WO1989009831A1

WO1989009831A1 - Lak cell cytotoxin

Info

Publication number: WO1989009831A1
Application number: PCT/US1989/001387
Authority: WO
Inventors: Evan M. Hersh; Philip Scuderi, Jr.; William Grimes; Sydney E. Salmon
Original assignee: Research Corporation Technologies, Inc.
Priority date: 1988-04-15
Filing date: 1989-04-05
Publication date: 1989-10-19
Also published as: EP0409901A4; JPH03503844A; EP0409901A1

Abstract

The present invention is directed to a unique group of cytokines produced by the stimulation of mammalian peripheral blood mononuclear cells with interleukin-2. More particularly, the present invention is directed to lymphokine activated killer cell cytotoxin (LCC), and to methods of production thereof, having unique cytotoxic and cytostatic activity against tumor cells. The present invention also contemplates a pharmaceutical composition comprising said cytokine and a pharmaceutically acceptable carrier useful in the treatment and prophylaxis of cancer and infectious diseases.

Description

LAK CELL CYTOTOXIN

Many cytokines are polypeptide molecules which directly or indirectly mediate host defense mechanisms and/or which mediate tissue growth differentiation. Cytokines have been recognized which mediate host defense against cancer and/or infection. Such cytokines include the interferons (IFN- α , IFN-β and IFN-ɤ ) , tumor necrosis factor (TNF-α ) , lymphotoxin (TNF- β ) , the interleukins (IL-1, 2, 3, 4, 5 and 6), leukoregulin, natural killer (NK) cell cytotoxic factor (NKCF), transforming growth factor (TGF), colony stimulating factors (CSF) such as macrophage (M-CSF), granulocyte (G-CSF) and macrophage, granulocyte-CSF (G, M-CSF) and oncostatin M. Each of the aforementioned cytokines has unique characteristics and a unique range of antiproliferative, cytostatic, antiviral or growth regulatory activity.-

Several cytokines are synthesized by leukocytes, commonly in response to stimulation by microorganisms, antigens or mitogens. This has been observed in vitro. Following this stimulation in cell culture, the supernatant fluid is retrieved and cytokine activity identified, isolated and further characterized. Seely and Golub, 1978, J. Immunol. 120:1415, described a phenomenon by which following stimulation by allogeneic cells, blood leukocytes become cytotoxic to cultured allogeneic cells. The cytotoxic nature of stimulated blood leukocytes was also shown to be augmented by interleukin-2 (IL-2) (Vose et al., 1978, Int. J. Cancer21: 588; Gillis et al., 1978, J. Immunol. 120:2027). Subsequently, Zarling et al., 1978, Nature (Lond) 274:269 showed that pooled fresh allogeneic cells could stimulate killing of fresh allogeneic target cells by peripheral blood mononuclear leukocytes. Later, Mills and Paetkau, 1980, J. Immunol. 125:1897 demonstrated that tumor cells in combination with IL-2 stimulated these mononuclear leukocytes into becoming cytotoxic to cultured allogeneic tumors. Lotze et al., 1981, Cancer Res. 41:4420 demonstrated that peripheral blood mononuclear cells stimulated by IL-2, expressed the ability to kill fresh autologous in addition to fresh allogeneic tumor cells. These effector cells, i.e. IL-2 stimulated peripheral blood mononuclear cells, are referred to as lymphokine activated killer (LAK) cells and the exhibited cytotoxic (i.e. cell killing) characteristic as the LAK cell phenomenon.

Agah et al., 1987, Cancer Immunol. Immunother. 24:247 has proposed that the LAK cell phenomenon in killing tumor cells is mediated by direct interaction with the target cells and the subsequent release of the contents of LAK cell- associated granules into the target cell. LAK cells are induced by IL-2 but their induction is regulated by IFN-ɤ and TNF- α. Many of the characteristics inherent in target cells sensitive to the LAK cell phenomenon have been elucidated. For example, autologous and allogeneic tumor cells and fresh and cultured tumor cells are considered sensitive targets as are altered or transformed normal cells. Additionally, target cells are usually trypsin sensitive. A major and unique characteristic of the LAK cell is that it will exhibit cytotoxicity to autologous and allogeneic fresh tumor cells in four hours as measured by Cr⁵¹ release.

Importantly, the LAK cell phenomenon is thought not to be mediated by any of the known and previously described cytokines.

Accordingly, the present invention identifies a previously unknown cytokine associated with the LAK cell phenomenon, thereby providing a molecule with unique cytostatic and cytotoxic activity useful in the therapy of malignant and certain infection diseases.

The present invention is directed to a group of cytokines having unique cytotoxic and cytostatic activity against tumor cells.

More particularly, the present invention relates to a cytokine, characterized by being associated with the LAK cell phenomenon and having unique cytotoxic and cytostatic activity against tumor cells, prepared by the process comprising the steps of contacting mammalian peripheral blood mononuclear cells (PBMC) with a cytokine-inducing effective amount of interleukin-2, preferably in the presence of a buffered medium, for sufficient time for said cytokine to be synthesized.

Specifically, the present invention is directed to mammalian LAK cell cytotoxin (LCC) and to the method for production thereof. Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of LCC and a pharmaceutically acceptable carrier useful in the therapy of cancer and certain infectious diseases.

Still another aspect of the present invention relates to antibodies specific to LCC useful in diagnostic assays for LCC.

Yet another aspect of this invention relates to arecombinant DNA molecule encoding LCC thereby providing a convenient source of recombinant LCC.

In the accompanying drawings, Figure 1 is a graphical representation of LCC activity detected in FPLC fractions of supernatant fluid obtained after peripheral blood mononuclear cells were incubated either without interleukin-2 (graph A) or in the presence of 1000 u/ml of interleukin-2 (graph B) for five days.

The present invention relates to a cytokine, characterized by being associated with the LAK cell phenomenon and having unique cytotoxic and cytostatic activity against tumor cells, produced by the process comprising the steps of contacting mammalian peripheral blood mononuclear cells (PBMC) with a cytokine-inducing effective amount of interleukin-2, preferably in the presence of a buffered medium, for sufficient time for said cytokine to be synthesized. In accordance with the present invention as disclosed in the specification and claims herein, the LAK cell phenomenon refers to the characteristic of peripheral blood cells (PBMC) , following stimulation by interleukin-2 (IL-2), to exhibit cytotoxicity (i.e. cell killing properties) against tumor cells wherein said IL-2 stimulated PBMC are referred to as lymphokine activated killer (LAK) cells. Accordingly, it has been surprisingly discovered that the cytokines of the subject invention are associated with the LAK cell phenomenon and hence, represent a heretofore unknown group of cytokines. The present invention is best described by reference to one specific example of said cytokines which represents the most useful molecule in the practice of this invention at the present time. This is done, however, with the understanding that all such cytokinesare encompassed by the subject invention. Hence, the present invention, in its preferred embodiment, is directed to the cytokine designated LAK cell cytotoxin from human PBMC. Hereinafter, this cytokine will be referred to as LCC. In accordance with the present invention, LCC exhibits a different spectrum of antitumor cytostatic and cytotoxic activity than LAK cells alone and possesses a different kinetics of activity.

In accordance with the subject disclosure, LCC is produced as follows: Human PBMC are placed in flasks at a concentration of from about 1×10² cells/ml to 1×10¹² cells/ml in buffered medium comprising RPMI 1640 medium containing a nutrient solution selected from the group comprising fetal calf serum, normal human serum or AIM5 serum free medium. In a preferred embodiment, from about 1×10⁴ to about 1x108

PBMC/ml but most preferably about 1×10⁶ PBMC/ml are used.

Incubation then proceeds in the presence of an LCC-inducing effective amount of IL-2, said effective amount determined herein to be in the range of about 800 u/ml to about 5000 u/ml but preferrably 1000 u/ml of IL-2. By LCC-inducing effective amount is meant the amount of IL-2 required to initiate, stimulate, promote and/or maintain the synthesis of

LCC in the cells. The incubation proceeds from about two to about ten days with about five days being a preferred length and time. The supernatant is then harvested and stored at -20°C for future use. As defined herein, the supernatant fluid contains essentially LCC activity and hence, said supernatant fluid is referred to herein as a crude preparation of LCC.

LCC activity is measured in vitro using a microwell assay system. An alternative system involving antibodieswill be discussed below. With respect to the microwell assay system, tumor cells are placed in wells of 96 well plates in about 200 ul of complete medium and a volume of supernatant containing LCC is added. Wells are evaluated for residual viable tumor cells after various periods of time. Using this procedure, LCC is found to be cytostatic in that it inhibits the proliferation of MCF-7 breast cancer cell line cells over 144 hours in culture. Interestingly, LCC mediated activity against MCF-7 is not exhibited until after at least 24 hours of culture (Table 1). Furthermore, the cytotoxic nature of LCC is evident against MCF-7 cells when added to a confluent monolayer thereof (Table 2). Accordingly, LCC can exhibit one or both of cytostatic and cytotoxic characteristics against tumor cells.

Other methods useful in determining the cytotoxic and cytostatic activity of LCC include the Biorad assay described by Bradford, 1976, Anal. Biochem. 72:248 which measures the solubilized protein of residual tumor cells (Tables 1 and 2); the method of Kleinerman et al., 1983, Cancer Res. 43:2010 which measures the release of incorporated ¹²⁵IUDR from tumor cells preincubated in this isotope (Table 3); and the inhibition of colony growth of cells plated as single cells in soft agar (Table 4) as described by Salmon and Trent (Eds), 1984, Human Tumor Cloning - Proceedings of the Fourth Conference on Human Tumor Cloning, Tucson, Arizona, USA.

Using the above-mentioned methods a wide variety of cell lines have been tested for sensitivity to LCC, comparing this sensitivity to LAK cells alone. Accordingly, LCC sensitive cells include A375M (melanoma), A375P (melanoma), Hs294T (melanoma), MCF-7 (breast), C480 (colon) and HEY (ovarian). LCC-resistant cell lines include Daudi (B-cell), K562 (myeloid), fetal fibroblasts, adult fibroblasts, PHA-lymphoblasts and IL-2-lymphoblasts. LCC also inhibits fresh human tumor cells as demonstrated by the clonogenic assay in soft agar (Table 6). This is one of many significant advantages of the present invention since this assay detects the growth of clonogenic tumor stem cells. Inhibition in this assay in vitro correlates with inhibition of tumor growth in vivo in the patient donor of the tumor (Salmon and Trent, supra). LCC appears not to inhibit or kill normal cells. Thus, it neither inhibits adult or fetal fibroblasts (Table 7) nor does it inhibit normal lymphocytes responding to phytohemagglutinin (PHA) or IL-2 stimulation (Table 8). Accordingly, the LCC of the present invention has a wide spectrum of activity against cancer and tumor cells and a normal and neoplastic cells. Hereinafter, the word tumor will be used to define such cell types including infectious diseases causing transformation of said cell types. Hence, as defined herein, tumor or tumor cells is meant to encompass all LCC-sensitive cells and LCC-target cells.

In accordance with the present invention, many of the characteristics of LCC have been defined. Thus, it is produced by LAK cells and can diffuse through a membrane (0.2 microns) and act on tumor cells at a distance (Table 9). It can be generated in serum free medium (Table 10). It is not generated in the absence of IL-2 (Table 10). It is generated by non-adherent cells (Table 11). It is non-dialyzable (Table 12). Its heat and acid stability characteristics have been defined (Tables 13 and 14).

A series of studies have been done to determine therelationship of LCC to any known cytotoxic or cytostatic cytokine. No such relationship has been found to exist. Therefore, by a series of direct or indirect assays, the LCC activity is unique. By direct assay, LCC containing supernatants have no IFN- α activity or IFN-ɤ and no TNF-α activity (after dialysis). LCC can be shown to be cytostatic to MCF-7 cells which are not sensitive to IFN- o( or TNF-β (lymphotoxin) (Table 15). The TNF resistant variant of L929 cells which is resistant to both TNF- α and TNF-β is sensitive to LCC (Table 16). By inference of sensitive and resistant cell lines to various reagents, LCC is distinct from (in addition to the above) IFN-β , IL-1, leukoregulin, NKCF, perforin, cytolysin, serine esterase, lysozomal arylsulfatase, TGFβ1, and oncostatin M. Some of these relate to the resistance of K562 cells to LCC (Table 17) which is known to be sensitive to NKCF, leukoregulin, perforin, cytolysin, etc. These concepts and findings are summarized in Tables 18 and 19.

LCC has been partially purified by FPLC, the supernatant of PBMC not stimulated with IL-2 (Figure 1A) lacks a peak (number 5) present in the supernatant FPLC fractionation of IL-2 stimulated PBMC (Figure 1B). This fraction also contains the LCC activity. Crude LCC was further purified and characterized. LCC is tightly associated with serum albumin. Passage of crude LCC over a Q-Sepharose (Pharmacia) column yields two fractions LCC-1 and LCC-2. LCC-1 is the flow through fraction and has potent cytotoxic activity while being devoid of albumin. LCC-2 is eluted from the column by treatment with a solution having a high salt concentration. It contains most of the albumin and has potent cytotoxic .activity as well. The salt solution consists of an amount and type of salt having an anion effective to displace LCC from the Q-Sepharose resin to yield the fraction designated LCC-2. Preferably, the salt solution is about 0.5 M to about 1.5 M NaCl, and most preferably is 1.0 M NaCl.

Any of the fractions LCC-1 to LCC-2, as well as crude LCC, may be subject to affinity purification or other purification means to remove unwanted cytokines or other contaminants such as TNFα or IFNX which may be present. In a preferred embodiment, any LCC-containing preparation is passed over an anti-TNFα or anti-IFNɤ antibody affinity resin to remove these activities from the LCC preparation. After passage through both columns, crude LCC contains no measurable TNF and only 22.5 u/ml, and LCC-1 contained no TNFα or IFNɤ activities. An amount of IFN_( of 22.5 u/ml (or even up to 1000 u/ml) has no effect on MCF-7 cells. LCC-1 retains its cytotoxic activity after removal of TNFα or IFNɤ and is useful for the final purification of LCC. TNFα- and IFNɤ -free LCC-1 and any purified fraction demonstrating LCC activity is useful in the generation antibodies, especially monoclonal antibodies.

The cytokine LCC is contemplated herein to be useful in the regression and pallation of some cancer, tumor and infectious diseases in mammals and preferably in humans. Such diseases where LCC is considered most useful are those where cell cytostasis or cytotoxicity (cell killing) of target cells is desired. A target cell is used herein to describe all cells (e.g. tumor and cancer) associated with said disease states.

Accordingly, the subject invention contemplates a method for inducing regression or inhibition of growth of said target cells in mammals (i.e. cancers and tumors) by administering a pharmaceutical composition containing an effective amount of said LCC. Additionally, a method for inducing regression -or inhibition of growth of cancer in a mammal is contemplated in which a nucleic acid molecule encoding the LCC contemplated herein is introduced into an affected (i.e., cancerous or transformed) cell in such a manner that said nucleic acid molecule is expressed intracellularly but extrachromosomally of said cell or following integration into the genome of said cell. In this case, the nucleic acid molecule is carried to said affected cell and transferred into said cell by a second nucleic acid molecule (e.g., various viruses). The first nucleic acid molecule is manipulated such that it contains the appropriate signals for expression. That is, in accordance with the present invention, a method of inducing regression or inhibition of growth of cancer in a mammal is contemplated comprising administering a first nucleic acid molecule encoding LCC, said nucleic acid being contained in a pharmacologically acceptable second nucleic acid carrier molecule such that said first nucleic acid enters a target cell and is either maintained extrachromosomally or integrates into the genome of said target all in such a manner that said first nucleic acid is expressed so as to produce an effective amount of LCC.

The active ingredients of the pharmaceutical compositions comprising LCC, are contemplated to exhibit excellent and effective therapeutic activity, for example, in the treatment of some cancers and tumors or infectious diseases. Thus, the active ingredients of the therapeutic compositions including LCC exhibit antitumor activity when administered in amounts from about 0.5 ug to about 2000 μq per kilogram of body weight per day. This dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A decided practical advantage is that the active compound may be administered in a convenient manner such as by the oral, intraveneous (where water soluble), intramuscular or subcutaneous routes.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Thepreventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 μg . Expressed in proportions, the active compound is generally present in from about 0.5 to about 2000 μg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The present invention also relates to antibodies to LCC. Such antibodies are contemplated to be useful indeveloping detection assays (immunoassays) for said LCC, especially during the monitoring of a therapeutic regimen and in the purification of LCC. The antibodies may be monoclonal or polyclonal. Additionally, it is within the scope of this invention to include any second antibodies (monoclonal or polyclonal) directed to the first antibodies discussed above. The present invention further contemplates use of these second antibodies in detection assays and, for example, in monitoring the effect of an administered pharmaceutical preparation. Furthermore, it is within the scope of the present invention to include antibodies to the glycosylated regions of LCC, and to any molecules complexed with said LCC. Thus, in accordance with this invention, an antibody to LCC encompasses antibodies to LCC, or part thereof, and to any associated molecules (e.g., glycosylated regions, lipid regions, carrier molecules, and the like).

The LCC, or parts thereof, considered herein are purified, as for example using FPLC, then utilized in antibody production. Both polyclonal and monoclonal antibodies are obtainable by immunization with the polypeptides, and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the purified polypeptide, or part thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product.

The use of monoclonal antibodies in the present immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard, J. Y. and Hoffman, T., "Basic Facts About Hybridomas", in Compendium of Immunology, Vol. II, L. Schwartz (Ed.) (1981); Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); European Journal of Immunology, Vol. 6, pp. 511-519 (1976), Koprowski, et al., U.S. Patent 4,172,124, Koprowski et al., U.S. Patent 4,196,265 and Wands, U.S. Patent 4,271,145, the teachings of which are herein incorporated by reference.

Unlike preparation of polyclonal sera, the choice of animal is dependent on the availability of appropriate immortal lines capable of fusing with lymphocytes thereof. Mouse and rat have been the animals of choice in hybridoma technology and are preferably used. Humans can also be utilized as sources for sensitized lymphocytes if appropriate immortalized human (or nonhuman) cell lines are available. For the purpose of the present invention, the animal of choice may be injected with from about 1 mg to about 20 mg of the purified LCC, or part thereof. Usually the injecting material is emulsified in Freund's complete adjuvant. Boosting injections may also be required. The detection of antibody production can be carried out by testing the antisera with appropriately labeled antigen. Lymphocytes can be obtained by removing the spleen or lymph nodes of sensitized animals in a sterile fashion and carrying out fusion. Alternatively, lymphocytes can be stimulated or immunized in vitro, as described, for example, in C. Reading J. Immunol Meth., 53: 261-291 1982.

A number of cell lines suitable for fusion have been developed, and the choice of any particular line for hybridization protocols is directed by any one of a number of criteria such as speed, uniformity of growth characteristics, deficiency of its metabolism for a component of the growth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work better than interspecies fusions. Several cell lines are available, including mutants selected for the loss of ability to secrete myeloma immunoglobulin. Included among these are the following mouse myeloma lines: MPC₁₁-X45-6TG, P3-NS1-1-Ag4-1. P3-X63-Ag8, or mutants thereof such as X63-Ag8.653, SP2-0-Ag14 (all BALB/C derived), Y3-'Ag1.2.3 (rat), and U266 (human).

Cell fusion can be induced either by virus, such as Epstein-Barr or Sendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is the most efficacious agent for the fusion of mammalian somatic cells. PEG itself may be toxic for cells, and various concentrations should be tested for effects on viability before attempting fusion. The molecular weight range of PEG may be varied from 1,000 to 6,000. It gives best results when diluted to from about 20% to about 70% (w/w) in saline or serum-free medium. Exposure to PEG at 37°C for about 30 seconds is preferred in the present case, utilizing murine cells. Extremes of temperature (i.e., above 45°C) are avoided, and preincubation of each component of the fusion system at 37°C prior to fusion gives optimum results. The ratio between lymphocytes and malignant cells is optimized to avoid cell fusion among spleen cells and a range of from about 1:1 to about 1:10 gives good results.

The successfully fused cells can be separated from the myeloma line by any technique known by the art. The most common and preferred method is to choose a malignant line which is hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient, which will not grow in an aminopterin- containing medium used to allow only growth of hybrids and which is generally composed of hypoxanthine 1×10^-4M, aminopterin 1×10⁵M, and thymidm. e 3×10^-5M, commonly known as the HAT medium. The fusion mixture can be grown in the HAT-containing culture medium immediately after the fusion or up to 24 hours later. The feeding schedules usually entail maintenance in HAT medium for two weeks and then feeding with either regular culture medium or hypoxanthine, thymidine-containing medium.

The growing colonies are then tested for the presence of antibodies that recognize the antigenic preparation. Detection of hybridoma antibodies can be performed using an assay where the antigen is bound to a solid support and allowed to react to hybridoma supernatants containing putative antibodies. The presence of antibodies may be detected by "sandwich" techniques using a variety of indicators. Most of the common methods are sufficiently sensitive for use in the range of antibody concentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth in selected medium. Cloning can be performed by cell limiting dilution in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilution, cell suspensions are diluted serially to yield a statistical probability of having only one cell per well. For the agarose technique, hybrids are seeded in a semisolid upper layer, over a lower layer containing feeder cells. The colonies from the upper layer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue culture flasks, yielding supernatants with variable concentrations of antibodies . In order to obtain higher concentrations, hybrids may be transferred into animals to obtain inflammatory ascites. Antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection. The ascites contain a higher concentration of antibodies but include both monoclonals and immunoglobulins from the inflammatory ascites. Antibody purification may then be achieved by, for example, affinity chromatography.

The presence of LCC contemplated herein, or antibodies specific for same, in a patient's serum or tissue can be detected utilizing antibodies prepared as above, either monoclonal or polyclonal, in virtually any type of immunoassay. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. This, of course, includes both single-site and two-site, or "sandwich", assays of the non-competitive types, as well as in traditional competitive binding assays. Sandwich assays are among the most useful and commonly used assays and are favored for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized in a .solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and then added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent.

In the typical forward sandwich assay, a first antibody having specificity for LCC, or part thereof. contemplated in this invention, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generallyconsist of cross-linking, covalently binding or physically adsorbing the molecule to the insoluble carrier. Following binding, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated at 25°C for a period of time sufficient to allow binding of any subunit persent in the antibody. The incubation period will vary but will generally be in the range of about 2-40 minutes. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten. By "reporter molecule," as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes). In the case of an enzyme immunoassay (EIA), an enzyme is conjugated to the second antibody, generally be means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, B-galactosidase and alkaline phosphates, among other. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1, 2-phenylenediamine, 5-aminosalicyclic acid, or tolidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the ternary complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that the foregoing can be used to detect directly or indirectly (i.e., via antibodies) the LCC of this invention.

Another aspect of this invention relates to a recombinant nucleic acid molecule, said molecule defined herein to be DNA or RNA, encoding LCC or part thereof. In one embodiment, the recombinant nucleic acid molecule is complementary DNA (cDNA). It is considered within the scope of the present invention to include the cDNA molecule encoding mammalian LCC, but preferably human LCC, or to regions or parts thereof including any base deletion, insertion or substitution or any other alteration with respect to nucleotide sequence or chemical composition (e.g. methylation and glycosylation). LCC encoded by cDNA is referred to herein as recombinant LCC.

Methods considered useful in obtaining recombinant LCC cDNA are contained in Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pp. 1-545. Briefly, polyadenylated mRNA is obtained from stimulated LAK cells and fractionated on agarose gels. Aliquots of mRNA are then injected into Xenopus laevis oocytes for translation and assayed for LCC activity using the methods contained herein. The enriched fraction of mRNA translating into LCC active molecules is then used as template for cDNA synthesis. Libraries of cDNA clones are constructed in the Pst1 site of the vector pBR322 (using homopolymer tailing) or in a variety of other vectors (e.g. the Okayama-Berg cDNA cloning vectors. Messing cDNA cloning vectors and the like). Specific cDNA molecules in a vector in said library is then selected by using specific oligonucleotides designed, based on amino acid sequences contained within LCC, to encode at least part of said sequence. Once identified, cDNA molecules encoding all or part of recombinant LCC are then ligated into expression vectors. Additional genetic manipulation is routinely carried out to maximize expression of the cDNA in the particular host employed.

Accordingly, LCC is synthesized in vivo, inserting said cDNA sequence into an expression vector, transforming the resulting recombinant molecule into a suitable host and then culturing or growing the transformed host under conditions requisite for the synthesis of the polypeptides . The recombinant molecule defined herein should comprise a nucleic acid sequence encoding a desired polypeptide inserted downstream of a promoter, a eukaryotic or prokaryotic replicon and a selectable marker such as resistance to an antibiotic. The recombinant molecule may also require a signal sequence to facilitate transport of the synthesized polypeptide to the extracellular environment. Alternatively, the polypeptide may be retrieved by first lysing the host cell by a variety of techniques such as sonication, pressure dissintegration or toluene treatment. Hosts contemplated in accordance with the present invention can be selected from the group comprising prokaryotes (e.g., Escherichia coli. Bacillus sp., Pseudomonas sp.) and eukaryotes (e.g., mammalian cells, yeast and fungal cultures, insect cells and plant cultures). The artisan will also recognize that a given amino acid sequence can undergo deletions, substitutions and additions of nucleotides or triplet nucleotides (codons). Such variations are all considered within the scope of the present invention.

The scope of the present invention, therefore, encompasses recombinant LCC and the cDNA and mRNA encoding LCC including any host carrying and optionally expressing recombinant LCC cDNA and mRNA.

The following examples further illustrate the present invention.

EXAMPLE 1 Generation of LCC LCC is generated as follows: PBMC are placed in T75 flasks at an approximate concentration of 1×10⁶ cells/ml in 20 ml RPMI 1640 medium containing 10% (v/v) fetal calf serum or normal human serum or alternatively in AIM5 serum medium. The incubation medium additionally contains 1000 u/ml IL-2 for five days. The supernatant is then harvested and stored at -20 °C. The supernatant is essentially crude LCC.

EXAMPLE 2 Effect of LCC on MCF-7 Cells Using the methods described herein, the effect of LCC on MCF-7 cells are examined. The data is shown in Tables 1 and 2.

LCC is cytostatic in that it inhibits the proliferation of MCF-7 breast cancer cell line cells over 144 hours in culture (no activity is detectable before 24 hoursof culture of tumor cells with LCC). LCC is also cytotoxic in that it will kill MCF-7 cells when added to a confluent monolayer thereof. Again, no activity is seen until at least 24 hours of culture.

TABLE 1

EFFECT OF LAK CELL CYTOTOXIN ON MCF-7 CELLS OVER 144 HOURS INCUBATION

LCC Containing

Supernatant Percent Cytotoxicity After

Concentration Indicated Hours of Exposure

V/V 72 96 144

0 0 0 0

.1 0 3 9

1.0 4 15 44

2.0 13 17 85

5.0 5 24 95

10.0 37 27 95

50.0 78 95 99

TABLE 2

EFFECT OF LAK CELL CYTOTOXIN ON CONFLUENT MCF-7 CELLS

% LAK **

Supe * Optical Density at Indicated Day of Culture

Added 3 5 7

0 1.13 1.09 1.04

10 0.93 0.75 0.35

25 0.88 0.53 0.12

50 0.64 0.40 0.02

* Supe: supernatant of IL-2 stimulated. ** : the optical density (O.D.) of the culture correlates directly with the number of residual viable tumor cells.

EXAMPLE 3 LCC Cytotoxic and Cytostatic Activity Using the methods contained herein, the cytotoxic and cytostatic activity of LCC is determined. The results are shown in Table 3 (release of ¹²⁵IUDR from tumor cells preincubated with this isotope) and Table 4 (inhibition of colony growth of cells plated as single cells in soft agar.

TABLE 3

EFFECTS OF LAK CELL CYTOTOXIN ON ¹²⁵IUDR LABELLED MCF-& BREAST CANCER CELLS

Cytotoxicity % Cytotoxicity at Indicated Supe Conc Measured By 5 10 25 50

¹²⁵IUDR 15 29 67 70 release

¹²⁵IUDR 60 82 99 100

Residual Cells

TABLE 4

EFFECT OF LAK CELL CYTOTOXIN ON MCF-7 IN THE CLONOGENIC ASSAY

Reagents Added To Tumor Cells Colonies Per Plate

Day 8 Day 13

None 31 333

LAK Cells (100:1) 53 141

LAK Supe (33%)

EXAMPLE 4 Sensitivity of cells to LCC

A wide variety of cells have been tested for sensitivity to LCC by the above-described or other methods and compared to their sensitivity to LAK cells (Table 5). Multiple solid tumor cell lines are sensitive. Several leukemia and lymphoma cell lines while resistant to LCC are sensitive to LAK cells. This indicates unique methods bywhich target cells are killed in this sytem.

LCC can also inhibit fresh human tumor cells as demonstrated by the clonogenic assay in soft agar (Table 6). This is very important because this assay detects the growth of clonogenic tumor stem cells. Inhibition in this assay in vitro correlates with inhibition of tumor growth in vivo in the patient donor of the tumor.

LCC appears not to inhibit or kill normal cells. Thus, it does not inhibit adult or fetal fibroblasts (Table 7) nor does it inhibit normal lymphocytes responding to phytohemagglutinin or IL-2 stimulation (Table 8).

TABLE 5

LAK CELL CYTOTOXIN SENSITIVE AND RESISTANT TARGETS

Sensitive Resistant

A375M (Melanoma)* Daudi (B Cell)*

A375P (Melanoma)* K562. (Myeloid)*

Hs294T (Melanoma)* Fetal Fibroblasts*

MCF-7 (Breast)* Adult Fibroblasts*

C480 (Colon)* PHA-Lymphoblasts

HEY (Ovarian)* IL-2 Lymphoblasts

* LAK cell sensitive TABLE 6

EFFECT OF LAK CELL CYTOTOXIN ON THE GROWTH OF FRESH HUMAN TUMORS IN THE CLONOGENIC ASSAY

Tumor Number Inhibited/ Percent Inhibition Type Number Tested In Individual Cases

Breast 1/1 57.8

Cancer

Colon 1/2 40.0

Cancer

Melanoma 4/5 99.9

98.4 96.9 56.1

Myeloma 1/3 43.4

Ovaian 2/6 69.8

Cancer 50.0

Sarcoma 0/2 -

Note: LAK supernatant tested only at 10% v/v

TABLE 7

EFFECT OF LAK CELL CYTOTOXIN ON FIBROBLASTS

LAK Cell O.D. of Culture

Supe Adult Fetal MCF-7 Concentration Fibroblasts Fibroblasts Cells

0 .20 ≠ .02 .32 ≠ .03 1 .09 ≠ .02

10 .24 ≠ .04 .41 ≠ .05 .56 ≠ .05

25 .29 ≠ .04 .43 ≠ .04 .14 ≠ .07

50 .33 ≠ .03 .52 ≠ .04 .04 ≠ .02

TABLE 8

EFFECT OF LAK-CELL CYTOTOXIN ON MITOGEN RESPONSES

SUPERNATANT MITOGEN RESPONSE CONCENTRATION (CMP/CULTURE X 10^-3) (VOLUME %) -- PHA IL-2

0 0.5 178 35

10 0.6 170 28

25 0.4 163 32

50 0.9 140 33 EXAMPLE 5 Characterization of LCC Many of the characteristics of LCC have been defined. Thus, it is produced by LAK cells and can diffuse through a membrane (0.2 microns) and act on tumor cells at a distance (Table 9). It can be generated in serum free medium (Table 10). It is not generated in the absence of IL-2 (Table 10). It is generated by non-adherent cells (Table 11). It is non-dialyzable (Table 12). Its heat and acid stability characteristics have been defined (Tables 13 and 14).

TABLE 9

GROWTH OF MCF-7 I N THE COSTAR TRANSWELL SYSTEM

Inner Well Contents Outer Well O.D. % Growth

LAK-Cell (2 X 10⁴ MCF-7 Cells)* Inhibition Cytoxin PBMC IL-2

- - - .68 ± .02 -

25% - - .14 ± .01 80.1 - 7 X 10⁵ - .54 ± .01 19.7 - 7 X 10⁵ 1000u/ml .29 * .04 55.7

Initial E:T, 35:1

TABLE 10

GENERATION OF LAK CELLS AND LAK CELL CYTOTOXIN IN AIM-5 SERUM FREE MEDIUM (% GROWTH INHIBITION)

Effector Effector Ratio or Concentration

Reagent 5:1 10:1 50:1

PBMC -14.0 - 5.0 -10.7 LAK Cells 64.5 79.3 75.2

10% 25% 50%

PBMC 13.7 2.5 - 5.9 supe

LAK Cell 75.6 91.9 95.3 supe

TABLE 11

LAK CELL CYTOTOXIN IS MADE BY NON ADHERENT CELLS

Source Percent Growth Inhibition of MCF-7 of at Indicated Supe Concentration

Supe 10 25 50

Adherent - .2 1 2.9 Cells

Non Adherent 25.7 48.3 69.4 Cells

PBMC (Non - 1.0 32.9 55.2 fractionated) TABLE 12

LAK CELL CYTOTOXIN IS NON DIALYZABLE

Percent Growth Inhibition of MCF-7

Reagent at Indicated Supe Concentration 10 25 50

Dialyzed 9.5 - .3 -31.8 Medium

Dialyzed 34.3 60.1 71.1 Supe

Non Dialyzed 41.6 67.9 78.7 Supe

TABLE 13

HEAT SENSITIVITY OF LAK CELL CYTOTOXIN

Temp Time % Cytotoxicity at Indicated

Supernatant Concentration

2 10 25

- - 21.2 71.0 87.8

56° 30' 15.5 60.4 85.4

65° 30' 0.0 21.2 47.0

80° 30' 1.8 20.0 37.2

80° 60' 0.0 0.0 3.4

TABLE 14

ACID (pH2) SENSITIVITY OF LAK CELL CYTOTOXIN

Time of % Cytotoxicity at Indicated Exposure Supernatant Concentration (min) 2 10 25

- 37.1 88.1 97.2

30 20.4 75.8 91.5

60 16.7 61.6 89.3

EXAMPLE 6 Comparison of LCC to Other Known Cytokines A series of studies were conducted to determine the relationship of LCC to any known cytotoxic or cytostatic cytokine. These results indicate that no relationship apparently exists. Therefore, by a series of direct or indirect assays, the LCC activity is unique. By direct assay LCC containing supernatants have no IFN-α activity, no TNF-α activity (after dialysis), and a minimal, non-cytostatic level of IFN-ɤ . LCC was shown to be cytostatic to MCF-7 cells which are not sensitive to TNF- α or TNF- β (lymphotoxin) (Table 15) . The TNF resistant variant of L929 cells which is resistant to both TNF-α and TNF-β is sensitive to LCC (Table 16). By inference of sensitive and resistant cell lines to various reagents, LCC is distinct from (in addition to the above) IFN- β, IL-1, leukoregulin, NKCF, perforin, cytolysin, serine esterase, lysozomal arylsulfatase, TGFβ1, and oncostatin M. Some of these relate to the resistance of K562 cells to LCC (Table 17) which is known to be sensitive to NKCF, Leukoregulin, perforin, cytolysin, etc. These concepts and findings are summarized in Tables 18 and 19.

TABLE 15

SENSITIVITY OF THE MCF-7 BREAST CANCER CELL LINE TO VARIOUS REAGENTS (% GROWTH INHIBITION)

Reagent Percent Growth Inhibition at Indicated Reagent Concentration

LAK Cell Supe (%) 10* 25 50

LAK Cells (E:T) 5:1* 10:1 50:1

THFα/β (u/ml) 1* 10 100

LAK Cell Supe 43.8** 60.4 71.7

LAK Cells 50.4 83.1 93.5

TNFα 0.0 - 0.7 6.2

TNFβ (LT) - 2.5 - 1.3 6.2

* concentration ** percent growth inhibition

TABLE 16

SENSITIVITY OF THE TNF RESISTANT L929 CELL LINE TO VARIOUS REAGENTS (% GROWTH INHIBITION)

Reagent Percent Growth Inhibition at Indicated Reagent Concentration

LAK Cell Supe (%) 10* 25 50

LAK Cells (E:T) 5:1* 10:1 50:1

TNFα/β (u/ml) 1* 10 100

LAK Cell Supe 6.6** 31.9 56.6

LAK Cells 26.0 60.7 86.1

TNFα -10.1 2.1 0.3

TNFβ (LT) 0.0 4.1 9.1

* concentration ** percent growth inhibition

TABLE 17

EFFECT OF LAK-CELL CYTOTOXIN ON THE PROLIFERATION OF DAUDI AND K562 CELLS

SUPERNATANT ³H-THYMIDINE INCORPORATION CONCENTRATION (CPM X 10^-3) (VOLUME %)

DAUDI K562

0 50.6 18.0

10 59.2 18.4

25 51.6 19.2

50 64.8 19.4 TABLE 18

ANALYSIS OF LAK CELL CYTOTOXIN FOR CYTOKINES

Cytokine Direct Cytokine Resistant Cytokine Sensitive Assay Line is LCC Line is LCC Sensitive Resistant

IFNα negative MCF-7, A375P -

IFHβ ND MCF-7, A375P -

IFN7 low* MCF-7 - (<40 u/ml)

TNFα low L929R - (<100 u/ml)

TNFβ (LT) ND L929R, MCF-7 -

IL-1 ND MCF-7 _

NKCF ND - K562

Leukoregulin ND - K562

Note: Dialyzed supe TNFα negative

TABLE 19

OTHER CYTOLYTIC OR CYTOSTATIC FACTORS

Factor Characteristic different than LCC

NKCF Lyses K562

Perforin Lyses K562 rapid acting (min)

Cytolysin Lyses K562 rapid acting (min) Serine esterase Contact release only

Lysozomal arylsulfatase Contact release only

Leukoregulin Lyses K562 MW 40,000 (TLS)

TGFβ1 Inhibits PHA stimulated blasts Oncostatin M Does not inhibit L292. MW 28,000 produced by purified T cells over 3-28d.

EXAMPLE 7 Partial Purification of LCC LCC was subject to purification by FPLC. Figure 1 is a graphical representation of LCC activity in FPLC fractions. Figure 1A represents fractions derived from supernatant of PBMC incubated in the absence of IL-2. No peak is seen at number 5. In difference, supernatant from IL-2 incubated cells has a peak at number 5. This also contains LCC activity.

EXAMPLE 8 Fractionation and Further Characterization of LCC

To purify LCC and remove the serum albumin tightly associated with it, crude LCC (LAK cell supernatent made in AIM 5 medium) was concentrated 10-fold to 100 ml using an Amicon Spiral Centrifuge (MW cut-off 10,000) and exchanged with 1000 ml of 50 mM MES buffer (pH 6.0) using the same spiral centrifuge technique until the ultrafiltrate reached pH 6.0. The concentrated LCC was loaded onto a Q-Sepharose column (50 ml bed volume, equilibrated with 50 mM MES, pH 6.0) with a peristaltic pump operating at a flow rate of 3 ml/min. The flow-through was collected, concentrated in an Amicon ultrafiltration cell (50 ml capacity, PM-10 filter) and exchanged with RPMI medium. The final volume was 30 ml and is designated LCC-1. LCC-1 has potent cytotoxic activity, is devoid of albumin but contains transferrin.

The proteins bound to the Q-Sepharose column were eluted with 1 M NaCl and exchanged into RPMI medium by Centriprep 30 ultrafiltration. This fraction is designated LCC-2, contains almost all the serum albumin and has potent cytotoxic activity.

Crude LCC, LCC-1 and LCC-2 contain small but measurable amounts of known cytokines including TNF and IFNɤ. These activities are measured in crude LCC, LCC-1 and LCC-2 by ELISA. These various fractions were subjected to affinity purification to remove contaminants using columns coated with anti-TNFαor anti-IFN ɤ antibodies. The results shown in Table 20 indicated that albumin-free LCC-1 retains much of its cytotoxic activity after removal of TNFαor IFNɤ while albumin-containing LCC-2 loses much of its activity with the same treatment. After passage through both columns, crude LCC contained no measurable TNFα and only 22.5 u/ml IFNɤwhereas LCC-1 contained no TNFα or IFNɤ. An amount of IFNɤ of 22.5 u/ml (or even 1000 u/ml) has no effect on MCF-7 cells.

TABLE 20 Purification and Characterization of LCC

Controls Crude LCC LCC-1 LCC-2

% cytotoxicity* 88.8 89.5 91.1 u/ml TNFα 17.7 17.9 18.0 u/ml IFN ɤ 100.0 100.0 54.0 after passage through anti-TNFα column % cytotoxicity* 60.8 91.7 23.5 u/ml TNF α 0.0 0.0 0.0 u/ml IFNɤ 100.0 100.0 71.1 after passage through anti-IFNɤ column

% cytotoxicity* 16.9 52.6 5.8 u/ml TNFα 0.0 0.0 2.8 u/ml IFNɤ 22.5 0.0 2.8

* to MCF-7 cells in a 3 day cell viability assay.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing a cytokine associated with the LAK cell phenomenon having unique cytotoxic and cytostatic activity comprising the steps of contacting mammalian peripheral blood mononuclear cells (PBMC) with a cytokine-inducing effective amount of interleukin-2 for a time and under conditions sufficient to provide said cytokine.

2. The process according to Claim 1 wherein said mammalian PBMC are human PBMC.

3. The process according to Claim 1 or 2 wherein the PBMC and interleukin-2 are contacted in a buffered medium.

4. The process according to any of Claims 1 to 3 wherein the cytokine-inducing effective amount of interleukin-2 is in the range of from about 200 u/ml to about 5000 u/ml.

5. The process according to any of Claims 1 to 4 wherein sufficient time for said cytokine to be synthesized is from about 2 to about 10 days.

6. The process according to any of Claims 3 to 5 wherein the buffered medium used is RPMI 1640 containing a nutrient solution selected from 10% (v/v) fetal calf serum, normal human serum or AIM5 serum-free medium.

7. The process according to any of Claims 1 to 6 wherein said cytokine has the characteristics of being able to diffuse through a membrane of 0.2 microns, can be generated in serum free medium and is generated by non-adherent cells.

8. The process according to any of Claims 1 to 7 wherein said cytokine has the identifying characteristics of LCC.

9. A pharmaceutical composition useful in inducing regression or inhibition of growth of tumors and cancer in a mammal comprising an effective amount of a cytokine being LAK cell cytotoxin from mammalian PBMC, and a pharmacologically acceptable carrier.

10. A pharmaceutical composition according to Claim 9 wherein the cytokine is derived from a nucleic acid encoding said cytokine.

11. The composition according to Claim 9 having a unit dosage form containing of from about 0.5 μg to about 2000 jig of said cytokine.

12. An antibody to a LAK cell cytotoxin from mammalian PBMC.

13. The antibody according to Claim 12 wherein said antibody is a monoclonal or polyclonal antibody.

14. The cDNA encoding LAK cell cytotoxing from mammalian PBMC.

15. The cDNA according to Claim 14 operably linked to a recombinant DNA molecule, said molecule capable of replicating in a eukaryote or a prokaryote and optionally capable of directing expression of said cDNA.

16. A host transformed with the cDNA according to Claim 15.

17. Isolated LAK cell cytotoxin (LCC) from mammalian PBMC.

18. Isolated LAK cell cytotoxin (LCC) wherein said LCC is recombinant LCC.

19. Isolated LCC according to Claim 17 wherein said PBMC are human PBMC.