COMPOSITIONS AND THE USE OF INTERLEUKIN-2 AND/OR INTERFERON-BETA AND TUMOUR NECROSIS FACTOR FOR COMBINATION THERAPY OR IN PROVIDING MEDICAMENTS OR FORMULATIONS

Abstract: Anti-tumor activity in mammals can be augmented by administering to the mammalian host a synergistically effect­ ive amount of TNF and IL-2 or of TNF and IFN-β, or of TNF, IL-2 and ITN-β in combination. The composition of TNF and IL-2 and/or IFN-β may be prepared in vitro or administered separately to the host. If the TNF and IL-2 are administered sequentially, the TNF must be administered prior to the IL-2 to obtain synergy. The composition is useful for treating such cancers as mastocytoma, melanoma, leukemia, and lym­ phoma.

Description: This invention relates to a combination of interleukin-2 (IL-2) and tumor necrosis factor (TNF) and the use of this combination as an anti-tumor therapeutic agent. Interleukin-2, a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan, D.A., et at., Science (1976), 193 :1007-1008. Then called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes, it is now recognized that in addition to its growth factor properties, it modulates a variety of functions of immune sytem cells in vitro and in vivo and has been renamed interleukin-2 (IL-2). Interleukin-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for example, U.S. Patent No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi, T. et al., Nature (1983), 302 :305-310 and Devos, R., Nucleic Acids Research (1983), 11 :4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms. U.S Patent No. 4,518,584 describes and claims muteins of IL-2 in which the cysteine normally occurring at position 125 of the wild-type or native molecule has been replaced with a neutral amino acid, such as serine or alanine. An oxidation-resistant mutein such as IL-2 which is biologically active may be prepared wherein each methionine residue of the protein from which the mutein is derived which methionine is susceptible to chloramine T or peroxide oxidation is replaced with a conservative amino acid such as alanine. These IL-2 muteins possess the biological activity of native IL-2. U.S. Patent Nos. 4,530,787 and 4,569,790 disclose and claim methods for purifying recombinant native IL-2 and muteins thereof, as well as purified forms of IL-2. PCT W085/04328 discloses an IL-2 composition suitable for reconstituting in a pharmaceutically acceptable aqueous vehicle composed of oxidized microbially produced recombinant IL-2. The IL-2 is noted as useful in combination with cytotoxic chemotherapy or irradiation or surgery in the treatment of malignant or pre-malignant diseases in a direct therapeutic or adjuvant setting or in combination with other immune-modulating drugs, lymphokines (e.g., IL-1, IL-3, CSF-1 and IFNs) naturally occurring or inducible anti-cellular toxins in treating malignant diseases. Various therapeutic applications of human IL-2 have been investigated and reported by S. Rosenberg and colleagues (see Mule et al., Science (1984), 225 :1487 and S. Rosenberg et al., New England Journal of Medicine (1985), 313 :1485-1492, for example). Tumor necrosis factor (TNF) was first described by Carswell et al., PNAS (USA) (1975), 72 :3666-3670 as an endotoxin-induced serum factor which causes necrosis of chemically transformed tumor cells when growing in mice. Purified preparations of murine TNF have been tested against murine and human cell lines in vitro . K. Haranaka and N. Satomi, Japan J. Exp. Med. (1981), 51 :191. In contrast to normal cells, tumor cell lines from both species were susceptible to the cytotoxic activity of the mouse TNF. Furthermore, the murine TNF was reported to be toxic against both human- and mouse-transplanted tumors in nude mice. See K. Haranaka et al., Int. J. Cancer (1984), 34 :263-267. Human TNF is also known to be cytotoxic to neoplastic cells, and has been produced in recombinant form. See Pennica et al., Nature (1984), 312 :724-729; Shirai et al., Nature (1985), 313 :803-806; Wang et al., Science (1985), 228 :149-154. The cloning of rabbit TNF is disclosed in EP 146,026, published June 26, 1985 (Dainippon Pharmaceutical Co., Ltd.) and EP 148,311, published July 17, 1985 (Asahi Kasei Kogyo Kabushiki). The cloning of human TNF having 151 and 155 amino acids (2 and 6 less than the native form) is disclosed in EP 155,549, published September 25, 1985 (Dainippon Pharmaceutical Co., Ltd.), and human TNF having 155 amino acids is disclosed in EP 158,286, published October 16, 1985 (Asahi Kasei Kogyo Kabushiki Kaisha) and corresponding GB 2,158,829A, published November 20, 1985. The cloning of mature TNF (157 amino acids) and various modified forms (muteins) thereof is disclosed in EP 168,214, published January 15, 1986 (Genentech) and PCT US85/01921, filed October 3, 1985, published April, 1986 (Cetus Corporation). Combination chemotherapy using two or more anti-cancer drugs to treat malignant tumors in humans is currently in use in research and in the clinic. The anti-cancer drugs may be antimetabolites, alkylating agents, antibiotics and general poisons. Combinations of drugs are administered in an attempt to obtain a synergistic cytotoxic effect on most cancers, e.g., carcinomas, melanomas, lymphomas and sarcomas, and to reduce or eliminate emergence of drug-resistant cells and to reduce side effects to each drug. It is known that Type I and Type II interferons may be combined to produce a synergistic biological effect. See, for example, Fleishmann, W. R., Cancer Res . (1982), 42 :869-875 and DeClercq. E., et al., Cancer Letters (1982), 15 :223-228 (mouse IFNs), and European Patent Publ. 107,498 published May 2, 1984 (human IFN-γ and IFN-α or -β). U.S. Patent 4,518,584 to Mark et al. (Cetus Corporation) discloses the combination of IL-2 muteins with gamma-interferon, B cell growth factor, and IL-1. In addition, it has been disclosed that IL-2 may be used with IFN-γ to treat tumor-bearing hosts with synergistic results (European Patent Publ. 149,551 published July 24, 1985 (Genentech) and German Patent Publication 3411184 published October 31, 1985 (Deut Roten Kreuzes)) or with augmentation of natural killer activity (Svedersky et al., J. Immunol. (1984), 133 :714-718 and Shalaby et al., J. Interferon Res. (1985), 5 :571-581.) Lopez-Botet et al., Eur. J. Immunol. (1984), 14 :1137-1141 reported, however, that IL-2 and IFN-γ are not sufficient in combination to induce natural killer-like activity in human T cell clones. It is also known from Dempsey et al., J. Immun. (1982), 129 :2504-2510 that the combination of IFN-α and IL-2 is more effective than IFN-α or IL-2 alone in causing natural killer cell activation. Also, Dr. Talmadge of the Preclinical Screening Lab., BRMP has reported in 1986 the augmented effect of using TNF and IFN-γ to treat metastatic disease in mice. EP 131,789, published January 23, 1985 (Sloan-Kettering Institute for Cancer Research) and EP 168,214 published January 15, 1986 (Genentech) disclose the synergistic effect of TNF and IFN-γ to treat various tumors in mice. EP 170,843, published June 20, 1985 (Boehringer Ingelheim) discloses the synergistic effect of TNF and IFN-α, β and/or γ on cancerous growth, particularly mixtures containing TNF and IFN-γ. See also Williamson et al., Proc. Natl. Acad. Sci. (1983) 50 :5397-5401 for in vivo effects of human TNF and human IFN. The effect of a combination of IL-2 and TNF on tumor-bearing animals has not been investigated. Accordingly, the present invention provides a composition suitable for parenteral or subcutaneous administration to mammalian hosts for therapeutic or prophylactic treatment of cancer comprising a mixture of TNF and IL-2 in synergistically effective amounts, wherein the TNF and IL-2 are from a mammalian species. In a further aspect the invention includes products containing TNF and IL-2 as a combined preparation for simultaneous, separate or sequential use in the therapeutic or prophylactic treatment of cancer in human patients. The invention also provides the use of both TNF and IL-2 in providing a medicament for the prophylaxis or treatment of cancer. Also included in the invention is a method of making a therapeutic or prophylactic formulation for administration to mammals for therapeutic or prophylactic treatment of cancer comprising formulating together, whether by mixing or providing separate doses or otherwise, synergistic amounts of mammalian TNF and IL-2. Although not part of the invention as such, the invention permits a method to be derived for therapeutic treatment of cancer in mammalian hosts comprising administering to the host a synergistically effective amount of TNF and IL-2, wherein the TNF and IL-2 are from mammalian species, and wherein if the TNF and IL-2 are administered sequentially, the administration of TNF precedes the administration of IL-2. Preferably the TNF is rabbit or human TNF and the IL-2 is human IL-2 and all proteins are recombinant, microbially produced proteins. The combination of IL-2 and TNF is found to provide a surprising synergism in treating various forms of cancer such as melanomas, leukemia, mastocytoma, and lung cancer. As used herein, the term "therapeutic" treatment refers to administration to the host of the TNF and IL-2 after the host has contracted cancer, as determined by any means. The treatment is not considered therapeutic if after treatment a tumor appears or an existing tumor burden is not decreased or eliminated. The effect of the dose will diminish with time, with from 5-7 days after the tumor is visible being typically the maximum period in which treatment can be given, depending mainly on the type of tumor and dosage levels. As used herein, the term "cancer" refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer and gastrointestinal or stomach cancer. Preferably, the cancer is leukemia, mastocytoma, melanoma and lymphoma. As used herein, the term "synergistically effective amount" as applied to IL-2 and TNF refers to the amount of each component of the mixture which is effective for survival of the host and which produces a survival level which does not intersect, in a dose-response plot of the dose of TNF versus dose of IL-2 versus host survival, either the dose TNF axis or the dose IL-2 axis. The dose response curve used to determine synergy herein is more fully described by Sande et al., p. 1080-1105 in A. Goodman et al., ed., the Pharmacological Basis of Therapeutics, MacMillan Publishing Co., Inc., New York (1980). For purposes of synergy, cure is defined as cure of the host after 14 days from the date of tumor implantation into the host for Meth A tumors and after 60 days for all other tumors. The optimum synergistic amounts can be determined, using a 95% confidence limit, by varying factors such as dose level, schedule and response, and using a computer-generated model which generates isobolograms from the dose response curves for various combinations of the IL-2 and TNF. The highest survival rates on the dose response curve correlate with the optimum dosage levels. As used herein, the term "recombinant" refers to TNF or IL-2 produced by recombinant DNA techniques wherein generally the gene coding for the TNF or IL-2 is cloned by known recombinant DNA technology. For example, by using the human TNF or IL-2 cDNA as a template, the gene showing complementarity to the human TNF or IL-2 cDNA is inserted into a suitable DNA vector such as a bacterial plasmid, preferably E. coli plasmid, to obtain a recombinant plasmid, and the plasmid is used to transform a suitable host. The gene is expressed in the host to produce the recombinant protein. Examples of suitable recombinant plasmids for this purpose include pBR322, pCR1, pMB9 and pSC1. The transformed host may be eucaryotic or procaryotic, preferably a procaryotic host. As used herein, the term "pharmaceutically acceptable" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the hosts to which it is administered. The method of therapy/prophylaxis which is not part of this invention but which is derived therefrom, involves administering to a mammalian host, preferably a human host, a synergistically effective amount of TNF and IL-2 and TNF may be combined in vitro before administration or separately administered to the host. IL-2 and TNF may be administered either simultaneously or by administering TNF followed by IL-2, with any second administration generally after the first administration is completed. Administration of IL-2 before the TNF did not result in synergism, and IL-2 may reduce the sensitivity of the tumor to subsequent TNF treatment. The administration(s) may take place by any suitable technique, including subcutaneous and parenteral administration, preferably parenteral. Examples of parenteral administration include intravenous, intraarterial, intramuscular, and intraperitoneal, with intraperitoneal administration(s) being preferred (for convenience) with murine models. The dose and dosage regimen will depend mainly on whether the IL-2 and TNF are being administered separately or as a mixture, the type of cancer, the patient, and the patient's history. The amount must be effective to achieve a tumor reduction which is synergistic. The doses may be single doses or multiple doses. If multiple doses are employed, as preferred, the frequency of administration will depend, for example, on the type of host and type of cancer, dosage amounts, etc. For some types of cancers or cancer lines, daily administration will be effective, whereas for others, administration every other day or every third day will be effective, but daily administration will be ineffective. The practitioner will be able to ascertain upon routine experimentation which route of administration and frequency of administration are most effective in any particular case. The dosage amount which appears to be most effective herein is one which results in no tumor appearance or complete regression and is not toxic to the host. This optimum level will depend on many factors, for example, on the type of host and type of cancer, route, schedule of administration, existing tumor burden, the type of IL-2 and TNF, and the definition of toxicity. Toxicity to the host may be defined by the extent and type of side effects or by the amount of body weight loss or by death after a certain period of time. If body weight loss is the criterion for toxicity, typically a loss of from 10-20% by weight will be tolerated, with greater than 20% loss being considered toxic. If body weight loss of greater than 20% is considered toxic, if the host is murine, if the route of administration is intraperitoneal via a mixture prepared in vitro and is every day or every other day, the dosage level at each administration of recombinant, microbially produced TNF and IL-2 is preferably 230-260 µg TNF per kg host weight (more preferably 250 µg), and 15,000-15 million units IL-2 per kg host weight, where 1000 units is 1 µg (more preferably 15,600-625,000 units). For parenteral administration the IL-2 and TNF will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion), preferably in a pharmaceutically acceptable carrier medium which is inherently non-toxic and non-therapeutic.Examples of such vehicles include saline, Ringer's solution, dextrose solution, mannitol and normal serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. The carrier medium may contain minor amounts of additives such as substances which enhance isotonicity and chemical stability, e.g., buffers and preservatives. The IL-2 and TNF will typically be formulated in such carriers at a concentration of 0.1 mg/ml to 100 mg/ml of each, preferably 0.2 to 1 mg/ml of each. Alternatively, the IL-2 and TNF may be made into a sterile, stable lyophilized formulation in which the purified IL-2 and TNF are admixed with a water-soluble carrier such as mannitol, which provides bulk, and a sufficient amount of a surfactant such as sodium dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water. The formulation is suitable for reconstitution in aqueous injections for parenteral administration and it is stable and well-tolerated in human patients. The IL-2 formulation method is more completely described in PCT W085/04328. In yet another alternative, the mixture of IL-2 and TNF may be administered in an adoptive immunotherapy method, together with isolated, lymphokine-activated lymphocytes in a pharmaceutically acceptable carrier, where the lymphocytes are reactive to tumor when administered with the TNF and IL-2 to humans suffering from the tumor. This method is described more fully in S. Rosenberg et al., New England Journal of Medicine (1985), 313 :1485-1492. In another alternative, described in S. Rosenberg et al., Science , 233 :1318-1321 (1986), tumor-infiltrating lymphocytes (TIL) expanded in IL-2 may be adoptively transferred for the therapeutic treatment, particularly in combination with cyclophosphamide. The TIL approach of Rosenberg et al. may also be used herein. As mentioned above, the IL-2 and TNF herein may be any IL-2 and TNF prepared from tissue cultures or by recombinant techniques, and from any mammalian source, such as, e.g., mouse, rat, rabbit, primate, pig, and human. Preferably the TNF is derived from rabbit or human sources, more preferably human, and the IL-2 is dervied from a human source. More preferably, the IL-2 and TNF are recombinant human IL-2 and recombinant human TNF. The recombinant IL-2 may be obtained as described by Taniguchi et al., Nature , 302 :305-310 (1983) and Devos, Nucleic Acids Research , 11 :4307-4323 (1983) by cloning the native human IL-2 gene and expressing it in transformed microorganisms. It may also be an IL-2 mutein as described in U.S. Patent No. 4,518,584, in which the cysteine normally occuring at position 125 of the wild-type or native molecule has been replaced by a neutral amino acid such as serine or alanine, or an IL-2 mutein in which the methionine normally occurring at position 104 or the wild-type or native molecule has been replaced by a neutral amino acid such as alanine. Preferably, the IL-2 is an unglycosylated protein which is produced by a microorganism which has been transformed with the human cDNA sequence or a modified human cDNA sequence of IL-2 which encodes a protein with an amino acid sequence at least substantially identical to the amino acid sequence of native human IL-2, including the disulfide bond of the cysteines at positions 58 and 105, and has biological activity which is common to native human IL-2. Substantial identity of amino acid sequences means the sequences are identical or differ by one or more amino acid alterations (deletions, additions, substitutions) which do not cause an adverse functional dissimilarity between the synthetic protein and native human IL-2. Examples of IL-2 proteins with such properties include those described by Taniguchi et al., Nature (1983 ), 302 : 305-310; Devos, Nucleic Acids Research (1983), 11 :4307-4323; and by European Patent Publication Nos. 91,539 and 88,195; and in U.S. Patent 4,518,584, supra , as well as IL-2 ala104ser125 . Most preferably, the IL-2 is the des-ala₁-IL-2 ser125 mutein in which the initial terminal alanine to deleted and the cysteine at position 125 to replaced by a serine residue and IL-2 wherein any combination of up to 5 of the first 5 N-terminal amino acid residues are deleted. The IL-2 may be produced and purified to clinical purity by the method described and claimed in U.S. Patent No. 4,569,790, issued February 11, 1986. In an alternative formulation, the IL-2 may be solubilized, not by a detergent, but by reacting the IL-2 with an activated polymer selected from polyethylene glycol homopolymers and polyoxyethylated polyols such as polyoxyethylated glycerol. The polymer preferably has a molecular weight of from 300 to 100,000 daltons, more preferably 350 to 40,000 daltons. The polymer is activited by conjugation with a coupling agent having terminal groups reactive with both the free amine or thiol groups of the IL-2 and the hydroxyl group of the polymer. Examples of such coupling agents include hydroxynitrobenzene sulfonic ester, cyanuric acid chloride, and N-hydroxysuccinimide. This modification eliminates the necessity for adding detergents to solubilize the IL-2 at physiological pH. The IL-2 to then formulated directly with the water-soluble carrier and buffer as described above, the formulation is lyophilized, and the lyophilized mixture may be reconstituted as described above. The recombinant human TNF may be obtained as described by Pennica et al., Nature (1984), 312 :724-729; Yamada et al., J. Biotechnology (1985), 3 :141-153; Wang et al., Science (1985), 228 :149-154; EP 155,549 published September 29, 1985; EP 158,286 published October 16, 1985; EP 168,214 published January 15, 1986; and PCT US 85/01921 published April, 1986. The recombinant rabbit TNF may be obtained as described to EP 146,026 published June 26, 1985 and EP 148,311 published July 17, 1985. Preferably the TNF is a human TNF mutein wherein the first eight amino acid residues have been deleted, or the TNF is a cysteine-depleted mutein prepared analogously to that described in U.S. Patent No. 4,518,584. The various aspects of the invention are further described by the following examples. In these examples all parts for solids are by weight and all percentages for liquids and gases are by volume, unless otherwise noted, and all temperatures are given in degrees Celsius. EXAMPLE 1 A. General Treatment Mice Female BDF1, C57B1 and Balb/c mice and CD rats were employed in the in vivo tests. Mice were weight matched and randomized such that treatment groups (5 or 10) averaged 20 g ± 3 g. All animals were held for quarantine observation for seven days after arrival, maintained in microisolator cages and fed standard laboratory diets with drinking water ad lib. IL-2 The recombinant IL-2 employed in this example was des-ala₁-IL-2 ser125 described by Wang et al., Science (1984) 224 :1431-1433. The amino acid sequence of this IL-2 differs from the amino acid sequence of native human IL-2 in that it lacks the initial alanine of the native molecule, and the cysteine at position 125 has been changed to serine. Samples of E. coli that produce this IL-2 have been deposited by Cetus Corporation in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md, USA on September 26, 1983 under accession number 39,452 and on March 6, 1984 under accession number 39, 626 under the provisions of the Budapest Treaty. The IL-2 was processed and purified as described in the text and Figure 1 of PCT W085/04328, except that the oxidation was carried out using copper chloride, as described in U.S. Patent No. 4,572,798 rather than o-iodosobenzoate. When the IL-2 was recovered from the chromatography step(s) it was lyophilized and resuspended in a neutral aqueous buffer containing the reducing agent (DTT) to keep the IL-2 in a reduced state and a solubilizing agent to keep it in solution. The purity of the recombinant IL-2 after the chromatography step(s) was at least about 95% and the IL-2 contained less than about 0.02 ng/ml endotoxin as determined by the Limulus amebocyte assay. The purified IL-2 (3-5 x 10⁶ units/mg) was produced as a lyophilized powder in sterile vials and reconstituted using sterile phosphate buffered saline within 4 days prior to use and formulated at a concentration of 0.3 mg/ml with 50 mg/ml mannitol. In an alternative formulation, the IL-2 was formulated as by reaction with polyethylene glycol which was conjugated using N-hydroxysuccinimide. The conjugated protein was formulated directly in water (hereinafter called IL-2-PEG). TNF A mutein of human TNF having the first eight amino acids deleted from the N-terminus was prepared as described by Wang et al., Science (1985) 228 :149-153. Briefly, TNF was induced from HL-60 cells and purified and sequenced. Then an intronless sequence encoding human TNF was prepared by producing enriched mRNA, constructing a cDNA library, selecting a probe and probing the library to recover the sequence. Then an ATG start codon was introduced immediately preceding the GTC sequence encoding N-terminal valine of the mature protein by site-directed mutagenesis. Clones were selected and strands ligated into expression vectors to obtain procaryotic expression of the mutein. The mutein was then purified by column purification, recovered in the purification buffer, and produced as a lyophilized powder in sterile vials. Finally, it was reconstituted and suspended using sterile phosphate buffered saline within four days prior to use, and stored, if at all, at 4°C. The TNF contained less than 0.001 to 0.006 ng endotoxin/mg protein depending on production lot. Cancer Cell Lines The target cells employed were murine tumors L1210 (leukemia), P388 (leukemia), P815 (mastocytoma), and EL-4 (lymphoma), all obtainable from the American Type Culture Collection, Rockville, MD, and B16W10 (melanoma), which is a subclone of the Fidler line F10 (melanoma murine line) obtained by passage ten times in vitro and in vivo of the Fidler line, and which is described by Winkelhake et al., Cancer Res. (1979) 39 :3058-3064. All cell lines were passed twlce in tissue culture (37°C, 8% CO₂ in RPMI 1640 medium with 10% fetal bovine serum, 2 mM L-gln) from frozen stocks just prior to implantation. All tumors and cell lines were negative in tests for mycoplasma and for mouse anti-viral-antibody-production. Subcutaneous Tumor Injections The tumor cells were harvested from culture suspensions or monolayers. For subcutaneous tumors, the cells (5 x 10⁵-10⁶) were injected in the suprascapular region. For intraperitoneal (ip) tumors, 10⁵ cells were inoculated into the mice. For the B16W10 melanoma intravenous (pulmonary, iv) metastasis model, the cells were removed from tissue culture plates using trypsin-EDTA, rinsed twice in phosphate buffered saline, and 10⁴ cells were injected into the lateral tail vein in 0.2 ml volume. If the mice were not treated with any lymphokine, they all died within 20-30 days after inoculation, whether ip, iv., or sq. Experimental Methods Groups of five mice per dose were utilized except for the B16W10 iv model, where group sizes were 10. Animals received tumor challenges on Day 0 unless otherwise stated and all treatments were ip, initiated on the indicated day after tumor challenge and continued once per day for 14 days. For subcutaneous models, tumors were measured using linear calipers in three orthogonal directions by the same measurer throughout each experiment. While there is inter-individual variability when this technique is applied, repeat measurements performed by the same individual showed less than 5% error. All tumors studied were allowed to grow to volumes of approximately two cubic cm, at which point further measurements were difficult and animals were sacrificed. For ip tumors, animals were observed daily for survival. As all tumors that were studied are lethal to the mice within approximately 30 days of implant, observations for prolongation of lifetime were performed for at least 60 days. For iv-administered B16 model, animals were sacrificed 17-21 days after cell inoculations, and lung colonies were counted. B. Results 1. Table I indicates the results obtained when TNF alone, IL-2 alone, and various mixtures of TNF and IL-2 (prepared in vitro ) were administered per kilogram mouse weight intraperitoneally to five female BD2F1 mice per group implanted sq with 2 x 10⁶ P815 cells, beginning one day after tumor implantation (Day 1), continuing every day for 20 days. The control was injected only with PBS daily for 20 days. In the table, the "palp." abbreviation refers to palpable tumors. The results indicated that the subcutaneous model P815 mastocytoma (which was responsive to 250 µg/kg TNF every day for 14 days ip beginning Day 1 and was unresponsive to up to 10 million units/kg of the same regimen of IL-2) was responsive to a 5-fold lower dose of TNF when the IL-2 was administered concomitantly. In addition, no tumors appeared when the TNF and IL-2 were administered together at 250 µg and 625,000 units/kg, respectively. 2. Table II indicates the results obtained when TNF alone, IL-2 alone, and various mixtures of TNF and IL-2 (prepared in vitro ) were administered per kg mouse weight intraperitoneally to 10 female BDF1 mice (24 ± 3 g weight) per group implanted sq with 10⁶ L1210 cells, beginning one day after tumor implantation (Day 1), continuing every day for 13 days. The control was injected with PBS daily for 13 days. The results showed that the L1210 tumor did not respond to 250 µg/kg TNF (the maximal tolerated dose) or up to 5 million units/kg of IL-2 when these agents were administered alone. A dose of either over about 260 µg/kg TNF or 937,500 units/kg IL-2 resulted in a body weight loss of over 20%, indicating toxicity. When administered together, the IL-2 and TNF treatment produced no tumors, except if 250 µg TNF per kg was combined with only 9,990 units of IL-2 per kg host weight. 3. Table III indicates the results obtained when TNF alone, IL-2 alone, and various mixtures of TNF and IL-2 (prepared in vitro ) were administered per kg mouse weight intraperitoneally to five female BDF1 mice per group implanted sq with 1 x 10⁶ B16 cells, beginning one day after tumor implantation (Day 1), continuing every day for 14 days. The control was injected with PBS daily for 14 days. The combination of TNF and IL-2 prevented tumor growth, whereas IL-2 or TNF alone did not prevent it. The murine B16 melanoma is very similar to the human melanoma, and therefore many studies have been done on this cell line. The fact that the TNF and IL-2 combination is effective for B16 cells indicates that it may be effective in treating human melanoma. It was found that the murine tumors L1210, P388 and B16 were basically refractory to TNF whether the tumors were located intraperitoneally or subcutaneously. A marginal reduction in tumor size was observed with L1210. This refractoriness existed whether the TNF treatments were 1, 5 or 10 days after tumor implantation. 4. This experiment was conducted to help define the optimum schedule for administering the combination of IL-2 and TNF. The most rigorous model with which the tumors did not appear (the latest day after tumor implant for successful therapy) was determined. In this experiment, L1210 tumor cells were implanted in groups of animals and treatment was initiated either 1, 3, 7, 10 or 14 days afterward. Table IV indicates the results obtained when a mixture of 250 µg/kg TNF and 39,060 units/kg IL-2 was administered intraperitoneally to five female BDF1 mice per group implanted sq with 5 x 10⁶ L1210 cells, beginning 1, 3, 7, 10 or 14 days after tumor implantation, continuing every day up to 20 days from initial implantation. The control was injected with PBS daily for 19 days. Groups 3-5 and the PBS control had palpable tumors on Day 7. The data show that the most rigorous model is either a 3- or 5-day one (tumor growth cannot be prevented if treatment is initiated 7 days after tumor implant). 5. Table V indicates the results obtained when TNF alone, IL-2 alone, and various mixtures of IL-2 and TNF per kg mouse weight were administered intraperitoneally to five female BDF1 mice per group implanted sq with 3 x 10⁶ P388 leukemia cells, beginning one day after tumor implantation (Day 1), continuing every day for 14 days. The control was injected with PBS daily for 14 days. All tumors grew progessively from Day 15 and were too large and irregular to measure by Day 21. Therefore, the daily dose of the combination of TNF and IL-2 did not work in the P388 tumor model. 6. In this experiment, IL-2-PEG was used in place of IL-2 and a dose every other day was administered rather than a daily dose. Table VI indicates the results. The results indicated that the treatment with IL-2 and TNF on days 1, 3 and 7 was most effective, whereas daily treatment was not effective. 7. Table VII indicates the results obtained when a mixture of 12,500 units of IL-2 and 5 µg TNF (prepared in vitro ) was administered per kg mouse weight ip to five female BDF1 mice per group implanted sq with 1 x 10⁶ EL-4 mouse lymphoma cells, beginning one day after tumor implantation (Day 1), continuing every day for 14 days. the control was injected with PBS daily for 14 days. The combination of TNF and IL-2 prevented tumor growth of EL-4 lymphoma, whereas the control did not prevent it. 8. Table VIII indicates the results obtained when a mixture of 12,500 units IL-2 and 5 µg TNF (prepared in vitro ) was administered per kg mouse weight ip to ten female BDF1 mice per group implanted sq with 1 x 10⁶ B16 cells, beginning at 1, 3, 5, 7 and 10 days after tumor implantation (Day 1), continuing every day for 20 days. The results indicate that only small tumor burdens are cured when the combination therapy is employed. 9. Table IX indicates the results obtained when a mixture of 12,500 units IL-2 and 5 µg TNF (prepared in vitro ) was administered per kg mouse weight ip to five or ten female BDF1 mice per group implanted intraperitoneally (ip) or intravenously (iv) with 1 x 10⁵ B16W10 cells, beginning one day after tumor implantation (Day 1), continuing every day for at least 14 days. The controls were injected ip or iv with PBS daily for at least 14 days. After 14 days the animals which were injected iv were sacrificed and their lung colonies counted as black nodules, indicated as metastases per set of lungs. The results indicate that for intravenously implanted tumors there is no artificial pulmonary metastasis. For intraperitoneally implanted tumors there is a significant prolongation of life over the control. Therefore, this experiment indicates that the administration of IL-2 and TNF works with tumor cells located anywhere in the body, not just at subcutaneous locations. EXAMPLE 2 When the target cells implanted into the mouse host were a methylcholanthrene-induced sarcoma (Meth A) (Balb/c) (obtained as an ascites-passed tumor from Dr. Lloyd Old, Memorial Sloan Kettering, frozen as stock, and passed at least twice in ascites prior to use), it was found that 50 µg/kg mouse weight TNF alone or 15,625 units IL-2/kg mouse weight alone, injected ip daily for several days, caused complete regression of the tumors. However, within 60 days of implantation the tumors grew back in 80% of the mice. In contrast, when a mixture of 50 µg TNF/kg mouse weight and 15,625 units IL-2/kg mouse weight prepared in vitro was injected into the Meth-A mice ip daily for the same number of days, within 60 days after implantation none of the tumors grew back. The same IL-2 and TNF were used as employed in Example 1. The results show that the mixture of TNF and IL-2 gave a complete cure, whereas either component alone gave only a 20% cure in the Meth-A regression model, which is generally more sensitive to therapeutics than the models of Example 1. EXAMPLE 3 The experiments of Examples 1 and 2 were repeated several times (except no using P388) to generate data for between 10 to 50 animals per dose group. The maximum tolerated dose (MTD) was defined in these studies as the maximum amount of lymphokine(s) that could be injected such that no deaths occurred and body weight loss during and for five days after therapy was less than 5%. For TNF this MTD was found to be 250 µg/kg (5 µg/20 g mouse). For IL-2, a maximal soluble dose, 8 mg/ml, was utilized at a volume that maintained 0.1 ml for all therapeutic injections. Thus, IL-2 doses were 500-800 µg/kg (10-16 µg/20 g mouse) administered ip on a daily basis for 14 days. For purposes of this study, "significant" prolongation of life for ip tumor models is defined as time-to-death of greater than 150% of control (PBS treated) groups. Complete block of tumor take ("cure") is defined in the sq models as no measurable tumors evident for 60 days after initial tumor challenge. The results show that all animals developed sq tumors in the L1210, P815, B16W10 and EL-4 models, while 95% of the animals consistently developed sq Meth-A tumors. When the two lymphokines were evaluated for therapeutic efficacy as single agents, some initial growth inhibition was observed (notably with L1210 and P815) with TNF treatments when therapy was started one day after tumor challenge if TNF was administered at the MTD. A similar marginal effect was seen for some non-Meth-A tumor models (notably P815) when IL-2 was administered as a single agent daily for 14 days-again only when therapy was initiated within a day of tumor implant. In the Meth-A model, the lymphokines were more dramatically effective, because even a single dose of TNF resulted in regression of tumors that had been allowed up to 10 days to grow before therapy was initiated. Similar results were seen for 7-10 day old Meth-A tumors with high doses of IL-2 therapy. When either TNF or IL-2 was given as a single agent at repeated dosage over the first 14 days to animals bearing tumors only one day old, however, Meth-A tumor growth would be delayed about 30 days after cessation of therapy for a significant number of the animals, but a majority developed tumors by day 45. The results in non-Meth-A models showed that animals receiving an MTD of TNF simultaneously with an optimal (soluble) dose of IL-2 within 1 day after tumor challenge did not develop tumors. Interestingly, while the IL-2 dose in the combination could be cut back in some cases to 1% of the optimal, the amount of TNF in the mixture could not be reduced more than 50% in order to block tumor "takes." For a definition of susceptible tumor take periods for the various models using the IL-2 + TNF combination, a fixed combination dose (250 µg/kg TNF + 500 µg/kg of IL-2) that blocked tumor takes in the majority of models when treatment was initiated on day 1 was utilized and then the amount of time each of the tumor types could be allowed to grow was investigated prior to initiating effective combination therapy. The maximal allowable time for tumor take that still allowed for effective TNF + IL-2 therapy averaged 3-5 days, although for B16W10, therapy had to be initiated on day 1. Conversely for Meth-A, 10 day tumors were truly "curable" and exhibited regression. In each of these models, combination treatments beginning after the optimal tumor take period resulted in tumor growth inhibition but not in cures. Interestingly, growth inhibitory effects were seen only early during week one of the two week treatment period (except, of course, for Meth-A where the regression and growth inhibition lasted much longer) and tumors in animals receiving less than totally efficacious TNF + IL-2 doses grew rapidly to control levels during weeks 2 and 3. The results of single agent and combination (TNF and IL-2 proteotherapy for intraperitoneal models of the 5 murine tumors was studied. In all cases, treatments were initiated one day after tumor cell inoculations. While the combination of TNF and IL-2 blocked tumor take in the subcutaneous models, there were no similar blockages in the intraperitoneal models when these lymphokines were administered in combination or alone. However, a significant prolongation of life was seen for the peritoneal B16 melanoma, EL-4 lymphoma, and Meth-A tumors when the combination of IL-2 and TNF was administered using the same protocol as that which totally blocked tumor takes in the subcutaneous models. Finally, combination IL-2 and TNF therapy was compared with single agent administration in animals inoculated intravenously with B16W10 melanocytes. Studies similar to those testing tumor cell burden-take period for the subcutaneous tumors were also performed so that the maximum amount of time that could be allowed for tumor growth prior to starting curative therapy could be more clearly defined. Treatments with IL-2 and TNF when administered concomitantly at an optimal dose were synergistic if treatment was initiated one day after tumor implant. When treatment was initiated three days after implant, the number of pulmonary metastases were significantly less than controls, but all animals had tumors. In conclusion, combination TNF + IL-2 therapeutic synergy was found to (a) require TNF at a maximal tolerated daily dose; whereas the amount of IL-2 in the daily regimen could be cut back as much as 99%, (b) be tied to tumor burden or the amount of time that implanted cells were allowed to take prior to initiating therapy, and this time varied depending on tumor type, and (c) be effective for subcutaneous and pulmonary tumors but not result in blocking the take of intraperitoneal tumors. The synergistic effects of TNF and IL-2 in these models are most certainly due to a complex set of interactions. In addition to an apparent dependence on the host's tumor burden for successful immunotherapy, synergy between TNF and IL-2 may be explained, without limitation to any one theory, by (a) direct TNF action on tumor cells, (b) an increase in cytolytic cell, IL-2 receptor expression perhaps as an indirect result of TNF action on heterogeneous cell populations (e.g., macrophage), causing the release of other lymphokines (e.g., IL-1 which then affects IL-2 receptor expression), or (c) by direct activation of cytolytic cells by both IL-2 and TNF. It is possible, in fact, that the combination hyperactivates T cells or initiates LAK-like activities. The effects reported here are most likely due to such effector cell phenomena, as evidenced by the fact that the identical combination of TNF and IL-2 does not block tumor take for these same tumors when grown in nude or NIH-3 (Beige-nude-XID) mice that are CTL and LAK and CTL deficient, respectively. EXAMPLE 4 In this example the sequence of administration of IL-2 and TNF was evaluated to determine optimum protocol. The following experiments were performed: A. Meth-A Tumors 1. TNF Following by IL-2 Using the Meth-A tumor model wherein the tumor was subcutaneous, groups of five Balb/c mice bearing the tumor for seven days (or eleven days in one case of PBS + IL-2) were randomized, earnotched and then treated (Day 0) with TNF, IL-2, TNF followed by IL-2, PBS, or PBS followed by IL-2. The usual termination of tumor volume, body weight and tumor weight measurements was on Day 14. As noted, some groups in these experiments were held for 43 days to assess the frequency of long-term cures (i.e., where the tumor was completely eradicated). The protocols for the experiments are given below; all agents were delivered intraveneously in 0.2 ml volumes (ku = kilounits). The results are shown in Table X, where ΔBW is the ratio of the mean body weight at day 14 to the mean body weight at day 0 within a single group of mice, and where ΔTW is the ratio of the mean tumor volume at day 14 to the mean tumor volume at day 0 within a single group of mice. In conclusion, the administration of TNF followed by IL-2 significantly enhanced the anti-tumor efficacy as compared to either agent alone, resulting in long-term cures in the group treated with 20 ku/dose IL-2. At doses of 5 and 20 ku/dose with either a day 7 or day 11 tumor, IL-2 or TNF alone had little/no effect on efficacy. 2. IL-2 Followed by TNF Using the Meth-A tumor model wherein the tumor was subcutaneous, groups of five Balb/c mice bearing the tumor for seven days (or eleven days in the case of PBS + IL-2) were randomized, earnotched, and then treated (Day 0) with TNF, IL-2 followed by PBS, PBS followed by TNF, PBS/SDS, or IL-2 followed by TNF. The termination of tumor volume, tumor weight and body weight measurements was on Day 14. The protocols for these experiments are given below; all agents were delivered intravenously in 0.2 ml volumes (ku = kilounits). The results are shown in Table XI, where ΔBW and ΔTW are defined for Table X above. The results indicate that no enhancement in efficacy was observed when IL-2 was administered prior to TNF. Without limitation to any one theory, there was a hint of reduction of the TNF killing when IL-2 was administered first, as if IL-2 modulated the sensitivity of the tumor, or of the host, to TNF so as to render this tumor more resistant to TNF killing. B. L1210 Model 1. TNF Followed by IL-2 Using the L1210 tumor model described in Example I, groups of five BD2F1 mice implanted sq with 3 x 10⁶ L1210 cells on Day 0 were treated intraperitoneally on Day 3 with TNF and IL-2 together, TNF followed by IL-2, PBS, or IL-2 followed by TNF. The results are shown in Table XII. At Day 27, and after 60 days, Group 2 still had no evidence of tumor formation in this rigorous tumor model. The difference in results between Groups 1, 2, 4 and 5 indicates that scheduling and dosing as well as sequencing of administration are important in obtaining good response in the L1210 model. EXAMPLE 5 The combination of TNF and IL-2 administered ip dally for 14 days and in various 14-day sequences has shown efficacy against the B16 subcutaneous tumor. This experiment was designed to test whether the administration of TNF and IL-2 in a "clinical" schedule (e.g., weekends off) shows an equivalent effect. In addition, the combination of IL-2 given intramuscularly (im) and TNF given ip daily for 14 days was tested for efficacy. In this experiment BDF1 female mice, 5 per group, were injected subcutaneously with 5 x 10⁶ B16 cells per mouse on day 0. Treatment began on day 1. All injections were given ip except where noted. Tumor measurements were taken on days 10, 14, 21, 28, 35 and 42. Each group of mice was treated according to the following schedule, with 0.25 mg/kg TNF and 1 mg/kg IL-2 administered each time where noted. The endpoint was taken when the tumor volumes reached greater than 2000 mm³ or when there were no tumors after more than 60 days. The results are shown in Table XIII. The results show that none of the TNF/IL-2 dosing schedules mimicking a weekend off used in this study showed any efficacy. It appears that in the B16 subcutaneous tumor model, administrations of either TNF, IL-2 or the combination must occur within a 24 hour period, and for a duration of greater than 7 days to be efficacious. All of the mice receiving TNF ip and IL-2 im were dead by the eighth dose. In addition, control animals that were given an equal volume of saline im died after nine injections. It appears then that the test group could not tolerate the actual injection and that death was not related to the test material. EXAMPLE 6 This experiment tested previously efficacious combinations against ten-day tumor burden to determine their effectiveness in a more rigorous model. In this example BDF1 female mice, 5 per group, were injected subcutaneously with 5 x 10⁶ B16 cells per mouse on day 0. Treatment began on day 11. All injections were given ip and tumor measurements were taken on days 10, 14, 21 and 28. Each group of mice was treated according to the following dosage and schedule. The endpoint was taken when the tumor volumes reached greater than 2000 mm³ or when there were no tumors after more than 42 days. The results are shown in Table XIV. The results show that the same dose and sequence schedule of TNF (0.25 mg/kg day 1-3) and IL-2 (1 mg/kg day 4-14) that was efficacious against a 1-day tumor was also effective in the more rigorous 10-day B16 subcutaneous model (Group 2). The alternating TNF and IL-2 schedule (Group 3) was not efficacious. The same amount of IL-2 and TNF given ip simultaneously daily for the first three days and continued for the next 11 days only worked in mice bearing tumors 1 or 3 days old, not in mice bearing tumors 10 days old (the mixture of IL-2 and TNF after 3 days was not as good as sequential administration). This suggests that the sequence may be better than a mixture. EXAMPLE 7 A. Experimental design: 1. Species: rat, CD strain 2. Duration of the treatment: daily for 14 days 3. Route of administration: I.V. 4. Dose levels excipient control, TNF alone at 50 µg/kg, IL-2 alone at 0.5 or 1.0 mg/kg, TNf/IL-2 combined at 50 µg/kg TNF/0.5 mg/kg IL-2 or 50 µg/kg TNF/1.0 mg/kg IL-2. 5. Number of animals per dose level: 5 males and 5 females. 6. Parameters evaluated: Mortality Body weights and body weight changes Clinical sign observations Gross necropsy findings Hematology Possible histophathologic evaluations B. Results: Body weight gain was reduces at both TNF/IL-2 combined groups when compared with TNF or IL-2 alone group. Except for 3 female rats that died after one injection of TNF/IL-2 combined dose level of 50 µg/kg TNF/1.0 mg/kg IL-2, all other study animals survived the 14 daily infections, "Bloody stool/diarrhea" was noted for 2 of 3 animals before they were found dead and all three animals had "fluid-filled G.I. tract" at necropsy. All three animals died of apparent TNF toxicity because "bloody stool/diarrhea" or "fluid-filled G.I. tract" was the typical finding for TNF toxicity in the rat. All animals surviving until 14 days of the study had isolated episodes of "bloody stool/diarrhea" only at day 1 and 2 of the study and they had no signs of either IL-2 or TNF toxicity at necropsy. Elevated leukocyte, neutrophil, lymphocyte, and eosinophil counts were noted in both male and female rats at TNF-IL-2 combined dose level of 50 µg/kg TNF/0.5 mg/kg IL-2 or 50 µg/kg TNF/1.0 mg/kg IL-2. Significant depressed erythrocyte count, hemoglobin concentration, and % hematocrit were also noted in both male and female rats in a dose-related fashion for both TNF/IL-2 combined dose levels. C. Summary: Based upon the results of this study, the maximum tolerated dose (MTD) was established at 50 µg/kg TNF/0.5 mg/kg IL-2 when combined TNF and IL-2 was administered intravenously to the rat for 14 consecutive days. This MTD was comparable with the MTD when TNF was treated alone and somewhat lower than when IL-2 was treated alone (the MTD for IL-2 alone was at 1.0 mg/kg). No different toxicity findings were observed when combined TNF and IL-2 were administered under this testing condition when compared to when TNF or IL-2 was given alone. Me true "no observable effect level" (NOEL) was not established from the results of this study because of the reduced body weight gain, elevated leukocyte and differential leukocyte counts, and decrease in erythrocyte counts and related parameters. In summary, the present invention is seen to provide a combination of TNF and IL-2 which has anti-tumor activity and, furthermore, which does not cause significant increased toxicity in mammalian hosts. It is unexpected that TNF, which kills some cells in human tumor models in vitro but not in nude mouse xenograft models of those cells tested to date in vivo , nor in the classical murine tumor models in vivo , would be an effective anti-cancer agent when combined with small amounts of IL-2. It is also unexpected that the TNF and IL-2 cytokine mixture does not cause significant increases in toxicity (because it is known that the combination of IL-2 and IFN-γ is significantly more toxic than either agent alone).

Claims: 1. A composition for parenteral or subcutaneous administration to mammalian hosts for therapeutic or prophylactic treatment of cancer comprising a mixture of TNF and IL-2 in synergistically effective amounts, wherein the TNF and IL-2 are from a mammalian species. 2. A composition according to claim 1 further comprising a pharmaceutically acceptable carrier medium for the TNF and IL-2. 3. A composition according to claim 1 or 2 wherein the TNF is human or rabbit TNF and/or the IL-2 is human IL-2. 4. A composition according to claim 3 wherein the TNF is a mutein with the first eight amino acids deleted and the IL-2 is desala₁-IL-2 serl25 . 5. A composition according to claim 4 wherein the amount of TNF is 230-260 µg TNF per kg of host weight and the amount of IL-2 is 15,000-800,000 units IL-2 per kg of host weight. 6. A composition according to any one of claims 1-5 wherein the cancer is leukemia, melanoma, mastocytoma or lymphoma. 7. Products containing TNF and IL-2 as a combined preparation for simultaneous, separate or sequential use in the therapeutic or prophylactic treatment of cancer in human patients. 8. Products according to claim 7 wherein TNF and IL-2 are to be administered sequentially and the administration of TNF precedes the administration of IL-2. 9. Products according to claim 7 or claim 8 and further defined by the specific feature(s) of any one or more of claims 3-6. 10. The use of both TNF and IL-2 in the production of a medicament for the prophylaxis or treatment of cancer. 11. A method of making a therapeutic or prophylactic formulation for administration to mammals for therapeutic or prophylactic treatment of cancer comprising formulating together, whether by mixing or providing separate doses or otherwise, synergistic amounts of mammalian TNF and IL-2.

Inventors: WINKELHAKE, JEFFREY LEE, ZIMMERMAN, ROBERT
Applicants: CETUS CORPORATION
Application year: 1987
Main classification: A61K37
IPC codes: A61K38, A61K45, A61P35