WO1986004069A1

WO1986004069A1 - Human endogenous cancer regulatory factors

Info

Publication number: WO1986004069A1
Application number: PCT/JP1986/000011
Authority: WO
Inventors: Tadashi Obara; Hisanori Ezoe; Toshiyuki Takemoto; Tetsuo Morinaga; Katsuyuki Haranaka; Nobuko Satomi
Original assignee: Yamanouchi Pharmaceutical Co., Ltd.
Priority date: 1985-01-14
Filing date: 1986-01-13
Publication date: 1986-07-17
Also published as: NO863657L; EP0216929A1; KR870700072A; ES8705466A1; DK436786D0; DK436786A; PT81823B; NO863657D0; PT81823A; ES550833A0

Abstract

Human endogenous cancer regulatory factors useful as anti-tumor agents having a molecular weight in the range from 17,000 to 30,000, and pharmaceutical compositions containing the same. The low-molecular-weight, human endogenous cancer regulatory factors of the present invention are obtained by growing human monocytic cells or cloned strains therefrom in a tissue culture medium to form human endogenous cancer regulatory factors having a 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 a reducing agent and/or a protein denaturing agent.

Description

DESCRIPTION

HUMAN ENDOGENOUS CANCER REGULATORY FACTORS

Field of the Invention

This invention relates to new, low-molecular-weight, human endogenous cancer regulatory factors, method of preparing the same, and pharmaceutical compositions containing the same.

Background of the Invention

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

Patent Application No.128, 893 / 1983 and No.99, 370

/1984]. KBS of the latter type was further divided into three substances of different isoelectric point: KBS-α ( pH approximately 6.5 ), KBS-β ( pH approximately 7.0 ) and KBS-γ ( 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.

Unexpectedly, further studies have led us to find that a novel biologically active substance could be obtained from this KBS by treatment with reducing agent and/or protein denaturing agent. This new biologically active substance derived from KBS has a molecular weight in the range from

17,000 to 30,000 and shows anti-tumor activity, with a fraction having a molecular weight in the range from 20,000 to 30,000, particularly in the range of 22,500 ± 1,500, being the most preferred. We named it low-molecular-weight, human endogeous anti-tumor substance ( nieder Krebs Statika; n-KBS ) because of its lower molecular weight compared with KBS.

Disclosure of the Invention n-KBS, the low-molecular-weight, human endogenous cancer regulatory factors of this invention, are novel proteins having a relative molecular weight in the range from 17,000 to 30,000 and showing anti-tumor activity, and can be produced by treating KBS ( relative molecular weight 50,000 to 92,000, isoelectric point pH 6.0 to 8.5 ) with reducing agent and/or protein denaturing agent. Hence, 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. 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 In addition, there is valid reason to believe that 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.

Brief Description of the Drawings

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

(molecular weight 15,500); and 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 ).

Detailed Description of the Preferred Embodiments 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. To be more specific, 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.

Particularly preferred 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 ,

4B3 and others cloned from YKBS-7-1 5. As a typical example of cloning , the method used for selection of the desired clones from the established YKBS-7-1 5 cell line [ Japanese Patent Application No.239 , 355 / 1 983 ] wi ll be described below.

Cultured cells of YKBS-7-1 5 ( monocytic leukemic cells ) , derived from human leukemic peripheral leukocytes , were placed on a 96-well micrσplate, previously seeded with 1 x 10⁶ 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 % CO₂ and 95 % air. When 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. Similar operations were repeated while gradually scaling up the culture , eventually picking out clones 2A2 , 2A6 , 2C6 , 3A1 , 3B3 , 3B4 , 3C1 , 3D5 and 4B3 as strains of highest KBS productivity.

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

( ammonium chloride treatment for removal of erythrocytes ).

Of these, 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)

Pasteur Institute, France ( I-258 and 1-319, respectively ).

As other examples of cell lines of mαnocyte/macrophage series, may be mentioned Mono-1 and Mono-1 -207 [ Virchow

Arch. A. Pathol. Anat. Histol. 371 , 15 ( 1976 ) and 379, 269 ( 1978 )].

In addition, 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 ). For example, 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. In this formulation, penicillin, gentamycin, sisomicin and other antibiotics may also be used in place of kanamycin and streptomycin. It is also possible to effect the tissue culture in serum-free growth media, such as HB101 ( Hana Biologies Inc. ) or ISCOVE's medium ( Flow laboratories ) . In addition, 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. One example of the chemical substances that induce cellular differentiation is phytohemagglutinin ( PHA ) , but 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.

In addition, 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. As illustrative gram-positive bacteria may be mentioned 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. gardneri. 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. Although both stimulants ( first and second ) are commonly used for induction,. KBS could be produced by the action of either stimulant alone or even in the absence of any stimulant. Most commonly, however, 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. After cultivation over a period of 12 hours to three days, the second stimulant ( for example,

0.1 to 10 μg/ml of lipopolysaccharide derived from E. coli ) is added to induce the second stimulation, thus yielding,after further cultivation for a certain period, say 8 to 24 hours, KBS in the supernatant culture liquid. KBS thus produced, after being isolated or without being isolated, is treated with reducing agent and/or denaturing agent to give n-KBS of this invention. When KBS is to be isolated before the subsequent treatment, this can be done by using known separation and purification techniques, such as dialysis, salting out, ultrafiltration, centrifugation, concentration and freeze drying. If further purification is required, 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. Usually, 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. As examples of 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. 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 dσcecylsulfate ( SDS ), sorbitan fatty acid esters, glycerol fatty acid esters, pαlyoxyethylene 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. The 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. Alternatively, 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,

Sephacryl S-200, Biogel P-10, P-30, P-60, Ultrogel AcA44 and

AcA54 ) in the presence of reducing agent and/or protein denaturing agent. thus 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.

( 2 ) Main peptide fragment

The relative molecular weight of the main peptide fragment separated by cyanogen brom ide cleavage of n-KBS is approximately 1 8 ,500. ( 3 ) Partial amino acid sequence n-KBS has the following partial amino acid sequence :

Gin — His — Leu — Ala — Lys — Ser — A.sn — Leu- — X₁ — Pro —

Ala — X₂ —Ile — Pro — Tyr

( in which X₁ and X₂ each represent an unidentified amino acid residue) .

( 4) Amino acid composition n-KBS contains the following amino acids in addition to Cys and Trp:

Amino acid Content ( mol% )

Asp/Asn 9.9

Thr 5.6

Ser 8.3

Glu/Gln 11.3

Pro 9.3

Gly 9.8 Ala 7.6

Val 6.1

Met 1.5

He 3.6 Leu 9.0

Tyr 3.1

Phe 3.4

His 3.5

Lys 7.1 Arg 3.1

( The contents shown above are mole percentages of individual amino acid residues based on the total amino acid residues excluding Cys and Trp. Standard deviation is normally ±15%, although this level is exceeded in some cases. )

(5) Retention time in high-performance liquid chromatography 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.

The anti-tumor activity of n-KBS of this invention is described below. Cells ( 5 x 10⁵ ) of mouse-derived. methylchoranthrene-A-induced sarcoma ( Meth A ), serially maintained in Balb/C mice, were transplanted into the dorsal skin of Balb/C mice ( male, 7-week old ). After seven days, the size of tumors developed was measured and 0.5 ml of serial dilutions of n-KBS ( the sample prepared in Example 1 , molecular weight 22,500 ) were intravenously injected.

Tumor size measured nine days after administration revealed that complete cure was observed in all the test groups. For all the mice on which any degree of reduction in tumor size was noticed, tumor size continued to decrease tumor from then on, with the transplanted Meth Aifinally disappearing completely. For the control group, on the contrary, the size of the transplanted tumor increased to more than 1.5 cm after 19 days, and nearly all of the mice were dead

40 days later. The remarkable anti-tumor activity of n-KBS of this invention is apparent from the result of this experiment.

It was also demonstrated that 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 μg/ml of kanamycin and 25 μg/ml streptomycin or 50 unit/ml of gentamycin or sisomicin. 2.5 X 10⁴ L cells were incubated with serially diluted samples of equal volume

at 37°C for 48 hours in an atmosphere of air containing 5% carbon dioxide, and the cytotoxic effect was observed under a microscope. 1 unit ( U ) was defined by a reciprocal dilution of the sample to effect 50% killing of the L cells.

The cancer regulatory factor of this invention is administered in a form that allows full exhibition of its anti-tumor effect when used as medicine. Although it may be directly administered, it is also possible to offer it, according to pharmaceutical practice, as a mixture with pharmaceutically acceptable diluents and/or other pharmacologically effective substance (S) . Thus 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. Typically, 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.

The suitable dose of 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.

The following examples will further illustrate the invention but are not to be considered a limitation thereupon.

Formulation Example ( Injection ) 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.

Asp Aspartic acid Asn Asparagine Thr Threonine Ser Serine Glu Glutamic acid

Gin Glutamine Gly Glycine Ala Alanine Val Valine Met Methionine

He Isoleucine Leu Leucine Tyr Tyrosine Phe Phenylalanine Lys Lysine His Histidine

Arg Arginine

Pro Proline

Cys Cysteine Trp Tryptophan

Asp/Asn Aspartic acid and asparagine

Glu/Gln Glutamic acid and glutamine

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 HB101™ medium

( Hana Biologies Inc. ) at 37°C for a sufficient period of time with agitation. TPA ( 5 ng/ml ) was added to effect the first stimulation; 1 ng/ml of endotoxin ( lipopolysaccharide derived from E. coli 0111-B4 ) was added 36 hours later for the second stimulation, cell culture was continued for an additional 16 hours, and the culture supernatant was collected by centrif ugation. This supernatant was concentrated about 20-fold by

Pellicon Cassette and saturated aqueous ammonium sulfate was added at 4°C to a final concentration of 50% (v/v) . After thorough aging at 4°C, 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

AcA44 column, giving an active fraction with a relative molecular weight of 60,000 ± 10,000. The overall yield so far 25%. was about [This fraction, when subjected to chromatofocusing by means of high-performance liquid chromatography for prousing teins ( FPLC, Mono P column, Pharmacia AB ) to effect isoelectric fractionation using the linear gradient method , gave three KBS-activity peaks in the vicinity of pH 6.5 ( KBS-α ), pH 7.0 ( KBS -β ) and 8.0 ( K3S-λ ). ] b) The active fraction thus obtained ( relative molecular weight 60,000 ± 10,000 ) was extensively dialyzed, and subjected to disk polyacrylamide gel electrophoresis (PAGE) in the presence of 2mM 2-mercaptoethanol and 0.1% sodium dodecyl sulfate (12.5% T; disk: 8 cm) according to the method of Laemmli [Nature, 227, 680 (1970)]. After electrophoresis, the gel was cut into 1mm slice, protein was eluted, and KBS-activity was assayed as described above. KBS-activity was recovered in fractions corresponding to the relative molecular weight range of 22,500 ± 1,500. The result is summarized in Figure 1. The yield of n-KBS thus obtained was about 25%.

This sample of n-KBS was submitted to the following tests. (1) Amino acid composition n-KBS (0.5 to 1.0 μg) 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 )

Amino acid Content ( mol% )

Asp/Asn 9.9 ± 1.1

Thr 5.6 ± 0.3 Ser 8.3 ± 1.3

Glu/Gln 11.3 ± 0.7

Pro 7.3 ± 0.6

Gly 9.8 ± 0.8

Ala 7.6 ± 0.1 Val 6.1 ± 0.6

Met 1.5 ± 0.5

He 3.6 ± 0.6

Leu 9.0 ± 0.7

Tyr 3.1 ± 0.3 Phe 3.4 ± 0.8

His 3.5 ± 0.3

Lys 7.1 ± 0.5

Arg 3.1 ± 0.1

( The contents shown above are mole percentages of individual amino acid residues based on the total amino acid residues excluding Cys and Trp. Standard deviation is represented by the value folowing symbol ±. ) ( 2 ) Partial amino acid sequence (a) Cyanogen bromide cleavage and isolation of peptide fragments Cyanogen bromide cleavage was performed by treating one part of n-KBS with ten parts of cyanogen bromide in 88% formic acid at room temperature for 24 hours. At the end of reaction, the mixture was diluted with distilled water, the resulting solution was concentrated to dryness under reduced pressure, part of the dry sample thus protein obtained was subjected to slab SDS/PAGE, and the bands developed were checked by the silver staining method.

It was found that, as a result of cyanogen bromide protein cleavage, the band corresponding to a molecular weight of

22,500 disappeared and new bands appeared in its place

( a main band corresponding to a molecular weight of about

18,500 and some others which are lower in intensity ). dried SDS

The rest of the sample was subjected to PAGE in the presence of 2mM 2-mercaptoethanol

( 15% T; disk: 15 cm ), and the main band corresponding to a molecular weight of about 18,500 was cut out.The protein was eluted from the gel slice by means of electrophoretic elution . Figure 2 shows a silver-stained profile of SDS/PAGE of this main peptide fragment and n-KBS.

Standard molecular-weight protein markers (Bio-rad Laboratories Ltd.) are also indicated in the Figure. b) Determination of partial amino acid sequence The amino acid sequence of the main peptide fragment obtained above was examined by applying the automatic Edman degradation method using a gas-phase protein sequenator ( Model 470A, Applied Biosystems ). The analysis revealed fifteen amino acids arranged in the sequence shown below.

1 2 3 4 5 6 7 8 9 H — Gin — His — Leu — Ala — Ly s — Ser — Asn — Leu — X₁ — 10 11 12 13 14 15 Pro — Ala — X₂ —Ile — Pro — Tyr

( in which X₁ and X₂ each represent an unidentified amino acid residue).

(3) Retention time in high-performance liquid chromatography 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.

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 μg/ 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.

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. a-i) This unadsorbed fraction was concentrated by means of Pellicon Cassette, the salt concentration was adj usted to 0.5M sodium chloride, and the resulting solution was subj ected to af finity 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 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 α-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-β ) and at pH higher than 8.0.

Each of these active fractions was collected and separately treated as follows. KBS-β 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-β ) as a single, sharp peak at pH of about 7.0. The KES-active fraction at pH higher than 8.0, on the other hand, 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-λ ) 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 10⁴ units for KBS-β and to 1.0 x 10⁴ units for KBS-λ.

Part of each of the two active fractions was added to a column ( ø5 x 1400mm ) packed with Sephadex G-200 ( Phar macia AB ) for determination of relative molecular weight.

The relative molecular weight obtained was 60,000 ± 10,000'for KBS- 8 and 62,000 ± 10,000 for KBS-γ. 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. The resulting solution was subjected to affinity chromatography usihg a Con-A Sepharose column in the same manner as in a-i), and the fraction adsorbed on Con-A was concentrated, dialyzed overnight, and subjected to chromatofocusing on FPLC, Mono P column ( linear gradient method over the pH range from 8.1 to 5.0 ) to effect isoelectric fractionation, affording a fraction with KBS activity at pH about 6.5 ( KBS-α). The total cytotoxic activity of KBS-α against L cells was 2.5 x 10⁴ units. Part of the active fraction obtained above was added to a column ( ø5 x 1400mm ) packed with Sephadex G-200 ( Pharmacia AB ) for determination of relative molecular weight. The relative molecular weight of KBS-α turned out to be 60,000 ± 10,000. b) n-KBS having a relative molecular weight of 22,500 ± 1,500 was obtained from the fraction of KBS-β 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-α.

Example 3 a) YKBS-4B3 a cloned strain of YKES-7-15 was grown in 8 liters of serum-free HB101™ medium at 37°C for a sufficient period of time with agitation. TPA ( 5 ng/ml ) was added to effect the first stimulation, 1 μg/ml of endotoxin ( lipopolysaccharide derived from E. coli 0111-B4 ) was added 36 hours later for the second stimulation, cell culture was continued for an additional 16 hours, and the culture supernatant was collected after centrifugation. This supernatant was concentrated about 20 -fold by Pellicon Cassette, and saturated aqueous ammonium sulfate was added at

4°C to give a final concentration of 50% (v/v). After thorough aging at 4°C, 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 adsorbed on DEAE-Sephadex A-50 anion-exchanger loaded on a Buchner funnel and eluted with 20mM Tris-HCl buffer solution containing 0.08M sodium chloride ( pH 7.8 ).

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-β and KBS-Ɣ. These were subjected to chromatofocusing ( FPLC, Mono P column; Pharmacia ) to effect isoelectric fractionation by the linear concentration gradient method, affording peaks with KBS-activity at pH of about 7.0 ( KBS-β ) and at pH of about 8.0 ( KBS-Ɣ ) . b ) n-KBS having a relative molecular weight of 22 , 500 ±

1 , 500 was obtained from the fractions containing KBS-β and KBS-Ɣ in a manner similar to that of Example 1-h) .

Claims

1. Low-mclecular-weight, human endogenous cancer regulatory factors having a molecular weight in the range from 17,000 to 30,000.

2. Low-molecular-weight, human endogenous cancer regulatory factors as defined in claim (1), said molecular weight being in the range of 22,500 ± 1,500.

3. Low-molecular-weight, human endogenous cancer regulatory factors as defined in claim (1) or (2), said cancer regulatory factors releasing, when subjected to cyanogen bromide cleavage reaction, a main peptide fragment with a molecular weight of about 18,500 and having the partial amino acid sequence

Gin — His — Leu — Ala — Lys — Ser — Asn — Leu — X₁ — Pro — Ala — X₂ — He — Pro — Tyr

( in which X₁ and X₂ each represent an unidentified amino acid residue).

4. A method of preparing low-molecular-weight, human endogenous cancer regulatory factors which comprises growing human monocytic cells capable of producing said cancer regulatory factors, or cloned strains therefrom, in a produce tissue culture medium to human endogenous cancer regulatory factors having a 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.

5. A method of preparing low-molecular-weight, human endogenous cancer regulatory factors which comprises treating human endogenous cancer regulatory factor having a 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 with reducing agent and/or protein denaturing agent.

6. A Pharmaceutical composition containing low-molecular- weight, human endogenous cancer regulatory factor as defined in calim 1, 2 or 3