYTOKINE ANTIBODY FOR THE TREATMEI~IT OF SEPSIS
This invention is in the area of imrnunologytbiocher,nistry, and presents cytokine antibody, preferably interleukin 6 tIL-6) and macrophage colony stimulating fac~or (M-CSF) antibody alone, or in combination, for the prophylactic or therapeutic 5 treatment of sepsis. Antibody may be polyclonal, or monoclonal antibody, or fragments derived therefrom, or recombinant constructs having the binding acdvity of such antibody.
In the United States alone nosocomial bacteremia develops in about 194,000 patients, and of these about 7S,00~ die. Maki, D.G., 1981, ~s~mi~L~.
10 (Dikson, R.E., Ed.), page 183, Yrke Medical Books, U.S.A.. Most of these deaths are attributable to six major gram-negative bacilli, and these are Pse~d~lonas ae~ginos~, Es~herlchia coli. Prote~ls, Klebsie]la, ~glgk~ and ~. The current treatrnent for bacteremia is the administration of antibiotics which, unfortunately, have limited effectiveness. Although the precise pathology of bacteremia 1; is not completely elucidated, it is believed that bacterial endotoxins, lipopolysaccharides (LPS), are the primary causative agents. LPS consist of at least three significant antigenic regions, the lipid A, core polysaccharide, and O-speci~lc polysaccharide. The latter is also referred to as O-specific chain or simply O-antigen. The O-specific chain region is a long-chain polysacchalide built up from repeating polysaccharide units. The 20 number of polysaccharide units differs among different bacteIial species and may vary from one to as many as six or seven monosacchalide units. While the O-specific chain varies among different gram-negative bacteria, the lipid A and core polysacchandes are similar if not identical.
Since l,PS plays a key role in sepsis, a variety of approaches have been pursued25 to neutralize its activity. Presently, there is considerable work which suggest that antibody to LPS will soon be a valuable clinical adjunct to the standard antibiotic therapy.
I.PS initiates a cascade of biochemical events that eventually causes the death of the patient. It is widely believed that the second event, after the introduction of LPS, is 3~j the production of tumor necrosis factor (INF) as a result of LPS slimulation of rnacrophage cells.
- - Tumor Necrosis Factor (INF) is a cytokine which is known to have cytolytic and cytostatic anti-tumor activity. Carswell, et al., 1975, Proc. Nat'l~i~
36S6-3670; Williarnson, ~ al., 1983, oc. Nat'l A~d. S~ 0: 5397-5401. In 35 addition, it has recently been shown to be a mediator in the immunoinflammatory cascade and play a key role in sepsis. Beutler, et al., 1985, ~i~a~. ;~22: 869, .

2~71872 W~ 91/08774 Pcr/us9O/0741 l reported that in a murine model the lethal effect of endotoxin can be reduced bypolyclonal rabbit anti-murine TNF antibody. It is likely that antibody tQ TNF will have valuable clinical applications. Tracey, ~., 1987, ature, 3r~0:662.
In addition to TNF, another cytokine that is elevated in sepsis patients is interleukin-6 (IL-6). IL-6 is also called hybridoma growth factor, interferon B-2, B-cell stimulatory factor 2, 26-Kd protein and hepatocyte stimulating factor. The rnolecule has pleiotropic affects, apparently stimulates hepatic protein synthesis during the acute phase of an infection, and acts as an endogenous pyrogen.
Hack, Ç.t ~1., 1989, B~ , 74: 1704, have shown that a significant number of patients with sepsis display increased plasma levels of IL-6, and that the amount of IL-6 correlates with the symptoms of shock and with clinical prognosis. In the sepsis patients shown in that report, serum IL-6 levels were on the order of 1,000 U/ml.
Native IL-6 has a molecular weight of 19-30 kD and an imrnunoreactive species of 60-70 kD has also been reported (see Kelfgott, ç.l al., 1989, J. ImmunQL,1~:948 and Jablon, ~ al., 1989, J. lrn~uno~ 1542). The gene coding for a human IL-6 polypeptide has been cloned and expressed as shown by the following European patent applications: EPA 0 220 574, published May 6, 1987, to Revel, M, ~ al., entitled"Human interferon beta2A and interferon-beta2B, vectors containing genes coding for said interferons, cell lines producing same and use of said interferons as phannaceuticals"; EPA 0 254 399, published January 27,1988, to Clevenger, W., Çl.al.. entitled "B-cell stimulating factor"; EPA 0 257 406, published March 2, 1988, to Kishimoto, T., ~ al., entitled "Recombinant B-cell differentiation factor"; EPA 0 261 625, published March 30, 1988, to Honjo, T., ~ al., entitled "Human B-cell differentiation factor and process of producing said factor"; EPA 0 267 779, published May 18, 1988, entitled "Human pleiotropic immune fac~or and muteins thereof"; and PCI' WO 88/00206, published January 14, 1988, to Clark, S., ÇI .al-. entitled "Production and use of IL-6".
Antibody tO IL-6 has been described both in certain of the foregoing patent applications that show IL-6 (see, for example, EP 257,406), and in the scientific literature.
Another molecule hithertofore unsuspected of being involved in sepsis is macrophage colony stimula~ng factor (M-CSF), also known as CSF- 1. Ihis moleculecauses the selecdve dif~erentiation and proliferation of macrophages, and has been purified from a number of sources. Stanley, E.R., ~ , 1977, J Biol CherD, ~:4305 describes the purif~cation from muline L929 cells ~,vith a specific activity of abnut 1 x 108 units/mg. Das, S.J., et a1., 1982, J B~o] Chem., ~: 13679 reported tha human uIinary M-CSF has a specific activity of 5 x 107 units/mg and generates ~ ~ ' r~ v ~

`:: W O 91/08774 PC~r1US90/07411 predominately macrophage cells i.n vitrn. Stanley, E.R. and Gilbert, L.J., 1981,Journal of ImrnunologLcal MethQds, 42:253 also describe methods for the purification of M-CSF in low yield. Das, S.K. _~ al., 1981, ~ 6:30 describe partial purification of human urinary M-CSF. Wu, N., et al., 1979, LBiol (:~hem~ ,, ~L:6226 describe the purification of a CSF that prirnarily stirnulates the formation of macrophages. More recently, M-CSF has been purified in milligrarn amounts using 10,000 liters of human uIine as starting matenal.
M-CSF has been cloned, and expressed in a number of host cells, and consequently, recombinant M-CSF (rM-CSF) is av~ulable for use as an immunogen toelicit antibodies. Indeed, to date, human rM-CSF (hrM-CSF) cDNA clones having three different lengths have been identified, herein denoted a"B and y. (See Cerretti, et ah, 1988, Mol. Immunol, 25:761). They have been isolated from cells expressing the single M-CSF gene. The a"B and y clones contain M-CSF DNA sequences that encode unprocessed proteins having 224, 522 and 438 amino acids, respectively.
Recombinant M-CSFs have been expressed in active forrn, and thus these molecules may be used to generate suitable monoclonal antibodies. The preferred rM-CSF is that described in U.S. Patent No. 4,847,201, to E. Kawasaki et al., issued July 11, 1989. Therein is shown the expression, in both prokaryotes and eukaryotes, of the a forrn of M-CSF.
In addition to being defined by their biological activities, M-CSF and IL-6 may also be defined by their chemical structures. The DNA and arnino acid sequences of IL-6 are known. In contrast, the precise structure of naturally produced M-CSF is not clearly apparent from a reading of the scientific literature. For instance, human M-CSF
purified from urine is thought to consist of two essentially identical subunits with an apparent molecular weight of 25-35 kilodaltons. M-CSF purified from a pancreaticcarcinoma cell line, MIA PaCa-2, was reported to consist of two subunits, but with apparent mo}ecular weights of about 23-laO kilodaltons. I'hese differences may be due to differences in glycosylation, or rnay arise as a result of alternative splicing of the M-CSF mR~A transcIipt, as described above. See also Stradle, et al., 1989, J. Cell.
Biochem., 40:91. The M-CSF proteins precursor from the B clone is thought to give rise to a 70-90 kilodalton glycoprotein, believed to be a dimer with 35 15 kilodalton subunits, possibly having about 223-224 amino acids. The smaller precurs~r yields a 40-50 kilodalton glycoprotein, also comprising a dimer with a subunit rnolecular weight of about 20-25 kilodaltons, and possibly with about 158 amino acids. See Halenbec~, 3s R., et ah, 1988, Biotechnologv J., 8:45. Thus, it should be apparent that within the strucmral definition of M-CSF there exists a set of related proteins of valying molecular ~ U ~
WO 91/08774 PCr/US90/07411 i weights. Il should be further apparent from the foregoing discussion that the definition of M-CSF is not restricted tO proteins with the above-described molecular weights. It is to be anticipated, in light of the existence of multiple mRNAs coding for M-CSF, that proteins with molecular weights different from those discussed above will be 5 discovered, and thus are intended to come within the definition of M-CSF.
It will further be appreciated with regard to M-CSF or IL-6, that their precise structu~re depends on a number of factors. As all proteins contain ionizable amino and carboxyl groups it is, of course, apparent that they may be obtained in acidic or basic salt form, or in neutral form. It is further apparent, dlat the plirnary arnino acid 10 sequence may be augmented by derivatization using sugar molecules (glycosylation~ or by other chemical derivatizations involving covalent, or ionic attachment of, for example, lipids, phosphate, acetyl groups and the like, often occunring through association with saccharides. These modifications may occur in vitro, or ~ ViVQ, the latter being pefforrned by a host cell through post-translational processing systems. It 15 will be understood that such modifications, regardless of how they occur, are intended to come within the definition of M-CSF, and IL-6 so long as the activity of the protein, as defined above, is not destroyed. It is to be expected, of course, that such modifications may quarltitatively or qualitatively increase or decrease the biological activity of the molecule, and such chemically modified molecules are also intended to ~ 20 come within the scope of the definition of M-CSF, and Il-6 since these molecules would be expected to elicit medically useful antibodies.
A first object of the invention is a description of cytokine antibody, preferably IL-6 and/ or M-CSF antibody for the prophylactic or therapeutic treatment of sepsis.
A second object of the invention is a description of Il.-6 and/or M-CSF
25 antibody, or antibody fragments derived therefrom, for the prophylactic or therapeutic treatment of sepsis wherein the antibody is polyclonal, monoclonal, or recombinant constructs having the binding activity of such antibody or antibody fragments.
A third object of the invention is a description of a mixture of andbodies fo~ the prophylactic or therapeutic treatrnent of sepsis consisting of IL-6 and M-CSF antibody, 30 antibody fragments derived therefrom, or recombinant constructs having the binding activity of such antibody or antibody fragrnents.
A four~ object of the invention is a description of methods for adrninistrating IL-6 and/or M-CSF antibody for the prophylactic or therapeutic t~eatrnent of sepsis.
These, and other objects of the invention, will be more fully understood after a35 consideration of the following description of the invention.
Figure 1 shows L-6 levels in baboon plasma after a lethal or sublethal dose of E. ~.

- W O 91/08774 P ~ /US~0/07411 Figure 2 shows that IL-6 monoclonal antibody con siderably exlends the lifetime of a baboon administered a lethal dose of 1~
Figure 3 shows the effect of administering n -6 monoclonal antibody, 8M70, to a baboon on various physiologically pararneters prior to the baboon receiving a lethal 5 dose of ~. ~.
Figure 4 shows an increase in M-CSF levels in baboons administered a lethal dose of ;~
The present invention is directed to the production and utilization of cytokine antibody, preferably IL-6 and/or M-CSF antibody for the prophylactic or therapeutic 10 treatment of sepsis. Several patents/patent applications and scientific ~eferences are referred to below that discuss various aspects of the matenal and methods used to realize the invention. Because the invention draws on these materials and methods, it is thus intended that all of the references, in their entirety, be incorporated by r~ference.
To more clearly define the present invention, particular terms herein will be 15 employed accor~ing to the following definitions generally consisient with their usage in ~he art.
"Sepsis" is herein defined to mean a disease resulting from bacterial infection due to the bacterial endotoxin, lipopolysaccharide (LPS). It can be induced by at least the six major gram-negative bacilli and these are Pseudomo!las ~ Lnosa, ~scherichia 20 ~Q~, Proteu~, Klebsiella, Enterobacte~: and Serratia. It is expected that sepsis induced by gram-positive organisms may also be beneficially treated with the approaches described herein.
"Monoclonal antibody" re~ers to a composition of antibodies produced by a clonal population (or clone) denved through rnitosis from a single antibody-producing 25 cell. A composition of monoclonal antibodies is "substantially free of other antibodies"
when it is substantially free of antibodies that are not produced by cells ~om the clonal population. The terrn "substantially free" means approximately 5% (w/w) or fewercontaminating andbodies in the composi~on. Also intended to come within the scope of the defimition are modificaions to antibody that increase its effectiveness. A preferred 30 modification includes conjugation of a water soluble polymer. Preferably the water soluble polymer is polyethylene glycolt or a ~unc~ionally related molecule such as, for example, polypropylene glycol homopolymers, polyoxyethylated polyols, and polyvinyl alcohol. Derivaization of antibody with such water soluble polymers increases its in vjvQ half-life, reduces its immunogenicity, and reduces or eliminates 35 aggregation of ~he protein and may reduce its immunogenicity and aggregation that might occur when it is introduced ~ ~. Delivatization of proteins generally, or antibody specifically, with water soluble polyrners such as those described above are :
: ` 2071872 WO 91/08774 Pcr/US9O/0741 ~ ` ~

presented in U. S. Patent Nos. 4,179,337, issued December 18, 1~79, to Davis ~ al., entitled "Non-irnmunogenic polypeptides"; and 4,732,863, ;ssued March 22, 1988, to Tornasi, ~ al., entitled "PEG-modified antibody with reduced affinity for cell surface Fc receptors", respectively.
An "antibody-producing cell line" is a clonal population or clone derived through mitosis of a single antibody-producing cell capable of stable growth in vitro for many generations.
"Tumor Necrosis Factor" or "TNF" as used herein refers to both native and recombinant forms of this known, mammalian cytokine. TNF has been referred to by other names in the literature, including "Cachectin" and "TNF-a". "Recombinant TNF" or "rTNF" refers to proteins, including muteins, produced by expression of recombinant DNA that have the same or substantially the sarne amino acid sequence as native TNF (or ponions thereof), and retain both Ihe in vitro and in v,vo biological activity of TNF. The isolation and production of both native and recombinant mammalian TNF, including human TNF, is known in the art. See, e.g., Carswell et aL., 1975, Proc. Nat'l Acad. Sci. USA, 72: 3666-3670; Williamson et ah, 1983, Proc.
Nat'l Acad. Sci. U$A, 80: 5397-5401; Wang et ah, 1985, Science, 228:149-154;
Beutler ~ al., 1985, Science, 229: 869; Beutler et al., 1985, Nature, ~: 552; Pennicia et al., 1984, Nature, ~:724; Aggarwal et ah, 1985, J~ Biol. Chem., 260: 2345.
"Recombinant antibody" refers to antibody wherein one portion of each of the amino acid sequences of heavy and light chain is homologous to corresponding sequences in antibody derived from a particular species or belonging to a particular class, while the remaining segment of the chains is homologous to corresponding sequences in another. Most commonly, in a recombinant antibody the va~iable region of both light and heavy chain rr~ilrors the variable regions of antibody derived from one species of marr~mals, while the constant regions are homologous to the sequences in antibody derived from another. However, this is not necessarily always the case; for exarnple, Ward, et al., 1989, Nature, ~.:544, have shown that variable chain alone can be expressed in bacteria with significant andgen binding activity.
Two antibodies are "cross-blocking" or have a "shared epitope" when each antibody effectively blocks the binding of the other antibody in a binding inhibition assay. Thus, if antibodies A and B are cross-blocking, antibody A would not bind to its antigen when the andgen had been preincubated with antibody B, and antibody B
would not bind to its antigen when the an~igen had been preincubated with antibody A.
3s The telm "binding affinity" or "Ka" of an antibody to its epitope, as used herein, refers to a binding affinity calculated according to standard methods by the .~ , .~ . ' ~7~872 `' ' ` `'` WO 91/08774 PCr/US9û/074l1 .:

formula Ka = 8/3(It-Tt), where It is the total molar concentration of inhibitor uptake a 50% tracer, and Tt is the total molar concentration of tracer. See Muller, 1980, L
ImmDn~l~, 34: 345-352.
As used herein, the terrn "incubation" means contactmg antibodies and antigens under conditions that allow for the formation oi andgen/antibcdy complexes (e.g., proper pH, temperature, time, medium, etc.). Also as used herein, "separating"
refers to any rnethod, usually washing, of separadng a composition from a test support or irnrnobilized andbody, such that any unbound antigen or antibody in the composition ar~ removed and any antigen/antibody complexes on the support remain m~ac~ The selection of the appropnate incubation and separation techniques is within the skill of the art.

I. IL-6/M-C~SF Antibndv In a preferred embodiment of the invention, L-6 or M-CSF antibody producmg immunologic cells are isolated from a mammal immunized with IL-6 and/or M-CSF, and immortalized to yield antibody secreting cell lines e.g. hybridomas, triomas etc.
Cell lines that secrete the desired antibody can be identified by assaying culture supernatants for antibody activity. Thus, the invention can be broken down into three secdons, and each section discussed separately. That is, the immunization procedure, the cell irnmortalization procedure, and the identificaiion of IL-6 and/or M-CSFantibody.

A. Immunization with IL-6/1~-C~F
IL-6 and M-CSF, alone or in combination, may be used tO imrnunize an appropriate host animal. Preferably, the host animal is immunized with IL-6 described by Brakenhoff ~ al., 1987, Journal Qf Immu~olo~y, 1~9:4116, or in EP 257,406, orM-GSF described in U.S. Patent No. 4,847,201. A suitable adjuvant may be used tOenhance the immune ~sponse. A variety of distinguishable immunization protocols may be employed, and may consist of a primary intravenous, subcutaneous, or intr~peritoneal immunization followed by one or more boosts. The precise ~nunization schedule is generally not critical, and dete~ninative of which procedure is employed, is the presence of M-CSF or IL-6 antibody in the host animal as measured by a suitable assay, described below.
Alternatively, lymphocytes may be immunized in vi~ro. For example, irnmuniza~ion of peripheral blood cells may be achieved as deseribed by Boss, Methods of zymQlQ~ ~;L(l), and in EPA 86106791.6. Note particularly in yitrQ
immuniza~on techniques that can be used ~o produced either murine or human .
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W O 91/08774 PC~r/US~0/07411 monoclonal (Procedures for Transforrning Cells, pages 18-32, 140-174, MethQ~f ~YmQ~ vol.L~., part l). Such techniques are also described by Luben, R. and Mohler, M., l9X0, ~, 17:635, Reading, C. ~elhods in ~nzym~y, ~. (Part One):18, or Voss, B., 1986, ~h~Z~L~Zym~ ~.:27.
5 A number of in vitro irnmunization systems have been shownto be effective for sensitiziDg human B-cells. Reading, C., 1982, L~mmYn..~hQ~. ~:261.
It will be appa~nt to those skilled in the ar~, that in lieu of immuniung individuals directly with IL-6 or M-CSF, lymphocy~es may be isolated from individuals that are experiencmg, or have experienced a bacteremic attack. A fracuon of these 10 lyrnphocytes may be sensitized to these molecules and can be used to produce perrnanent antibody secreting hybrid cell lines. For exarnple, irnmunocompromised human patients are generally susceptible to bacterial infections, particularly those suffering from various malignancies, ëxtensive burns, etc., and Iymphocytes isola~ed therefrom may be a source of antibody secreting cells. -' In lieu of using IL-6 and M-CSF as imrnunogens, an alternative approach is to synthesize IL-6 or M-CSF peptides. and use these as immunogens. For example, a particularly useful peptide to produce antibody that binds to IL-6 is described in Japanese patent application No. 62102157. The methods for making antibody to peptides are well known in the art and generally require coupling the peptides to a 20 suitable carrier molecule, such as serum alburnin. The peptides can be made by techniques well known in the art, such as, for example, the Merrifield solid-phase method described in Sci~ce, ~:341-347 (1985). The procedure may use cornmercially available synthesizers such as a Biosearch 9500 automated peptide machine, with cleavage of the blocked amino acids being achieved with bydrogen 2s fluoride, and the peptides purified by preyarative HPLC using a Waters Delta Plep 3000 instrurnent, on a 15-20 ~Lm Vydac C4 PrepPAK column. Once clones are identified that secrete anti-peptide antibody, the antibody can be screened for binding and neutralizing activity to either L-6 or M-CSF.

B . 11,-61M-~SF Antibody Antibody to IL-6 or M-CSF may be either polyclonal, monoclonal, or ~agrnents derived ~erefrom . The antibody is preferably human or humanized, although non-human antibody will perform satisfactory. Additionally, recombinantconstructs having the antibody binding specificity of antibody to IL-6 or M-CSF may be produced.

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. 2071872 WO 91/08774 Pcr/usso/o74l 1 The preparation of high-titer neutralizing polyclonal antibody can be realized by immunizing a variety of species and employing one of several different irnrnunization - regirnes. The preferred method of the instant invention is to imrnunize rabbits with IL-S or M-CSF prepared in complete Freund's adjuvant by injection into ax~al Iymph 5 nodes. The animals are subsequently subjected to multiple boosts (containing about half the original amount of IL-6 or M-CSF) in incomplete ~reunds adjuvant at about 21-day intervals. About 10 days following each 21-day interval, 20~30 ml of blood is removed, the serurn isolated and antibody isolated therefrom. This procedure may be carAed out for a period of several months.
Monoclonal antibody may be produced using L-6, M-CSF, or peptides/peptide conjugates of these molecules as described above, and using theprocedures described by Kohler, G. and Milstein, C., 1975, l~ature, ~:495, or modifications thereof that are known in the art. Using the screening assays described below, the specificity of antibody produced can be discerned.
The inilial work of Kohler and Milstein, above, involved fusing murine Iymphocytes and drug selectable plasmacytomas to produce hybridomas.
Subsequently, the technique has been applied to produce hybrid cell lines ~hat secrete hurnan monoclonal antibodies. The latter procedures are generally described in Abrams, P., 1986, ethods in Enzvmolo~,v, ~ :107, but other modifications are 20 known to those skilled in the art. Regardless of whether murine or human antibody is produced, the antibody secreting cells are combined wi~h the fusion partner and the cells fused with a suitable fusing agent, preferably polyethylene glycol, and more preferably polyethylene glycol 1000. The latter is added to a cell pellet containing the antibody secreting cells and the fusion partner in small amounts over a short period of 25 time accompanied vith gentle agitation. After the addition of the fusing agent, the cell mL~ture is washed to remove the fusing agent and any cellular debris, and the cell mixture consisting of fused and unfused cells seeded into appropriate cell culture charnbers containing selective growth media. After a period of several weeks, hybrid cells are apparent, and may be identified as to antibody production and subcloned to .
30 ensure the availability of a stable hybrid cell line.
The prefe~d antibody is human monoclonal antibody which can be prepared fr~m Iymphocytes sensitized with IL-6/~-CSF either in ~i~Q or in ~rQ by irmnortalization of antibody-producing hybrid cell lines, thereby rnalcing available a permanen~ source of the desired antibody, using the cell fusion techniques described 35 above. Alternatively, sensitized Iymphocytes may be immortalized by a combination of two techniques, viral ~ansfolTnation and cell fusion. The pre~erred combina~on consist of transforming antibody secreting cells with Epstein-barr virus, and subsequently ` 2 ~ 7 2 WO 9l/08774 PC~/US90/0741 1 , 10 fusing the transformed cells to a suitable fusion partner. Such fusion partners are known in the art, and exemplary partners may be a mouse myelorna cell line, a heteromyeloma line, or a hurnan myeloma line, or other immortalized cell line. P~r Patent Application No. 81/00957; Schlom Çt al., 1980, P~S USA, 77:6841; Croce 5 ~., 1980, Nature, ~:4BB. The preferred fusion par~er is a mouse-human hetero-hybrid, and more preferred is the cell line designated F3B6. This cell line is on deposit with the American Type Culture Collection, Accession No. HB87B5. It was deposited April 18, 1985. The procedures for genera~ing F3B6 are described in European Patent Application, Publication No. 174,204.
Techniques applicable to the use of Epstein-Barr virus transformation and the production of immortal antibody secreting cell lines are presented by Roder, J. e~
1986, Methods in ~nzymo~Q~y, 12 l :140. Basically, the procedure consist of isolating Epstein-Barr virus from a suitable source, generally an infected cell line, and exposing the tar~et antibody secreting cells to supernatants containing the virus. The cells are washed, and cultured in an appropriate cell culture medium. Subsequen~y, virallytransformed cells present in the cell culture can be identified by the presence of the Epstein-Barr viral nuclear antigen, and transformed anibody secreing cells can be identified using sta~ dard methods known in the art.
It will be apparent to those skilled in the art, and as mentioned above, that while the preferred embodiment of the instant invention is neutralizing Il-6 or M-CSF
monoclonal antibody, singly or in combination, that the andbody(s) may be altered and still rnaintain biological activity. Thus, encompassed within the scope of the invention is antibody modified by reduction to various size fragments, such as F(ab')2, Fab, Fv, or the like. Also, the hybrid cell lines that produce the antibody may be considered to be a source of the DNA that encodes the desired antibody, which may be isolated and transferred to cells by known genetic techniques to produce genetically engineered antibody. An e~ample of the latter would be the production of single chain antibody having the andbody combining site of the hybridomas described herein. Single chain antibody is described in U.S. Patent No. 4,704,692.
A second example of genetically engineered antibody is recombinant, or chirneric antibody. Methods for producing recombinant antibody are shown in U.S.Patent No. 4,X16,567, to Cabilly, et al.; Japanese patent applica~ion, Serial No.
84169370, filed August 15, 1984; British patent application 8422238, filed on September 3, 1984; and Japanese patent application, No. 85239543, filed October 28, 3s 1985. Also, British patent application, No. B67679, filed March 27, 1986, describes methods for producing an altered antibody in which at least parts of the complementary deterrnining regions (Cl:)Rs) in the light or heavy chain valiable domains have been -,. . . . . .. . . .
'' , ' . -, ' ' ' ' : ' , '. - ' , ,' , ' ' . ' ' ' ', ' ' : ........ - ~ ,:

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20~18~2 - WO 91/08774 Pcr/uS90/0741 1 Teplaced by analogous parts of CDRs from an antibody of different specificity. Using the procedures described therein it is feasible tO construct recombinant antibody having the CDR region of one species grafted onto antibody from a second species that has its CDE~ region replaced Regardless of the type of antibody, polyclonal or monoclonal etc., it is desirable to purify the antibody by standard techniques as is known in the art, or as described by Springer, 1980, MonQ~Lonal And~odi~s,:194, (Eds. Kennett, T. McKearn and K.
Bechtol, Plenum Press, New York. Generally this consists of at least one amrnonium suLfate precipitation of the antibody using a 50% amrnonium sulfate solution. Antibody l o affmity columns may also be used.
The preferred IL-6 antibody is denoted 8M70, and methods for obtaining it are described below in the Example section.

C. S~ Qf ~ E en~QS~.y Cell lines that secrete IL-6 or M-CSF antibody can be identified by assaying culture supernatants, ascites fluid etc., for antibody. The preferred screening procedure consists of two sequential steps. First, hybridomas are identified that secrete antibody;
and second, the antibody is assayed to determine if it exhibits neutralizing activi~y.
As applied to cell culture supernatants, the initial screening step is preferably done by RIA or ELISA assay. Bo~h assays are known in the art, and consists of binding IL-6 or M-CSF to a solid ma~ix, and assaying for antibody binding to these molecules as revealed by a second, labelled antibody. For as description of the ELISA
assay method see Langone, J. and Van Vinakis, H., 1983, ~ethQd~Q~nzym~L~
2'~. Part E, and for a description of RIA assay see Miller et ~1., 1983, ~hQ~
Enzvm~, 121:433 Part I. If peplides are used as imrnunogen, the initial screening step ~5 determines if the antibody binds to peptide conjugates bound to a solid matrLx.
An additional assay for L-6 or M-CSF an~ibody is to screen for antibody that imrnunoprecipitates IL-6 or M-CSF from solution. For example, supernatants beingtested for the presence of antibody may be incubated with labelled Il,-6 or M-CSF for an appropriate time to allow antigen/antibody complexes to folm. The complex may be washed to removed any unreacted reagents, and next the antibody complexes incubated with anti-xenotypic or anti-isotypic antibodies specific for the monoclonal antibody being screened. These anti-xenotypic or anti-isotypic antibodies may be immobilized, for example, on a plastic bead. Thus, if the monoclonal antibody being screened is IL-6 antibody, then labelled IL-6 will be indirectly bound to the bead and thereby immunoprecipitated. IL-6 antibody can then be quantitated using suitable detection methods ~own in the art dependent on the nature of the label use~ Also, the material .
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```20~ 72 W O 91/08774 P ~ /US90/07411 can be dissociated from the bead using standard techniques and identified by gelelectrophoresis, as is known in the art.
The preferred electrophoresis proceclure is Western .Blot gel analysis as , described by Burnette, 1981, ~!.1~, ~:195. The Western blots are blocked, washed, and probed preferably in 10 mM sodium phosphate buffer containing 150 mM sodium chloricle (pH 7.4), with 0.1% bovine serum albumin (wlv), and 0.1%ovalbumin (w/v). In addition, a detergent is preferably employed such as Tween 20 at a concentlation of about 0.1%. Sodium azide rnay also be included in the solution at a concentration of 0.02%. The blots are preferably first probed with either hybricloma culture supematant, or dilute ascites fluid containing IL-6 or M-CSF antibody, washed, and then an~ibody binding revealed with l2sI-protein A for about 30-60 rninutes. The blots are washed, and subjected to autoradiography using X-ray film.
To expedite the time it takes to assay for IL-6 and M-CSF antibody, several culture supematants may be combined and assayed simultaneously. If the mixture is positive, then media from each well may subsequently be assayed independently toconfirm the presence of antibody.

II. ImmunoassaY
In another embodiment, the presen~ invention is directed to an immunoassay which can be used to detect IL-6 and~or M-CSF levels which are indicative of theprognosis of sepsis patients. The concentrations that are detectable will be in the range of less than 1 ng/ml to about 1 ug/ml of either molecule. The imrnunoassay of the present invention is preferably a sandwich assay employing the antibodies disclosed herein, although other assay formats known in the art may also be used.
- 25 In practicing the immunoassay method of the present invention, labelled IL,-6 or M-CSF antibody is incubated with the fluid test sample taken from a patient containing unknown concentrations of these molecules. Af~er allowing for a suitable period of incubation for antigen-andbody complexes to form, ~he mLxtu~e is washed and incubated with an indicator solution containing a solid matrix to which is boundantibody that binds IL-6 and/or M-CSF antibody, and such Mtibody may be either -~_, monoclonal or polyclonal. I his second antibody is incuba~ed for a sufficient penod of time to allow antigen-antibody complexes to form between the labeled antibody and~
6 andlor M-CSF. After this incubation, the imrnobiiized complex is separated from any unbound ~eactants, and the arnount of label bound tothe immobilized antibody is 35 measured. (See, for example, Shadle, et ah, 1989, Exp,Hematol., ~7:154).

.. ....
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W O 91/08774 PC~r/US90/07411 It will be appreciated by those skilled in the art that while IL-6 and M-CSF maybe measured independendy in different sample aliquots, that they may also be measured simultaneously in the same iluid. If they are measured simul~aneously, the same general procedures as described above may be followed and i.urther incorporatingtechnical means whereby the concentrations of IL-6 and M-CSF can be distinguished.
Such means are well known in the art and may consists of differentially labelling IL-6 and M-CSF antibody.
The antibodies employed in the present invention can be imrnobilized on any appropriate solid test support by any appropriate technique. The solid test support can be any suitable insoluble carrier material for the binding of antibodies and immunoassays. Many such rnaterials are known in the art, including, but not limited to, nitrocellulose sheets or filters; agarose, resin, plastic (e.g. PVC or polystyrene) latex, or metal beads; plastic vessels; and the like. Many methods of immobiliang antibodies are also known in the art. See, e.g., Silman ~ ~., 1966, Ann. ~ev.
] .s ~iochem., ~: 873; Melrose, 1971, Rev. Pure 8~ Ap~. Chem., ~: 83; Cuatrecaas çt al., 1971, Meth! Enzvm.,~. Such methods include covalent coupling, direct adsorption, physical entrapment, and attachment to a protein-coated surfaee. In the latter method, the surface is first coated with a water-insoluble protein such as zein, collagen, fibrinogen, keratin, glutelin, etc. The antibody is attached by simplycontacting the protein-coated surface with an aqueous solution of the antibody and allowing it to dry.
Any combination of support and binding technique which leaves the antibody irnmunoreactive, yet sufficiently imrnobilizes the an~body so that it can be retained with any bound antigen during a washing, can be employed in the present invention. A
preferred solid test support is a plastic bead.
As discussed above, the assay of the present invention employs a labelled antibody. The label can be any type that allows for the detection of the antibody when bound to a support. Generally, the label directly or indirectly results in a signal which is measurable and related to the amount of label present in the sample. For example, directly measurable labels can include radio-labels (e.g. 125I, 35S, 14C, etc.). A
preferred direc~y measurable label is an enzyme, conjugated to $he antibody, which produces a color leaction in the presence of the appropriate substrate. (e.g. horseradish peroxidase/o-phenylenediamine). An example of an indirectly measurable label is antibody that has been biotinylated. The pIesence of this label is measu~ed by contacting it with a solution containing a labeled avidin complex, whereby the avidin becomes bound to the biotinylated andbo~y. The label associated with the avidin is then measuled. A prefe~red example of an indirect label is the avidin/biotin system . . . .
. . . . . .
' . , .' : ' ' : :':
.. ..

207~87~
WO 91/08774 PCl/US90~0741 1 employing an enzyme conjugated to avidin, the enzyrne producing a color reaction as described above~
Whatever label is selected, it results in a signal which can be measured and is related to the amount of label in a sample. Comrnon signals are radiation levels (when 5 radioisotopes are used), optical density (e.g. when enzyme color reactions are used) and fluorescence (when fluorescent compounds are used). It is preferred to employ a nonradioactive signal, such as optical density (or color intensity) produced by an enzyme reaction. Numerous enzyme/subs~ated combinations are known in the immunoassay art which can produce a suitable signal. See, e.g., U.S. Patent Nos.I0 4,323,647 and 4,190,496, the disclosures of which are incorporated herein.

III. ~almç~
The IL-6 or M-CSF antibodies described herein, alone or in combinanon, may be used to passively irnmunize a host organisrn suffering from bacteremia or sepsis, or at risk with respect to bacterial infection. Either anti-L-6 alone, anti-M-C~F along, or 1, preferably anti~ 6 and anti-M-CSF will be administered. Treatment will generally consist of adrninistering the antibodies parenterally, and preferably intravenously. The dose and administranon regime will be a function of whether the antibodies are being admmistered therapeutically or prophylactically, and the patient's medical history.
Typically, the arnount of antibody adrniniste}ed per dose will be in the range of about 2a o.l to 25 mg/lcg of body weight, with the preferred dose being about 0.1 to 10 mg~cg of patient body weight. For parenteral adminis~ation, the antibodies will be formulated in an injectable forrn combined with a pharmaceutically acceptable parenteral vehicle.
Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dex~rose solution, and solutions consisting of small amounts of the human serum alburnin. The vehicle may contain ~ninor amounts of additives that maintcun the isotonicity and stability of the antibody. The preparation of such solutions is within the ski~l of the art. Typically, the antibodies will be formulated in such vehicles at a concentration of about 2-8.0 mg/ml to about 100 mg/ml.
The effec~veness of the subject IL-6 and M-CSF antibodies in the t~eatment of sepsis an be demons~ated in one of several animal model systems. The pleferred animal model system is baboon, and is described by Taylor, ~ 1-. 1987, J. Q~ini~a .. ~2:918, and by Taylor, Q~ al., 1988, C~i~b~. ~:227. Briefly, this consist of infusing a lethal dose of E. çoli, about 4 x 1010 organisms per kilogram of body weight administered over a 2-hour period. This is sufficient to ~11 100% of the 35 test animals in a period ranging from 16-32 hours. The animals are anesthe~ized with sodium pentobarbital in the cephalic vein through a percu~aneous catheter. They are .

.. . . - .
.. , : . .:
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. .

WO 91/Q8774 PCr/US90/0741 1 also orally intubated and positioned on their right side on a heating pad. Blood sarnples are removed from the femoral vein which is aseptically canmllated in the hind limb.
The percutaneous catheter is used to infuse the E. ~ organisms. Blood samples a;e taken at desired time intervals and assayed for white blood cells hetnatocrit, platelet 5 levels, and fibrinogen. Additionally, mean systemic arterial pressure (MSAP) may be monitored with a transducer (St.atnam P2306, Porter ) pressure gauge. Changes inthese parameters may 'oe prognostic of a patients ability to withstand exposure to a lethal dose of bacteria.
Having described what the applicants believe their invention to be, the 10 following examples are presented to illustrate the invention, and are not to be construed as limiting tne scope of the invention. For example, variation in tne source, type, or method of producing antibodies; different labels and/or signals; test supports of different materials and configurations; different immobilization methods may be employed without depar~ing from the scope of the present invention.

Example I
Preparation of L-6. M-CSF or L-6~ M-C$F Peptidelrr~uno~s A. -6. M-t:SF
n.-6 was prepared as described by Brakenhoff ~ al-, 1987, lo~urrL~al of Immunolngy. 13~:4116. Alternative methods are shown in EPC patent application publication no. 261,625 and PCT patent application international publication number WO 88/00206. Recombinant M-CSF was produced as described in U.S. Patent No.
4,847,201. The latter patent describes the expression of monomeric M-CSF which, in the mamma~an expression system shown, spontaneously recombines and refolds to yield biologically active M-CSF dimers. Preferably, in lieu of using spontaneously refold M-CSF, the monomers may be refolded as described in PCI' patent application, ~nternational Publica~on No. W088/OB003. Additionally, M-CSF may be produced using the methods described in U.S. Patent Nos. 4,868,119, and 4,879,227.

B. ~Di~
Based on the h~own amino acid sequences of IL-6 and M-CSF, peptides are synthesized and tested ~or immunogenic ac~vity, binding to IL-6 and M-~SF, and neut}aliza~on of the biological activity of these molecules. Peptides may be synthesized using the solid-phase method, descnbed in detail in Merrifield R.B., 1985, ~, ;~:341-347, on a Biosearch 9500 automated peptide machine, cleaved with hydrogennuoride, and purified by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-20 ~m Vydac C4 PrepPAK column. The preferred pep~ide for producing .. .... - , .: :
.
.

207~7``2 " W O 91/08774 PC~r/US90/07411 an~body to IL-6 is described in Japanese Patent No. 6210~157 and has the amino acid sequence: Pro-Val-Pro-Pro-C}ly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala.
M-CSF peptides that may be employed are those taken from asnino acids 4-150 of the mature molecule. That is, M-CSF which lacks the leader sequence. Exemplary 5 pepddes include; Thr-Ala-Pro-Gly-Ala-Ala-Gly^Arg-Cys-Pro-Pro-Thr, and Met-Ile-;'`' Gly-Ser-Gly-His-Leu-Gln-Ser-Leu-Gln-Arg-Leu-ne-Asp-Ser.
Before using tbe pep~ides to make antibody they are conjugated to a suitable carrier molecule to enhance eliciting an antibody response. These procedures aredescribed in U.S. Patent No. 4,762,706, inventors McCormick, et al.. Suitable 10 camers are keyhole limpet hemocyanin (KLH) or bovine serum alburnin (BSA). The conjugation is achieved via a sulfhydryl group of a cysteine residue that, if necessary, is added to the amino or carboxyl terrninal end of the peptides. A heterobifunctional crosslinking reagent, N-maleimido-~amino caproyl ester of 1-hydroxy-2-nitro-benzene-4-sulfonic acid sodium salt, is prepared by the following procedure.
One molar equivalent (2.24 g) of 4-hydroxy-3~nitro-benzene sulfonic acid sodium salt (HNSA) is mixed together with one molar equivalen~ (2.06 g) of dicyclohexylcarbodiimide and one molar equivalent (2.10 g) of N-maleimido-~
aminocaproic acid in 25 ml of dimethylformamide ~DMF) at room temperature overnight. A white precipitate of dicyclohexyl urea is formed. The precipitate is 20 filtered and 300 ml diethyl ether is added to the mother liquor. After about 10 rninutes to 4 hours a gurnmy solid precipitated from the mother liquor is formed. This solid will contain 58% of active HNSA ester and 42% of free HNSA.
The analysis consists of dissolving a small amount of the precipi~ate in phosphate buffer at pH 7.0 and measuring the absorbance at 406 nm; this reading 25 provides the amount of unreacted free HNSA which is the contarninating material in the HNSA ester preparation. Addition of very small arnounts of concentrated strong base (such as ~N NaOH) instantly hydrolyses the ester formed and a second reading is taken. Subtraction of the first reading from the second yielded the amount of ester in the original material. The solid is then dissolved in DMF and placed on a LH20 30 Sephadex column and eluted with DMF so that the ester is separated from the contamina~ing free HNSA. The progress of purification is monitored by t~ain layer chromatography using eluting solvents of chloroform, acetone and ace~ic acid (6:3~
` ! Vovvol)~ The product is positively identified as mal-sac HNSA ester by its reactivity with amine. The yield of the pure ester is estimated to be approximately 30% of 35 theoretical; ~he purified material consists of 99% ester.
The ester thus obtained is found to dissolve fully in water and is stable in water for several hours, provided no nucleophiles are added. When placed in lN ammonia , . . .
. , ~, . .
: .
.i . ' ' ' . : , ~ U ~ 1 0 I ~
WO 91/08774 pcr/us9o/o741 the ester produces the corresponding amide with a portion hydrolyzed to free acid~ The purified ester is found to be stable for extended periods when stored dessicated.
About 0.5 mg of the purified mal-sac HNSA ester is dissolved in 1 ml of distilled water. A 10 ~1 aliquot of this solution is diluted into 1 ml of }0 rnM phosphate 5 buffer at pH 7Ø The absorbance at 406 nm is used to calculate the concentration of free HNSA as described above. When 50 ~Ll of 4.8N sodiurn hydroxide solution is added to the diluted aliquot of ester and mixed, the absorbance of the solution at 406 nm increases significantly, indicating that the hydroxide nucleophile rapidl~hydrolyses the ester to component acid and free HNSA anion.
The difference between the post-base and initial free HNSA concentration represents the concentration of ester. From the actual concentration of ester and protein amino groups the amount of ester to be added to the protein solution to achieve the desired degree of substitution can be calculate~
The purified ~SA ester is then reacted with BSA as follows (the reaction wilh l 5 KL~ is similar to this procedure):
A total of 22 mg (20 ~Lmoles) of BSA (of molecular weight 66,296) is dissolved in 2.0 ml of 0.I M phosphate buffer at pH 7.5 to yield a total amine concentration of 1.0 x 10-2 moles per-liter (assuming 59 lysines/BSA molecule). A calculated amount ( I I mg, 2.35 x 10-5 moles) of the above-prepared mal-sac HNSA ester (97.7% pure) in 20 powder form is dissolved in 2.0 rnl of BSA solution. The reaction is carried out at room temperature. Ten 111 aliquots are removed from the solution at timed intervals and are each diluted into 1.0 ml of 0.01 M phosphate buffer at pH 7Ø The spectrurn of each diluted aliquot is recorded using a Hewlett-Packard spectrophotometer and the absorbance at 406 nm measured. A total of 50 ~11 of 4.8N NaOH is then added to each 25 aliquot, each aliquot is mixed and its spectrum retaken, and the absorbance at 406 nm measured.
From the absorbance at 406 nm before and after addition of base the concentration of ester remaining arld the percent ester that reacts are determined for the reaction mixtures. The results show that the reaction rate is essentially linear over a 15-30 minute period.
After 15 rninutes of seaction time, the reacion is stopped by applying thereaction rnL~ture tO a PD10 desalting Sephadex G-25 colurnn (Pharmacia, Inc.) equilibrated with 0.1 M phosphate buffer at pH 6Ø It is found that 2.6 x 10-3 moles/l of the ester reacts, and thus 25.9% of the 59 epsilon-arnino groups of BSA are 35 presurnably substituted~ Thus, the product contains 16 mal-sac groups per molecule.
The product of the first reaction, mal-sac-BSA (or mal-sac-KLH), is isolated by applying the reaction rnixture to a PD10 desalting Sephadex G-25 column equilibrated .. . . . .
.
' .
.

r~l V ~

W O 91/Q8774 PC~r/US90/07411 18 with 0.1 M phosphate buffer at pH 6Ø The column is eluted with 0.1 M phosphatebuffer in 1.0 ml fractions. The column elution is followed by moni~oring the absorbance spectrum, and peak fractions containing the mal sac BSA are pooled.
The peptides synthesized as described above are added and the pooled mixture 5 is stirred at room temperature overnight. The conjugates are subjected to extensive dialysis against distilled water and Iyophilization, and in sorrle cases are analyzed for changes in amino acid composition. These peptide conjugates may be used to irrlrnunize animals, or Iymphocytes ~ ~EQ to produce the desired antibody.

X~
IrnmunizatiQn with IL~ SF Qr Pe~tide ~mmun Produ~tion of H~ridomas A. Munnç Monoclonal Antibody The following describes~the imrnunization of mice with M-CSF with the ~ im of isolating immunized Iymphocytes and producing murine hybridomas. lllis procedure is 15 also applicable to generating antibody against TL-6. It will be further appreciated that the procedure can be employed to produce antibody against M-CSF, or TL-6, or M-CSF peptides, synthesized and conjugated as described above.
Generally, the procedures described in the following references are followed for. generating hybridomas. Shulman et a]., 1978, Nature, ~:269; Oi ~ ~., in ~
20 Me~ho~inG~llularTln~u~QlQov,p351 (Mischell~LSchiigieds. 1980). Foung~al., 1983, Proc. Nat'l Acas~ci. ~A,79:7484. Further references include, Gerhard et al., 1978, proc. Nat'l Acad. Sci.llSA, ~: 1510; MQ~Q~lonal Antibodie (R. Kennett, T. McKearn, & K. Bechtol eds. 1980); Schreier~ al., 1980, ~vbridoma Techniques;
Mnnoe~lonal Annbodiçs ~d T-CelLHvbnc1- m~s (G. Hamrnerling, U. Hammerling, &
25 J. Kearney eds. 1981); Kozbor ~ ~1., 1982, Pro . ~at'l A~ad~Sci. USA, ~2: 6651;
Jonak~ al., 1983, ~i~ : 124; ~
~ (R. Kennett, K. Bechtol, & T. McKearn eds. 1983?; Kozbor et ~1., 1983, 1mm~, 4:72-79; Shulman ~ al, 1982, ~a~, ~: 269-270; Oi et al., in Selected MethQ~ ~. Cell~l~ Ir~nunolo~y, pp. 351-371 (B. Mischell & S. Schiigi 30 eds. 1980); Foung ç.t al-. 1983, QC- NatlQcad. Sci. USA, 79:7484-7488.
Balb/.^ mice were immuni~ed w~th recombir..ant M-CSF (rM-CSF).
~nmunization consisted of a primaly intrapeAtoneal irnrnunization of 4011g of rM-CSF
in complete Freunds adjuvant, followed by two subsequent intraperitoneal injections without complete Freunds adjuvant, consisting of 20 ~Lg rM-CSF each. The firs~
35 imrnunization consisting of 20 ~lg was administered about ~hree weeks after the primary immunization, and the second 20 ~lg boos~ was administered about one week later.

.

- 2071~
~ . .
W O 91/08774 PC~r/US90/07411 About five and one half weeks after the second 20 llg boost, a final immunization was conducted, consisting of administering 10 ~Lg of rM-CSF intravenously. Three days later, spleens from immunized rnice were removed and the splenocytes fused to a murine myeloma cell line.
The fusion procedure that was followed is descnbed by Kohler & Milstein, 1975, ~n,a~, 2~:495, as modified by Fendly ~ ~1., in ~Q~, ~:359 (1987).
Briefly, rnice were sacrificed and splenocytes teased from imrnuniæd spleens, and washed in serum free Dulbecco's Modified Eagles medium. Similarly, SP 2/OAgl4 myelorna cells were washed, and combined with the splenocytes in a ~:1 ratio of spleen cells to myeloma cells. The cell m~xture was pelleted, media removed and fusion affected by the addition of 1.0 ml of 40% (vlv) solution of polyethylene glycol 1500 by dropwise addition over 60 seconds at room temperature, followed by a 60-second incubation at 37-C. To the cell suspension with gentle agitation was added 9 ml of Dulbecco's Modi~led Eagles medium over 5 minutes. Cell clumps in the mixture were gently resuspended, the cells washed to remove any residual PEG and plated at about 2 x 105 cells/well in Dulbecco's Modified Eagles medium supplemented with 20% fetal calf serum. After 24 hours, the cells were fed a 2x solution of hypoxanthine anda~aserine selection medium. The cells were plated in a total of 15.5 microtiter plates, which corresponded to 1488 wells. About 2.4 weeks later, 684 wells exhibited good cell growth, and these were screened for antibody to M-CSF. Several neutralizingantibodies were identified such as those secreted by hybridomas 382-5H4, 382-3Fl, and 382-4B~ or their subclones. These antibodies were punfied using standard methods, and may be used below for the treatment of sepsis.
The preferred IL-6 antibody, 8M70, was generated essendally using the procedures to generate the M-CSF antibody with the following differences.
Recombinant ~6 was used as ~e immunogen, and it was produced using the procedures described by Brakenhoff et al., Journal of Irnrnunology, 1987, vol. 139, page 4116. Eight separate fusions were conducted before the antibody was identified using RIA screening techniques. Using standard biochemical methods, it was deterrnined to have a Kd of about 10~
The hybAdoma cell line, 8M70, also termed CLB-IL-6-8, was cultured in 1 liter roller-botdes in IMDM media supplemented with 2% fetal calf serLtm, ~0 uM 2-mercaptoethanol, and penicillin and streptomycin. The cells were grown to a density of about 106/ml, and a week later the supematants were collected, and concentrated using 3s a hollow fiber device. To purify the antibody on a protein A colurnn (Pharmacia) solid NaCl was added ~o the concentrate to a final concentration of 3 M. This solution was diluted 1:1 with a solution consisting of 3 M

. . . .
- . , . . :
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.

2~7~872 - ```
WO 91/08774 PCl/US90/07q~1 ~0 NaCl and 1.5 M glycine, pH 8.9. The protein A column was equilibrated with the latter buffer, and the concentrate added to the column, the column washed, and ~mtibodyeluted off the colurnn with 100 rnM sodium citrate buffer, pH 6Ø Those peaks containing antibody were pooled, dialyzed against phosphate buffered saline, and5 stored undl used.

B ~man MQnnclonal ~r~
Peripheral blood Iyrnphocytes are isolated from septic patients, and then infected with Epstein-Barr virus and the inf~ted Iymphocytes immonalized by fusion to a selectable myeloma cell line, and t~ybrid cell lines so generated isolated and 10 characterized as to antibody producti~. More specifically, mononuclear cells are separated on Ficoll-hypaque (Pharrnacia), and monocytes depleted from the mixture by adherence to plastic. Standard laboratory techniques are utilized to effect these procedures. Next, nonadherent cells are enriched for antibody producers by antigen-specific panning. Panning is a technique generally known in the art, and involves 15 incubation of a population of antibody secreting cells on a plastic surface coaled with the appropriate antigen, in this instance IL-6, M-CSF or peptide imrnunogens derived from these moleculcs, and produced as described in Exarnple 1. Those cells that express antibody on their surface bind antigen, and consequently a&ere to the plastic surface, whereas cells that do no~ express cell surface antibody, do not adhere and can 20 be removed by washing. Thus, specific antibody secreting cells are enriched for by this technique.
More specifically, 6-well plates (Costar) are coa~ed with 1-20 llg of IL-6, M-CSF or peptide irnmunogens per well in phosphate buf~ered saline at 4 C overnight.
The wells are blocked after the ove~night ~ncubation period with phosphale buffered 25 saline containing 1% bovine serum albumin for at least 1 hour at 4~C, and subsequently washed with phosphate buffered saline/BSA. Next, 107 Iymphocytes in 1 ml of PBS/BSA are added to each well of the six well plates. The lymphocytes are allowed to incubate on the plates for 70 minutes, after which any nonadherent cells are removed by aspiration. The adherent cells are incubated with cell culture medium (IMDM, 30 Sigma Chemical Co., St. Louis, Missouri) containing 10% fetal calf serum.
'rhe adherent cells are subjected to Epstein-Barr virus transforn~tion by addingan equal amouDt of cultuue media obtained from growing the Epstein-Barr virus infected marmoset cell line, B95-8, or similar cell line, and thus containing the virus, to media bathing the adherent cells. The cells are cultuled in this environment at 37 C for 35 3 hours, and in this way the Iymphocytes in the adherent cell populaùon are subjected to Epstein-Barr infection. Following ~he infecion period, the cells are washed and . ~ .

~ V ~
~VO 91/08774 PC~r/US90/07411 plated onto 96 well microtitre plates at a density of about 104 -105 cells/well in IMDM
medium, plus 10% fetal calf serum, and 30% conditioned medium. The latter is derived from a Iymphoblastoid cell line, preferably JW5. The mediurn also contains S x 10-5 M
2-mercaptoethanol, ~0 ~ug/ml gentarnycin sulfaee ~Sigma), ancl 600 ng/ml cyclosporme A (Sandirnmune, Sandoz, Basel, Switzerland).
After about 14 to 21 days of incubation, cell culture supernatants are combined and screened for the desired antibody binding activity as described below. Positive hybridomas are subcultured at low density, retested for activity, and grown up and fused to the cell line F3B6 using po}yethylene glycol and the plate fusion technique 0 known in the art. The latter technique is described by Larrick, J.W., 1985, in ~luman Hybridomas and Monoclonal Antibodies, E.G. Engleman, S.K.H. Foung, J.W., Larrick, and A.A. Raubitschek, Editors, Plenum Press, New York, page 446. F3B6 is a heteromyeloma cell line that is sensitive to growth in media containing 100 ~Mhypoxanthine, S llg/ml aaserine and 5 ,uM ouabain. It is on deposit with the American Type Culture Collecdon with accession no. HB878~. Finally, the resulting hybrids are again screened to insure that they produce the desired antibody.

Example m IL-6 Antibodv for the Treatment of Sepsis The effectiveness of the IL-6 antibody, 8~170, in a baboon sepsis model system was tested çssentially as described by Taylor, et al., 1987, J. of Clinical ~nv., 79:918, and by Taylor, et al., 1988, Circulat~:y Shock, 26:227. Briefly, this consisted of first measuring I:L-6 levels in baboon plasma in response to a lethal or sublethal dose of E.
coli., and secondly, determining if IL-6 antibody was effective in trea~ing sepsis by preventing the death, or prolonging the lives of septic animals. A lethal or sublethal dose of E. ~ consisted of approximately 4 x 101 and 0.4 x 1010 organisms, respectively.
Figure 1 shows that after administradon of a lethal dose of E. coli, IL-6 levelsstart to increase after 1 hour, at which titne it is about l,jOO pg/rnl, and continue to increase for at least up to 6 hours to about 9,000 pg/ml. In contrast, Figure 1 also shows that thele is little perceptible increase in IL-6 after administration of a sublethal dose of E. coli. Baboons that receive a lethal dose of E. ~!i invanably die within 16-32 hours. Taylor, et ~1., 1987, J. of Cllinical lnv., 79:918, and by Taylor, et al., 1988, C~irculatorv Shock, 26:227.
The effectiveness of the IL-6 monoclonal, 8M70, in preventing the death or prolonging the life of baboons was tested using two administration rou~ines wherein the antibody was delivered in physiological saline. In the first, 5.9 mg of an~ibody per kg '' - ' . ,. ~ ~, ' : . ' : , . :. :
, ~2`~7~`872 ~-WO 9~/08774 Pcr/US9O/07411 `

of body weight was administered in three separate doses at 24, 22, and 71 hours before a lethal challenge of bacteria. Alternatively, 5.0 mg of antibody per kg of body weight was administered in a single dose simultaneously with Ihe bacterial challenge.
Figure 2 shows that in both instances IL-S monuclonal antibody considerably extends 5 the lifetime of the baboons that received the multiple or single dose of antibody and survived for 48, and 60 hours, respectively. Recall that baboons that receive a lethal dose of E. ~ invariably die within 16-32 hours.
Figure 3 shows the effect of L-6 antibody given simultaneously with a lethal dose of bacteria on various physiological parameters including white blood cells10 (WBC) hematocrit (HCT), platelet (Plat~ and fibrinogen (Fibr) levels, and rnean systemic arterial pressure (MSAP). With the exception of MSAP, the antibody had little effect. The extent of decrease of the MSAP was about half that observed for control animals, and thus may reflect the life-prolonging activity of IL-6 antibody.
. .
Exampl~lV
~l~CSF Antibo~f~h~reatment of Sepsis The effect of M-CSF antibody for the treatment of sepsis can be deterrnined by first measuring the tevels of M-CSF in baboons administered a lethal dose of ~ çQ~., and secondly, showing that blocking or reducing the increase in M-CSF with antibody .
prevents the death, or extends the lifetime of the arumals. The baboon was 2 o administered ~, ~ as described in the preceding exarnple. and M-CSF was measured using a RL~ assay as described in U.S. Patent No. 4,847,201, or by Halenbeck et al., Journal of Biotechnology, 1988, vol. 8, page 45.
Figure 4 shows M CSF plasrna levels in a baboon treated with a lethal dose of . M-CSF was about 25 ng/ml after 1.3 hours, and increased to about 60 ng/ml after 4 hours. Thereafter, from 4 hours to 16 hours, the level fell tO about 45 ng/ml.
The effectiveness of M-CSF antiWy would be demonstrated as described in the preceding exarnple by administering the antibody prior to, or sirnultaneous with the bacteria. A dose of 5.0-15 mg per kg of animal body weight, with 15 mg per kg preferred, would be effective in extending the lifespan of treated animals when administered simultaneously with the bacteria. However, it Will be appreciated that a fully protective dosing regime can be determined empiIically by those skilled in the art, and can consists of multiple doses of antibody.

.

f~U ~'10 I f~
` ~0 91/08774 PCr/US90/0741 1 ~m~Qy Treatnlent of Se~nrbdv to IL-6 and/or M~F
Purified 8M70 antibody to Il,-6, and M-CS~ antibody ( i.e. antibody secreted by hybridomas 382-SH4, 382-3F1, and 382-4B5 or their subclones are administered 5 alone, in combination, or sequentially to human patients for the therapeutic or prophylactic treatment of sepsis. Prophylactically the dose would be given just prior to surgery, and repeated at least once thereafter. Therapeutically ~he dose would be given every 24-48 hours until remission of the disease is apparent. I`he initial therapeutic dose would be 5-1~ mg per kilograrn of patient body weight, and then reduced to 5-10 10 mg per kilograrn.
Variations of the above embodirnents will be readily apparent to those of ordinary skill in the art without departing from the scope of the present invention, as described in the following claims.

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Claims (19)

WE CLAIM:

1. A method for treating sepsis in an organism comprising administering to said organism a composition comprising effective amounts of IL-6 antibody and M-CSF antibody.

2. A method as described in claim 1, wherein said antibody to IL-6 is selected from the group consisting of polyclonal, monoclonal, single chain, bispecific, or recombinant antibody.

3. A method as described in claim 1, wherein said antibody to M-CSF is selected from the group consisting of polyclonal, monoclonal, single chain, bispecific, or recombinant antibody.

4. A method as described in claim 2, wherein said antibody to IL-6 comprises monoclonal antibody.

5. A method as described in claim 3, wherein said antibody to M-CSF
comprises monoclonal antibody.

6. A method as described in claim 4, wherein said IL-6 antibody comprises human or humanized antibody.

7. A method as described in claim 5, wherein said M-CSF antibody comprises human or humanized antibody.

8. A method for treating sepsis in an organism comprising administering to said organism a composition comprising an effective amount of IL-6 antibody.

9. A method as described in claim 8, wherein said antibody to IL-6 is selected from the group consisting of polyclonal, monoclonal, single chain, bispecific, or recombinant antibody.

10. A method as described in claim 9, wherein said antibody to IL-6 comprises monoclonal antibody.

11. A method as described in claim 10, wherein said IL-6 antibody comprises human or humanized antibody.

12. A method for treating sepsis in an organism comprising administering to said organism a composition comprising an effective amount of M-CSF antibody.

13. A method as described in claim 12, wherein said antibody to M-CSF is selected from the group consisting of polyclonal, monoclonal, single chain, bispecific, or recombinant antibody.

14. A method as described in claim 13, wherein said antibody to M-CSF
comprises monoclonal antibody.

15. A method as described in claim 14, wherein said M-CSF antibody comprises human or humanized antibody.

16. A composition useful for the prophylactic or therapeutic treatment of sepsis comprising an effective amount of antibody to M-CSF and IL-6

17. A composition as described in claim 16, wherein said antibody is selected from the group consisting of polyclonal, monoclonal, single chain, bispecific, or recombinant antibody.

18. A composition as described in claim 17, wherein said antibody comprises polyclonal antibody.

19. A composition as described in claim 17, wherein said antibody comprises monoclonal antibody.