WO1996007673A1 - Proteines inductibles par la chimiotaxine des monocytes - Google Patents

Proteines inductibles par la chimiotaxine des monocytes Download PDF

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Publication number
WO1996007673A1
WO1996007673A1 PCT/DK1995/000362 DK9500362W WO9607673A1 WO 1996007673 A1 WO1996007673 A1 WO 1996007673A1 DK 9500362 W DK9500362 W DK 9500362W WO 9607673 A1 WO9607673 A1 WO 9607673A1
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Prior art keywords
mcip
mcaf
peptide according
peptide
mcp
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PCT/DK1995/000362
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English (en)
Inventor
Christian Grønhøj LARSEN
Christian VESTERGÅRD
Kristian Thestrup-Pedersen
Steen Sindet-Pedersen
Steen Lindkaer Jensen
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Nycomed Dak A/S
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Priority to AU33805/95A priority Critical patent/AU3380595A/en
Publication of WO1996007673A1 publication Critical patent/WO1996007673A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to three novel proteins called Monocyte ' Chemotaxin Inducible Proteins (MCIP- ⁇ -, 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.
  • 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.
  • 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.
  • 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) .
  • C-X-C chemo ⁇ kines the C-X-C chemo ⁇ kines
  • MCAF/MCP-1 can be produced by almost all nucleated cells and is strongly in ⁇ substituted 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.
  • 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 ⁇ -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.
  • 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) .
  • this monocyte chemotactic and activating factor MCAF/MCP-1
  • 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) .
  • 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.
  • MCAF/MCP-1 indu ⁇ ces the production of cytokines with known direct effect on tumour growth but neutralizing antibodies towards IL-l ⁇ and IL-13, TNF ⁇ 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.
  • Valante and coworkers see Valante et al., 1988
  • MM-LDL minimally modi ⁇ fied low-density lipoprotein
  • MCAF/MCP-1 induces secondary protein production
  • 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 ⁇ fonocyte Chemotaxin Jnducible Proteins (MCIP- ⁇ , MCIP-0 and MCIP- ⁇ ) , 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 TNF ⁇ and are unlikely to be identical with interferon- ⁇ . The existence of these proteins was visualized and proved by 2 dimensional gel electrophoresis as described below.
  • 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.
  • 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 ⁇ trans technique incorporating 20% and 15% SDS-PAGE running gels.
  • 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" .
  • the substance or peptide is used in substantially pure form.
  • purification of the peptide may be required.
  • 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.
  • 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- ⁇ , MCIP-/3 and/or MCIP- ⁇ 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.
  • the pharmaceutical compositions are admini ⁇ stered intravenously.
  • the invention provides composi ⁇ tions which comprise an MCIP- ⁇ , MCIP-/J and/or MCIP- ⁇ 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- ⁇ , MCIP-jS and/or MCIP- ⁇ agonist or antagonist may also be administered with a second biologically active agent, such as a standard chemotherapeutic agent.
  • a second biologically active agent such as a standard chemotherapeutic agent.
  • agents include but are not limited to vincristine, daunorubicin, L- asparaginase, mitoxantrone and amsacrine.
  • 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- ⁇ , MCIP-/J and/or MCIP- ⁇ 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.
  • the dose for continuous infusion will typically be in the range of about 500 ng to about 800 ⁇ g per day for a 70 kg patient, preferably between about 10 ⁇ g and about 300 ⁇ g.
  • the dose will typically be between 700 ng/kg/day and 16 ⁇ g/kg/day.
  • the concentration of MCIP- ⁇ , MCIP-J and/or MCIP- ⁇ agonist or antagonist in the pharmaceutical formulations can vary wide ⁇ ly, i.e. from about 10 ⁇ g to about 5 mg/ml, preferably be- tween about 100 ⁇ g 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.
  • 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- ⁇ -, MCIP-/J and/or MCIP- ⁇ agonist or antagonist.
  • 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.
  • 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- ⁇ , MCIP-3 and/or MCIP- ⁇ agonist or antago ⁇ nist substance, preferably 25-75%.
  • the MCIP- ⁇ , MCIP-0 and/or MCIP- ⁇ agonist or antagonist is preferably supplied in finely di ⁇ vided form along with a surfactant and propellant.
  • Typical percentages of MCIP- ⁇ , MCIP- ⁇ and/or MCIP- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the MCIP- ⁇ ., MCIP- ⁇ and/or MCIP- ⁇ 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.
  • the MCIP- ⁇ , MCIP- and/or MCIP- ⁇ 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.
  • 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.
  • diseases where MCAF/MCP-1 plays a central role such as immune-inflammation, cancer, atherosclerosis and surgically induced tissue injuries.
  • the present invention relates to the use of one or several of these three novel proteins (MCIP- ⁇ , MCIP-/3 and MCIP- ⁇ ) 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.
  • 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- ⁇ and/or MCIP-/3 and/or MCIP- ⁇ 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.
  • immune-incompetence such as HIV-infections, mononucleosis or other infections
  • 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.
  • the invention relates to the use of MCIP- ⁇ 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- ⁇ - and/or MCIP-3 and/or MCIP- ⁇ may be therapeutically beneficial
  • Bacterial infections including Salmonella T., Pseudomonas
  • Non-malignant disorders of growth such as psoriasis, papillomas of the skin, the gastro-intestinal tract, and the vesical bladder
  • the invention furthermore relates to the use of synthetic or natural (antibodies and/or cytokines and/or steroids) MCIP- ⁇ , MCIP-/3 and/or MCIP- ⁇ 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.
  • antagonists or inhibitors examples include 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.
  • an 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.
  • 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.
  • 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.
  • 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.
  • diseases selected from the group consisting of disseminated intravascular coagulation, sepsis, thrombo- sis, atherosclerosis, glomerulosclerosis, glomerulonephritis,
  • 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- ⁇ may be therapeutically beneficial
  • 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- ⁇ and/or MCIP-S and/or MCIP- ⁇ is believed to play a patho- physiological role, based on the findings that IL-10 regu ⁇ lates MCIP- ⁇ , MCIP-/3 and MCIP- ⁇ auto-induction.
  • IL-10 interleukin-10
  • IL-10 agonists in the treatment of diseases where MCIP- ⁇ and/or MCIP-S and/or MCIP- ⁇ is believed to play a patho- physiological role, based on the findings that IL-10 regu ⁇ lates MCIP- ⁇ , MCIP-/3 and
  • hMCAF/MCP-1 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
  • Monocytes were purified from heparinized fresh donor blood with LymphoprepTM (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.
  • LymphoprepTM LymphoprepTM
  • FCS sterile-filtered heat-inacti ⁇ vated fetal calf serum
  • the cell concen ⁇ tration of the monocytes adhering to the plastic was calcu ⁇ lated to 3 x 10 6 /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.
  • S-methionine Amers- ham, Cat.No. SJ.1015
  • the monocytes were then stimulated for 72 hours with MCAF/- MCP-1 100 ng/ml, either alone or in combination with rhIL-10 15
  • 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 ⁇ l of gel lysis buffer (Celis et al., ' 1992).
  • the cells were resuspended in 100 ⁇ l of lysis buffer without further treatment.
  • the samples were stored at -80°C until analysed.
  • 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.
  • 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.
  • 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 MCAF/MCP-1.
  • the arrows in Fig. 1 indicate proteins identified as MCAF/MCP-1.
  • Fig. 2 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.
  • 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 ⁇ Ci 35 S-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.
  • 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 ⁇ Ci 35 S- 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- ⁇ , MCIP-J and MCIP- ⁇ ) .
  • MCIP- ⁇ and MCIP-/J have molecular weights of about 17 kD and an isoelectric point of about 5.5, while the size of MCIP- ⁇ is about 22 kD and its isoelectric point about 5.0, see Table 3 below.
  • 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- ⁇ and MCIP-0. EXAMPLE 2
  • Buffy Coat blood was obtained from Clinical Immunological Department, Skejby Hospital, Aarhus, Denmark. After Lympho- prepTM (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 10 6 cells were added per Nunc culture bottle (175 cm 3 ) 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 10 6 /- 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 cm 3 ) were used.
  • the last 24 hours 25 ml of RPMI 1640 without methionine (Gibco Cat.No. 041-90454) was used with 2% FCS and 35 S-methionine (500 ⁇ Ci, SJ 1015, Amersham) in each bottle.
  • Supernatants were collect ⁇ ed for protein isolation and kept at -80°C.
  • 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) .
  • 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. 10 4 cells were cultured in 100 ⁇ l 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).
  • McCoys 5a Gibco 26600023
  • FCS sterile filtered calf serum
  • penicillin 10,000 IU/ml
  • streptomycin 10 mg/ml
  • gentamycin 2.5 mg/ml
  • MCIP- ⁇ One of these pro ⁇ teins, MCIP- ⁇ , proved to significantly suppress the growth of melanoma cells in vi tro . This means that MCIP- ⁇ 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- ⁇ is thus a likely candidate for the treatment of melanoma in human beings.
  • Fig. 1 shows basic monocyte supernatant proteins.
  • Fig. 1A shows the unstimulated monocytes,
  • Fig. IB the MCAF-stimulated monocytes.
  • 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.
  • 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.
  • 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
  • Fig. 3C is the supernatant from monocytes stimulated with MCAF and IL-10.
  • 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.
  • 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
  • Fig. 4C is the supernatant from monocytes stimulated with MCAF and IL-10.
  • MCAF is indicated by arrows.
  • 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.
  • 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. 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- ⁇ 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- ⁇ and MCIP-J.
  • 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- ⁇ .
  • the isoelectric point of MCIP- ⁇ - 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 35 S-methionine incorporation.
  • the arrow indicates the location of MCIP- ⁇ .
  • 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- ⁇ .
  • Fig. 8 shows Coomassie Brilliant Blue stained 2 D gel of purified human IFN- ⁇ (The Interferon Laboratory, Hj ⁇ rring, Denmark) . The gel is performed under parallel conditions to the gel shown in Fig. 7A. The isoelectric point of IFN- ⁇ 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- ⁇ . Thus, MCIP- ⁇ is not identical to IFN- ⁇ .

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Abstract

L'invention concerne l'utilisation de trois nouveaux peptides permettant d'obtenir une composition thérapeutique relative à des maladies dont la pathogenèse s'explique par la production et/ou le fonctionnement réduits de médiateurs immuno-stimulants, notamment des cytokines, par exemple IL-1, TNF, TNFη et MCAF/MCP-1 (facteur chimiotactique et activateur des monocytes), ou bien par une production accrue de médiateurs immuno-inhibiteurs, notamment des cytokines, par exemple IL10. Ceci vaut notamment pour des maladies sélectionnées dans le groupe des infections provoquées par immuno-incompétence, telles que les infections à VIH ou la mononucléose, ou d'autres infections, par exemple bactériennes, dues à Salmonella T. et aux Pseudonomas A. par exemple, ou pour la tendence aux hémorragies, les cancers tels que le mélanome malin, des troubles non malins de la croissance, tels que le psoriasis, les papillomes cutanés, ou ceux du tractus gastro-intestinal et de la vessie, et des troubles de la croissance induits par des infections virales telles que les verrues vulgaires et le condylome accuminé causés tous les deux par le virus du papillome humain. Ces peptides servent aussi d'adjuvants d'immunisation. Cette invention concerne en particulier un peptide d'un poids moléculaire d'environ 17kD et d'un point isoélectrique d'environ 5,5, et un peptide d'un poids moléculaire d'environ 22kD et d'un point isoélectrique d'environ 5,0, qu'on peut obtenir tous les deux par électrophorèse sur gel bidimensionnelle où l'électrophorèse sur gel en deuxième dimension d'isoélectrofocalisation intervient par une technique de gradients augmentant par paliers et incorporant 20 et 15 % de gels pour SDS-PAGE.
PCT/DK1995/000362 1994-09-09 1995-09-11 Proteines inductibles par la chimiotaxine des monocytes WO1996007673A1 (fr)

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AU33805/95A AU3380595A (en) 1994-09-09 1995-09-11 Monocyte chemotaxin inducible proteins

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6270758B1 (en) 1998-10-08 2001-08-07 Duke University Substantially non-toxic biologically active mucosal adjuvants in vertebrate subjects

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2556220A1 (fr) * 1983-12-13 1985-06-14 Hayashibara Biochem Lab Nouveau lymphokine et anticorps monoclonal specifique de ce lymphokine, leur production et leurs utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2556220A1 (fr) * 1983-12-13 1985-06-14 Hayashibara Biochem Lab Nouveau lymphokine et anticorps monoclonal specifique de ce lymphokine, leur production et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
K. MATSUSHIMA ET AL: "Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 169, pages 1485 - 1490 *
T. YOSHIMURA ET AL: "Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes", JOURNAL OF IMMUNOLOGY, vol. 142, no. 6, 15 March 1989 (1989-03-15), BALTIMORE US, pages 1956 - 1962 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6270758B1 (en) 1998-10-08 2001-08-07 Duke University Substantially non-toxic biologically active mucosal adjuvants in vertebrate subjects
US7041294B2 (en) 1998-10-08 2006-05-09 Duke University Substantially non-toxic biologically active mucosal adjuvants in vertebrate subjects

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