PT1007082E - Modified tumor necrosis factor - Google Patents

Description

1

DESCRIPTION " MODIFIED TUMOR NECROSIS FACTOR "

This invention is directed, inter alia, to polyethylene glycol-modified tumor necrosis factor having a molecular weight in the range of 10,000 to 40,000 and for the use of a therapeutically effective amount of the modified TNF for the preparation of a pharmaceutical composition for treating tumors . Malignant melanoma (stage 3) is a fatal disease that kills most patients within a year of diagnosis. The incidence of melanoma is increasing rapidly in the United States and is even higher in other countries, as is the case in Australia. Treatments are urgently needed for patients suffering from melanoma. Kidney cancer currently kills about 13,000 individuals per year in the United States. This form of cancer is often undetected until it is in a very advanced state. The only form of treatment that significantly affects the patient's prognosis is surgical resection of the affected organ. Unfortunately, because this type of cancer is extremely metastatic, total removal of all metastases is difficult, if not impossible. Colon cancer is one of the most prevalent forms of cancer and currently kills about 140,000 individuals per year in the United States. Although there are a large number of traditional chemotherapeutic drugs developed to treat this disease, long-term survival 2 (defined as the percentage of patients surviving five years or more) has not appreciably changed over the past four decades. In addition, all traditional chemotherapeutic drugs are extremely toxic, have deleterious and often fatal side effects, and are costly. A curative, non-toxic treatment for this disease is urgently needed.

A distinctive feature of melanomas, and of renal and colon tumors is that these tumors rapidly develop resistance to traditional chemotherapies. Although patients may respond initially to chemotherapeutic treatment, drug-resistant tumors develop rapidly and eventually kill the patient. An alternative way to treat these tumors would be to identify an " Achilles heel " in tumors and to develop therapies that selectively treat this target. Such a potential target has been identified. Specifically, it has been found that all these types of tumors require intense vascularization of each of the metastases for the cancers to grow. Therefore, it may be envisaged that a therapeutic agent that inhibits the vascularization of these tumors may provide a single means for treating these tumors. Tumor necrosis factor (TNF) is one of many cytokines, which are a group of different proteins produced by leukocytes and related cells, with immunostimulatory activity. TNF was originally designated by its ability to cause tumor necrosis. There are at least two different mechanisms through which TNF is believed to kill tumors. The first is through a direct effect on the tumor itself. 3

Alternatively, TNF can selectively abolish tumor vascularization. In an early manuscript describing TNF, Carswell and Old reported that METH A tumor cells were completely resistant to TNF in vitro. J. Proc. Natl. Acad. Sci., 72: 3666-3670 (1975). However, METH A tumors in mice were extremely sensitive to TNF in vivo. This is thought to be due to the fact that TNF selectively abolishes the vascularization of these tumors. It has been shown later that some as yet unidentified factor is released by some tumors that make normal vascular endothelial cells adjacent thereto susceptible to death by TNF. Briefly, TNF kills these tumors not by direct death of tumor cells but rather by the death of normal vascular endothelial cells lining the blood vessels that supply the blood, oxygen and other nutrients necessary for the tumor to live and grow.

Unfortunately, the initial clinical trials attempted to develop TNF as a direct tumoricidal agent. When used in this way, TNF must be injected in high doses since it abandons rapidly (less than 20 minutes) the circulation. In addition, these high doses of TNF induce symptoms of the " shock " characterized by a dizzying drop in blood pressure. An alternative way to use TNF would be to formulate it so that it remained in circulation. In doing so, the TNF would have more time to interact with the vasculature of the tumor and would have enough time to nullify the blood supply to the tumor. Several other proteins have been formulated with polyethylene glycol (PEG) so that they circulate for longer periods and remain in the vasculature. These proteins include asparaginase, adenosine deaminase and superoxide dismutase. See, for example, Harras, J.M., in " Polyethylene Glycol Chemistry: Biotechnical and Biochemical Applications, " Plenum Press (1992). One group of Japanese researchers previously described that TNF could be formulated with certain PEGs and that the resulting material had considerably greater circulating half-lives and increased antitumor activity. Tsutsumi, Y., et al., Jap. J. Cancer Res., 85: 9-12 (1994); Tsutsumi, Y., et al., Jap. J. Cancer Res., 85: 1185-1188 (1994); Tsutsumi, Y., et al., Jap. J. Cancer Res., 87: 1078-1085 (1997). These investigators used only PEG with a molecular weight of 5000 coupled to primary TNF amines with a succinimidyl succinate linker group.

It has now surprisingly been found that modification of TNF with polyethylene glycol (PEG) having a weight average molecular weight in the range of 20,000 to 40,000, preferably in the range of 20,000 to 30,000, significantly increases the circulating half-life of TNF and also enhances the tumoricidal activity of TNF. For example, by this modification, serum half-life of TNF has increased from as little as 20 minutes for up to fifteen days, and the antitumor ED50 decreases by as much as 1000-3000 IU to as little as 10-50 IU. As a result of the modification, smaller doses of TNF can be administered to effectively treat the tumors, with fewer harmful concomitant side effects to the patient. 5

Accordingly, this invention relates to modified TNF, wherein said modification comprises covalently attaching to said TNF, either directly or through a biocompatible binding agent, one or more PEG molecules having a weight average molecular weight in the range from 20,000 to 40,000. TNF is modified with five to twelve of the PEG molecules, preferably with about five to nine PEG molecules.

This invention also relates to the use of a therapeutically effective amount of modified TNF for the preparation of a pharmaceutical composition for treating a patient suffering from a tumor.

This invention further relates to a method for increasing the circulating half-life of TNF which comprises modifying said TNF by covalently attaching between about five and twelve PEG molecules with a weight average molecular weight in the range of 20,000 to 40,000 .

This invention further relates to a method for increasing the tumoricidal activity of TNF which comprises modifying said TNF by covalently attaching between about five and twelve molecules of PEG having a weight average molecular weight in the range of from 20,000 to 40,000. Figure 1 is a graph depicting the circulating half-life of native TNF-α (blank circles), PEG-SS MM 5,000-TNF-a (closed circles) and PEG MM 20,000-TNF-a (blank triangles) in mouse serum. Figure 2 is a graph depicting the circulating half-life of native TNF-α (blank circles), PEG-SS MM 5,000-TNF-Î ± (closed circles), PEG-SS MM 12,000-TNF-Î ± (triangles PEG NHS MM 12,000-TNF-Î ± (filled squares) and PEG NHS MM 20,000-TNF-Î ± (blank squares) in mouse serum . " Tumor necrosis factor " or " TNF " as used herein encompasses either naturally occurring proteins such as isolated human or mouse TNF proteins or proteins produced using recombinant technology such as recombinant murine TNF and recombinant human TNF. Although the TNF-α protein is preferred, the term " TNF " also covers the TNF-β protein. The terms also encompass TNF proteins that have been mutated by amino acid deletion or alteration without significantly weakening biological activity (for example, as non-restrictive examples, amino acid deletion 212-220 or lysine alteration at 248, 592, 508 for alanine ). " Polyethylene glycol " or " PEG " refers to mixtures of condensation polymers of ethylene oxide and water in a branched or straight chain represented by the general formula H (OCH2 CH2) n OH. " Polyethylene glycol " or " PEG " is used in conjunction with a numerical suffix to indicate the approximate average molecular weight of the latter. For example, PEG 5,000 refers to polyethylene glycol having a weight average molecular weight of about 5,000; PEG 12,000 refers to polyethylene glycol having a weight average molecular weight of about 712,000; and PEG 20,000 refers to polyethylene glycol having a weight average molecular weight of about 20,000. These polyethylene glycols are accessible from various commercial sources and are routinely referred to by the weight average molecular weights as indicated above. &Quot; melanoma " may be a malignant or benign tumor originating in the melanocytic system of the skin and other organs, including the oral cavity, esophagus, anal canal, vagina, leptomeninges, and / or the conjunctiva or eye. The term " melanoma " includes, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, lentigo malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial disseminated melanoma. " Sick " refers to an animal, preferably a mammal, more preferably a human. " Biocompatible " refers to materials or compounds which are generally non-detrimental to biological functions and which will not give rise to any unacceptable level of toxicity, including allergic and pathological conditions. " Circulation half-life " refers to the time period after injection of the modified TNF in the patient until the TNF amount has been eliminated at levels that are half the maximum initial serum level. The circulating half-life may be determined in any relevant species, including humans or mice. &Quot; Covalently linked " as used herein refers to a covalent linkage binding the TNF protein to the PEG molecule either directly or through a linking group.

According to this invention, TNF is modified with polyethylene glycol having a weight average molecular weight in the range of 20,000 to 40,000, preferably in the range of 20,000 to 30,000. Generally, polyethylene glycol having a molecular mass of 30,000 or more is difficult to dissolve and the yields of the formulated product are very low. The polyethylene glycol may be branched or straight chain, but is preferably a straight chain.

Polyethylene glycols may be attached to TNF through biocompatible linking groups. As discussed above, " biocompatible " indicates that the compound or group is non-toxic and can be used in vitro or in vivo without causing injury, disease, disease or death. The PEG may be attached to the linking group, for example via an ether linkage, an ester linkage, a thiol linkage or an amide linkage. Suitable biocompatible linking groups include, for example, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a maleimide group (including, for example, succinate of succinimidyl (SS), succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxysuccinimide (NHS), an epoxide group, an oxycarbonylimidazole group (including, for example, nitrophenyl group (NPC) or trichlorophenyl carbonate (TPC)), a trisylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine. In another preferred embodiment, the biocompatible linker is an ester group and / or a maleimide group and binds to TNF via a primary amine in the TNF protein. More preferably, the linking group is SS, SPA, SCM, SSA or NHS; most preferred of all the.

Alternatively, TNF can be coupled directly to PEG (i.e., without a linking group) through an amino group, a sulfhydryl group, a hydroxyl group or a carboxyl group.

Methods for covalently attaching TNF to PEG either directly or via a biocompatible linking group are known in the art, as described for example in Harras, J.M., in " Polyethylene Glycol Chemistry: Biotechnical and Biochemical Applications, " Plenum Press (1992), the disclosure of which is hereby incorporated by reference. It is preferred that the TNF protein is covalently attached to five to twelve PEG molecules. Methods for determining the number of PEG molecules bound to the protein are known in the art, for example, Habeeb, A.F.S.A., Anal. Biochem., 14: 328-339 (1966); Harras, J.M., supra., Incorporated herein by reference. The number of PEG molecules bound to TNF will vary according to the linking group used, the duration of the reaction and the molar ratios of TNF and PEG used in the reaction.

As a person skilled in the art will recognize, the modified TNF of this invention may be administered in a number of ways, for example, orally, intranasally, intraperitoneally, parenterally, intravenously, intrathecally, intratumorally, intramuscularly, interstitially, intraarterially, subcutaneous, intraocular, intra-synovial, transepithelial and transdermal. A therapeutically effective amount of one of the modified compounds of the present invention is an amount effective to inhibit tumor growth and that amount may vary according to the method of administration. In general, effective doses should be in the range of about 0.001 to 0.1 mg / kg once a week. Modified TNF can be formulated with pharmaceutically acceptable carriers and diluents, as is known in the art. For example, for intravenous administration, the modified TNF may be mixed with a solution of phosphate buffered saline or any other suitable solution known to those skilled in the art prior to injection. Assays have shown that modified TNF is particularly effective in the treatment of melanoma, colon cancer, renal cancer and breast cancer tumors. The invention is further explained in the following examples, which are provided for the purpose of illustration and are not intended to restrict the scope of the present invention. The TNF used in the experiments described below was of the type found in mice and in humans. Human TNF (whole protein) was produced in E. coli and murine TNF as well as human TNF mutants were produced in Pichea pastoris. Recombinant TNF was produced in E. coli or Pichea using methods similar to those described in Pennica, D., et al., Nature, 312: 724-729 (1981); Streekishna, K., et al., Biochemistry, 28: 4117-4125 (1989). Mouse TNF was produced in E. coli and Pichea. 11

Example 1

Specific Activity of TNF-α

Prior to pegylation mature tissue necrosis factor (TNF-α) (human recombinant) was evaluated using an L929 cytotoxicity assay as described below. The specific activity of TNF-α was 106 units U.I. per milligram. Densitometry of an SDS-PAGE gel showed that the material was 99% pure.

The material (1 ml at 16.8 mg / ml, determined by the method of Bradford, MM, Anal. Biochem., 72: 248-254 (1976)) was concentrated with a bovine serum albumin (BSA) standard, with the modification introduced by Peterson) to approximately 0.1 ml using a Centricon with a 3,000 kDa limit. This material was then diluted to 4 ml with 100 mM phosphate buffer, pH 8.0 and concentrated again to 0.1 ml. This procedure was repeated twice, and the resulting material was finally collected in a total volume of 1 ml of the same buffer.

The protein concentration was then determined by the Bradford method, supra. BSA was used as a standard and about 0.6 mg of protein was recovered.

Pegylation

PEGylation reactions were performed using the general methods described in Harras, J. M., quoted above. To the TNF (1 mg / ml in 100 mM phosphate buffer, pH 7.2-7.5) the SS-PEG or NHS-PEG was added to an excess of 10 to 50 molar and mixed for one hour at room temperature. The pH and molar ratio used varied with the PEG reactivity and had to be determined empirically. PEG-TNF was separated from unreacted PEG and TNF by ultracentrifugation using a filter with a 100 kDa limit. In each of the modifications referenced in this example, TNF was modified with five to nine PEG molecules. PEG-TNF purity was assessed by SDS-PAGE and the percentage of primary amines modified by this procedure was determined using florescamine as described by S.J. Stocks (Anal.Biochem 154: 232 (1986)). SDS-PAGE results indicated that very little, if any, native TNF-α remained in the preparation after pegylation.

In vitro activity of PEG-TNF-α PEG-TNF-α was evaluated in terms of cytotoxic activity in vitro using the L929 cytotoxicity assay performed according to the procedure described below, except that cell number was not known. The specific activity of the TNF-α starting material was 1.5 x 106 units / mg or about half of the measurement of the original specific activity. Differences in specific activity were attributed to the use of a method of determining the different protein and the number of unknown passages of the cells. The specific activity of PEG-SS MM 5,000-TNF-a was 0.7 x 10 6 units / mg or about half of the specific activity of the native material. The specific activity of PEG-SS MM 20,000-TNF-a was 0.8 x 106 units / mg, a value similar to PEG-SS MM 5000-TNF-a. 13

Lethality of PEG-TNF-α

As a screen, two C57 bl6 (female, 20-25 g) mice were injected intraperitoneally (i.p.) either with native TNF-α or with PEG-SS-TNF-α and the survival of the animals was monitored. The doses used were 1, 5 and 10,000 units of activity.

With native TNF-α the following results were obtained: 10,000 U.I. - both mice were dead the next morning 5000 U.I. one of the mice was dead the next morning; the second mouse was in obvious difficulties (rough hair and little movement) and died after 2 days 1,000 U.I. one of the mice was dead the next morning; the second mouse was in trouble (rough hair and little movement) and in such a bad state that it was euthanized after 2 days

With PEG-SS-TNF-α, all mice at all doses remained healthy for two weeks post-injection. The behavior was normal, as was eating behavior and drinking. There was no change in the hair (the hair was not bristling). All mice were euthanized 15 days after injection.

Determination of serum half-life of PEG-TNF-α

An ELISA assay for human TNF obtained from Genzyme was used. The case was used as suggested by the manufacturer. Mice were either injected with either TNF-α or PEG-α (100 units) i.p. and approximately 25 μΐ serum was collected from retroorbital bleedings at the 14 times indicated in Fig. 1. A total of 5 mice (females, C57 bl6 mice, 20-25 g) were collected in each group. Native TNF-α (open circles) was eliminated very quickly and the only point above baseline was at 30 minutes post injection. PEG-SS MM 5,000-TNF-a (closed circles) had a half-life of about 4 days. The half-life of PEG MM 20,000-TNF-α (blank triangles) was > 15 days.

This experiment was repeated using the treatment groups listed below, with the results shown in Fig. 2: native TNF-α (blank circles); PEG-SS MM 5,000-TNF-Î ± (filled triangles); PEG-SS MM 20,000-TNF-a (blank triangles); PEG NHS MM 12,000-TNF-α (filled squares). The serum half-life for the different treatment groups was > 15 days for PEG NHS MM 20,000-TNF-Î ± and PEG-SS MM 20,000-TNF-Î ±; approximately 4 days for PEG-SS MM 5,000-TNF-a; approximately 6 days for PEG-SS MM 12,000-TNF-a; approximately 8 days for PEG NHS MM 12,000-TNF-a; and 30 min post injection for native TNF-α. Briefly, each PEG-TNF-α exhibited a much longer half-life than native TNF-α; however, PEG NHS MM 20,000-TNF-a and PEG-SS MM 20,000-TNF-a had significantly longer half lives (> 15 days) than the lower molecular weight PEG-modified TNF-α.

Antitumor Activity of PEG-TNF-Î ± 15

The B16 melanoma model was used. C57 bl6 mice (females, 20-25 g) were injected with one million B16 cells, s.q. on the flank. The tumors were allowed to grow for a week before starting treatment. Each treatment group consisted of 5 mice.

Treatment groups included: control of phosphate buffer (pH 7.5), native TNF-α (10 IU and 100 IU) in phosphate buffer (pH 7.5) and PEG-SS-TNF-α (10 IU , 100 IU and 1000 IU) in phosphate buffer (pH 7.5). The mice were injected with 0.1 ml i.p. once a week on Day 7, Day 14 and Day 21. The survival of the animals was recorded.

After the 2nd Week (one week after the first treatment), the tumors in the control animals were well visible. None of the animals treated with PEG-SS-TNF-a appeared to have growing tumors.

On Day 22 the results presented in Table 1 were obtained:

Table 1

Treatment Group Results Control 4 killed, 1 with large tumor native TNF-α 10 U.I. all dead animals 100 U.I. 4 killed, 1 with large tumor PEG-SS MM 5000-TNF-a 10 U.I. 2 dead, 3 with large tumors 100 U.I. 2 dead, 2 with tumors, 1 without tumor 1000 U.I. 1 killed, 3 with small tumors, 1 without tumor PEG-SS MM 20000-TNF-a 10 U.I. 0 dead, 1 with small tumor, 4 without tumor 100 U.I. all animals without tumors 1000 U.I. all animals without tumors 16

Table 2

Treatment Group Mean Survival Time Control 18, 18, 20, 21, 24 days 20.2 days Native TNF-Î ± 10 U.I. 17, 18, 19, 21, 21 days 20.2 days 100 U.I. 16, 18, 19, 19, 23 days 19.0 days PEG-SS MM 5000-TNF-a 10 U.I. 20, 22, 24, 26, 27 days 23.6 days 100 U.I. 21, 22, 24, 26, 27 days 35.0 days 1000 U.I. 21, 49, 53 days; 2 live animals PEG-SS MM 20000-TNF-a 10 U.I. 38 days, 4 live animals 100 U.I. all live animals 1000 U.I. all live animals

These results indicate that modification of TNF with PEG does not only reduce TNF lethality but that PEG-modified TNF with a molecular mass of approximately 20,000 exhibited surprisingly increased circulating half-lives and surprising and significantly increased antitumor activity.

These results are surprising for a number of reasons. There was no way to predict that the TNF modification with high molecular weight PEG would increase the circulating half-life of TNF. In fact, the rate of elimination of proteins in general can not be predicted based on their molecular mass. That the TNF modification with the high molecular weight PEG not only does not decrease, but in fact increases, the tumoricidal activity of TNF is surprising in view of the steric hindrance that should be created by the high molecular weight modifier. Furthermore, it was expected that the modified TNF, due to its increased circulation half-life, would be even more toxic than native TNF, which was not the case. The following is a description of the cytotoxicity assay used in this example. A. Materials

Fibroblasts L929 ATCC # CCL1 NCTC clone 929.

Essential Media Modified by Dulbecco (DMEM)

Fetal Serum Bovine (GIBCO Laboratories, Grand Island, NY # 16000-010) 1M HEPES Buffer in Normal Physiological Serum (BioWhittaker, Inc. # 17-737E)

(GIBCO Laboratories, # 25300-021) Trypsin Blue Developer (GIBCO Laboratories, # 15250-012) Gentamicin Sulfate (BioWhittaker, Inc., # 17-518Z)

Tissue Culture Balloons, 150cm2, 75cm2, 25cm2 (Corning, Inc., Corning, NY, # 25120, # 25110, # 25100)

96 well plates for cell culture, flat bottom (Corning Inc., # 25861)

12 Channel Pipetter (Titertek)

Sterile tips for pipettor (Intermountain Scientific Corp., Bountiful, UT, # P3250-8)

Tanks for Sterile Reagents (Costar Corp., Cambridge, MA, # 4870)

Tumor Necrosis Factor-Recombinant Human α (TNF-a) (prepared in the laboratory)

Albumin, bovine, 10% in IMEM (Boehringer Mannheim Corp., Indianapolis, IN, # 652237)

Phosphate Buffered Saline (PBS)

Dulbecco's Phosphate Buffered Saline (D PBS) (GIBCO Laboratories, # 3104190) 18

Microparticle Plates Reader (Molecular Devices Corp., Menlo Park, CA, Emax) B. L929 Fibroblast Spread: 1. Keep the fibroblasts passing twice a week in DMEM / 10% FBS as follows. Aspirate the medium of the flask, leaving the cells adherent. Wash the cell monolayer with 5 ml trypsin-EDTA and aspirate trypsin-EDTA. Incubate 1 to 2 minutes at 37 ° C. Add 15 ml of DMEM / FBS to wash the monolayer and stop the enzymatic activity of trypsin. 2. Count the cells and assess viability by exclusion with Trypan Blue. Inoculate 30 ml of DMEM / FMS without antibiotics in a T75 flask with 1 x 101 2/3 cells. Incubate in a humidified incubator at 37 ° C and 5% CO 2. 1

For the assay, inoculate 60 ml of DMEM / FBS into a T150 flask with 2 x 104 cells. Incubate the flask for 3 to 4 days until the cells approach confluence (80-290%). 3 C. In Vitro TNF-α Cytotoxicity Assay First Day: Preparation of Cells 1. Add trypsin to cells and dilute with DMEM / FBS to a final concentration of 1.22 x 104 cells / ml. Add gentamicin sulfate to a final concentration of 50 μg / ml. A cell count of 2 x 10 4 cells is required for each plate, and 1.75-2 x 19 may be expected. 2. Apply the L929 fibroblasts on the plates by adding 150 μl of the cell suspension to each well of the bottom 96 well plate plan for tissue culture. Incubate overnight in a humidified incubator at 37øC and 5% CO 2.

Second Day: Addition of Samples 1. Using an inverted microscope, check the confluence of each well before proceeding. Each well must be equally confluent so that the assay is reproducible. The outer wells of the plate are not used for assay calculations because of irregular cell growth in these wells. Cells must be at a confluence of 75 to 90% for best sensitivity. As the number of cell passages increases, the number of hours for duplication of cells decreases. The number of cells / well can be adjusted accordingly to obtain the correct confluence. 2. Prepare a working solution of the TNF-a standard. Store at 4 ° C until use. 3. Add 150 μΐ of the TNF working solution to the first well of columns 2 and 3. Add 150 μΐ of sample to the first well of Columns 4 through 10. Add DMEM / FBS / gentamycin to Columns 1, 11 and 12. Prepare dilutions in series to one half by mixing the contents of the first wells and transferring 150 μΐ to the second wells of the columns using a 12-channel pipettor. Finally, the contents of the 8 wells (row H) were mixed and 150 μΐ of each well was discarded. At this point, all wells should contain 150 μΐ. 4. Incubate the plates for 20 hours in a humidified incubator at 37øC, 5% CO2.

Third day:

The viability of the cells was determined by adding 20 μl of 3 [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) (25 mg / ml in phosphate buffered saline, pH 7.4 ) to each well of the culture dish and incubating the cultures at 37 ° C for four hours. After this time, the culture supernatants were discarded and 150 μl of DMSO was added to each well. The absorbance of each well was determined at 570 nm using a microtiter plate reader. Wells having an A540 close to 50% of the arithmetic mean of the control are considered to represent 50% lysis (1 unit) of the L929 cells.

D. SOLUTIONS

TNF-a standard (10 μ9)

Thaw the frozen TNF-α pattern on ice. Transfer aliquot to cryotubes with screw cap at 2 μg / tube and store at -80 ° C.

TNF-a Standard (256 ng / ml) stock solution To a tube (2 g) of TNF-α standard, add 7.81 ml of 1% albumin in D PBS / gentamicin. Transfer an aliquot into 1 ml tubes and keep at 4 ° C. The solution is typically used for six months.

TNF-α Working Solution (0.8 ng / ml)

In two plates, add 3.12 μΐ stock solution to 997 μΐ DMEM / FBS / gentamicin. (The standard solution will be diluted 1: 2 in the first well of the multiwell plate to give a final concentration of 0.4 ng / ml). Store at 4 ° C until use.

Physiological Serum Buffered with Phosphate 1L (PBS, lx)

Dissolve 0.2 g of KCl, 0.2 g of KH2 PO4, 8 g of NaCl and 1.14 g of Na2 HPO4 in 900 ml of water. Adjust the pH to 7.4 Q.B. for 1 L. Autoclave.

Example 2

Additional samples of PEG-modified human recombinant TNF (according to general methods described in Harras, Supra) were prepared and evaluated for serum half-life and for the conservation of specific activity. In each case, TNF was modified with about five to nine PEG molecules. The data (Table 3) surprisingly show that PEG-modified TNF with a mean molecular weight of about 10,000 to 40,000, preferably about 20,000-30000, exhibits significantly greater serum half-life while retaining a high specific activity.

The half-lives of the various TNF-IN-SERUM formulations were determined using ELISA assays obtained from Genzyme (Cambridge, MA), as suggested by the manufacturer. Serum samples from the retroorbital plexus were collected using 50 μ cap capillary tubes with heparin. A pre-treatment blood sample was collected immediately prior to i.v. injection with TNF or PEG-TNF formulations. Additional blood samples were collected at 30 minutes, 24 hours as well as at 3, 7, 12 and 15 days after treatment.

The samples were centrifuged and the resulting supernatant was preserved at -20 ° C until dosed.

Table 3

Serum half-life (days) Specified Retained Activity (%) PEG-SS 5000 TNF-Î ± (2 assays) 4, 55-58 PEG-SS 12000 TNF-Î ± (2 assays) 8,832,538 PEG-SS 20000 TNF-a 16 56 PEG-SS 30000 TNF-a 17 54 PEG-SS 40000 TNF-a 17 55 PEG-SS 5000 TNF-a 5 51 PEG-SS 20000 TNF-a 8 53 Branched chain succinimide-PEG 5000 TNF -a 7 49 Branched chain succinimide-PEG 20000 TNF-a 16 52 Branched chain succinimide-PEG 40000 TNF-α 18 54

Example 3

Additional samples of human recombinant TNF, modified with PEG were prepared and the serum half-life and the conservation of the specific activity were evaluated. The data presented in Table 4 show that the half-life and the specific activity may vary depending on the linking group used. 23

Table 4

PEG-SCM 5000 TNF-Î ± (5-9 PEG) 5 54 Amino acid succinimidyl ester-PEG 5000 TNF-Î ± (6-8 PEG) Serum half-life (days) ) PEG 8000 TNF-a epoxide (primary amine binding site) (9-12 PEG) 6 PEG 8000 epoxide TNF-α (hydroxyl attachment site) (10-20 PEG) 50 Ether glycidyl PEG 5000 TNF-a (15 PEG) 12 0 Nitrophenyl PEG 5000 TNF-a (6-9 PEG) 5 21 Trichlorophenyl carbonate PEG 5000 TNF-α (12-15 PEG) 5 11 PEG 5000 TNF-a (10-12 PEG) 2 8 PEG aldehyde 5000 TNF-α (1 PEG) <1 100 PEG aldehyde 20000 TNF-α (1 PEG) 2 100 PEG 5000 isocyanate TNF-α (5-12 PEG) 9 19 Vinylsulfone of PEG 5000 TNF-α (4 PEG) 3 12 Maleimide of PEG 5000 TNF-α (3 PEG) 3 43

Example 4

Additional tests were performed to evaluate the effect of recombinant human PEG-modified TNF in mice injected with several varieties of tumor cells. The TNF used in these assays was modified with PEG-SS 20000 according to a method which was described by Harras, Supra. The mice were injected with 1 x 106 tumor cells and injected two weeks later i.p. with PEG-TNF once a week for three weeks. Cure was defined as the percentage of animals that survived five times more than untreated animals. The results are presented in Table 5 and indicate that the modified TNF of this invention is effective in the treatment of melanoma tumors, renal tumors, colon tumors and breast tumors.

Table 5

Tumor Type Cell Line PEG-TNF Dose U.I. Cure (%) Melanoma BI 6 10 75 30 100 100 100 Kidney G401 10 80 30 80 Colon HT2 9 10 40 30 60 100 80 Breast MCF7 10 0 30 0 100 20 Brain SW1088 100 0 Leukemia L2110 100 0 Hepatoma Hep3B 100 0

Example 5 TNF of a variety The results are from

PEG-modified assays were carried out to evaluate origins. presented in Table 6.

Table 6

Protein TNF Specific Activity LD50 ED50 Hypotension ED50 Antitumor Recombinant murine TNF produced in Pichea 2.1 x 10 7 U.I./mg 20 pg 1 μg &gt; 1000 U.I. &quot; -modified with PEG-SS MM 20000 1.0 x 107 U.I./mg 100 μg 2 μg 20 U.I. Recombinant murine TNF produced in E. coli 2.0 x 10 7 U.I./mg 70 pg 1 μg &gt; 3000 U.I. &quot; -modified with PEG-SS MM 20000 1.0 x 107 U.I./mg 300 pg 4 μ5 20 U.I. Human TNF mutated by deletion of amino acids 212-220 2.1 x 107 U.I./mg 60 pg 1 μg &gt; 2000 U.I. &quot; -modified with PEG-SS MM 20000 0.9 x 107 U.I./mg 100 μg 5 μg 10 U.I. Human TNF mutated by changing lysine 248, 592, 508 to alanine 1.5 x 107 U.I./mg 300 μg 100 μg &gt; 1000 U.I. &quot; -modified with PEG-SS MM 20000 0.5 x 107 U.I./mg &gt; 1000 pg &gt; 100 μg 5 U. I.

Lisbon, October 31, 2006

Claims (17)

Modified TNF, comprising TNF covalently linked to between about five and twelve PEG molecules with a weight average molecular weight in the range of 20,000 to 40,000. The modified TNF of claim 1 wherein said PEG has a weight average molecular weight in the range of 20,000 to 30,000. The modified TNF of claim 1 wherein said PEG is covalently linked to said TNF through a biocompatible linking group. The modified TNF of claim 1 wherein said linking group is N-hydroxysuccinimidyl succinate. The modified TNF of claim 1 wherein said linking group is N-succinimidyl propionate. The modified TNF of claim 1 wherein said TNF is covalently linked to about five to nine PEG molecules. The modified TNF of claim 1 wherein said TNF is covalently linked to said PEG molecules through primary amines in TNF. The modified TNF of claim 1 wherein said TNF is TNF-a. The modified TNF of claim 1 wherein said TNF is isolated from human TNF. 1 2 The modified TNF of claim 1 wherein said TNF is recombinant human TNF. A method of increasing the circulating half-life of TNF comprising modifying said TNF by covalently attaching between about five and twelve PEG molecules with a weight average molecular weight in the range of from 20,000 to 40,000. A method of increasing tumorigenic TNF activity comprising modifying said TNF by covalently attaching between about five and twelve PEG molecules with a weight average molecular weight in the range of from 20,000 to 40,000. A method of using a therapeutically effective amount of the modified TNF of claim 1 for the preparation of a pharmaceutical composition for treating a patient suffering from a tumor. The use of claim 13 wherein said tumor is a melanoma. The use of claim 13 wherein said tumor is a colon cancer. The use of claim 13 wherein said tumor is a renal cancer. The use of claim 13 wherein said tumor is a breast cancer. Lisbon, October 31, 2006 2