WO1996007673A1

WO1996007673A1 - Monocyte chemotaxin inducible proteins

Info

Publication number: WO1996007673A1
Application number: PCT/DK1995/000362
Authority: WO
Inventors: Christian Grønhøj LARSEN; Christian VESTERGÅRD; Kristian Thestrup-Pedersen; Steen Sindet-Pedersen; Steen Lindkaer Jensen
Original assignee: Nycomed Danmark AS
Current assignee: Takeda Pharma AS
Priority date: 1994-09-09
Filing date: 1995-09-11
Publication date: 1996-03-14
Anticipated expiration: 1997-03-09
Also published as: AU3380595A

Abstract

The present invention relates to the use of three novel peptides for the manufacture of a composition for the treatment of diseases, the pathogenesis of which is related to the decreased production and/or function of immuno-stimulating mediators, especially cytokines, e.g. IL-1, TNF, TNFη and MCAF/MCP-1, and/or is related to an increased production of immuno-inhibitory mediators, especially cytokines, e.g. IL-10, in particular a disease selected from the group consisting of immune-incompetence, such as HIV-infections, mononucleosis or other infections such as bacterial infections, e.g. Salmonella T. and Pseudomonas A., tendency of bleeding, cancer such as malignant melanoma, non-malignant disorders of growth, such as psoriasis, papillomas of the skin, the gastro-intestinal tract, and the vesical bladder, and growth disorders induced by viral infections such as common warts and condyloma accuminatum, both caused by human papilloma virus, and for use as adjuvants in connections with immunization. In particular, the invention relates to a peptide which has a molecular weight of about 17 kD and an isoelectric point of about 5.5 and a peptide which has a molecular weight of about 22 kD and an isoelectric point of about 5.0, both obtainable by two-dimensional gel electrophoresis where the IEF second dimension gel elctrophoresis is carried out using a stepwise gradient technique incorporating 20 % and 15 % SDS-PAGE running gels.

Description

MONOCYTE CHEMOTAXIN INDUCIBLE PROTEINS

FIELD OF INVENTION

The present invention relates to three novel proteins called Monocyte^' Chemotaxin Inducible Proteins (MCIP-α-, MCIP-3 and CIP- ) , assumed to be novel cytokines, to a pharmaceutical composition comprising one or several of these proteins, and to the use of one or several of these proteins for the manu- facture of a pharmaceutical composition for the prevention and/or the treatment of diseases, the pathogenesis of which is related to the decreased production and/or function of immuno-stimulating mediators, especially cytokines, e.g. IL-1, TNF, IFN-7 and MCAF/MCP-1 and/or is related to an in- creased production of immuno-inhibitory mediators, especially cytokines, e.g. IL-10. In particular, the invention relates to the use of a substance of the invention for the manufac¬ ture of a pharmaceutical composition for the prevention and treatment of cancer, infections and immunodeficiency syn- dromes.

BACKGROUND OF THE INVENTION

Research from the last two decades has shown that the ini- tiation, regulation and ending of inflammatory reactions as well as the regulation of growth and differentiation within the mammalian organisms are under tight control by a special group of signal peptides generally called cytokines. Cyto¬ kines are peptides which can be produced by most nucleated cells and which transmit regulatory signals between cells, thus forming a communication network between identical or different cell types of the organism. The cytokines are extremely potent mediators and active at concentrations down to 10^"15 M. Cytokines are also key factors for the develop- ment of cellular immune reactions, which in turn form the basis for the clinical manifestations of inflammation due to cancer, infection, allergy, trauma, graft vs. host reactions and auto-immune diseases. The allergic and auto-immune dis- eases are explained by abnormalities in the immune system, especially in the T lymphocyte-mediated immunity, but gene¬ rally these diseases are of unknown etiology. In vi tro stu¬ dies, animal experiments and clinical experimental studies have shown that cytokines play important pathophysiological roles for the inflammatory reactions related to cancer, auto¬ immune diseases, allergy, ischemia, reperfusion injury, trauma, infections, and are important for the development of atherosclerosis, pregnancy and fetal development, bone homeostasis.

Chemokines are chemotactic cytokines belonging to a particu¬ lar super-gene family of proteins which are characterized by being related by a four-cysteine motif. The superfamily is subdivided into two branches based upon whether the first two cysteines in the motif are either adjacent (termed the C-C branch) or spaced by an intervening residue (the C-X-C chemo¬ kines) . There is a vast amount of literature concerning the discovery of the most prominent member of the C-X-C family, namely the roonocyte chemotactic and activating factor, MCAF. This factor is also called MCP-1 and will therefore in the following be referred to as MCAF/MCP-1. MCAF/MCP-1 can be produced by almost all nucleated cells and is strongly in¬ duced by such pro-inflammatory cytokines as IL-1 and TNF. The MCAF cDNA open reading frame codes for a 99 residue protein with the last 76 residues corresponding to MCAF/MCP-1. The C-X-C family also includes such chemokines as LDt8 and RAN- TES. These genes are located close to one another on the Q 11-21 region of the human chromosome 17, and the gene for MCAF/ MCP-1 consists of 3 exons and 2 introns. Recombinant MCAF/MCP-1 has been shown to cause a rapid and transient increase of free cytosolic Ca++ ions. The increase is depen¬ dent on the influx of extracellular Ca++ through plasma mem¬ brane channels. MCAF/MCP-1 acts as a chemoattractant for monocytes and stimulate some other functions such as the release of superoxide anions and release of N-acetyl β-Gluco- ronaminidase. MCAF/MCP-1 also activates basophilic leukocytes being a potent histamine releasing agent. MCAF/MCP-1 activity seems so far to be specific for monocytes and basophilic leu¬ kocytes. MCAF/MCP-1 additionally induces cytostatic activity of monocytes towards tumour cells. Thus it has previously been observed that MCAF/MCP-1 stimulates cultured normal human monocytes to be growth inhibitory in vi tro towards several human tumour cell lines including colon carcinoma cells (A375) , rhabdomysosarcoma cells (HTB 82) , mammary tumour cells (MCF7) and leiomyosarcoma cells (HTB 88) (Matsu- shima et al. , 1989) . The effective dose yielding half maximal activity was similar to that of chemotactic activity. No sig¬ nificant inhibition of tumour cell growth was observed on three human glioma cell lines (HTB 16, U373 and HTB14) or two human bladder carcinoma cell lines (HTB 3 and HTB 4) . Addi¬ tionally, this monocyte chemotactic and activating factor, MCAF/MCP-1, also induces morphological changes in monocytes such as irregularity of shape and increased agglutination after 3 hours of stimulation in vitro. Addition of MCAF/MCP-1 to tumour cell lines alone did not cause any changes of growth in any tumour cell lines suggesting that the effect of MCAF/MCP-1 was through stimulation of monocyte activity (Oppenheim et al., 1991) .

MCAF/MCP-1 in disease

In vivo experiments have shown that tumour cells which were engineered to produce MCP-1 failed to grow in recipient nude mice, while the parent cells formed large tumours in each case. The cytotoxic capacity of MCAF/MCP-1 stimulated human monocytes may relate to the recently described ability of natural purified MCAF/MCP-1 to stimulate the release of superoxide anion and N-acetyl J-GIucoronaminidase from human monocytes. Another explanation could be that MCAF/MCP-1 indu¬ ces the production of cytokines with known direct effect on tumour growth but neutralizing antibodies towards IL-lα and IL-13, TNFα or IL-6 did not block the cytostatic effect of MCAF/MCP-1, and MCAF/MCP-1 did not induce an RNA production for these cytokines (Matsushima et al . , 1989) . It has also been shown, using rat models of lung injury, that MCAF/MCP-1 is upregulated and produced in lung tissue, and in a model of immune-complex-induced alveolitis it was revealed that anti-MCAF/MCP-1 antibody administration lessened the severity of the disease. MCAF/MCP-1 production has also been well scrutinized in rheumatoid arthritis, human idiopathic pulmonary fibrosis, and in atherosclerosis. The finding that chemokines in general and MCAF/MCP-1 in particular may play a role in atherosclerosis dates back to at least 1988 when Valante and coworkers (see Valante et al., 1988) purified what was later identified as MCAF/MCP-1 from baboon vascular smooth muscle cells. Later it was found that minimally modi¬ fied low-density lipoprotein (MM-LDL) induces MCAF/MCP-1 expression in both human endothelial cells and in smooth muscle cells. These observations led to the hypothesis that MCAF/MCP-1 is a key mediator of monocyte attraction in athe- rogenesis. This is supported by the fact that MCAF/MCP-1 expression has been localized to atheromatous plaques in animal models and in humans.

DETAILED DESCRIPTION OF THE INVENTION

MCAF/MCP-1 induces secondary protein production

In this application data are described demonstrating that MCAF/MCP-1 induces the production of three cytokine-sized hitherto unknown proteins by cultured normal human mono- nuclear cells. It is therefore suggested that these proteins, which are tentatively called Λfonocyte Chemotaxin Jnducible Proteins (MCIP-α, MCIP-0 and MCIP-γ) , are involved in mediat¬ ing the pathophysiological effects of MCAF/MCP-1. These three proteins do not bear physical properties similar to those of IL-1, IL-6, or TNFα and are unlikely to be identical with interferon-γ. The existence of these proteins was visualized and proved by 2 dimensional gel electrophoresis as described below. Two of these proteins showed similar size, about 17 kD (MCIP-α and MCIP-_/S) while a third protein molecule (MCIP-γ) showed a molecular size about 22 kD. The invention relates to these three novel proteins which are key elements in the pathophysiological properties exerted by MCAF/MCP-1. It is suggested that one or several of these novel proteins are responsible for the anti-tumour effect exerted by MCAF/MCP-1, and that one or several of these proteins may substitute for MCAF/MCP-1 in exerting this anti-tumour effect.

In its broadest aspects, the present invention thus relates to a peptide which has a molecular weight of about 17 kD and an isoelectric point of about 5.5 obtainable by two-dimensio¬ nal gel electrophoresis where the IEF second dimension gel electrophoresis is carried out using a stepwise gradient technique incorporating 20% and 15% SDS-PAGE running gels, and to a peptide which has a molecular weight of about 22 kD and an isoelectric point of about 5.0 obtainable by two- dimensional gel electrophoresis where the IEF second dimen¬ sion gel electrophoresis is carried out using a stepwise gra¬ dient technique incorporating 20% and 15% SDS-PAGE running gels.

Furthermore, the invention relates to substances having simi¬ lar effects as the peptides of the invention (defined e.g. as described in the experimental part) , in the following termed "agonists" .

In a preferred embodiment of the invention, the substance or peptide is used in substantially pure form. To obtain this, purification of the peptide may be required. Examples of the procedures employed for the purification of peptides are: (i) immunoprecipitation or affinity chromatography with anti¬ bodies, (ii) affinity chromatography with a suitable ligand, (iii) other chromatography procedures such as gel filtration, ion exchange or high performance liquid chromatography or derivatives of any of the above, (iv) electrophoretic proce- dures like polyacrylamide gel electrophoresis, denaturating polyacrylamide gel electrophoresis, agarose gel electrophore¬ sis and isoelectric focusing, (v) any other specific solubi- lization and/or purification techniques. Another aspect of the invention is a method of producing a peptide as defined above, comprising performing a two-dimen¬ sional gel electrophoresis of supernatants of cultured human monocytes, where the IEF second dimension gel electrophoresis is carried out using a stepwise gradient technique incorpo¬ rating 20% and 15% SDS-PAGE running gels, and isolating the peptide.

Furthermore, the invention relates to a pharmaceutical compo- sition comprising at least one of the peptides defined above, such as a pharmaceutical composition comprising a peptide as defined above or a substance being an antagonist or inhibitor as defined in the following, and a pharmaceutically accept¬ able excipient. The composition may comprise e.g. purified synthesized protein or a purified recombinant peptide, a monoclonal or polyclonal antibody.

The MCIP-α, MCIP-/3 and/or MCIP-γ agonist or antagonist used in this invention may be prepared as formulations in pharma- ceutically acceptable media, for example, saline, phosphate buffered saline (PBS), Ringer's solution, dextrose/saline, Hank's solution, and glucose. The compositions may contain ■ pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, deter¬ gents, and the like. Additives may also include additional active ingredients, e.g. bactericidal agents, or stabilizers. The amount administered to the patient will vary depending upon what is being administered, the purpose of the admini- stration, such as prophylaxis or therapy, the state of the host, the manner of administration, and the like.

The pharmaceutical compositions are typically intended for transdermal or parenteral administration, e.g. intravenously, subcutaneously, or intramuscularly. Orally administrative forms are also desired and can be provided by modifying the composition to bypass the stomach environment. The composi¬ tion can be used for prophylactic and/or therapeutic treat- ment. Preferably, the pharmaceutical compositions are admini¬ stered intravenously. Thus, the invention provides composi¬ tions which comprise an MCIP-α, MCIP-/J and/or MCIP-γ agonist or antagonist substance dissolved or suspended in an accept- able carrier, preferably an aqueous carrier. These composi¬ tions may be sterilized by conventional sterilization tech¬ niques, or may be sterile filtered.

The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being com¬ bined with a sterile aqueous carrier prior to administration. The MCIP-α, MCIP-jS and/or MCIP-γ agonist or antagonist may also be administered with a second biologically active agent, such as a standard chemotherapeutic agent. Such agents include but are not limited to vincristine, daunorubicin, L- asparaginase, mitoxantrone and amsacrine.

In therapeutic applications, the pharmaceutical compositions are administered to a patient in an amount sufficient to produce the desired effect, defined as a "therapeutically effective dose". The therapeutically effective dose of an MCIP-α, MCIP-/J and/or MCIP-γ agonist or antagonist will vary according to, for example, the particular use for which the treatment is made, the manner of administration, the health and condition of the patient, and the judgment of the pre¬ scribing physician. For example, the dose for continuous infusion will typically be in the range of about 500 ng to about 800 μg per day for a 70 kg patient, preferably between about 10 μg and about 300 μg. The dose will typically be between 700 ng/kg/day and 16 μg/kg/day.

The concentration of MCIP-α, MCIP-J and/or MCIP-γ agonist or antagonist in the pharmaceutical formulations can vary wide¬ ly, i.e. from about 10 μg to about 5 mg/ml, preferably be- tween about 100 μg and about 2 mg/ml. The concentration will usually be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administra¬ tion selected. Thus, a typical pharmaceutical composition for 8 intravenous infusion could be made up to contain 250 ml of dextrose/saline solution and 2.5 mg of MCIP-α-, MCIP-/J and/or MCIP-γ agonist or antagonist.

For solid compositions, conventional non-toxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium sac¬ charin, talcum, cellulose, glucose, sucrose, magnesium carbo¬ nate, and the like. For oral administration, a pharmaceuti- cally acceptable non-toxic composition is formed by incorpo¬ rating normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, an MCIP-α, MCIP-3 and/or MCIP-γ agonist or antago¬ nist substance, preferably 25-75%.

For aerosol administration, the MCIP-α, MCIP-0 and/or MCIP-γ agonist or antagonist is preferably supplied in finely di¬ vided form along with a surfactant and propellant. Typical percentages of MCIP-α, MCIP-β and/or MCIP-γ agonist or anta- gonists are 0.01-20% by weight, preferably 1-10%. The sur¬ factant must, of course, be non-toxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arbitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides may be employed.

The surfactant may constitute 0.1-20% by weight of the compo¬ sition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. Liquified propellants are typically gases at ambient conditions, and are condensed under pres¬ sure. Among suitable liquified propellants are the lower alkanes containing up to 5 carbons, such as butane and pro- pane; and preferably fluorinated or fluorochlorinated alka- nes. Mixtures of the above may also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided peptide(s) and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.

To enhance the serum half-life, the MCIP-α., MCIP-β and/or MCIP-γ agonist or antagonist may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended lifetime of the peptides. Thus, in certain embodi¬ ments, the MCIP-α, MCIP- and/or MCIP-γ agonist or antagonist may be encapsulated in a liposome. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. , 1980, or in US Patents Nos. 4,235,871, US 4,501,728, and US 4,837,028.

Furthermore, the invention relates to the use of specific synthetic agonists or antagonists, including antibodies, in the treatment of diseases where MCAF/MCP-1 plays a central role, such as immune-inflammation, cancer, atherosclerosis and surgically induced tissue injuries. For other examples, see Tables 1 and 2.

In its broadest aspects, the present invention relates to the use of one or several of these three novel proteins (MCIP-α, MCIP-/3 and MCIP-γ) as therapeutic agents, where an insuffi- cient production of MCAF/MCP-1 or an insufficient effect of MCAF/MCP-1 may play a role in maintaining a certain disease process, see e.g. the diseases mentioned in Table 1 below.

The invention thus relates to the use of one or several of these proteins for the manufacture of a composition for use in human anti-cancer therapy and as an adjuvant therapy of infectious diseases as well as to the use of one or several of these proteins for the manufacture of a composition for use in the treatment of bleeding, i.e. as a procoagulant therapy.

In particular, the invention relates to the use of at least one of the peptides according to the invention for the manu¬ facture of a composition for the treatment of diseases where MCIP-α and/or MCIP-/3 and/or MCIP-γ may be therapeutically beneficial, e.g. malignant diseases, or to the use of at least one of the peptides according to the invention for the manufacture of a composition for the treatment of a disease selected from the group consisting of immune-incompetence, such as HIV-infections, mononucleosis or other infections such as bacterial infections, e.g. Salmonella T. and Pseudo- monas A. , cancer and tendency of bleeding, or for use as adjuvants in connection with immunization, or to the use of at least one of the peptides according to the invention for the manufacture of a composition for use in adjuvant therapy of infectious diseases, or to the use of at least one of the peptides according to the invention for the manufacture of a composition for use in the treatment of bleeding, i.e. for use as a procoagulant.

The invention also relates to a method of treating and/or preventing one or more of the diseases mentioned in Table 1, the method comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a peptide according to the invention.

In particular, the invention relates to the use of MCIP-α for the treatment of malignant melanoma and other malignant dis¬ eases. MCIP- might also be used for the treatment of other non-malignant disorders of growth, such as psoriasis, papil- lomas of the skin, the gastro-intestinal tract, and the vesi- cal bladder. The use of MCIP-o- is also contemplated in the treatment of growth disorders induced by viral infections such as common warts, and condyloma accuminatum, both caused by human papilloma virus. Table 1 Diseases where MCIP-α- and/or MCIP-3 and/or MCIP-γ may be therapeutically beneficial

Immune-incompetence (due to HIV-infections, mononucleosis and other infections)

Bacterial infections (including Salmonella T., Pseudomonas

A.)

Tendency of bleeding Cancer such as malignant melanoma

Non-malignant disorders of growth, such as psoriasis, papillomas of the skin, the gastro-intestinal tract, and the vesical bladder

Growth disorders induced by viral infections such as common warts, and condyloma accuminatum, both caused by human papilloma virus

In another of its broad aspects, the invention furthermore relates to the use of synthetic or natural (antibodies and/or cytokines and/or steroids) MCIP-α, MCIP-/3 and/or MCIP-γ inhi¬ bitors for the treatment of diseases where MCAF/MCP-1 is ■ believed to play a pathophysiological role, see e.g. the diseases mentioned in Table 2 below.

Examples of such antagonists or inhibitors are peptide anta¬ gonists, natural antagonists, e.g. IL-10, cf. the results of experiments 2 and 4, and antibodies.

A specific antagonist against at least one of the peptides according to the invention, in particular an antagonist which is an antibody, thus constitutes an important embodiment of the invention.

The term "antibody" refers to a substance which is produced by a mammal or more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to a peptide antigen of the invention. In the present specifica- tion and claims "an antibody" is defined as consisting essen¬ tially of the specifically binding basic unit which consists of two heavy chains and two light chains. In its broadest aspect, however, the concept of an antibody should also include e.g. a dimer or pentamer of the basic unit.

The variant domain of an antibody is composed of variable and constant sequences. The variant part of the domain is called the idiotype of the antibody. This part of the antibody is responsible for the interaction with the antigen, the antigen binding. In the present context, the term antibody is under¬ stood as the whole antibody molecule or any fragments there¬ of. An antibody can be fragmented during and/or after the production. It can also be made in the fragmented form to begin with and used as such or used after joining different fragments. Especially interesting fragments are binding fragments of the antibodies of the invention, e.g. Fab or Fab' fragments.

Moreover, the invention relates to the use of a specific antagonist against at least one of the peptides according to the invention for the manufacture of a composition for use in the treatment of diseases selected from the group consisting of disseminated intravascular coagulation, sepsis, thrombo- sis, atherosclerosis, glomerulosclerosis, glomerulonephritis, granuloma formation, diabetes mellitus, allergy, bronchial asthma, allergic encephalitis, sarcoidosis, pulmonary fibro- sis, fulminant hepatic failure, red blood cell incompatibili¬ ty, graft v. host disease, periodontal disease, rheumatoid arthritis, psoriasis and aortic aneurysm formation.

The invention also relates to a method of treating and/or preventing one or more of the diseases mentioned in Table 2, the method comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of an antagonist against a peptide according to the invention. Table 2 Diseases where inhibitors of MCIP-c. and/or MCIP-S and/or MCIP-γ may be therapeutically beneficial

Disseminated intravascular coagulation

Sepsis

Thrombosis

Atherosclerosis

Glomerulosclerosis Glomerulonephritis

Granuloma formation

Diabetes Mellitus

Allergy

Bronchial asthma Allergic encephalitis

Sarcoidosis

Granuloma formation

Pulmonary fibrosis

Fulminant hepatic failure Reed blood cell incompatibility

Graft v. Host disease

Periodontal disease

Rheumatoid arthritis

Psoriasis Aortic aneurysm formation

The invention also relates to the use of interleukin-10 (IL-10) or IL-10 agonists in the treatment of diseases where MCAF/MCP-1 is believed to play a pathophysiological role, based on the findings that IL-10 regulates MCAF/MCP-1 auto- induction, as well as to the use of interleukin-10 (IL-10) or IL-10 agonists in the treatment of diseases where MCIP-α and/or MCIP-S and/or MCIP-γ is believed to play a patho- physiological role, based on the findings that IL-10 regu¬ lates MCIP-α, MCIP-/3 and MCIP-γ auto-induction. EXAMPLE 1

IDENTIFICATION OF MCIP-α, MCIP-3 AND MCIP-7

Materials and Methods

Cytokines and antibodies

Recombinant hMCAF/MCP-1 (rh MCAF) was a kind gift from pro- fessor Kouji Matsushima, Kanazawa, Japan. Monoclonal anti- MCAF antibodies (clones MI-446 and ME-120) were a kind gift from K. Matsushima. Polyclonal rabbit anti-MCAF antibodies were a kind gift from Dr. A. Harada, Japan. Recombinant hlL- 10 was obtained from Pepro Tech Inc. NJ. (Cat.No. 20010) . The LPS-free culture medium RPMI 1640 was purchased from GIBCO

(Cat.No. 6187-010) , and medium without methionine from GIBCO (Cat.No. 041-90454) . Cells were cultured in 24 wells Nunc Micro Plates (Nunc, Denmark) .

Cell cul tures

Monocytes were purified from heparinized fresh donor blood with Lymphoprep™ (Nycomed Pharma, Oslo, Norway) , gradient centrifugation followed by 45 minutes of culturing in LPS- free RPMI 1640 containing 10% sterile-filtered heat-inacti¬ vated fetal calf serum (FCS) , penicillin 10,000 IE/ml, strep¬ tomycin 10 mg/ml, and gentamycin 2.5 mg/ml. The cell concen¬ tration of the monocytes adhering to the plastic was calcu¬ lated to 3 x 10⁶/well and after three washes with Hanks buffer solution, the cells were incubated in microwells with 1 ml of RPMI 1640 without methionine and containing 1% ste¬ rile-filtered heat-inactivated FCS including penicillin 10,000 IE/ml, streptomycin 10 mg/ml and gentamycin 2.5 mg/ml. To evaluate de novo protein synthesis ³⁵S-methionine (Amers- ham, Cat.No. SJ.1015 ) was added in portions of 200 μCi/ml. The monocytes were then stimulated for 72 hours with MCAF/- MCP-1 100 ng/ml, either alone or in combination with rhIL-10 15

100 ng/ml added 0 hour and 24 hours or monoclonal anti-MCAF/- MCP-l antibodies, 2 μg/ml added at 0 hour and at 24 hours.

The monocytes and the supernatants were prepared for protein analysis by centrifugation of the cell culture at 2000 rpm for 10 minutes. The supernatants were freeze-dried and dis¬ solved in 100 μl of gel lysis buffer (Celis et al.,^' 1992). The cells were resuspended in 100 μl of lysis buffer without further treatment. The samples were stored at -80°C until analysed.

Two-dimensional gel electrophoresis and Western blotting

The preparation of gels as well as the performance of the one-dimensional isoelectric focusing (IEF) and non-equili¬ brated pH-gradient electrophoresis (NEPHGE) and the two- dimensional SDS polyacrylamide gels were performed as de¬ scribed in detail by Celis et al. , 1992. This technique was slightly modified. Briefly, NEPHGE second dimension gel elec- trophoresis was carried out using a stepwise gradient tech¬ nique (20% and 15% SDS-PAGE running gels) . Following electro¬ phoresis, gels were developed using silver-staining (Celis et al., 1992) revealing the protein content of cells or culture medium.

The extent of de novo protein production was evaluated by auto-radiography of gels and exposing the film (Kodak X- OMAT-S Denmark) for approximately 2 weeks.

Western blotting

Proteins from gels were transferred to Hybond C nitrocellu¬ lose membranes (Amersham, UK) and blocked with 1.5% hemoglo¬ bin (SIGMA) in Hanks balanced salt solution (HBSS) and then incubated with a polyclonal MCAF/MCP-1 antibody followed by a horse radish peroxidase-labelled second antibody (DAKO, Cat.No. P 217) and stained with Enhanced Chemiluminescence (ECL, Amersham, Cat.No. RPN 2106, UK) . The immunostaining was detected by exposing a film (Kodak X-OMAT-S, Denmark) for 90 seconds.

Results

Experiment 1: MCAF/MCP-1 autoinduction

Fig. 1 and Fig. 2 show the silver-stained gels and the auto- radiographies of supernatants of normal human monocytes, cultured for three days in the presence of 100 ng/ml of

MCAF/MCP-1. The arrows in Fig. 1 indicate proteins identified as MCAF/MCP-1. When comparing the autoradiographies (Fig. 2) of MCAF/MCP-1-stimulated cells and the control, one observes a significant increased staining of MCAF/MCP-1 corresponding to the MCAF/MCP-1 stimulated cells indicating that MCAF/MCP-1 induces the de novo production of MCAF/MCP-1, a so-called auto-induction.

Experiment 2: IL-10 regulates MCAF/MCP-1 autoinduction

Fig 3. and Fig. 4 show the silver-stained gels and the auto¬ radiographies of supernatants of normal human monocytes, cultured for one day in the presence of 100 ng/ml of MCAF/MCP-1 and 100 ng/ml IL-10 in a medium without radio- activity. After the first 24 hours, cells were washed and cultured for another 48 hours with IL-10 but without MCAF/MCP-1 and in the presence of 400 μCi ³⁵S-methionine. It is observed that the presence of IL-10 prevents MCAF/MCP-1 auto-induction. The localization, or the expected localiza- tion, of MCAF/MCP-1 is indicated with arrows.

Experiment 3: MCAF/MCP-1 induces de novo production of three acidic proteins

The autoradiographies shown in Fig. 5 show the 2 D gel ana¬ lysis of the supernatant of monocytes cultured for three days in the presence of 100 ng/ml MCAF/MCP-1 and 400 μCi ³⁵S- methionine. The arrows indicate the localization of the three proteins induced by MCAF/MCP-1, and from left to right they are tentatively named Monocyte Chemotaxin Inducible Proteins (MCIP-α, MCIP-J and MCIP-γ) . MCIP-α and MCIP-/J have molecular weights of about 17 kD and an isoelectric point of about 5.5, while the size of MCIP-γ is about 22 kD and its isoelectric point about 5.0, see Table 3 below. By comparing these cha¬ racteristics and the localization on the two-dimensional gel with the protein data bases made available by professor, Dr. J Celis, Institute of Medical Biochemistry, Aarhus Univer- sity, no known proteins matching MCIP-α, MCIP-/3 and MCIP-γ were found. It is therefore concluded that these proteins may be novel molecules. MCIP-α and MCIP-3 are likely to be diffe¬ rent forms of the same protein, the differences being due to different phosphorylation.

Table 3 Physical-chemical characteristics of Monocyte Chemotaxin Inducible Proteins

MW (kDalton) Isoelectric point

MCIP-α 17 5.5±0.1

MCIP-γ 22 5.0±0.1

± -= safety interval

Experiment 4: Inhibi tion of MCIP-a and MCIP-β production by IL-10

Fig. 6 shows the result of a study where monocytes were cul¬ tured for 24 hours in the presence of 100 ng/ml of MCAF/MCP-1 and 100 ng/ml of IL-10. The cells were washed and cultured for another 48 hours in the presence of 100 ng/ml IL-10. The radiography (Fig. 6) revealed that IL-10 inhibited the pro- duction of MCIP-α and MCIP-0. EXAMPLE 2

FURTHER EVALUATION OF MCIP-α

Materials and methods

Cell culture for protein purification (preparative scale)

Buffy Coat blood was obtained from Clinical Immunological Department, Skejby Hospital, Aarhus, Denmark. After Lympho- prep™ (Nycomed Pharma, Oslo, Norway) gradient centrifugation, the mononuclear cells were washed with HANKS solution and dissolved in RPMI 1640 (Gibco Cat.No. 61879-010) with 10% fetal calf serum (FCS), penicillin 10,000 IE/ml, streptomycin 10 mg/ml, and gentamycin 2.5 mg/ml. 200-250 x 10⁶ cells were added per Nunc culture bottle (175 cm³) in 100 ml medium .and were incubated at 37°C for 1 hour. The monocytes were iso¬ lated by plastic adherence. Non-adherent cells were removed. The monocyte concentration was calculated to be 60-80 x 10⁶/- bottle. 25 ml of fresh medium with 2% fetal calf serum (FCS) was added to each bottle and rh MCAF was added to a concen¬ tration of 100 ng/ml. Cells were stimulated for 48 hours. A total number of 5 bottles (175 cm³) were used. The last 24 hours 25 ml of RPMI 1640 without methionine (Gibco Cat.No. 041-90454) was used with 2% FCS and ³⁵S-methionine (500 μCi, SJ 1015, Amersham) in each bottle. Supernatants were collect¬ ed for protein isolation and kept at -80°C.

Protein purification

Supernatants after cell stimulation were kept on ice at 4°C and the proteins were fractionated by stepwise ammonium sul¬ phate precipitation. Ammonium sulphate (pro analysis quality (Merck, Germany) ) was added slowly and under rotation by an Orbital Rotor (Kem-En-Tec, Denmark) to 50% saturation and after mixing for 1 hour, the proteins were collected by cen¬ trifugation. Thereafter more ammonium sulphate was added to the supernatant until a saturation of 80% was achieved. After an extra hour the proteins were collected. The protein precipitates were dissolved in 2 ml of HANKS + 0.01% (w/w) sodium azide (Bie & Bernsen, Denmark) and extensively dia- lyzed by 5 times changes of 3 1 of milipore water (Spectra Pore dialysis tube 132107, Spectrum LA, California) .

Two dimensional gel electrophoresis and electroelution

One dimensional isoelectric focusing (IEF) and stepwise gradient second dimensional gel electrophoresis was carried out as described above (20% and 15% SDS-PAGE running gels) . Freeze-dried proteins after dialysis were dissolved in gel lysis buffer (1.5 mg/40 μl) from the 80% fraction.

Following electrophoresis gels were developed by Coomassie

Brilliant Blue staining. Proteins were cut out from the dried gels and collected from 8 different gels. The position of MCIP-o. was determined by autoradiography as described above. The gel pieces were put into an electroelutor (MOD 422 Electro-Eluter (Bio RAD, Solna, Sweden) ) . For control experi¬ ments, three other proteins were eluted (cf. Fig. 1 C) . After electroelution the proteins were dialyzed again in Spectra Pore dialysis tubes and freeze-dried.

Tumour growth inhibition assay of proteins originating from 2 D gels

Human malignant melanoma G 361 was purchased from the Euro¬ pean Collection of Animal Cell Cultures, Porton Down, Salis- bury Wilts SP4 0JG, Great Britain. Cells were cultivated in triplicates. 10⁴ cells were cultured in 100 μl of medium alone or with test proteins. Culture medium was McCoys 5a (Gibco 26600023) containing 10% sterile filtered calf serum (FCS), penicillin (10,000 IU/ml) , streptomycin (10 mg/ml) and gentamycin (2.5 mg/ml).

Cells were stimulated for 72 hours and the last 18 hours in the presence of 0.5 μCi ³H TdR (Amersham) . At the end of the stimulation, the cells were washed twice in culture medium and then collected in gel lysis buffer (as they grow in mono- layer) . 2 ml of Insta gel (Universal LSC Packard, Cat.No. 6013009, the Netherlands) was added. Samples were analyzed in a Packard 1600 TR Liquid Scintillation analyzer.

Results

Experiment 1

Tumour cell alone 46 000 + 2000 CPM Protein added 5 ng/ml 23 000 + 1000 CPM

Experiment 2

Tumour cell alone 101 000 + 11000 CPM Protein added 5 ng/ml 80 000 + 8000 CPM 0.5 ng/ml 92 000 + 6000 CPM 0.05 ng/ml 101 000 + 14000 CPM

For comparison three other cancer cell lines (Human colon carcinoma HT 29/219, Human malignant melanoma A 375 and Human lymphoma H 33HJ-JA1) were also purchased from ECACC and tested.

It was not possible to show significant regulation in tumour growth in these cell lines by MCIP-α. However, this does not exclude the possibility that MCIP-α can inhibit the tumour growth in these cell lines as it is necessary to perform a number of experiments to determine the time when the cell division is highest and at which time the thymidine should be added. Further experiments will be performed as outlined above to further evaluate these cell lines as well as other cell lines. In a similar manner, experiments with regard to MCIP-jS and MCIP-γ will be performed. In conclusion, a group of proteins have been observed and purified, said proteins being produced by cultured human monocytes when stimulated with MCAF/MCP-1. One of these pro¬ teins, MCIP-α, proved to significantly suppress the growth of melanoma cells in vi tro . This means that MCIP-α may in turn be involved in the natural response of the immune system when this system attempts to suppress the malignant transformation of melanocytes to melanoma. MCIP-α is thus a likely candidate for the treatment of melanoma in human beings.

LEGENDS TO FIGURES

Fig. 1 shows basic monocyte supernatant proteins. Fig. 1A shows the unstimulated monocytes, Fig. IB the MCAF-stimulated monocytes.

More specifically, Fig. 1A shows the silver-stained basic gel analysis of the supernatant from unstimulated monocytes in the region of the basic gel where MCAF is to be expected. The spots marked with arrows correspond to MCAF, and it can be distinguished that MCAF may consist of several forms. The asterisk indicates a protein which is probably IL-8.

Fig. IB shows the corresponding gel region, but with the supernatant from MCAF-stimulated monocytes. MCAF is marked by arrows, and it can be seen that MCAF is not equally distinct on the silver staining. Also, IL-8 is shown, indicated by an asterisk.

Fig. 2 shows basic monocyte supernatant proteins. Fig. 2A shows the unstimulated monocytes, Fig. 2B the MCAF-stimulated monocytes. Fig. 2A shows an autoradiography of the gel shown in Fig. 1A. The arrows indicate the spots representing the new synthesis of MCAF. Fig. 2B shows the film which was placed above the gel shown in Fig. IB, and the arrows indi¬ cate the proteins representing the new synthesis of MCAF.

When comparing Fig. 2A and Fig. 2B, it can be seen that the new synthesis of MCAF is upregulated in MCAF-stimulated mono- cytes, the coloration being stronger.

Fig. 3 shows regulation of MCAF in the supernatant of mono¬ cytes. Fig. 3A is the supernatant from non-stimulated mono¬ cytes, Fig. 3B is the supernatant from monocytes stimulated with MCAF, and Fig. 3C is the supernatant from monocytes stimulated with MCAF and IL-10. 23

Fig. 3A shows a section of the silver-stained basic gel ana¬ lysis of the supernatant from non-stimulated monocytes cul¬ tured as described in experiment 2, corresponding to the region where MCAF is located, marked with arrows. Fig. 3B shows the corresponding region, but from monocytes stimulated with MCAF and otherwise cultured in the same manner. MCAF is also marked with arrows here as in Fig. 3C which shows the result from monocytes stimulated with MCAF and IL-10.

When comparing Fig. 3B and Fig. 3C with Fig. 3A, a distinct upregulation of MCAF is seen whereas the difference between Fig. 3A and Fig. 3C is not that distinct on the silver stain¬ ing.

Fig. 4 shows regulation of MCAF in the supernatant of mono¬ cytes. Fig. 4A is the supernatant from non-stimulated mono¬ cytes, Fig. 4B is the supernatant from monocytes stimulated with MCAF, and Fig. 4C is the supernatant from monocytes stimulated with MCAF and IL-10.

The figures show autoradiographies of the gels shown in Fig. 3. MCAF is indicated by arrows. When comparing Fig. 4A with Fig. 4B, a stronger coloration of the proform can be seen in Fig. 4B, whereas it is more difficult to see a stronger coloration of the final form in Fig. 4B because of radioactive debris present just under the final form of MCAF. In Fig. 4C, a distinct and strong downregulation of the monomeric form of MCAF can be seen, but a downregulation of the dimeric form is also seen; however, this is not so dis- tinct.

Fig. 5 shows acidic monocyte supernatant proteins. Fig. 5A shows the unstimulated monocytes, Fig. 5B the MCAF-stimulated monocytes.

Fig. 5A shows an autoradiography of an "acidic" gel analysis of the supernatant from unstimulated monocytes cultured as described in experiment 3. Fig. 5B shows the same region but from the supernatant of MCAF-stimulated monocytes. Correspon¬ ding to the arrows, Fig. 5B shows three proteins whose new synthesis is upregulated. In Fig. 5A, the same positions are marked, but no proteins are seen here. The weight indications are approximate indications.

Fig. 6

Fig. 6A shows the gel analysis (autoradiography) of cultured unstimulated human monocytes. Fig. 6B shows that stimulation with MCAF/MCP-1 (100 ng/ml) induces the de novo production of two novel proteins (MCIP-α and MCIP-/3, see Table 3) . Fig. 6C shows the gel analysis of cultured human monocytes pre-incu- bated with recombinant IL-10 (100 ng/ml) and stimulated with MCAF/MCP-1 (100 ng/ml) demonstrating that IL-10 blocks the MCAF/MCP-1 induced production of MCIP-α and MCIP-J.

Fig. 7

Fig. 7A shows Coomassie Brilliant Blue stained 2 D gel of the purified proteins from the MCAF stimulated monocyte super¬ natant fraction. The arrow indicates the location of MCIP-α. The isoelectric point of MCIP-α- is 5.5 and the molecular weight is 17 kD (0.75 mg protein/gel is applied of the 80% ammonium sulphate fraction) .

Fig. 7B shows X-Ray Film superimposed on the 2 D gel and autoradiography of the fraction showing new synthesis of the proteins by ³⁵S-methionine incorporation. The arrow indicates the location of MCIP-α.

Fig. 7C shows Coomassie Brilliant Blue stained 2 D gels showing 4 different proteins called A, B, C and E. The abi¬ lity of these proteins to downregulate growth was compared in various cancer cell lines. Only protein E had significant ability and protein E is identical with MCIP-α. Fig. 8 shows Coomassie Brilliant Blue stained 2 D gel of purified human IFN-α (The Interferon Laboratory, Hjørring, Denmark) . The gel is performed under parallel conditions to the gel shown in Fig. 7A. The isoelectric point of IFN-α is 5-6.5 and the molecular weight is 19.2-19.7 kD. The lower arrow indicates the position corresponding to that of MCIP-α. Thus, MCIP-α is not identical to IFN-α.

REFERENCES

Celis, J.E., Rasmussen, H.H. , Madsen, P., Leffer, H. , Honore, B., Dejgaard, K. , Gesser, B., et al. Electrophore¬ sis 13, 893-959 (1992) Matsushima, K., Larsen, C . G . , DuBois, G.C. and Oppenheim, J.J. J^". Exp. Med. 169, 1485-1490 (1989)

Oppenheim, J.J., Zachariae, CO., Mukaida, N. and Matsu¬ shima, K. Ann . Rev. Immunol . 9, 617-648 (1991) Szoka et al., Ann. Rev. Biophys. Bioeng. 9, 467 (1980) Valante, R.J., Graves, D.T., Vialle-Valentin, C, Delgado, R. and Schwartz, C.J. Biochemistry 27, 4162-4168 (1988)

- US Patent No. 4,235,871

- US Patent No. 4,501,728

- US Patent No. 4,837,028

Claims

1. A peptide which has a molecular weight of about 17 kD and an isoelectric point of about 5.5 obtainable by two-dimen- sional gel electrophoresis where the IEF second dimension gel electrophoresis is carried out using a stepwise gradient technique incorporating 20% and 15% SDS-PAGE running gels.

2. A peptide which has a molecular weight of about 22 kD and an isoelectric point of about 5.0 obtainable by two-dimensio¬ nal gel electrophoresis where the IEF second dimension gel electrophoresis is carried out using a stepwise gradient technique incorporating 20% and 15% SDS-PAGE running gels.

3. A peptide according to claim 1 or 2 in substantially pure form.

4. A method of producing a peptide as defined in any of claims 1-3, comprising performing a two-dimensional gel electrophoresis of supernatants of cultured human monocytes, where the IEF second dimension gel electrophoresis is carried out using a stepwise gradient technique incorporating 20% and 15% SDS-PAGE running gels, and isolating the peptide.

5. A pharmaceutical composition comprising the peptide ac¬ cording to claim 1 or the peptide according to claim 2 or both.

6. Use of the peptide according to claim 1 or the peptide according to claim 2 or both for the manufacture of a compo¬ sition for the treatment of diseases, the pathogenesis of which is related to the decreased production and/or function of immuno-stimulating mediators, especially cytokines, e.g. IL-1, TNF, TNFγ and MCAF/MCP-1, and/or is related to an increased production of immuno-inhibitory mediators, espe¬ cially cytokines, e.g. IL-10.

7. Use of the peptide according to claim 1 or the peptide according to claim 2 or both for the manufacture of a compo¬ sition for the treatment of diseases where MCIP-α and/or MCIP-3 and/or MCIP-γ may be therapeutically beneficial, e.g. malignant diseases.

8. Use of the peptide according to claim 1 or the peptide according to claim 2 or both for the manufacture of a compo¬ sition for treatment of a disease selected from the group consisting of immune-incompetence, such as HIV-infections, mononucleosis or other infections such as bacterial infec¬ tions, e.g. Salmonella T. and Pseudomonas A., tendency of bleeding, cancer such as malignant melanoma, non-malignant disorders of growth, such as psoriasis, papillomas of the skin, the gastro-intestinal tract, and the vesical bladder, and growth disorders induced by viral infections such as common warts and condyloma accuminatum, both caused by human papilloma virus, and for use as adjuvants in connection with immunization.

9. Use of the peptide according to claim 1 or the peptide according to claim 2 or both for the manufacture of a compo¬ sition for use in adjuvant therapy of infectious diseases.

10. Use of the peptide according to claim 1 or the peptide according to claim 2 or both for the manufacture of a compo¬ sition for use in the treatment of bleeding, i.e. for use as a procoagulant.

11. A method of treating and/or preventing one or more of the diseases mentioned in Table 1, the method comprising admini¬ stering to a patient in need thereof a therapeutically or prophylactically effective amount of a peptide according to claim 1 or 2.

12. A specific antagonist against the peptide according to claim 1 or the peptide according to claim 2 or both.

13. An antagonist according to claim 12 which is an antibody.

14. Use of a specific antagonist against the peptide accord¬ ing to claim 1 or the peptide according to claim 2 or both for the manufacture of a composition for use in the treatment of diseases selected from the group consisting of dissemina¬ ted intravascular coagulation, sepsis, thrombosis, athero¬ sclerosis, glomerulosclerosis, glomerulonephritis, granuloma formation, diabetes mellitus, allergy, bronchial asthma, allergic encephalitis, sarcoidosis, pulmonary fibrosis, fulminant hepatic failure, red blood cell incompatibility, graft v. host disease, periodontal disease, rheumatoid ar¬ thritis, psoriasis and aortic aneurysm formation.

15. A method of treating and/or preventing one or more of the diseases mentioned in Table 2, the method comprising admini¬ stering to a patient in need thereof a therapeutically or prophylactically effective amount of an antagonist against a peptide according to claim 1 or 2.