WO1989000805A2

WO1989000805A2 - Treatment of infections with tumor necrosis factor

Info

Publication number: WO1989000805A2
Application number: PCT/US1988/002441
Authority: WO
Inventors: Kong Teck Chong
Original assignee: Cetus Corporation
Priority date: 1987-07-31
Filing date: 1988-07-20
Publication date: 1989-02-09
Also published as: AU2624988A; IL87182A0; EP0379523A1; JPH02504391A

Description

TREATMENT OF INFECTIONS WITH TUMOR NECROSIS FACTOR r

This invention relates to the use of tumor necrosis factor in the prophylactic or therapeutic treatment of infectious diseases.

It has long been known that microbial infection can be

5 inhibited by a variety of immunomodulating agents. These agents may be classified as: 1) crude immunosti ulant of microbial nature

(examples are ycobacterium boris strain BCG and the killed vaccine of corynebacterium parvum); and 2) chemically defined immunoadjuvants of bacterial origin (examples are 1 ipopolysaccharides). More recently,

10 various synthetic immunomodulating drugs have been shown to possess antiviral effects. The most promising of these, Inosiplex, has been tested in man and claimed to be effective against a variety of virus infections (Hepatitis A, recurrent Herpes Simplex, Herpes Zoster, influenza, rhinovirus-caused common cold). The response, however, has

15 not always been predictable and appears to vary with individual patients.

Cytomegalovi us (CMV) is one of the most important viral opportunistic infections in AIDS patients. The infection can often lead to severe and sometimes fatal disease, including

20 retinochoroiditis hepatitis, pneumonitis, esophagi tis, and disseminated disease. Indeed, CMV infection is so closely associated with the AIDS syndromes that the virus has been suspected of contributing to the immunosuppression that characterizes AIDS. See Acquired Immunodeficiency Syndrome (AIDS), ed. J.I. Gall in and A.S.

25 Fauci, Advances in Host Defense Mechanisms, Vol. 5, 1985. Heretofore, there is no known effective therapy.

Tumor necrosis factor (TNF) was first described by Carswell et al., PNAS (USA) (1975) _72_:3666-3670 as an endotoxin-induced serum factor that causes necrosis of chemically transformed tumor cells when 30 growing in mice. Human TNF is known to be cytotoxic to neoplastic cells, and has been produced in recombinant form. See Pennica et al . , Nature (London) (1984) 312:724-729 and Shirai et al., Nature (London) (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 asei ogyo Kabushiki). The cloning of human TNF having 151 and 155 ami no 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 U.S. Patent No. 4,677,063 issued June 30, 1987, (Cetus Corporation), the disclosures of which are incorporated herein by reference.

TNF has been reported by Mestan et al., Nature, 323:816-819 (1986), Wong et al . , Nature, 323:819-822 (1986), and Masayoshi et al . ,

Cell , 45_:659-666 (1986) to inhibit viral infection in various susceptible cells in vitro, when cells were pretreated in vitro with

TNF before virus challenge.

Accordingly, the present invention is directed to a method for prophylactic or therapeutic treatment of infections in mammalian hosts comprising administering to the host, before or after infection of the host, an effective amount of tumor necrosis factor (TNF) from a mammalian species, wherein the amount of TNF is a sufficient dose to achieve at least a 50% protection of the host. In a preferred embodiment the treatment is prophylactic for viral infections.

As used herein, the term "recombinant" refers to TNF produced by recombinant DNA techniques wherein generally the gene coding for the TNF is cloned by known recombinant DNA technology. For example, by using the human TNF cDNA as a template, the gene showing complementarity to the human TNF cDNA is inserted into a suitable DNA vector such as a bacterial plasmid, preferably an E. coli plasmid, to obtain a recombinant plasmid, and the plasmid is used to transform a suitable host. Tne gene is expressed in the host to produce the recombinant TNF protein. Examples of suitable recombinant plas ids for this purpose include pBR322, pCRl, pMB9 and pSCl. The transformed host may be eucaryotic or procaryotic, preferably a procaryotic host.

As used herein, the term "pharmaceutically acceptable" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the hosts to which it is administered.

As used herein, the term "prophylactic or therapeutic" treatment refers to administration to the host of the TNF either before or after infection. If the TNF is administered prior to exposure to the infecting agent, the treatment is prophylactic (i.e., it protects the host against infection), whereas if administered after infection, the treatment is therapeutic (i.e., it combats the existing infection). Preferably, the dose is administered from 18 hours before infection for prophylactic treatment and in early as well as in later phase of infection for therapeutic treatment.

As used herein, the term "infections" refers to any kind of infectious disease, including those caused by bacteria, fungi, viruses, protozoa or parasites. Examples of bacterial infections include j\_ aeruginosa, E. coli tetanus, Mycobacterium species, Streptococcal strains, diphtheria and Salmonella. Examples of fungal infections include cryptococcosis, histoplasmosis, and other infections due to Candida species. Examples of viral infections include Hepatitis A, recurrent Herpes Simplex, AIDS, Herpes Zoster, influenza, cytomegalovirus (CMV), and rhinoviruses. Preferably, the infection is viral, more preferably Herpes Simplex or CMV, or the infection is bacterial, more preferably Gram-negative, and most preferably P. aeruginosa and E. coli infection.

The method of this invention involves administering to a mammalian host, preferably a human host, an effective amount of TNF. The admιnistration(s) may take place by any suitable technique, preferably parenteral administration. Examples of parenteral administration include intravenous, intraarterial , intramuscular, and intraperitoneal , with intravenous being Dreferre . The dose and dosage regimen will depend mainly on whether the TNF is being administered for therapeutic or prophylactic purposes, the type of infection, the patient, and the patient's history. The amount must be effective to achieve a protection level of at least 50%, preferably at least 70%; dosages which do not achieve this minimal level of effectiveness may not be employed. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred. For purposes herein, a protection level of at least 50% means that at least 50% of the treated hosts exhibit improvement against the infection, including but not limited to improved survival rate, more rapid recovery, or improvement or elimination of symptoms. The preferred dosage of TNF is about 0.4- 2.0-^.g per kg body weight of TNF per dose.

For parenteral administration the TNF will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion), preferably in a pharmaceutically acceptable carrier medium that is inherently non- toxic and non-therapeutic or non- prophylactic. 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 that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The TNF will typically be formulated in such carriers at a concentration of about 0.1 mg/ml to 100 mg/ l , preferably 0.2 to 1 mg/ml.

As mentioned above, the TNF employed herein may be obtained 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 a human source and more preferably is a human recombinant TNF.

The recombinant human TNF may be obtained as described by Pennica et al . , Nature, _312_:724-729 t¹-⁹⁸⁴). Yamada et al . , J. Biotechnology, (1985) 2:141-153; 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 US85/01921 published April, 1986. The recombinant rabbit TNF may be obtained as described in EP 146,026 published June 26, 1985 and EP 148,311 published July 17, 1985. Preferably the TNF is a human TNF mutein that is N-terminally deleted, preferably wherein the first eight amino acid residues have been deleted, using the procedure described in U.S. Patent No. 4,677,063 issued June 30, 1987 or the TNF is a cysteine-depleted mutein described in copending U.S. Serial No. 698,939 filed February 7, 1985 and in U.S. Patent No. 4,518,584, supra (for IL-2 but applicable to TNF).

The various aspects of the invention are further described by the following examples, which are not intended to limit the invention in any manner. For example, the test used in the examples for % protection is % survival rate, which is only one test of many which may be employed to determine the extent of protection in the host. 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 - Use of IL-2 and TNF to Treat Gram-Negative Infections

TNF.

A mutein of human TNF having the first eight amino acids deleted from the N-terminus was . prepared as described in U.S. Patent No. 4,677,063. 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 li gated into expression vectors to obtain procaryotic expression of the mutein. The mutein was then suspended in a formulation buffer. IL-2 Preparation

The recombinant IL-2 employed in this example was des-ala^ IL-2_ser 25 - T e 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 £___ coli that produce this IL-2 have been deposited by Cetus Corporation in the American Type Culture Collection, 12301 Pa kl awn Drive, Rockv lle, 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 U.S. Patent No. 4,604,377, the disclosure of which is incorporated herein by reference, 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 sol ubil zing 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 was formulated at a concentration of 0.3 mg/ml with 50 mg/ml mannitol.

Bacteria E. col i SM18, a type 02 clinical isolate from a bacteremic patient at Saint Mary's Hospital, San Francisco, CA, was cultured overnight in brain-heart infusion broth (Difco Laboratories, Detroit,

MI) at 37 C, harvested by centrifuging at 6000 rpm for 15 minutes, washed in standard phosphate buffered saline (PBS) resuspended in brain-heart infusion broth containing 20% glycerol, aliquoted, and o stored at -70 C. The viability of the bacteria was determined by plating ten-fold serial dilutions onto trypti case- soy agar plates and counting the colony- forming units (cfu) after a 24-hour incubation at 37 C. The titre was expressed as the mean number of cfu/ml of stock bacteria. The bacteria were prepared for injection by diluting a freshly thawed vial in PBS at room temperature.

Experiment Female CD1 mice, 6-8 weeks old (Charles River Laboratories,

Inc., Wilmington, MA), randomly distributed into groups of 15, were injected intraperitoneally in a single dose with either saline, 0.01 g per dose of recombinant TNF described above, 10,000 units per dose of recombinant IL-2 described above, or a single dose of the TNF (at O.OLug) followed immediately by a single dose of the IL-2 (at 10,000 units). Four hours after this injection the mice were injected intraperitoneally with the LD₁₀₀ (6 x 10⁷ cfu) of _E^_ coli SM18 described above per mouse. The LD^QQ represents the minimum number of bacteria necessary to kill all the mice within a group. The results in Table I show mortality rates after seven days.

TABLE I

Treatment % Survival

TNF 17

The mice were observed daily and mortality was recorded for 4 to 7 days.

The statistical significance of the % survival numbers, indicated in the table below by parentheses, was computed using the standard one-tailed Fisher Exact test for all experiments. This test is described by W. J. Conover, Practical Nonparametric Statistics (J.

Wiley and Sons:New York, 1980).

The results show that the combination of IL-2 and TNF has a synergistic rather than additive effect on combating bacterial infections. EXAMPLE 2 - Use of TNF to Treat Herpes Simplex Virus

Outbred CD1 mice were treated with 2.0 <-g/kg of the TNF described in Example 1, intraperitoneally. Two doses were administered, at one day before and at four hours before infection challenge with herpes simplex virus at 1 x 10 pfu per mouse. The results showed that the mice treated with TNF showed significantly greater survival than saline- treated control animals (p 0.05). Thus, TNF may be used alone or in combination with another lymphokine in the treatment of viral infection. In summary, the present invention is seen to provide an effective therapeutic and prophylactic composition to combat infections that contains TNF.

Modifications of the above-descri ed modes for carrying out the invention that are obvious to those skilled in the fields of molecular and clinical biology, pharmacology, and related fields are intended to be within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for prophylactic or therapeutic treatment of infections in mammalian hosts comprising administering to the host, before or after infection of the host, an effective amount of tumor necrosis factor from a mammalian species, wherein the amount of TNF is a sufficient dose to achieve at least 50% protection of the host.

2. The method of claim 1 wherein the TNF is from a human source.

3. The method of claim 2 wherein the TNF is recombinant and the treatment is prophylactic.

4. The method of claim 3 wherein the TNF is produced from microbes.

5. The method of claim 4 wherein the TNF is an N-terminally deleted TNF mutein.

6. The method of claim 5 wherein the TNF mutein is -8 mutein.

7. The method of claim 1 wherein the amount of TNF is a sufficient dose to achieve at least 70% protection of the host.

8. The method of claim 1 wherein the host is human.

9. The method of claim 1 wherein the infection is a bacterial or viral infection.

10. The method of claim 9 wherein the bacterial infection is a Gram-negative infection.

11. The method of claim 9 wherein the viral infection is a herpes simplex virus or cytomegalovirus infection.

12. The method of claim 1 wherein the TNF is in admixture with a pharmaceutically acceptable carrier medium prior to administration.

13. The method of claim 1 wherein the dosage of TNF is about 0.4-2.0- .g per kg body weight of TNF per dose.