IE83792B1 - Monoclonal antibodies against human tumor necrosis factor alpha - Google Patents

Description

MONOCLONAL ANTIBODIES AGAINST HUMAN TUMOR NECROSIS FACTOR a This invention concerns a monoclonal antibody that has "high neutralizing activity against human Tumor Necrosis Factor~ a (TNE' a) and neutralizing- activity also against human Tumor Necrosis Factor B (TNF B), a hybrid ce11 line that produces the antibody and the use of the monoclonal antibody.

TNF d is a polypeptide of molecular weight 17500, whose primary and tertiary structure has been elucidated (Pennica, D. et al. Nature 312: 724 (1984) and Jones, E.Y. et a1. Na- ture 338: 225 (1989)). TNF a is biologically active as—; trimer. The main source of TNF a are cells of the monocytel macrophage lineage, but after appropriate stimulation it is produced by other cell types as well (e.g. T-lymphocytes).

TNF B is a polypeptide closely related to TNF 0.. molecules show a 28% homology at the amino acid level (see, Pennica, D. et al. gupgg and Gray, P.W. et al. Nature 312: 721 (1984)). The main source of TNF B are T—1ymphocytes.

TNF a was originally described as a molecule that induces haemorragic necrosis of tumors in mice. However, after large amounts of homogeneous TNF a became available upon cloning of its CDNA, it soon became clear that TNF a.nediates a large array of biological activities that allow the defini— tion of TNF a as a mediator of inflammation. See, generally, Beutler, B. and Cerami, A., Nature 320: S84 (1986).

TNF B mediates a qualitatively similar array of biologi- cal activities but quantitative differences as to the doses of TNF a or B required to mediate single biological activi- ties have been described.

TNF g plays, within the inflamatory response, a pivotal role in the host defense against invasion of the organism by noxious agents. However, under certain circumstances, like chronic or acute, systemic or localized hyperproduction, TNF a leads, in conjunction with other mediators of the in- , 83792 Both disease (Piguet, P.F., et al. J. Exp. Med. 166: 1280 -(1987)), in cerebral malaria (Grau, G.E., et al. Science : 1210 (1987)), rheumatoid arthritis (Brennan, F.M., et al., Lancet ii: 244 (1989)) and several other disease states.

Much less is known about the in vivo effects of TNF 8.

Given the fact, however, that it nediates in vitro, bio- logical activities similar to those of TNF a, it can be ar- gued that, in case of hyperproduction, TNF B as well may ‘contribute to the pathogenesis of some disease states.

These results have suggested that antibodies against TNF also antibodies against. TNF 8 could be in which a and, possibly, therapeutically’ useful in those disease states these polypeptides exert a pathogenic effect.

In order to be therapeutically useful antibodies against TNF a should be able to neutralize the toxic effects of TNF a in vivo. Polyclonal antibodies are easily obtainable from the serum of hyperimmunized animals. These polyclonal anti- body preparations, however, are not optimal for in vivo use because I 1 - they are a mixture of antibodies containing antibodies which do not neutralize TNF u, ~ they are a mixture of antibodies containing different antibodies having different affinities for the same epitope and — they are difficult to standardize in terms of potency because of lot—to—lot variations.

Monoclonal antibody technology is the tool of choice to by- pass these problems. It permits the in vitro ynoduction, under controlled conditions and in unlimited amounts, of monoclonal antibodies of reproducible specificity and af- finity against TNF a as against every immunogenic molecule.

Clearly, many different nmnoclonal antibodies can be ob- tained against one single antigen. They may vary from each other in terms of ~ antibody class and subclass, — epitope specificity, - binding affinity and - in vitro and in vivo neutralizing activity.

For therapeutic use it is.desirab1e to employ a monoclonal antibody against TNF a, as against any other antigen, which has high neutralizing activity. This would allow the admini- stration of lower doses of the monoclonal antibody in order to attain therapeutically’ effective levels in vivo, thus mitigating the possible, undesired side-effects caused by the monoclonal antibody.

EP4*035069O discloses fragments of the monoclonal antibody MAK 195 which is known to be specific for human TNF—alpha- ATh€S€ fragments (F(ab)2 and P(ab)) were shown to neutralize toxic effects of TNF alpha in vitro and in vivo. Accor- dingly, said antibodies and fragments thereof are useful in the treatment of diseases wherein TNF-alpha is involved.

EP4¥O3517&9C0ncerns‘1mproved monoclonal antibodies reactive with cachectin (human TNF) and a method for producing a mono- clonal antibody from splenocytes is disclosed. The anti- bodies obtained may be of all classes and subclasses of immunoglobulins, including bindung fragments. The anti- Fwbfldoma VE3,Na4,359370(198fl,reports on murine monoclonal antibodies which are able to neutralize the epitopes on TNF.

Evidence.is made that the monoclonal antibodies which are obtained by conventional methods neutralize the cytotoxity of TNF in vitro and in vivo.

WO together with an pharmaceutical products and compositions anti—neoplastic agent and/or an antimicrobial agent.

In this respect, the present invention provides a novel *monoclonal antibody or a binding fragment thereof which is able to neutralize human TNFa. The antibody according to the present invention has high TNFa neutralizing activity both in vitro as well as in vivo. Thus, human TNF d exerts half-maximal biological activity at about 0.15 ng/ml (3.3 x 10'12M, assuming TNF a is a trimer). In the presence of 1. ug/ml (6.25 x lO’9M) of said antibody, human TNF a exerts half-maximal biological activity at about 229 ng/ml (4.6 x 10‘9M, ratio of half—maximal biological activity in the presence vs absence of 1 ng/ml of said antibody is about 1527). In the presence of 100 ng/ml (6.25 X 1OT‘°M) of said antibody, human TNFa exerts half-maximal biological activity at about 26 ng/ml (5.2 x 10"1°M). In the presence of 10 ng/ml (6.25 x 10‘11M) of said antibody human TNFa exerts half-maximal biological activity at about 1.9 ng/ml (3.8 x l0‘11M)._ in vitro, Thusxhemonoclonal antibody or a binding fragment thereof? wowdedbythepm$entkwenfion!is able to neutralize human’Tumor Necrosis Factor alpha,andk;characterized in that said antibody is able to precipitate human TNF alpha forming high molecular weight antigen-antibody complexes, whereof the smallest antigen—antibody complexes formed with human TNF‘a1p_ha contain substantially three molecules of said antibody and two human TNF'alpha molecules and have a molecular weight of at least 400 kD.

In parliculaf, ‘the monoclonal antibody provided by the present inventionfis characterized by the fact that it neutralizes in vitro trimeric human TNF ci ata ratio : On 6 molar basis. In addition, said monoclonal antibody neutralizes and therefore recognizes the structurally related polypeptide humani mm: 13 as well.

Thus,in vitno, human TNF B exerts half-maximal biological activity at about 4 ng/ml (8 x l0"11M, trimer). In the presence of 1 ug/ml (6.25 x 10’9M) of said antibody, human TNF B exerts half-maximal biological activity at about 25.6 ng/ml (5 x 10‘1°M; half—maximal biological activity in the presence vs absence of».~1 dug/ml of -said antibody is. about 6.3). " the present invention shows that said , assuming TNF B is a ratio of As a second aspect, monoclonal antibody is able to precipitate human TNFo. as determined by double diffusion in two dimensions (Ouchterlony method), As a main aspect, the present invention shows that the smallest antigen~antibody complex '1 formed .upon incubation of said monoclonal antibody with human TNF a is a high molecular weight antigen-antibody complex, containing substantially,_at least three molecules of said monoclonal antibody and at least two Human TNF 0. moleculeis The term "high molecular weight antigen-antibody complex" is used to denote antigen—antibody complexes of at least 400 kD,typically from about 570 kD to about 600 kD.

Moreover said antibody provides complete protection in mice from an otherwise lethal dose of human TNF a at doses lower than about 1 ug/mouse, typically from about 0.4 to about 0.8 M9/mouse. It has to be noticed that 0.4 ug/mouse correspond to about 20 ug/kg body weight.

The invention further provides a stable hybridoma cell line and progeny thereof which produces such monoclonal an- tibody.

The invention additionally provides a process for the preparation of such monoclonal antibody.

The invention also provides a pharmaceutical composition’ comprising such monoclonal antibody which is able to neutra- lize both human TNF o. and human TN}? 8 and a pharmaceutically acceptable carrier and/or diluent.

A further aspect of the present invention is also to provide a method of:detecting"the Content of human TNF a in a body fluid; An object of the present invention is also to provide antiidig typic antibodies which recognize the human TNF a- and human TNF 8- receptors.

The monoclonal antibodies according to the present invention can be of any of the classes of immunoglobulins, such as IgM, JIgD, IgE, IgA or subclasses of IgG. The monoclonal antibody may be used intact, or as binding fragments, such as Fv, Fab, F(ab‘)2.

Preferably monoclonal antibodies according to the present invention are of the IgG class.

Part-icularly preferred monoclonal antibodies according to the present invention are those produced by hybridoma clone 78, as herein described, which are of IgG1 k isotype.

Preferred binding fragments of the monoclonal antibodies according to the present. invention are Fv, Fab, F (ab')2 fragments of said antibodies. in i As stated above the present invention provides a process for the preparation of -the monoclonal antibody of the in- vention or binding fragments thereof, which process com- prises culturing a hybridoma cell line or progeny thereof according to the invention and recovering the monoclonal antibody thus produced.

The monoclonal antibodies according to the present in- vention, or the fragments thereof, can be prepared by im- mortalizing lymphocytes of the B—cell lineage.

Immortalization can be accomplished through transformation with an oncogenic virus or through fusion with an already inmnortal cell line (e.g. myeloma. or lymphoblastoid cell line). The latter approach, which was originally described by Koehler and Milstein (Koehler, G; and Milstein, C., gI__a_; ture 256: 496 (1975)) givesrise to inmortal hybrid cell lines capable of unlimited growth and antibody production.

The irmnortal cell lines thus obtained may be cloned and screenedin such a way as to detect antibodies against the desired antigen in the cell supernatants. A large number of screening techniques have been described in the literature and are well—known to those skilled in the art. These tech- niques unequivocally allow the isolation of monoclonal an- / *’*'~.. tibodies against the desired antigen(s). However, if the operator wants to isolate a monoclonal antibody having par~ ticular properties (i.e. having high or low affinity for the antigen, being neutralizing" or non—neutralizing) the screening techniques have to be set up in such a way as to allow him to isolate monoclonal antibodies having the de- sired properties. The way these assays are set up depends on the property one is looking for, on the antigen against which the operator wishes to obtain the antibodies and is, generally speaking, not predictable and must be studied on a case by case basis. This step, however, is of crucial im- portance if the operator wants to isolate monoclonal anti- bodies with high activity.

If desired, once the cell lines producing the monoclonal _ antibodies with the desired properties against human TNF a have been isolated according to the present invention, they can be used as a source of the genes encoding the monoclonal antibodies.

These genes can be isolated by preparing first CDNA li- braries from messenger RNA. A single CDNA clone, coding for the immunoglobulin is then isolated and may be further ma- nipulated. The ultimate goal of these manipulations is usu— ally, but not exclusively, that of generating antibodies of reduced immunogenicity upon in vivo administration into a host different from the one from which the original anti- bodies were derived. This can be achieved by ligation of the nucleotides corresponding to the variable region genes of tin: original species to nucleotides corresponding txa the constant region genes of the species to which the antibodies have to be administered. Alternatively, nucleotides encoding the regions of the original monoclonal antibody determining complementariness may be substituted for corresponding nuc- leotides of the variable regions of antibody genes of the species to which the antibodies have to be administered.

Finally, the original or the modified CDNA clone may then be isolated and placed into suitable prokaryotic or eukaryotic /aax expression vectors and subsequently transfected into a host for ultimate bulk production.

In the accompanying diagram representations: Figure 1 shows the specificity for human TNF a of the monoclonal antibody, as determined in an ELISA that measures the residual binding to TNF a of 100 ng/ml of the antibody upon incubation with different doses of human TNF a or with different doses of unrelated antigens.

Figure 2 shows the.neutralizing activity of 1 ug/ml.(6.25 x 10‘"M) of the monoclonal antibody on different doses of human TNF a.

Figure 3 shows the neutralizing activity of 1 ug/ml of the monoclonal antibody on different doses of human TNF 3.

Figure 4 shows a Scatchard plot of the binding of human TNFa to said monoclonal antibody.

Figure 5 shows the dissociation of human TNF a from the monoclonal antibody of the present invention.i Figure 6 shows an Ouchterlony double immunodiffusion in In the central trough were added 20 ug of the upper right, lower agar. monoclonal antibody; in the upper left, left and lower right troughs 10 ug, 5 ug, 1.25 ug and 2.5 ug of human TNFG, respectively.

Figure 7 shows the. fast—pressure liquid chromatography size exclusion ‘profiles'of 2 x 10‘°M human TNF a of 6.25 x lO'5M of said monoclonal panel A), of a mixture of 2 x 10"°'M (FPLC) alone (-——- panel A), antibody alone ( human TNF a and 2 x 10"7M of the monoclonal antibody (panel B) and of a mixture of 10"1°M iodinated human TNF a and 1.25 x 1O"11M of the monoclonal antibody (panel C).

In the accompanying tables, Q Table 1 shows the parameters of the binding of human TNFa to the monoclonal antibody of the present invention.

Table 2 shows the concentrations of human TNF a giving half—maximal biological activity in the absence or presence of different concentrations of said monoclonal antibody.

Table 3 shows the effect on survival of the administra- tj_Qn of different doses of human.TNF a into mice.

Table 4 shows the protective effect of the monoclonal antibody of the present invention in mice treated with two lethal doses of TNF a.

Table 5 shows the protective effect of the monoclonal antibody’ of the present invention injected into Inice at different times before and after the treatment with two le- thal doses of TNF 0.. with respect to characterizing the claimed hybrid cell line, the terms "permanent" and "stable" mean viable over a prolonged time, typically at least about six months. The invention enables a stable, permanent hybridoma cell line to be provided which maintains the ability to produce the specified monoclonal antibody through at least 25 passages.

The term "monoclonal antibody" refers to an antibody selected fran antibodies whose population is substantially homogeneous, i.e. the individuals of the antibody population are identical except for naturally occurring mutations.

The term "antibody" is also meant to include intact molecules as well as fragments thereof, such as F5, Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of antibody, clear more rapidly front the circulation .and may’ have less non specific tissue binding than intact antibody. It will be appreciated that Fv, Fab, and-F(ab')2 and other fragments of the monoclonal antibody of the present invention may be used as well as the intact antibody for the same purposes, e.g. the detection of TNF a and treatment of those disease states in which TNF a has been shown to play a detrimental role.

The term "neutralizing" is used to denote the ability of antibody«containing solutions to block the capacity of TNF a and TNFB to exert their biological activities in vitro and/or in vivo.

The term "enhanced or high TNF a neutralizing activity in vitro" is used to denote the ability of a solution contain- ing a monoclonal antibody to neutralize human TNF a at a < :1 ratio on a weight basis and at a < 2:1 ratio on a molar basis at doses of monoclonal antibody 5 10 ng/ml.

The term "enhanced or high TNF a neutralizing activity in vivo" is used to denote the ability of a solution containing a monoclonal antibody to block, at doses of 5 20 ug/kg body weight, the capacity of TNF a to play a detrimental role in vivo.

The monoclonal antibody of the present indeed able to afford complete protection in mice from an invention is otherwise "lethal "dose "Bf TNF a at doses lower than 1 pg/mouse typically from about 0.4 to about 0.8 ug/mouse. It has to be noted that 0.4 ug/mouse corresponds to about 20 ug/kg body weight.

In a preferred embodiment according to the present in- . vention such a monoclonal antibody is secreted by a hybri- doma cell line which has been prepared using cells of an immortalizing cell line and cells derived from a mouse which had been immunized with human recombinant TNF a.

The immortalizing cell line is a cell line which, for practical purposes, can be maintained perpetually in cell culture. In other words, it is stable and permanent and, when fused with cells which do not exhibit these properties, is able to confer the properties on the fusion product.

Any appropriate immortalizing cell line may’ be used.

Typically, a plasmacytoma (myeloma) of mamalian origin may be employed. A preferred type of cell line is a murine hypoxanthine—phosphoribosyl-transferase (HPRT) deficient plasmacytoma. One such cell line is particularly preferred.

This is the NSC cell line, a well-known HPRT—deficient pla~ smacytoma of BALB/c origin which does not produce or secrete either* immunoglobulin or heavy or light immunoglobulin chains on its own (Clark, M.R. and Milstein, C. Somatic Cell Genet. 1: 657 (1981)).

The immortalizing cells are fused with cells, at least some of which produce antibodies which bind specifically to human TNF a. mice or rats).

The antibody-producing cells are generally obtained from a mouse immunised with respect to human TNF a. Commercially available human TNF a may be used for this purpose. After fusion, the fusion products are screened for those secreting the desired monoclonal antibodies.i In vitro they are first tested in an ELISA that detects antibodies against TNF d. Then they are tested for their ability to neutralize the cytotoxic activity of human TNF a on mouse LM cells.‘ ‘ antibodies can be then grown in vitro in suitable culture media in tissue culture flasks or in a 1arge~sca1e culture device (e.g. Acusyst, Endotronics, Coon Rapids, Minnesota) or in vivo as an ascitic_tumor in laboratory animals (e.g. the antibody can be separated from the as the case may be, by If desired, culture medium or the body fluid, techniques such as ammonium sulfate precipitation, ion ex- change chromatography, high-performance liquid ch- romatography or by other techniques known to those of or- dinary skill in the art. According to the present invention monoclonal antibodies with high neutralizing activity against TNF a and neutralizing activity also against TNF B are therefore provided, which are characterized in that : a) their population is substantially homogeneous, b) they are produced by immortal cells which are them‘ selves hybrids between an immortal cell line and an antibody-producing cell, c) they show enhanced TNF a neutralizing activity in vitro and in vivo, d) they are able to neutra1ize,thereby recognizing,human TNFB. e) they precipitate human TNFu, E) they form with human TNFa, in solution, complexes the _ smallest of which contain substantially at least flwee migoclpnal antibody molecules and have high molecular 9 ’tYDJ.Cally of. at least 400 kD. monoclonal antibodies of the present invention can be employed in mammals, including humans for prophylactic and/or therapeutic use in any disease state in which TNF a and/or TNF B exert a pathogenic effect. Typically such disease states are cachexia, septic shock, graft—versus—host disease, AIDS, cerebral malaria, rheumatoid arthritis, chronic and acute inflammatory di- seases, myocardial ischaemia and others in which it is already known or will be known in the future that TNF a and/or TNF B play’ a detrimental role. The dosage levels suitable for local or systemic administration ix) adult humans of the monoclonal antibodies according to the present invention, e.g. those produced by hybridoma clone 78, may range from about 20 ug to about 1 mg of antibody per kilogram of body iweight. Single or multiple administrations of the compositions can be carried out with dose levels and In any pattern being selected by the treating physician. the pharmaceutical formulations should provide an event, antibody quantity sufficient to effectively treat or prophylactically treat the patient.

The monoclonal antibody of, this invention may be formulated either alone or together with other pharmaceuti- cally active agents in pharmaceutical compositions by in- cluding appropriate amounts of the monoclonal antibody to- gether with a pharmaceutically acceptable carrier and/or diluent.

Alternatively, antibody of the present invention can be administered in a combined method of treatment; with a different. pharmaceutically’ active agent. that can be formulatedéwith or a monoclonal Pharmaceutically active agents, the monoclonal antibodies of the present invention, alternatively can be administered in a combined method of in particular thus can be for instance antibodies, against ‘ other antigens, containing a nmnoclonal antibody of treatment, monoclonal antibodies, providing a "cocktail" the present invention and one or more (monoclonal) are known tog ._a-4),: antibodies magainst other antigens involved in the pathogenesis of the relevant disease state.

Further active agents, that can be formulated with the monoclonal antibodies of the present invention, or alterna- tively can be administered in a combined method of treat- ment, especially in order toproducea.therapeutically useful effect, depend on the disease state to be cured and are, for instance, commercially available gamma globulin and immune iglobulin products, antibiotics, antimicrobial products, antibacterial and antitumor agents or a mixture of two or more of them.

Moreover the monoclonal antibodies of the present inven- tion can be used alone or in a combined method of treatment with antibacterial and, in particular, with anti~neop1astic agents, so as tr) prevent. or ameliorate the side effects arising therefrom. Typical side effects which can be treated are e.g. cachexia, nausea, vomiting, anorexia, alopecia, diarrhea and neutropenia.

Typically, the antimicrobial agents may include a penicillin in conjunction with an aminoglycoside (e.g. gentamycin, tobramycin). However several well known additional agents, e.g. cephalosporins, can be utilized.

The term "antineoplastic agent" is meant to comprise both single antitumor drug and "cocktails" ile. a mixture of such drugs, according to the clinical practice.

Antitumor agents that can be formulated with a compound of the invention or alternatively, can be administered in a combined method. of treatment e.g. doxorubicin, daunomycin, epirubicin, idarubicin, etoposide, fluorouracil, mephalan, cyclophosphamide, bleomycin, vinblastip and mitomycin or a mixture of two or more thereof.

The term "combined" method of treatment is meant to include both separate substantially contemporaneous administration of a pharmaceutical composition containing a —monoclonal antibody according to the invention and a phar— maceutical composition containing a different pharmaceuti- Cally active agent.» ’ Accordingly, a preferred object of the present invention is a combined method of treatment of cancer in mammals, including humans, in need of such treatment, said method comprising administering ) a monoclonal antibody according to the present invention or a binding fragment thereof, and 2) an antitumor agent, in amounts and close enough together in time sufficient to produce a therapeutically "Se?"-1 eff‘-."—°F:. - u, ‘ Object of the present invention is also to provide products containing a nwnoclonal antibody according to the present invention, or a binding fragment thereof and an antitumor agent as a combined preparation for simultaneous, separate or sequential use in anti-cancer therapy. i.The monoclonal antibodies of the invention and the binding fragments thereof can therefore be used in a treatment to ameliorate a cancer. They may be administered to a patient suffering from a cancer treatab1e,with an antitumor agent, for example an anthracycline glycoside such as doxorubicin, daunomycin, epirubicin or idarubicin as xnentioned. above.

An antibody according to the invention and an antitumor agent such as anthracycline glycoside can be adinistered to improve a leukaemia such as neuroblastoma, the condition of a patient having myeloblastic leukaemia, lymphoma, Wilm's tumor or malignant neoplasm of the bladder, breast, sarcoma, lung or thyroid.

Moreover the high neutralizing activity of the antibody according to the present invention, as shown e.g. in Figure 2, suggests that said antibody recognizes an epitope close to or within the receptor—binding site of human TNF a. Two TNF a receptors have been characterized (schall, T.J. et al.

Cell 61: 361 (1990) and Smith, C.A. et al. Science 248: 1019 (1990)).

Both receptors bind human TNF a and human TNF B with almost equal affinity when expressed on whole cells. Soluble forms of the two receptors, however, bind human TNF B with much lower affinity than human TN}? d. (Seckinger, P. et al. L Biol. Chem. 264: 11966 (1989) and Engelmann, H. et al. Q; aBiol. Chem. 265: 1531 (1990)). This result suggests that solubilization of the extracellular domain of the receptors induces a change in the relative affinities for human TNF a and human TNF B. If the monoclonal antibody of the present invention recognizes an epitope within the receptor-binding site~of human TNF'a then it should functionally behave like a TNF a receptor, i.e. it should recognize both human TNF a" as well as human TNF 8. Since said monoclonal antibody is a soluble molecule, it should behave functionally like a sol- uble TNF a receptor,ri;e. it should bind human TNF a with a much higher affinity than human TNF 8. Figure 3 shows that this is indeed the case. For human TNF B the ratio of half- maximal biological activity in the presence vs absence of 1 ug/ml of said antibody is 6.3. This suggests that said mon- oclonal antibody binds to human TNF a and human TNF 8 like a soluble TNF a receptor. It is therefore ideally suited for being used as an immunogen in order to obtain, according to standard techniques well—known to those skilled in the art (see, generally, Anti—idiotypes, receptors and molecular mimicry, Linthicmn, D.S. and Farid, N.R., Eds., Springer- -Verlag Heidelberg, 1988) antiidiotypic antibodies which bind to the monoclonal antibody of the present invention like human TNF a. Consequently, these monoclonal antii- diotypic antibodies that are able to bind to TNFa— receptors like TNFa and consequently mimic the biological activities of TNFa can be therapeutically useful in those diseases in which administration of TNFG is likely to play a benefical role. These diseases states are e.g. cancer, in which the antiidiotypic antibodies of the invention, similarly to TNFa, can be administered either alone or, preferably, as J. part of a combined method of treatment (Mule, J.J. et al- Exp. Med. 17 : 629 (1990)) and some autoimmune diseases (Jacob O.J. and McDevitt, H.0., Nature 331: 356 (1988)).

The monoclonal antibodies and the pharmaceutical compositions, according to the present invention can be ad- ministered parenterally by subcutaneous, intramuscular, in- traarterial or intravenous administration.

The pharmaceutical compositions containing the monoclonal antibodies of the present invention are usually prepared following conventional methods.

‘Generally speaking; carriers and/or diluents, according to the pharmaceutical composition of the present invention include alcoholic/aqueous solutions, emulsions, or suspen- sions, including saline or buffered media. Parenteral vehi- cles include sodium chloride solution, Ringer's . dextrose, dextrose and sodium chloride, lactated.Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as, for example, anti~ microbials, antioxidants, chelating agents, inert gases, and the like. See, Pharmaceutical Sciences, 16th Ed., Mack, eds., 1980. generally, Remington's Usually, in disease states in which TNF a exerts a pa- thogenic effect, TNF a levels in body fluids increase com- pared to the levels detectable in the same body fluids of healthy individuals. Therefore determination of TNF a levels in these body fluids can be of diagnostic and prognostic value.

A further aspect of the present invention is, therefore, to provide immunoassays where the biological properties of the monoclonal antibodies according to the present invention are particularly advantageous. For the purpose of the present invention, human TNF (L which is detected by the monoclonal antibody of the invention may be present in biological fluids or tissue. Any sample, obtained from an individual, containing an unknown amount of human TNF a can be used. Normally, the sample is a liquid, such as, for ex- ample, blood, serum, plasma and the like.

The monoclonal antibodies of the present invention are particularly suited for use in immunoassays wherein they may be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibody in these im- munoassays can be detectably labeled in various ways.

There are many carriers to which the monoclonal antibody of the present invention can be bound and which-can be used in detecting the presence of human TNF a. carriers include glass, polystyrene, polypropylene, poly- amylases, natural and rmmdified agaroses and magnetite. The ethylene, dextran, nylon, celluloses, polyacrylamides, nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present. invention.

Those skilled in the art will note many other suitable car- riers for binding monoclonal antibody, or will be able to ascertain the same by the use of routine experimentation.

There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates. Those of ordi- nary skill in the art will know of other suitable labels for binding to the monoclonal antibody, or will be able to as- certain the same by the use or routine experimentation. Fu- rthemore, the binding of these labels to the monoclonal an- tibody can be accomplished using standard techniques commonly known to those of ordinary skill in the arts One of the ways in which a monoclonal antibody of the present invention can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be Well-known ’ detected, for example, by spectrophotometric or fluorometric means. Examples of enzymes which can be used to detectably label the monoclonal antibodies of the present invention, include malate dehydrogenase, staphyloccocal nuclease, de- lta—V—steroid isomerase, yeast alcohol dehydrogenase, alph- a—glycerophosphate dehydrogenase, triose phosphate isom- erase, horseradish peroxidase, alkaline phosphatase, aspara- glucose cmidase, beta-galactosidase, ribonuclease, glucose~VI—phosphate dehydrogenase, ginase, urease,‘ catalase, glucoamylase and acetylcholine esterase.

The monoclonal antibody of the present invention can also ‘ be labeled with a radioactive isotope which can then be determined by such means as the use of a gammacounter or a scintillation counter" Isotopes which are particularly use- ful for the purpose of the present invention are: 3H, 1251, .311’ 32P’ 35S’ 14C’ Slcr’ 36C1’ 57Co’ SBCO’ 59Fe’ 75Se.

It is also possible to label the monoclonal antibody with a fluorescent compound. when the fluorescently labeled mo- noclonal antibody is exposed to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye. Among the most commonly used fluo- rescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, op— hthaldehyde and fluorescamine. .

The monoclonal antibody of the invention can also be de- tectably labeled using fluorescent emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DT- PA) or ethylenediaminetetraacetic acid (EDTA).

The monoclonal antibody of the present invention also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged mono- clonal antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent ,r(£s\_ labeling compounds are luminol, isoluminol, theromatic ac- ridinium ester, imidazole, acridinium salt and oxalate es- ter.

Likewise, a bioluminescent compound may be used to label the monoclonal antikxxdy of the ‘present invention. Biol- uminescence is a type of chemiluminescence found in biolog- ical systems in which a catalytic protein increases the ef- ficiency of the chemiluminescent reaction. The presence of a bioluminescent monoclonal antibody is determined by detect- ing the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Another technique which may also result in greater sen- sitivity when used in conjunction with the present invention _ consists of coupling the monoclonal antibody of the present invention to low molecular weight haptens. The haptens can then be specifically detected by means of a second reaction. it is common to use such haptens as biotin pyridoxal and For example, (reacting with avidin) or dinitrophenyl, fluorescamine (reacting with specific anti—hapten antibodies) in this manner.

The monoclonal antibodies of the present invention are ideally suited for the preparation of a kit- Such a kit may comprise a carrier means being compartmentalized to receive one or more container means such as vials, tubes and the like, in close confinement therewith, each of said container means comprising the separate elements of the immunoassay to be used. A similar kit may be prepared comprising compart- mentalized carrier means having one or more container means cxmmrising separate elements suitable for therapeutic use according to the present invention. 7 The types of immunoassay which can be used or incorpo~ rated in kit form are many. Typical examples of some of the immunoassays which can utilize the antibodies of the inven- tion are competitive assays and immunometric, or sandwich, immunoassays.

By. the term "immunometric_ assay" or "sandwich immun- oassay", it is meant to include simultaneous sandwich, for— ward sandwich and reverse sandwich immunoassays. These terms are well understood by those of ordinary skill in the art.

Those of ordinary skill in the art will also appreciate that the monoclonal antibody of the present invention may be useful in other variations and forms of immunoassays which are presently known or which may be developed in the future.

These’are intended to be included within the scope of the —present invention. .m.......J. ha a forward sandwich immunoassay, a sample is first incubated with a solid phase immunoabsorbent containing mo- noclonal antibody against human TNF a. Incubation is continued for a period of time sufficient to allw the an- tigen in the sample to bind to the immobilized antibody in - the solid phase. After the first incubation, the solid phase immunoabsorbent is separated from the incubation mixture and remove excess antigen and other interfering such as non—specific binding proteins, Solid. phase imobilized washed to substances, which also may be present noabsorbent containing human TNF a to the antibody is subsequently incubated for a second time with soluble labeled antibody or antibodies. After‘ the second incubation, another wash is performed to remove unbound labeled antibody(ies) from the solid phase immunoabsorbent and removing non—specifically bound labelled antibodyties).

Labeled antibody(ies) bound to solid phase immunoabsorbent is/are then, detected and the amount of labeled antibody detected serves as a direct measure of the amount of antigen Alternatively, ‘labeled in the sample. immu- present in the original sample. antibody which is not associated with the immunoabsorbent complex can also be detected, in which case the measure is in inverse proportion to the amount of antigen present in Forward sandwich assays are described, for 4,012,294 and the sample. example, in United States Patents 3,867,517; ,376,110. .nn\ In carrying out forward imunometric assays, the process comprises, in more detail: (a) first forming a mixture of the sample with the solid phase bound antibody and incubating the mixture for a time and under conditions sufficient to allow antigen in the sample to bind to the solid _ phase bound antibody. (b) after said incubation of step (a), adding to the Mdmixture the detectably labeled antibody or antibodies and incubating the new resultant mixture for a period of time and under conditions, which are sufficient to allow the labeled antibody to bind to the solid phase immunoabsorbent; (c) separating the solid phase immunoabsorbent from the mixture after the incubation in step (b); and (d) detecting either the labeled antibody or antibodies bound to the solid phase immunoabsorbent or detecting the antibody not associated therewith.

In ea reverse sandwich assay, the sample is initially incubated with labeled antibody, after which the solid phase immunoabsorbent containing multiple immobilized antibodies is added thereto, and a second incubation is carried out.

The initial washing step of a forward sandwich assay is not required, although. a wash is performed after the second incubation. Reverse sandwich assays have been described, for example, in United States Patents 4,098,876 and 4,376,110.

In carrying out reverse immunometric assays, the process comprises, in more detail: (a) first forming a mixture of the sample with the soluble detectably labeled antibody for a period of time and under conditions, which are sufficient to allow antigen in the sample to bind to the labeled antibody; ' (b) after the incubation of step (a), adding to the mixture the solid phase bound antibodies and incubating the new resulting mixture for a period of time and under conditions sufficient to allow antigen bound to the labeled antibody to bind to the solid phase antibodies; (c) separating the solid phase immunoabsorbent from the incubating mixture after the incubation in step (b); and ' (d) detecting either the labeled antibody bound to the solid phase immunoabsorbent or detecting the ‘labeled antibody not associated therewith. , _ In aesimultaneous sandwich assay, the sample, the immu- noabsorbent having multiple immobilized antibodies thereon and. labeled soluble antibody’ or antibodies are incubated simultaneously in one incubation step. The simultaneous assay requires only a single incubation and has a lack of washing steps. The use of a simultaneous assay is by far the Vpreferred method. This type of assay brings about ease of reproducibility, linearity of the The sample containing antigen, handling, homogeneity, assays and high precision. solid phase immunoabsorbent with imobilized antibodies and labeled soluble antibody or antibodies is incubated under conditions and for a period of time, which are sufficient to allow antigen to bind to the imobilized antibodies and to the soluble antibody. In general, it is desirable to provide‘ incubation conditions sufficient to bind as much antigen as since this maximizes the binding of labeled thereby increasing the signal. possible, antibody to the solid phase, Typical conditions of time and temperature are two hours at about 45°C, or twelve hours at about 37°C.

Antigen typically binds to labeled antibody more rapidly than to immobilized antibody, since the former is in solu- tion whereas the latter is bound to the solid phase sdpport.

Because of this, labeled antibody may be employed in a lower concentration than immobilized antibody, and it is also preferable to employ a high specific activity for the la- beled antibody. For example, a labeled antibody according to this invention can be employed at a concentration of about -50 ng/per assay, whereas immobilized antibody can have a concentration of 10-500 ng/per assay per antibody; Where radiolabeled, the antibody might have a specific activity with, for instance, one radioiodine per molecule, or as high as two or more radioiodines per molecule of antibody.

In carrying out the simultaneous immunometric assay on a sample containing 21 multivalent antigen, the process com- prises, in more detail: (aT"simu1taneous1y forming a mixture comprising the c c =sample, together with the solid phase bound antibody and the soluble labeled antibody or antibodies; (b) incubating the mixture formed in step (a) for a period of time and under conditions sufficient to allow antigen in the sample to bind to both immobilized and labeled antibodies; (c) separating the solid phase immunoabsorbent from the incubation mixture after the incubation; and (d) detecting either labeled antibody bound to the solid phase immunoabsorbent or detecting labeled antibody not associated therewith. 0 Of course, the specific concentrations cu? labeled. and immobilized antibodies, the temperature and time of incuba- tion as well as other assay conditions can be varied de- pending on various factors, including the concentration of antigen in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determina- tion by employing routine experimentation.

After the incubation, the solid phase immunoabsorbent is removed from the incubation lnixture. This can be! accom- plished by any of the known separation techniques, such as sedimentation and centrifugation. Detection can be performed by a scintillation counter, for example, if the label is a radioactive gamma—emitter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme .label, the detection can be accomplished by colorimetric nethods which employ a substrate for the en~ zyme. other steps such as washing, stirring, shaking, filtering and the like may of course be added to the assays, as is the custom or necessity for any particular situation.

There are many solid phase imunoabsorbents which have been employed and which can be used in the present inven~ tion. Well known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon and other materials and tubes formed fro or coated with such materi- als and the like. The immobilized antibodies can be either covalently or physically bound to the solid phase immuno- absorbent, by techniques such as covalent binding yia an amide or ester linkage, or by absorption. Those skilled in the art will know many other suitable solid phase immuno- absorbents and methods for imobilizing antibodies thereon, or will be able to ascertain such, using no more than rou- tine experimentation.

The the described. by the following example. intended to limit the invention in any manner. invention are further This example is not various aspects of days Eééflfikfi l. Immunization of mice with human TNF a Human TNF <1 (Esquire Chemie AG, Zuerich, diluted in phosphate-buffered saline (PBS) supplemented with complete Freund's adjuvant (CFA, Difco, Detroit, Michigan, 500 ul to’S00 ul PBS), is administered into the rear foot- pads of 5 BALB/c mice at a dose of 30 ug/mouse. 14 days‘after the first immunization human TNF a, diluted in PBS supp1emented"with CFA as above, is administered subcu- taneously to the same mice at a dose of 30 ng/mouse. 14 days after the second immunization human TNF a, diluted in PBS, is administered intraperitoneally to the same mice at a dose of 30 n9/mouse. the third immunization human TNF a is Switzerland), after administered to the same mice at the same doses and on the same conditions as for the third imunization.

After 3 days the mouse that has the highest titer of antiTNF a antibodies in the serum as determined in an ELISA is sa- crified.

. Immortalization of mouse spleen cells producing anti—TNFa antibodies.

The spleen is removed aseptically, homogenized and the resulting cell suspension centrifuged. At the same time NSO myeloma cells (>99% viable cells) are centrifuged as well.

Both pellets are then resuspended in complete RPMI medium.

Complete RPMI medium is RPMI 1640 (Flow, Opera, Italy) sup- plemented with ].ImM sodium pyruvate (Gibco, Paisley, Scot- land), nonessential amino acids (Gibco), 100 ug/ml of stre- ptomycin, 1.5 ug/ml of amphotericin B and additional glut- amine (2 mM). Both cell suspensions are combined at a spleen cell myeloma cell ratio of 10 : l. The resulting cell mixture is centrifuged for 10 min at 400 g. The medium is discarded and 1 ml of polyethylene glycol (PEG 1500, Serva, Heidelberg, west Germany, 40% w/v in complete RPMI medium) is carefully ‘added drop by drop over a 1 minute period in order to allow_fusion to take place. 5 ml of complete RPMI medium are then added over a 5 min period, then 10 ml over a min period and finally 15 ml over a 5 min period. The fi- nal suspension is centrifuged for 10 min at 400 g and the pellet resuspended in complete RPMI medium supplemented with % fetal calf serum (FCS, Flow, heat inactivated at 56°C for 30 min) so to have a final cell density of 5x105 cells/m}.—1spleen cells + NSO cells). 20012.1 of this suspen- sion are seeded into/teach" well of 96-well tissue culture plates (Falcon No. 3072, Becton Dickinson, Milan, Italy) that have been previously seeded with irradiated mouse per- itoneal cells (3000 Rad; 2.5 x 10‘ cells/well). Plates con- taining the fusion products are then incubated for 24 hours at 37°C, 5% CO2. Then 50 ul of complete RPMI medium supple- " mented with 20% FCS and hypoxanthine, aminopterin and thy- midine (HAT, respectively 10"M, 4 x 10"’M and 1.6 x 10‘5M final concentrations in the plate) are added. In this cul- ture medium only hybridomas can survive, since NSO cells are killed in the presence of aminopterin and unfused spleen cells have a limited lifespan.

The plates are then further incubated at 37°C, 5% CO2.

Cultures are fed every '2 days with fresh medium. ‘Growing hybrids are visible by the 4"‘ day. Aminopterin is removed from the medium after 3 weeks, and complete RPMI medium supplemented with 20% FCS is used starting from the 5"‘ week.

After -.10-14 days, cultures are tested for the production of antibodies binding to human TNF 0. in an enzyme—linked immunosorbent assay (ELISA) and of antibodies neutralizing one of the several biological activities of human*TNF 0..

Typically, because of its ease, the supernatants are tested for their ability to neutralize the cytotoxic activity of human TN}? 0. on mouse LM cells (for a detailed description of the assays, see below). Hybrid cells producing the antibod— ies with the strongest neutralizing activity are cloned by limiting dilution at an input of 0,5 cells/well of 96-well plates (Falcon) previously seeded with irradiated mouse pe— ritoneal cells (3000 Rad; 2.5 x 10‘ cells/well). After 2 weeks growth—positive wells are tested for the production of TNF <1-specific antibodies by means of the two assays men- tioned above .

One clone, producing antibodies with the strongest neutralizing activity, is selected and further expanded. The supernatant of this clone is tested in order to quantify exactly its neutralizing activity in" vitro and in vivo. For this purpose, the immunoglobulin content of the supernatant is first determined in a quantitative ELISA (for a detailed description of the assay, see below). In order to quantify exactly its neutralizing activity in vitro, 1 119/ml, 100 __ ng/ml or 10 ng/ml (6.25 x 10"9M, 6.25 X 10‘1°M or 6.25 X ‘1‘M) of the monoclonal antibody are coincubated with increasing doses of human TNF a. After 2 hours the residual cytotoxic activity’ of human 'rNF a. on. mouse LM cells is determined.

In order to see if the monoclonal antibody cross—reacts with the closely related polypeptide TNF B, 1 ug/ml of the monoclonal antibody is coincubated with increasing doses of human TNF B and after 2 hours the residual cytotoxic activity of human TNF B on [M cells is determined.

In order to quantify exactly its neutralizing activity in vivo different doses of the monoclonal antibody are then tested for their ability to protect mice from a lethal shock syndrome induced by TNF <1 coadministered with D-g- alactosamine (for a detailed description of the assay, see below). A The cells of the hybridoma clone are cloned again as de- scribed above in order to assure stability and homogeneity of the cell population. one clone (clone 78) is selected and expanded for further characterization. The monoclonal anti- body produced by this clone is found to have heavy chains of the IgG1 subclass and light chains of the k class.

, Verification of the specificity of a human TNF a—specific monoclonal antibody.

Figure 1 shows that the binding to human TNF a of the monoclonal antibody produced by hybridoma 78 is displaced only after preincubation with human TNF a but not with a series cfif unrelated. antigens. The unrelated antigens are epidermal growth factor (EGF), somatostatin, interleukin 6 (IL6), transferrin, thymic hormone factor (THF), insulin, rat imunoglobulin G antibodies.

. In vitro neutralizing activity of a monoclonal antibody with enhanced TNF a neutralizing activity and TNF B neutralizing activity.

Figure 2 shows the‘neutralizing activity of 1 ug/ml (6.25 p x 10‘9M) of the monoclonal antibody produced by hybridoma clone 78 on increasing doses of human TNF a. As can be seen human TNF a alone exerts 50% cytotoxicity at 0;15 ng/ml (3.3 x l0"12M). In the presence of 1 ug/ml of said nmnoclonal antibody human TNF a exerts 50% cytotoxicity at about 229 ng/ml (4.6 x lO"9M). This shows that 1 ug/ml of said monoclonal antibody completely neutralizes about 228 ng/ml of human TNF a. The ratio of half-maximal biological activ- ity in the presence vs absence of 1 ug/ml of said antibody is therefore about 1527.

Assuming that human TNF a is present in solution as a tri- mer, it can be deduced that the monoclonal antibody neu~ tralizes human TNF a at a ratio of about 1.3 : 1 on a molar basis.

Figure 3 shows that for human TNFB the ratio of ha1f~maxima1 biological activity in the presence vs absence of 1 ug/ml of the said antibody according to the invention is abouta6.3.

Table 2 shows the doses of human TNF a giving 50% cytotoxic activity on LM cells in the absence or in the presence of 6.25 x 10"9M, 6.25 x l0'1°M or 6.25 x 1O‘11M of said antibody. As can be seen, said antibody neutralizes, at all doses tested, human TNF a at a ratio < 2 on a molar basis.

AfV’\_ .. Affinity of a monoclonal antibody with enhanced TNF c. neutralizing activity.

The affinity of the monoclonal antibody according to the present invention is determined by Scatchard analysis.

For this purpose, 1 ng/ml -(6.25 x l0"2M) of said monoclonal antibody is incubated with different doses of iodinated human TN? 0. and the amount of antibody-bound human TNFO. is thereafter determined.

As ca'n—be seen from Figure 4, binding ‘data yield‘-‘a straight line. parameters of human TN}? 0. binding to antibody. As can be seen, the affinity constant (K°,,.) is 3.16 x l01°M‘1 and the highest amount of iodinated’ human TNF a. bindable by 6.25 x 10"12M of -said monoclonal antibody Scatchard plots of the Table 1 shows the said monoclonal , is 4.34 X 10‘1’M.

. Dissociation of human TNF 0. from a monoclonal antibody with enhanced TN}? 0. neutralizing activity. said antibody is (2.5 Dissociation of human TNF a. from determined by preincubating iodinated human TNF 0. ng/ml, S x 10‘11M) with the monoclonal antibody (2 ng/ml, .25 X l0"11M) excess unlabelled human TN)? 0. to prevent reassociation of Figure 5 shows the result of such an and then measuring dissociation by adding iodinated human TN}? 0:. experiment. As can be seen, -«the dissociation of iodinated human TNF a. follows the kinetics of a first order reaction _ by giving a staight—line plot. The k_1 value (dissociation rate constant) determined from this plot is 1.37 x 10"5 sec'1 (T % = 14 h). 7. Ouchterlony double immunodiffusion in agar. " Fig. 6 shows that the monoclonal antibody according to the present invention is able to immunoprecipitate human TN}? 0.. Precipitin bands are formed when 20 ug of said monoclonal antibody (central trough) are allowed to react with 10 pg (upper left trough), 5 ug (upper right) and 2-5 ug (lower right) of human TNF 0..

. Fast-pressure liquid chromatography (FPLC) size exclusion profiles of mixtures of human TNF 0. and a monoclonal antibody with enhanced-TNF 0. neutralizing activity.

Fig. 7 shows the FPLC size exclusion profiles of 100 ug/ml (2 X 10‘6M) human TNF 0. alone (—--—— panel A), of 100 ug/ml (6.25 x l0'5M) of said monoclonal antibody alone ( panel A), of a mixture of 100 ug/ml (2 x l0"‘M) human TNF o. and 30 pg/ml (2 x 10"M) of the monoclonal antibody (l0‘1°M) iodinated (panel B) and of a mixture of 5 ng/ml A human TNF Cl. and 2 ng/ml (1.3 x lO"11M) of the monoclonal antibody (panel C). As can be seen human TNF 0. alone elutes at a volume corresponding to 40 kD. This chromatographic behavior of human TNF <1 is in agreement with other studies where oligomeric TNF c. has been reported to exhibit an apparent molecular weight of 34 - 40 kD in gel-filtration (Kumitani, M.G., et al. J. Chromatogr. 443 : 205 (1988)).

On the other hand mixtures of human TNF <1 and the monoclonal antibody set up at reagent concentrations either in large excess of or close to the calculated KO," give rise to complexes that elute at a volume corresponding to 3 molecular weight of at least 400 kD,tYPi°a11Y 5704500 KD (panels B and C).

. Conclusion drawn from in vitro experiments performed with a monoclonal antibody with enhanced TNF 0. neutralizing activity.

Considering the molecular weight of the complexes (570 — 600 kD) formed upon interaction of human THE‘ (1. with said monoclonal antibody in view of the fact that the highest amount of human THE‘ . bindable and neutralizable by the monoclonal antibody is = 0.7 moles TNF..o./mole antibody, it can be concluded that each of said complexes is. a high molecularweight complex consistingsubstanr tially of at least three molecules pf said monoclonal antibody. (6 binding armqand two human TNF 0. molecu1e5(6 epitopes), Such complexes would be expected to elute, in gel-filtration, at a volume corresponding to 560 - 5807 kD, a value very close to the one observed. Since the results on binding stoichiometry were obtained from experiments performed in antigen excess we exclude that the human TNI-' o./monoclonal antibody complexes consist of alternating mAb78 — huTNFa molecules in an open—chain configuration.

Instead, we propose that human TNF <1/monoclonal antibody molecules combine to form ring-like structures. The energetic advantage and, consequently, the extreme stability of ring-like antigen — antibody complexes has been discussed and thoroughly studied in divalent hapten - antibody systems (Archer, B.G., and Krakaur, H; Biochemistry 16 : 618 (1977); Erickson, J.W. et al. Biochemistry 30 : 2357 (1991); Posner, R.G. et al. Biochemistg 30 : 2348 (1991) and Dembo, M. and Goldstein, B., Immunochemistry 15 : 307 (1978)). In fact, dissociation studies (Figure 5) show that the human TNFG./monoclonal antibody complexes are, indeed, very stable (K_1 = 1.37 x 10‘5 sec"). Such values are in the same order of magnitude of those reported for monoclonal antibodies binding to cell-surface antigens according to a monogamous bivalent mode of binding (Mason, D.W. and Williams, A.F., Biochem. J. 187 : 1 (1989)), another kind of antigen - antibody interaction leading’ to the formation of similarly Stable complexes. Moreover, if, through formation or such cyclic complexes, human TNF a and said monoclonal antibody are allowed to relax into the state of greatest stability, then this explains the apparently paradoxic result that, in antigen excess, the smallest antigen - antibody complex formed is in antibody excess.

In view of such a model, however, the question arises on how human THE‘ 0. and said monoclonal antibody can undergo precipitation. Given that in the described complexes no human _'.1'NF a. epitopes remain free to interact with said monoclonal antibody molecules, we exclude that precipitation results from cross-linking of such complexes. ‘Rather, we favor the possibility that formation of precipitating and cyclic complexes are 2 fundamentally different phenomena in competition with each other. At antigen excess and at low (close to the Rob.) reagent concentrations formation of complexes would predominate, at equivalence / cyclic antibody excess and at high reagent concentrations formation of complexes built up of alternating antibody — antigen molecules according to classic lattice theory.

Finally, monoclonal antibodies binding to human TNF a like the one described in the present invention may be of advantage in view of possible therapeutic applications in human TNF a — _related disease states.

In fact, complexes consisting of at least three monoclohal antibody molecules can be viewed as microaggregates. Cells of the monocyte - macrophage lineage should preferentially eliminate them from the circulation thereby preventing their deposition in Critical 01593115 (e the kidneyi and, thus, avoiding immune com- Plex " mediated inflammatory reactions in these districts.

. In vivo neutralizing activity of a monoclonal antibody_ with enhanced TNF a neutralizing activity.

In order to evaluate the in vivo neutralizing activity, a model system is used in which mice are injected intraperi— toneally with 36 mg D-galactosamine (Fluka AG, Buchs, Swit- zerland) and human TNF a. D-galactosamine renders the Tables 3 and 4 show the results of the in vivo protection studies that have been performed with the monoclonal anti- body according to the produced by hybridoma clone 78.

Table 3 in particular shows that 0.4 149/mouse of the monoclonal antibody are able to fully protect mice from the lethal effects of 0.1 ug/mouse of human TNF a coadministered with 36 mg of D-galactosamine. Employing a supralethal dose of 1 ug/mouse human TNF a, 4 pg/mouse of the monoclonal antibody fully protect mice.

Table 4 shows the results of time kinetic studies. As can present invention be seen 10 ug/mouse of the monoclonal antibody are able to- fully protect mice from the: lethal effects of 1 ug/mouse of TNF at either administered 1 hour and half prior to/or si- If administered 20 minutes after On the other multaneously with TN}? 0..

TN}? a, partial protection is still observed. hand 1 119/mouse of the monoclonal antibody fully protects mice if administered up to 40 minutes after 0.1‘ ug/mouse of TNF <1. If administered 2 hours after TN}? 0., partial‘ protec- tion is still observed. Cells of the hybridoma clone 78 have been deposited with the provisional ECACC accession number 90110707, on November 7, 1990,at the European Collection ofAnimal Cell Cultures (ECACC), Salisbury, United Kingdom.

. I 1,53,.

METHODS . ELISA for the detection of human TNF 0.-specific antibodies.

.Flat bottom microtiter plates 3912) are coated at 100 ul/well with 1 ug/ml of human TNF a. diluted in 100 mM bicarbonate buffer, pH 9.6. After an overnight incubation at 4°C, unabsorbed TNF a is discarded and PBS (Falcon No. suppleinented with 1% bovine serum albumin (BSA, Armour,- Kankakee, Illinois.) ..is. added to each well to saturate unoc- cupied plastic sites.

The plates are further incubated for 3 hours at room temperature. Thereafter plates are washed 3 times with washing buffer (WB), that is PBS supplemented with 0.1% Tween 20 (Merck, Schuchardt, Hohenbrunn, FRG) and 0.01% merthiolate (BDH , Pool , GB) . 5 0 ul of hybr idoma supernatants, diluted 1 : 5 in PBS supplemented with 1% BSA are added to each well, the plates incubated for 60 minutes at 37°C, and washed as above. Then 100 111 of peroxidase- conjugated goat-anti mouse immunoglobulin G (HRPaIgG, Bio- Rad, Richmond, California) diluted 1 : 3000 in WB are added to each well, the plates are incubated for 45 minutes at 37°C and washed as above. Then, 100 111 of peroxidase sub- strate (019132, Chemicon, SCI, Rome, Italy, 1 tablet dissolved —— in 5 ml citrate buffer, OPD4, Chemicon) are added to each well. The reaction is allowed to proceed for 2 minutes at room temperature,after which color development is stopped by the addition of 100 ul/well of 4M H2804. The extent of color development is read on a plate ELISA reader at 492 nm.

Supernatants giving optical density (OD) values > 0.5 are considered positive for the anti-TNF <1 antibodies. presence of ng/ml of a hybridoma'supernatant are coincubated with different concentrations of human TNF <1 or with different concentrations of unrelated antigens or with diluent alone (PBS supplemented with 1% BSA) for 2 hours at 37°C. At the end of the incubation period residual TNF a-binding is mea- sured in the previously described ELISA that allows the de- tecti9n_of human TNF a-specific antibodies.

. ELISA for the quantitative determination of mouse IgG antibodies. _ Flat-bottom microtiter plates are coated at 100 ul/well with goat—anti mouse Ig (organon Teknika, Turnhout, Belgium, ug/well final concentration) diluted in 40 mM phosphate buffer (pH 7.4). After an overnight incubation at 4°C, un- absorbed antibody is discarded and PBS supplemented with 1% BSA is added to each well to saturate unoccupied plastic sites.

The ‘plates are further incubated for -3 hours at room temperature and thereafter washed 3 times with WB. During this incubation period serial two—fold dilutions of a re- ference mouse IgG preparation (Pel-Freez, Rogers, Arkansas) are set up, typically from S to 0.25 ug IgG/ml. In parallel,: serial two-fold dilutions of supernatants containing an un- known quantity of IgG are set up, typically from 1:100 to 1:l2800. All dilutions are performed in PBS supplemented with 1% BSA. 50 ul of each dilution are added to a well, the plates incubated for 60 minutes at 37°C and then washed as above.

’Peroxidase substrate addition and color development is as for 1.

IgG concentrations in the supernatants containing an un— known quantity of IgG are calculated pmocessing the data with an ELISA—SOFT-PC program (Perkin—E1mer, Norwalk, Connecticut).

. ELISA for the typing of heavy and li ht chains of monoclonal antibodies.

For this purpose a HYBRIDOMA SUB ISOTYPING KIT (Calbio- chem, La Jolla, California) is used according to the’ manu- facturer's instructions. Typing is performed on hybridoma supernatants diluted 1 : 5 in PBS supplemented with 1% BSA. wells giving OD values > 0.4 are considered positive for the presence of a monoclonal antibody of a given Ig class, sub- class Tfd light chain.

. Determination of the neutralizing activity in vitro of anti—TNE' a monoclonal antibodies. A .1 of this suspension are added to the wells of flat bottom microtiter plates (Falcon 3072). Then dilutions of human TN}? (1 or human TNF B (Omnia Res, Cinisello Balsamo, Italy) are set up in complete MEM so to have final concen- trations, after addition to the wells, of l ug/ml to 25 P9/ml .

Antibody-containing supernatants or solutions are diluted -1 in complete MEN so to have a final concentration, after addition to the wells, of 1 ug/ml, 100 ng/ml or 10 ng/ml monoclonal antibody. 50 ul of each TNF 0. or TNF B dilution are coincubated with 50 ul of monoclonal antibody or with S0 111 complete MEM for 2 hours at 37°C. During this incubation period’ a solu- tion containing actinomycin ‘D (Fluka) in complete MEM is set up. The final concentration of actinomycin D after addition to the wells is l ug/ml; At the end of the incubation period each antibody-TNF 0. or -TNF 8 mixture and 50 111 of acti- nomycin D are added to the wells of the microtiter plates.

To some wells 100 ul of each dilution containing TNF a or TNF B alone are added. To some control wells only actinom~ ycin D plus complete MEM is added. Thereafter the plates are incubated for 24 hours at 37°C. - Atthe end of this ‘incubation period 20 111 of a thiazolyl blue solution (MTT, Calbiochem, 5 mg/ml in PBS) are added to each well. The plates are further incubated for 4 hours at 37°C. Then, supernatants are discarded, 200 pl of dimethylsulfoxide (DMSO, Farmitalia Carlo Erba) are added to each well and the extent of color development is read on a plate ELISA reader at 570 nm. _ The OD values obtained for each antibody—TNF a or —TNF B mixture are compared with those obtained for TNF a or TNF 3 alone. The doses of TN? d or TNF 3 giving, in the absence or presence of monoclonal antibody, OD values which are 50% of those obtained for the control wells (50% cytotoxicity) are determined. Monoclonal antibodies with the highest neutralizing activity are defined as those which determine the highest increase of the TNF a dose required to give 50% cytotoxicity.

. Determination of the affinity of anti-TNF a monoclonal antibodies.

In order to determine the affinity of anti-TNF a mono— , clonal antibodies the following experiment is performed. ng/ml (6.25 x 10"‘2M) of said. monoclonal antibody or diluent alone (PBS supplemented with 0.5% BSA) are incubated with different concentrations of iodinated human TNF a (NEN, Wilmington, DE, concentrations ranging typically from 0.5 x ‘11M to 4 x 10"1°M) in Eppendorff tubes for 4 hours at room temperature. At the end of the incubation period 1:5 diluted Imunobeads (170-5104, Bio-Rad, Segrate, Italy) are added to each tube and the tubes are further incubated for l centrifuged through a (1:1.5 vol/vol, hour. Then, the mixtures are phthalate~dibutyl phthalate oil mixture Fluka, AG). Immunobead~associated radioactivity is counted in a gammacounter. Data thus obtained are processed by means of an equilibrium binding ‘data analysis program (EBDA, Elsevir Science Publishers, Amsterdam, Netherlands).

. Determination of the dissociation rate constant (k_1) of human TNF a bound to an anti-TNF a monoclonal antibody. 2 ng/ml (1.25 x 10"11M) of the monoclonal antibody produced by hybridoma 78 or buffer (PBS + 0.5% BSA) alone are infifibated with 2.5 ng/ml (5 x 10‘11M) iodinated human TNFa for .4..hours. at room temperature. Thereafter 1 : 5' diluted Immunobeads are added for 1 hour and the mixtures are then centrifuged twice. The resulting pellets are resuspended in 250 ng/ml (S x 10"M) "cold" human TNF a to compete for rebinding with any iodinated human TNF a that dissociates. After different times of incubation (see Figure ) at room temperature the pellets are centrifuged through an oil mixture as descibed in the previous section. Residual iodinated human TNF a specifically bound to the monoclonal antibody is then determined and the results are plotted as percentage radioactivity bound versus log time. The time for 50% dissociation (T %) is read from this graph and used to calculate k_1 : 0.693 K-1 '1‘% . Ouchterlony double immunodiffusion in agar.

In order to see if the monoclonal antibody produced by hybridoma 78 precipitates human TNF <1 a double diffusion assay is‘performed. For this purpose 1% agarose gel in 0.9% Nacl is used. The samples (human TNF a and the monoclonal antibody) are placed in circular wells. Patterns are allowed to develop for at least 24 hours at 4°C before being photographed.

. Determination of the FPLC size exclusion profiles of mixtures of human TNF a and an anti—TNF a monoclonal antibody.

For this purpose mixtures of human TNF a or iodinated human TNF a and purified anti-TNF a monoclonal antibody in human TNF a excess are set up (e.g. 100 ng/ml human TNF a and 30 ng/ml of monoclonal antibody or 5 ng/ml iodinated human TNF a and 2 ng/ml of monoclonal antibody) in PBS (for mixtures of 100 ng/ml human TNF a. and 30 ng/ml of the monoclonal antibody) or in PBS + 0.5% BSA (for mixtures of 5‘ ng/ml iodinated human TNF <1 and 2 ng/ml of monoclonal antibody). The mixtures are allowed to incubate for 4 hours at room temperature. Thereafter the mixtures, TN}? 0. alone or the monoclonal antibody alone are chromatographed on a 10 x 300 mm Superose 6 FPLC column (Pharmacia LKB Biotechnology, Uppsala, Sweden) to generate A280 absorbance profiles. The column. is preequilibrated and eluted with 50 mm sodium phosphate, 150 mM Nacl, pH 7,2 buffer. Molecular weight standards are, typically, blue dextran (mw 2 x 106), human IgM (mw 900000), bovine thyroglobulin (mw 670000), bovine IgG (mw 158000), chicken (mw 44000), horse myoglobin (mw 17000), vitamin B12 (mw 1350). ovalbumin Determination of the neutralizing activity in vivo of anti-TNF a.monoclonal antibodies.’ In order to evaluate the neutralizing activity in vivo of anti-TNF a monoclonal antibodies a model system for human TNF a-induced lethal shock in mice is set up. For this pur- pose mice are injected intraperitoneally with different doses of human TNF a (typically 0.1 ug or 1 ug/mouse diluted in 0.25 ml PBS) and D-galactosamine (typically 36 mg/mouse diluted in 0.25 ml PBS). Mice injected with lethal doses of human TNF a die within 24-48 hours.

The neutralizing activity in vivo of the anti-TNF a mono- clonal antibody is evaluated injecting mice intravenously with different doses of the monoclonal antibody (diluted in .25 ml PBS) and 90 minutes later intraperitoneally with a lethal dose of human TNF (1.

In another set of experiments the neutralizing activity in vivo of the anti-TNF on monoclonal antibody over time is evaluated. For this purpose the monoclonal antibody is administered at different time before and after the admin- istration of a lethal dose of human TNF 0..

TABLE 1 Parameters of the binding of huTNFa to mAb78 Measured Maximal Ratio affinity binding Bmax/mAb78 constant capacity Kobe Bmnx (x10‘1°M"1) (x1O‘2M) .16 1 0.7* V 4.34 1 0.46 0.71 1 0.

*Results (i SD) were obtained in 3 independent experiments.

Concentrations of huTNFa giving 50% cytotoxicity on LM cells in the absence or presence of different concentrations of TABLE 2 mAb78. __ Concentration Concentration of Neutralization of mAb78 huTNFa giving Ratio * 50% cytotoxicity* - . i 0.4 X 10"12M - 6.25 X 10‘9M . t 0.2 X 10"9M 1.36 6.25 X 10"1°M i 0.5 X 10"1°M 1.21 1 X 10"11M 1.8 .25 X 10‘*lM L0 0 W H‘ * Results (1 SD) were obtained in at least 3 independent experiments * Neutralization Ratio = Ratio between concentration of mAb78 and concentration of neutralized huTNFa. firs.» ‘ TABLE 3 Letha1ity of mice treated with human recombinant TNF a and D—ga1actosamine.

Dose of Dose of D-galaétosamine" TNF a " Lethality 36 mg/mouse — 0/4 " 0.01 ug/mouse 2/4 " 0 . 1 119/mouse 4/4 " 1 pg/mouse 4/4 "D~ga1actosamine and TNF a were coadministered intraperitoneally.

TABLE 4 Effect of monoclonal antibody 78 (mAb 78) on mice treated with human recombinant TNF a and D-galactosamine.

Dose of Dose of Dose of D~ga1actosamine‘ TNF a " mAb78 " Lethality mg/mouse 0.1 n9/mouse - 19/21 " " 0.5 ug/mouse 0/5 " " 0 .4 " 0/5 " " 0 . 3 " 4/13 " " 0 . 2 " 4/13 " " 0. 1 " 3/5 " 1 ug/mouse — 12/12 " " 5 ug/mouse 0/12 " " 4 " 0/8 " " 3 " 1/8 " " 2 " 4/8 " " 1 " 6/8 "See Table 1 "mAb was administered intravenously 1 h 30' before TNF d and D—ga1actosamine. .7». 1 TABLE 5 Effect gf [monoclonal antibody 78 (mAb78) administered at dif- ferent times to mice treated with human THE‘ 0. and D—ga1actosaxnine .

Time of mAb78 and/or THE‘ 0. " administration Lethality -1 h 30' 0 +20’ +40‘ +1 h + 2 11 +3 h 1 119 THE‘ 31/33 ug 78 1 119 THE‘ 1/24 1 ug ‘DIP 0/19 + mg 78 5/15 1 319 INF 10 119 78 13/15 1 ug ‘INF 10 pg 73 V _ 15/19 1 £19 TNF 10 ug 78 11/14 1 119 TNF 1 10 119 78 8/9 0.1 ug TN? ' 15/15 1 pg 78 0.1 ug ‘INF 0/5 + 1 11g 78 0/10 0.1 ug TNF 1 119 78 0/5 0.1 ug 1'13? 1 ug_ 78 0/5 0.1 119 TN}? 1 :19 '18 3/15 0.1 119 TN}? 1 11g 78 2/10 0.1 ug ‘INF — 1 pg 78 5/5 ‘TNF was administered intraperitoneally together with 36 mg D—ga1actosamine/mouse, Nab 78 was administered intravenously .

Claims (22)

1. A monoclonal antibody or a binding fragment thereof which is able to neutralise human Tumor Necrosis Factor alpha, __characterised in that said antibody is able to precipitate human TNF alpha forming high molecular weight antigen—antibody complexes, whereof the smallest antigen- antibody complexes formed with human TNF alpha contain substantially three molecules of said antibody and two human TNF alpha molecules and have a molecular weight of at least 400 kD.

2. The monoclonal antibody or a binding fragment thereof according to claim 1, which is able to neutralise in vitro human TNF alpha at a ratio of about l.3:l on a molar basis.

3. The monoclonal antibody or a. binding fragment thereof according to claim 1, which is able to provide complete protection in mice from an otherwise lethal dose of human TNF alpha at doses lower than 1 pg/mouse.

4. The monoclonal antibody or a binding fragment thereof according to claim 1, which is able to recognise a1§o human Tumor Necrosis Factor beta.

5. The monoclonal antibody according to claim 1 which represents a Fv, Fab or F(ab')2 fragment.

6. A stable hybridoma cell line and a progeny‘ thereof which secrete a monoclonal antibody according to claims 1 to 4.

7. The hybridoma cell line 78 (ECACC provisional accession number 90110707) and a progeny thereof.

8. A process for the preparation of a stable hybridoma cell line according to claim 6, which process comprises fusing immortalising cells with cells derived from a mouse that has been immunised with human TNF alpha and selecting those hybridoma cell line secreting a monoclonal antibody according to claim 1.

9. The monoclonal antibody or a binding fragment thereof according to claims 1 to 4, as obtainable from the hybridoma cell line 78.

10. A pharmaceutical composition comprising as an active agent a monoclonal antibody or a fragment thereof according to claims 1 to 5 and 9 and a pharmaceutically acceptable carrier and/or diluent.

11. A monoclonal antibody or a binding fragment thereof according to claims 1 to 5 and 9 for use as a prophylactic and/or therapeutic agent in a disease state in which human TNF alpha and/or human TNF beta exerts a pathogenic effect.

12. A monoclonal antibody or a binding fragment thereof according to claims 1 to 5 and 9 for use in the prevention or treatment of a side effect arising from antibacterial or antineoplastic therapy.

13. Products containing a monoclonal antibody or a binding fragment thereof according to claims 1 to 5 and 9, and an antifiiotic, antimicrobial or antibacterial agent, or a mixture of two or more of them, as a combined preparation for simultaneous, separate or sequential use in antimicrobial and antibacterial therapy.

14. Products containing a monoclonal antibody or a binding fragment thereof according to claims 1 to 5 and 9, and an antitumor agent, as a combined preparation for simultaneous, separate or sequential use in anitcancer therapy.

15. A method of detecting the content of human TNF alpha in a sample of a body fluid, which method comprises contacting the sample with a monoclonal antibody or a fragment thereof, according to claims 1 to S and 9.

16. .A monoclonal antibody or E1 binding fragment thereof according to any one of claims 1 to 5 or 9, substantially as described herein by way of example and/or with reference to a the accompanying drawings.

17. A stable hybridoma cell line and. a. progeny' thereof according to claim 6 or 7, substantially as described herein 20 K) by way of example and/or with reference to the accompanying drawings.

18. A process according to claim 8, substantially as described herein by way of example and/or with reference to the accompanying drawings.

19. A‘wpharmaceutical composition according to claim 10, 2 \ v ‘ substantially as described herein by way of example and/or with reference to the accompanying drawings.

20. Use according to claim 11 or 12, substantially as described herein by way of example and/or with reference to the accompanying drawings.

21. A product according to claim 13 or 14, substantially as described herein by way of example and/or with reference to the accompanying drawings.

22. A method according to claim 15, substantially as described herein by way of example and/or with reference to the accompanying drawings. Tomkins & Co.