Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity

Definitions

• This invention relates to new, low-molecular-weight, human endogenous cancer regulatory factors, method of preparing the same, and pharmaceutical compositions containing the same.
• the inventors formerly isolated human endogeous cancer regulatory factors ( Krebs Statika; KBS ), novel biologically active substances, by growing human monocytes or cloned strains therefrom in a tissue culture medium.
• the KBS thus obtained was of two types: one having a relative molecular weight of 82,000 ⁇ 10,000 and an isoelectric point of pH 6.5 ⁇ 0.5 and the other having a relative molecular weight in the range from 50,000 to 72,000 and an isoelectric point in the pH range from 6.0 to 8.5 [ Japanese
• KBS of the latter type was further divided into three substances of different isoelectric point: KBS- ⁇ ( pH approximately 6.5 ), KBS- ⁇ ( pH approximately 7.0 ) and KBS- ⁇ ( pH approximately 8.0 ).
• KBS is a group of biologically active substances derived from human cells, and is considered, because of the powerful activity, both in vitro and in vivo, against many kinds of tumor , to be amongst the substances that are produced in very small amounts in the bodies of patients who have spontaneously recovered from cancers.
• n-KBS the low-molecular-weight, human endogenous cancer regulatory factors of this invention
• KBS relative molecular weight 50,000 to 92,000, isoelectric point pH 6.0 to 8.5
• n-KBS of this invention may be considered to be one of the subunits of KBS, which has been separated from KBS as a result of treatment with reducing agent and/or protein denaturing agent through at least partial destruction of its higher-order structure or at least partial scission of its disulfide linkages.
• n-KBS Like KBS, n-KBS has powerful activity against many kinds of tumor , both in vitro and in vivo, and in vivo. also shows tumor necrotic activity.
• n-KES which is derived from human cells like KBS, is not recognized as heterologous in human bodies and therefore causes little, if any, antigen-antibody reaction. It is also possible to reconstruct KBS from n-KBS of this invention through protein renaturation or restoration of disulfide linkages.
• Figure 1 is a KBS-activity elution profile of n-KBS after being subjected to electrophoresis ( Example 1 ) , in the recovered f rom which the ordinate represents KBS activity each gel slice and the abscissa denotes fraction number.
• the arrows indicate the elution positions of reference proteins [ TF, human transferrin ( molecular weight 76,000 ); HSA, human serum albumin ( molecular weight: 67,000 ); OVA, ovalbumin ( molecular weight 43,000 ); SBTI, soybean trypsin inhibitor ( molecular weight 21,500 ); and HG, hemoglobin
• Figure 2 shows a SDS-polyacrylamide gel electrophoresis ( SDS-PAGE in the presence of 2-mercaptoethanol ) of the main peptide fragment and n-KBS ( Example 1 ).
• n-KBS of this invention can be obtained by growing human monocytic cells capable of producing KBS, or a cloned strain therefrom, in a tissue culture medium to give KBS having a relative molecular weight in the range from 50,000 to 92,000 and an isoelectric point in the pH range from 6.0 to 8.5, followed by treatment with reducing agent and/or protein denaturing agent.
• human, normal or leukemic cells capable of producing KBS, or a cloned strain therefrom are grown in a tissue culture medium and undergo first and second stimulation, affording KBS having a relative molecular weight in the range from 50,000 to 92,000 and an isoelectric point in the pH range from 6.0 to 8.5, and KBS thus obtained is in turn treated with reducing agent and/or protein denaturing agent.
• the normal or leukemic monocytic cells used in this invention include normal monocytes, macrophage cells and leukemic cells thereof, and those which can be easily proli ferated in a tissue culture medium are more preferable.
• leukemic cells of monocyte/macrophage series are leukemic cells of monocyte/macrophage series.
• Typical examples include HL-60, which are leukemic cells of monocytic macrophage series; YKBS-7-15, YKBS-7-16 and YKBS-7-17 which are monocytic leukemic cells selected from clinically established peripheral leukocytes of monocytic leukemia origin; and cloned strains 3A1, therefrom, such as clones 2A2, 2A6 , 2C6 , 3B3 , 3B4 , 3C1 , 3D5 ,
• YKBS-7-1 5 monocytic leukemic cells
• YKBS-7-1 5 monocytic leukemic cells
• derived from human leukemic peripheral leukocytes were placed on a 96-well micr ⁇ plate, previously seeded with 1 x 10 6 mouse thymocytes as feeder cells , at a concentration of 1 to 2 cell/well ( limiting dilution. method ) , and grown at 37°C under an atmosphere of 5 % CO 2 and 95 % air.
• colonies were visible, subculture was carried out by successively transf errring them into a 24-well microplate , and colonies highly capable of KBS production were selected.
• the established call lines YKBS-7-15, YKBS-7-16 and YKBS-7-17, are monocytic leukemia cell lines which the inventors and the Institute of Medical science, University of Tokyo have succeeded in isolating by culturing the buffy coat leukocytes from the peripheral blood of a patient suffering acute monocytic leukemia in conventional manner including
• YKBS-7-15 and YKBS-4B3 which is an isolated clone from YKBS-7-15 have been deposited with the C.N.C.M. of (February 29, 1984) (July 25, 1984)
• cells capable of differentiation into monocytic cells may of course be used in this invention. These cells eventually exhibit the properties of monocytic leukemia, and malignant myeloma cells are one example.
• the cells listed above are subjected to tissue culture usually in a growth medium containing fetal calf serum ( FCS ).
• FCS fetal calf serum
• YKBS-4E3, a clone of YKBS-7-15 used in Example 1 can be successfully grown in RPMI-1640 medium ( Gibco Co. ) containing 10% FCS, 15mM HEPES ( Wako Junyaku Co. ), 50 mg/l kanamycin and 25 mg/l streptomycin.
• tissue culture in serum-free growth media, such as HB101 ( Hana Biologies Inc. ) or ISCOVE's medium ( Flow laboratories ) .
• the cells may also be proliferated intraperitoneally or subcutaneously in non-human, warm-blooded animals like nude mice and juvenile hamste rs.
• the tissue culture media as used in this invention include such in vivo growth media.
• Examples of the first stimulant used in this invention include conditioned media with lymphocytes or lymphoblasts , chemical susbstances or natural extracts that induce cellular differentiation, and mixtures thereof.
• Another example is the supernatant which is obtained by culturing sens itized cells in animals administered with a certain chemical susbstance such as Trichothecene mycotoxin.
• PHA phytohemagglutinin
• macrophage activating substances such as muramyl dipeptide ( MDP ) , 1 2 -0- tetradecanoylphorbo l - 1 3 -acetate ( TPA ) , 12, 1 3-phorbol butyrate, dimethylsulfoxide (DMSO ) and mezerein are particularly preferred.
• substances capable of activating the reticuloendothelial system may also be used as the first stimulant in this invention.
• These are ordinary Grampositive bacteria, fungus-produced materials, protozoans and yeast, which are used in the form of living cells, dead cells ( after heat or formalin treatment, for example ) or cell extracts.
• Propioni bacteria such as P. acnes ( Corynebacterium parvum ) and P. granulocum ( Corynebactarium granulocum ); Mycobacteria, such as Bacillus Calmette-Guerin ( B.C.G. ) and M. segmentis; and Mocardia, such as N. erythropolis and N.
• Illustrative fungus-produced materials are toxins produced by Fusarium. Typical protozoans are Plasmodium and Toxoplasma. A commonly used yeast is Zymosan extracted from Saccharomyces cereviciae or the like. Certain synthetic polymers such as pyran copolymers may also be employed as the first stimulant.
• the second stimulant used in this invention is an endotoxin produced by Gram-positive or Gram-negative bacteria.
• Typical examples include lipopolysaccharides derived from E. coli, Pseudomonas aeruginosa and typhoid bacillus. Lipopolysaccharides from Gram-positive bacteria can also be used with good results.
• KBS can be produced by growing normal human monocytic cells or monocytic leukemic cells in a growth medium or a common tissue culture medium containing serum and other nutrients, and adding the first stimulant before, during or after cultivation, followed by addition of the second stimulant.
• both stimulants first and second
• KBS could be produced by the action of either stimulant alone or even in the absence of any stimulant.
• the cells are fully proliferated in a usual tissue culture medium, and the first stimulant (for example, 0.1 to 100 ng/ml of TPA ) is then added to induce the first stimulation.
• the second stimulant for example,
• KBS lipopolysaccharide derived from E. coli
• these techniques may be combined with such additional operations as adsorption on ion exchangers followed by elution therefrom, gel filtration, affinity chromatography using concanavaline A or a suitable antibody supported on Sepharose, isoelectric fractionation, high-performance liquid chromatography, chromatofocusing by means of high-performance liquid chromatography for proteins, ion exchange (for example, on FPLC/Mono-P and Mono-Q columns; Pharmacia AB ), and slab or other types of electrophoresis on polyacrylamide gel.
• the supernatant containing KBS is separated from the culture medium by centrifugation and then treated with an anion exchanger, followed by purification by other techniques.
• Suitable anion .exchangers include DEAE-Sephadex A-50, DEAE-Sepharose CL-6B, DEAE- Sephacal, QAE-Sephadex A-50 ( products of Pharmacia AB ), AIECDE 52 ( Wattman Co. ), Servacel AS ( Serva Co. ) and Cellex QAE ( Bio-rad Laboratories ).
• KBS thus produced is then treated with reducing agent and/or denaturing agent to give n-KBS of this invention by using techniques commonly employed in this technical field.
• the reducing agent may be mentioned thiol compounds, such as 2-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acid, monothiophosphoric acid, cysteine, N-acetylcystein and reduced-form glutathione; tertiary phosphines, such as tri-n-butylphosphine and trisdiethylaminoethylphosphine; sulfites, such as sodium sulfite; and sodium borohydride.
• thiol compounds such as 2-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acid, monothiophosphoric acid, cysteine, N-acetylcystein and reduced-form glutathione
• Typical protein denaturing agents include urea, guanidine hydrochloride, guanidine sulfate, and surface-active agents ( anionic, cationic, amphcteric and nonionic ).
• Illustrative examples of the surface-active agents are sodium d ⁇ cecylsulfate ( SDS ), sorbitan fatty acid esters, glycerol fatty acid esters, p ⁇ lyoxyethylene nonylphenyl ethers, sodium polyoxyethylene alkyl sulfates, sodium di-2-ethylhexylsulfosuccinate, dodecyltrimethylammonium bromide and deoxylysolecithin.
• the treatment is carried out under mild conditions, for example, by adding reducing agent and/or protein denaturing agent to a solution of KBS in water or a suitable buffer, or to a fraction containing KBS.
• concentration of reducing agent and/or protein denaturing agent, treating temperature ( normally room temperature ), treating time, pH aftd other treatment conditions are properly selected according to the concentration of KBS used and other factors.
• KBS can be converted to n-KBS by polyacrylamide gel electrophoresis ( PAGE ) in the presence of reducing agent and/or protein denaturing agent. It is also possible to obtain n-KBS from KBS by subjecting it to gel filtration using gel particles such as dextran, polyacrylamide and agarose (e.g., Sephadex G-50, G-75,
• n-KBS produced and then recovered and purified has the properties enumerated below: ( 1 ) Molecular weight
• the relative molecular weight o f n-KBS as measured by di sk polyacrylamide gel electrophoresis ( PAGE ) in the presence of 2-mercaptoethanol and sodium dodecylsulfate ( SDS ) is 1 7, 000 to 30, 000.
• n-KBS has the following partial amino acid sequence :
• n-KBS contains the following amino acids in addition to Cys and Trp:
• n-KBS appeared at a retention time of 28 minutes as a single peak when measured on TSK gel G2000 SW column ( 7.5mm ID x 60cm ) at room temperature using 0.1 M sodium phosphate(pH 6.0)/5.5M guanidine hydrochloride as eluent at a speed of 0.5 ml/min.
• n-KBS The anti-tumor activity of n-KBS of this invention is described below.
• Meth A methylchoranthrene-A-induced sarcoma
• n-KBS of this invention has remarkable tumor necrosis activity when tested on nude mice transplanted with human-derived, gastric cancer cell line MKN-45 ( serially maintained on nude mice ) according to the method of Haranaka, et al. [ The Japanese Journal of Clinical Medicine, 40, No.8, pp. 186-193 ( 1982 )]. n-KBS also proved effective against Sarcoma 180, Ehrlich solid tumor, P388 solid tumor and Lewis lung carcinoma.
• the biological activity ( KBS activity ) of the low-molecular-weight, human endogenous cancer regulatory factor ( n-KBS ) of this invention was evaluated by an in-vitro cytotoxicity test using normal or malignant mammalian cells according to the procedure reported by Carswell, et al. [ Proc. Nat. Acad. Sci. USA, 72, No.9, 3666-3670 ( 1975 )]. The test was conducted using a 96-well microplate ( Nunk Inc. of Denmark ), Eagle's minimum essential medium ( MEM) containing 10% fetal calf serum, 50 ⁇ g/ml of kanamycin and 25 ⁇ g/ml streptomycin or 50 unit/ml of gentamycin or sisomicin. 2.5 X 10 4 L cells were incubated with serially diluted samples of equal volume
• n-KBS may assume any desired form suitable for oral or parenteral administration, such as powders, granules, tablets, sugar-coated tablets, capsules, pills, suppositories, suspensions, liquids, emulsions, injections and aerosols.
• the factor is previously lyophilized, and administered to patients intravenously, subcutaneously or intramuscularly after being dissolved, prior to use, in physiological saline, sterile water or aseptic isotonic solution for injection. It is also possible to add, prior to lyophilization, suitable stabilizers such as mannitol and human serum albumin, as well as solubilizing agents such as glycine.
• n-KBS of this invention may vary depending on patient sensitivity, age, sex, body weight, and conditions, on route, timing and frequency of administration, on the type and properties of the formulation, and on other factors. Therefore, the dose level shown below should be considered a guide figure; a lower dose may serve the purpose in some cases, and a higher dose may be necessary in some other cases. Normally, however, the minimum daily dose for human adults is 10,000 units.
• n-KBS of this invention ( 1,000,000 units ) was dissolved in 100 ml of physiological saline, the solution was filtered germ-free, and the filtrate was dispensed into vials ( 1 ml in each ) and freeze-dried.
• Amino acids may hereinafter be represented by abbreviations specified by the Commission on Biochemical Nomenclature ( CBN ) under IUPAC-IUE, or by trivial abbreviations commonly used in this particular field. L-isomer is meant, unless otherwise specified, for amino acids in which optical isomerism can exist.
• Example 1 a) YKBS-4B3 a cloned strain of YKBS-7-15 ( monocytic leukemia cells ) which is an established cell line derived from peripheral buffy coat leukocytes of a monocytic leukemia patient was grown in 8 liters of serum-free HB101TM medium
• TPA 5 ng/ml
• endotoxin lipopolysaccharide derived from E. coli 0111-B4
• Pellicon Cassette and saturated aqueous ammonium sulfate was added at 4°C to a final concentration of 50% (v/v) .
• the precipitate was collected by centrifugation, dissolved in a small amount of 20mM Tris-HCl buffer solution containing 0.04M sodium chloride ( pH: 7.8 ), and dialyzed against the same buffer as above.
• the dialyzate was applied to DEAE-Sephadex A-50 anion-exchanger (loaded on a Buchner funnel and previously equilibrated with the same buffer as above ) and eluted with 20mM Tris-HCl buffer solution containing 0.03M sodium chloride ( pH 7.8 ).
• the eluate was again concentrated by Pellicon Cassette, the concentrate was thoroughly dialyzed against 50mM aqueous solution of ammonium bicarbonate,hen the dialyzate was freeze-dried.
• the dry powder thus obtained was dissolved in a small amount of 20mM Tris-HCl buffer solution containing 0.04M sodium chloride, and the solution was subjected to gel chromatography on an Ultrogel
• n-KBS This sample of n-KBS was submitted to the following tests.
• Amino acid composition n-KBS (0.5 to 1.0 ⁇ g) was hydrolyzed in 0.1 ml of 6N-HCl, at 110°C for 24, 48 and 72 hours in a sealed tube, and the hydrochloric acid was removed under reduced pressure, and 0.30 to 0.35 ml of 0.02N-HCl was added to prepare test specimens.
• Amino acid analysis was perfomed by the fluorescent method using o-phthalaldehyde on a Hitachi Amino Acid Analyser Model 835 (Hitachi, Ltd.). Amino acid composition determined on the basis of two experiments is shown in Table 1 below. ( Table 1 )
• FIG. 1 shows a silver-stained profile of SDS/PAGE of this main peptide fragment and n-KBS.
• Example 2 a) YKBS-4B3, a cloned strain from YKBS-7-15, was grown in 8 liters of RPMI 1640 medium ( Gibco Co. ), which is a commonly used, basic growth medium containing 10% fetal calf serum, at 37°C for a sufficient period of time with agitation, 5 ng/ml of 1 2-O-tetradecancylphorbol-1 3-acetate ( TPA ) was added to induce the first stimulation, 1 ⁇ g/ ml of endotoxin ( lipopolysaccharide derived from E. coli 01 1 1 :B4 ) was added as the second stimulant 36 hours later, cell culture was continued for an additional 1 6 hours , and the culture supernatant was collected by centrifugation.
• RPMI 1640 medium Gibco Co.
• TPA 2-O-tetradecancylphorbol-1 3-acetate
• This supernatant was diluted with 20mM Tris-HCl buffer solution ( pH 7.8 ) until the final salt concentration fell below 0.04M, and the diluted solution was allowed to f low batchwise through a Buchner funnel loaded with DEAE-Sephadex A-50 anion-exchanger, previously equilibrated with 20mM Tris-HCl buffer solution ( pH 7.8 ) containing 0.0414 sodium chloride, to recover the unadsorbed fraction.
• the column was thoroughly washed with the same buffer solution as above to remove unadsorbed substances , the fraction adsorbed on Con-A was eluted with 20mM Tris-HCl buffer solution ( pH 7.8 ) containing ⁇ -methylmannoside and 0.5M sodium chloride, and the eluate was concentrated and dialyzed overnight against 25mM triethanolamine/ imino diacetic acid buffer solution ( pH 8.1 ).
• the dialyzate was then subjected to chromatofocusing by means of high-performance liquid chromatography for proteins ( FPLC, Mono P column, Pharmacia AB ) to effect isoelectric fractionation ( linear gradient method over the pH range from 8.1 to 5.0 ), affording separate KBS-activity peaks at pH of about 7.0 ( KBS- ⁇ ) and at pH higher than 8.0.
• KBS- ⁇ fraction was dialyzed overnight against 25mM triethanolamine/iminodiacetic acid buffer solution ( pH 8.1 ), and the dialyzate was subjected to chromatofocusing on FPLC, Mono P column in the same manner as above, giving the active fraction ( KBS- ⁇ ) as a single, sharp peak at pH of about 7.0.
• the KES-active fraction at pH higher than 8.0 was dialyzed overnight against 25mM diethanolamine-HCl buffer solution, and the dialyzate was subjected to chromatofocusing on FPLC, Mono P column ( linear gradient method over the pH range from 9.5 to 6.8 ), affording the active fraction ( KBS- ⁇ ) as a single, sharp peak at pH of about 8.0.
• the total cytotoxic activity of the active substances thus recovered against L cells amounted to 4.0 x 10 4 units for KBS- ⁇ and to 1.0 x 10 4 units for KBS- ⁇ .
• the relative molecular weight obtained was 60,000 ⁇ 10,000'for KBS- 8 and 62,000 ⁇ 10,000 for KBS- ⁇ .
• a-ii) The fraction adsorbed on DEAE-Sephadex A-50 was eluted with 20mM Tris-HCl buffer solution containing 0.1 M sodium chloride ( pH 7.8 ), the eluate was concentrated, and the salt concentration was adjusted to 0.5M sodium chloride.
• n-KBS having a relative molecular weight of 22,500 ⁇ 1,500 was obtained from the fraction of KBS- ⁇ in a manner similar to that of Example 1-b). The yield was approximately 28%. Similarly, n-KBS could also be obtained from the fraction of KBS- ⁇ .
• the eluate was again concentrated by Pelli con Cassette the salt concentration was adj usted to 0.5M sodium chloride, and the resulting solution was subj ected to affinity chromatography using a Con-A Sepharose column previously equilibrated with 20mM Tris-HCl buffer solution ( pH 7.8 ) containing 0.5M sodium chloride.
• the column was washed with the same buffer solution as above to collect the unadsorbed fraction, which was concentrated and dialyzed overnight against 25mM triethanolamine/ iminodiacetic acid buffer ( pH 8.1 ) , af fording fractions containing KBS- ⁇ and KBS- ⁇ .

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• 1986
  • 1986-01-10 PT PT81823A patent/PT81823B/pt not_active IP Right Cessation
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  • 1986-01-13 WO PCT/JP1986/000011 patent/WO1986004069A1/en not_active Application Discontinuation
  • 1986-01-13 EP EP86900636A patent/EP0216929A1/en not_active Withdrawn
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