FI68080C - FOER FARING FOR CARCINOSTATICS AND IMMUNOSISCULATION OF SUBSTANCES - Google Patents

Description

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C (45) '1' 'J 1 ~ ^' --j (51) Kv.lk. * / Lnt.CI. * C 12 P 1/04 FINLAND —FINLAND (21) Patent application —Patentansöknlng 80264] (22) Application date— Ansöknlngsdag 21.08.80 (23) Starting date - Giltighetsdag 21.08.80 (41) Has become public —Blivit offentlig 25.02.81

National Board of Patents and Registration Date of display and publication. 29/3/85

Patent- och registerstyrelsen v 'Ansökan utlagd och utl.skrifteri publicerad (32) (33) (31) Pyydetty etuoikeus — Begärd priori te t 24.08.79 24.08.79, 24.08.79, 24.08.79, 06.08.80, 06.08. 80, 06.08.80, 06.08.80, 06.08.80 Japan i-Japan (JP) 107814/79, 107817/79, 107818/79, 107819/79, 108120/80, 108121/80, 108122/80, 108123 / 80, 108l24 / 80 (71) Toyama Chemical Co., Ltd., 2-5, 3-chome, Nishishinjuku, Shinjuku-ku, Tokyo, Japan-Japan (JP) (72) Kenzo Tamai, Kanazawa-shi, Isamu Saikawa , Toyama-shi,

Takashi Yasuda, Toyama-shi, Shohachi Murakami, Toyama-shi,

Toyoo Maeda, Kanazawa-shi, Hisatsugu Tsuda, Toyama-shi,

Hiroshi Sakai, Takaoka-shi, Masatoshi Sugita, Toyama-shi,

Yoshiko Yamamoto, Namerikawa-shi, Hisashi Minami, Toyama-shi,

Takako Hori, Toyama-shi, Japan-Japan (JP) (74) Oy Koister Ab (54) Method for the preparation of carcinostatic and immunostimulatory substances - Förfarande för f ramstä11 Ning carcinostatic and immunostimulatory substances

The invention relates to a process for the preparation of new substances having a carcinostatic and immunostimulatory effect. These substances, called TF substances, are obtained by culturing a TF-producing bacterium belonging to the genus Fusobacterium under anaerobic conditions and recovering the substances formed from the culture or its supernatant. The method produces the following TF materials: TF-100, TF-110, TF-120, TF-130, (TF-1316, TF-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF- 136), TF-140 and TF-150.

In recent years, a form of treatment has been increasingly introduced for patients with various types of cancer, which involves increasing the patient's immunological function and obtaining a carcinostatic effect through immunological action. In such treatment, antitumor agents obtained from various bacterial organisms or cultures of bacteria or polysaccharides obtained from Basidiomycetes have been used. However, these antitumor agents are not effective enough, have harmful side effects, and are difficult to prepare.

The organism and its supernatant obtained by culturing bacteria belonging to the genus Fusobacterium are also known, for example Fusobacterium K031-3B, Fusobacterium fusifomis W-12, Fusobacterium girans 1012 and Fusobacterium nucleatum 1010, / Dental Surgery, 23, 322- (1974) and 21, 534-539 (1972) (Japanese). However, the components obtained from the culture or supernatant and their pharmacological properties have not been previously studied.

It has now been found that the supernatant obtained by culturing bacteria belonging to the genus Fusobacterium and removing organisms from the culture contains components having a carcinostatic effect. These components have virtually no inhibitory effect on cancer cell colony formation in the colony flotation assay and do not have a carcinostatic effect based on the destruction of cancer cells but an indirect carcinostatic effect based on increased patient-mediated antitumor activity or patient-mediated antitumor activity. The toxicity of the component is very low.

The invention relates to a process for the preparation of a carcinostatic and immunostimulatory TF substance which is TF-100, TF-110, TF-120, TF-130, (TF-1316, TF-132a, TF-132b, TF-133a, TF -133b, TF-1323, TF-136), TF-140 or TF-150. The method is characterized in that the Tus-producing bacterium belonging to the genus Fusobacterium is cultured under anaerobic conditions at pH 5-8.5 and at 30-45 ° C for 1-5 days in a culture medium containing nitrogen, carbon and vitamin sources as well as reducing agents and inorganic salts with or without agar; a precipitant consisting of a hydrophilic organic solvent is added to the produced culture or its supernatant after adjusting the pH of the culture or supernatant to 2-7 so that the concentration of the precipitant is 30-70 3 68080% by volume, or to the culture or supernatant adding a precipitant consisting of barium ions from a barium ion source so that the barium ion concentration is 0.005-0, IM at a pH of 10-13; the precipitate formed is recovered; the precipitant is removed from the precipitate; The fraction containing TF is separated as a water-soluble fraction from the precipitate; and the TF material is recovered from the water-soluble fraction.

The properties of the TF substances obtained by the process are shown below.

Any Tus-producing bacterium belonging to the genus Fusobacterium can be used in the method, and Fusobacterium nucleatum species, in particular Fusobacterium nucleatum TF-031 (Farm 5077; ATCC 31647), is preferably used.

The bacteriological properties of Fusobacterium nucleatum TF-031 are as follows: (I) Form (1) Cell shape: shuttle-shaped (Fig. 1) (2) Cell diversity; missing (3) Mobility: absent (4) Spores: absent (5) Gram species: gram-negative (6) Acid resistance: negative (II) Growth conditions in culture medium (1) TF agar plate and oblique surface culture medium External shape: round shape Size: approx. 1 mm

Swelling: hemispherical structure: dewdrop-like Surface: smooth Edges: smooth Color: milky yellowish white Transparency: opaque 4 68080 (2) TF liquid culture medium Growth ratio: strong Turbidity: coagulate Precipitation: absent

Surface growth: none, no growth to a depth of approx. 5 mm Gas: none (III) Physiological properties (1) Hydrogen sulphide production: + (2) Nitrate reduction: - (3) Butyric acid production: + (4) Indole production: + (5) Urease: - (6) Catalase: - (7) Hydrolysis of starch: - (8) Oxygen behavior: anaerobic (9) Ammonia production: + (10) Carbon dioxide production: +

(11) Growth range: pH 5 to 8.5, temperature 30 to 45 ° C

(12) Gas production from saccharides: L-arabinose (-), D-xylose (-), D-glucose (-), D-rannose (-), D-fructose (-), D-galactose (-) , malt sugar (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerol (-) and starch (-).

The novel TF agents of this invention are produced, for example, as follows.

5 68080

Culturing of TF-producing bacteria belonging to the genus Fusobacteriuin ___I_____

Culture or ______supernatant * 1

Hydrophilic _______ "~ '' - ^ organic i - ~ -----“ solvent | Let stand

Soluble ^ verse _f ----------------- T_SE ° fc! 5_ _] | Sakka |

Phosphate buffer / ______________ ...... (sodium chloride) or water__________

Insoluble) "~~~; verse <---------- Difference ----! DryingV ------ [Water soluble" --- ^ ------- 'verse x 2

TF-100 I

______L_______________________ ----- Dialysis or ultra- '' '

External solution \ Filtration__

Drying Internal solution x 3 TF-110

No adsorb- ^ Treatment ion- and ionized iae fcr— - change foot __ | ^ ___________________. phosphate buffer

Desalted Boy - ^ - (Sodium Chloride) I Eluate —-- '

Drying L ... ...,.

__I jVakevomti Ψ | —— TF-12 0 J Desalination

Drying TF-130 (TF-1316, 132a, 132b, 133a, 133b, 1323, 136)

___ - TT

6 68080

Culture or its supernatant x -------- χ2

Water soluble l. 1 v I · —- ^ Drying ---------> TF-100 j Jae_ 'I -------- 1 ^ - ^ Formation of barium salt x --- X ^ Barium ions

Ρ = ΓΓ_ —I

I Erotus_I 'Solution

Barium salt! __I____

I Removal of barium I |

Difference 1_ \ Fixed --1--1 Λ ai ne__

Solutions

Dialysis or ultra-filtration j Concentration j Γ; jDrying j TF-140 7 68080 x 2

Water soluble fraction --x3

Internal. T,. "T

,. - ^ Drying --- v TF-110 solution Λ ___] __Treatment quaternary /

Soluble iae C- rise; 4ä anraonium "* -J-lx with salt r .......................

Quaternary ammonium-_______ complex

Sodium chloride ----------- solution ________

Dissociation process ~ 4 · _____

| Washings ^ J

Soluble verse

Hydrophilic organic __v solvent T ·, · · --- Separation

Soluble iae_ \ __

Sediment

Dry

V

TF-150 68080 8

The above production process is illustrated as follows: (1) Bacterial culture

Bacteria belonging to the genus Fusobacteriura are cultured by any conventional method for culturing anaerobic bacteria. This means that the culture medium containing a nitrogen source, such as calf brain core extracts, various peptones, etc .; a vitamin source such as yeast extract, etc .; an inorganic salt such as sodium chloride, etc .; a carbon source such as glucose, lactose, etc .; a reducing agent such as L-cystine, sodium sulfite, thioglucolate, etc. is adjusted to a pH between 6-8.5, preferably between 7.2 and 8.2, and the bacteria are inoculated into this culture medium, after which the unchanged culture is allowed to grow under anaerobic conditions At 35 ° to 42 ° C, preferably at 36 to 38 ° C for 1 to 5 days, preferably 24 to 72 hours. In particular, it is desirable to use the culture medium described in Table 1 (hereinafter referred to as TF culture medium). However, brain-heart extract, which is calf brain-heart extract, is not always necessary as a carbon source and can be replaced by heart extract which is calf heart extract, meat extract, fish extract, corn dissolution broth, etc. Of the various peptones, protease-peptone and phyton-pep tones are not always necessary and the trypticase peptone can be replaced by a polypeptide.

When agar is not used, it is desirable to use a mixed culture.

9 68080

table 1

Culture medium TF-a TF-b TF-c components (g / l) trypticase-peptone 17 17 17 phyton-peptone 3 3 1,5 protease-peptone 10 55 brain-heart extract 35 17.5 heart extract - - 25 yeast extract 3 33 sodium chloride 7.5 7.5 7.5 glucose 6 66 lactose 5 55 L-cystine 0.25 0.5 0.5 sodium sulphite 0.1 0.1 0.1 thioglycolate 0.5 0.5 0.5 agar 0 or 0.7 0 or 0.7 0 or 0.7 (2) Recovery of the supernatant from the culture (removal of organisms).

The organisms are removed from the culture obtained above to obtain a supernatant. For the removal of organisms, a conventional method can be used, for example, centrifugation and a filtration method using a filter aid such as Hyflo Super Cel, and in particular, the centrifugation method is advantageous in terms of work steps, degree of organism removal and supernatant yield. The organisms are preferably removed in this step, but may be removed in the next step (3).

(3) Recovery of carcinostatic agent TF-100

A hydrophilic organic solvent is added to the supernatant or culture obtained above and the precipitate formed is collected. At this point, the supernatant or culture is preferably adjusted to pH 2-7. The hydrophilic organic solvent is, for example, an alcohol such as ethanol, methanol, etc., or a ketone such as acetone, etc., although alcohols, especially ethanol, give the best results. The hydrophilic organic solvent is suitably added in a concentration of 30-70%, in particular 50-70% by volume. After the addition of the hydrophilic organic solvent, the resulting mixture is allowed to stand at a low temperature, preferably at 5 ° C, for several hours to many days to achieve complete precipitation.

The precipitate thus formed is separated by conventional methods such as decantation, centrifugation or filtration.

Water is then added to the precipitate in an amount of 10-15 times the precipitate / water can be replaced with phosphate buffer or sodium chloride-containing phosphate buffer to obtain TF-120 or 130 (1316, 132a, 132b, 133a, 133b, 1323 or 136) as described below. and / and the water-insolubles formed are removed by a conventional method such as centrifugation, filtration, etc., after which the water-soluble fraction is collected. When a culture is used, the organisms are removed by the above-mentioned methods. The water-soluble fraction thus obtained is dried by lyophilization or the like to obtain the carcinostatic agent TF-100.

The TF-100 thus obtained has the properties shown in Table 2.

(4) Assembly of carcinostatic agent TF-110

The water-soluble fraction obtained in (3) above is subjected to dialysis or ultrafiltration; or alternatively, TF-100 is dissolved in water in an amount 10-15 times the amount of TF-100, and the resulting solution is subjected to dialysis or ultrafiltration. Small molecule substances such as amino acids and inorganic substances are removed by the treatment described above. The internal solution obtained by dialysis or ultrafiltration is then collected and dried to obtain the carcinostatic agent TF-110.

The TF-110 thus obtained has the properties shown in Table 2.

(5) Assembly of carcinostatic agent TF-120

The water-soluble fraction obtained in (3) above, or, if necessary, an internal solution by dialysis or ultrafiltration (internal solution 11 68080 obtained in (4) above), or a solution of TF-110 powder in a small amount of water, is treated with an ion exchanger. . As the ion exchanger, a weakly basic ion exchanger or a weakly basic ion exchanger having molecular sieving ability is used, and, for example, diethylaminoethyl-Sephadex A-50 (registered trademark of Pharmacia Co., Ltd) is preferably used. The column packed with an ion exchanger is equilibrated, for example, with a phosphate buffer having a pH of about 8, after which the above-mentioned treatment solution is passed through the column, and the eluted solution is collected and then dried by lyophilization or the like; or alternatively the same buffer as above and the solution to be treated is placed in a vessel containing an ion exchanger, and the contents are then mixed and filtered, after which the filtrate is dried; whereby a carcinostatic agent TF-120 having the properties described below can be obtained. TF-120 can also be obtained by desalting the eluted solution or filtrate, for example by dialysis using a cellophane tube or the like.

Cr), using Sephadex G-25v (registered trademark of Pharmacia Co., Ltd), or by ultrafiltration and then drying.

The TF-120 thus obtained has the properties shown in Table 2.

(6) Assembly of carcinostatic agent TF-130

The column containing the adsorbed components from which TF-120 has been removed as discussed in (5) above is treated with a phosphate buffer, preferably a phosphate buffer having a sodium chloride content of between 0.1 and 0.6 M and a pH of n. 8 (hereinafter, a phosphate buffer having a sodium chloride content of 0.1 to 0.6 M is referred to as a 0.1 to 0.6 M sodium chloride phosphate buffer); or alternatively, the water-soluble fraction obtained in (3) above is subjected, if necessary, to dialysis or ultrafiltration, and then treated with an ion exchanger using 0.1 to 0.6 M sodium chloride-phosphate buffer; to obtain a fraction which has not eluted with phosphate buffer free of sodium chloride but eluted with 0.1 to 0.6 M sodium chloride phosphate buffer.

This method can be implemented with either a column system or a batch system. The desired fraction thus obtained, which is eluted with sodium chloride phosphate buffer, is dried by lyophilization or the like to obtain the carcinostatic agent TF-130. The eluate containing the desired fraction eluting with the above-mentioned sodium chloride-phosphate buffer can be desalted, for example by dialysis using a cellophane tube, by molecular sieving, using Sephadex G-25, or by ultrafiltration and then dried.

As used herein, the symbol "TF-130" means the collectively desired eluted fractions obtained by treating the water-soluble fraction obtained in (3) above or, if necessary, the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration with an ion exchanger, which fractions do not elute with phosphate buffer free of sodium chloride but elute with 0.1-0.6 M sodium chloride phosphate buffer. Concretely, TF-130 comprises, for example, the following fractions.

(i) Fraction not eluted with sodium chloride-free phosphate buffer but eluted with 0.6 M sodium chloride-phosphate buffer in the above ion exchange treatment (the fraction is designated TF-1316).

(ii) The fraction which has not eluted with 0.1 M sodium chloride-phosphate buffer but has eluted with 0.2 M sodium chloride-phosphate buffer in the above-mentioned ion exchange treatment / fraction is designated TF-132a and b; TF-132a is obtained by washing the ion exchanger and containing the above-mentioned adsorbed component with 0.1 M sodium chloride-phosphate buffer, treating the ion exchanger with 0.2 M sodium chloride-phosphate buffer, and then collecting the fraction eluted with 0.2 M sodium chloride-phosphate buffer; and TF-132b is obtained by treating the aqueous fraction obtained in (3) above or the internal solution component (TF-110) obtained in (4) above with an ion exchanger.

(iii) The fraction not eluted with 0.2 M sodium chloride-phosphate buffer but eluted with 0.3 M sodium chloride-phosphate drop buffer in the above ion exchange treatment / fraction is designated TF-133a and b; a and b have the same meaning as defined above for TF-132a and b, respectively.

(iv) The fraction which has not eluted with 0.1 M sodium chloride-phosphate buffer but has eluted with 0.3 M sodium chloride-phosphate buffer in the above-mentioned ion exchanger treatment / fraction is designated TF-13237.

(v) A fraction which has not eluted with 0.5 M sodium chloride-phosphate buffer but has eluted with 0.6 M sodium chloride-phosphate buffer in the above-mentioned ion exchanger treatment / "the fraction is designated TF-1367 ·

Because TF-1316, 132a, 132b, 133a, 133b, 1323, and 136 have common properties in the following ratios, compounds having common properties are designated TF-130. This means that the TF-130 obtained as described above has the following properties: (a) Off-white to light brown powder.

(b) It inhibits the further growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 cancer and has an immunostimulatory effect.

(c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether.

(d) It has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C.

(e) Its infrared absorption spectrum obtained by the KBr tablet method has absorption bands close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm the ultraviolet absorption spectrum of the aqueous solution gives a strong absorption at the absorption limit and gives an absorption peak in the vicinity of 256-280 nm.

(g) It is positive in the Molisch reaction, phenol-sulfuric acid reaction, anthron-sulfuric acid reaction, i.indol i-hydrochloric acid reaction and Lowry-Folin reaction, and negative in the ninhydrin reaction.

(h) Elemental analysis values C: 27.5-39.8%, H: 3.2-5.7%, N: 2.8-6.3%.

(i) Its saccharide content, as measured by the phenol-sulfuric acid method, is from about 19.0% to about 64.0% by weight (as glucose), and its 68080 protein content, as measured by the Lowry-Folin method, is from 6.0 to In the vicinity of 28.0% by weight (as calf serum albumin).

The above treatment with an ion exchanger is explained in more detail below. As the ion exchanger used in the following methods, a weakly basic ion exchanger or a weakly basic ion exchanger having molecular sieving ability and, for example, diethylaminoethyl Sephadex A-50® used in 5) above are suitable for use, and the following method can be carried out in a batch system.

(i) 0.6 M sodium chloride-phosphate buffer, preferably at a pH of about 8, is passed through an ion exchange column containing the absorbed component through which the solution to be treated has been passed in (5) above and the eluted fraction is collected, optionally concentrated and desalting by dialysis, ultrafiltration or the like and then drying by lyophilization or the like to obtain the carcinostatic agent TF-1316.

The TF-1316 thus obtained has the properties shown in Table 2.

(ii) -1) 0.1 M sodium chloride-phosphate buffer, pH

is preferably about 8, is passed through an ion exchange column containing the absorbed component and through which the solution to be treated is passed in (5) above to wash the column, followed by 0.2 M sodium chloride-phosphate buffer, preferably having a pH of about 8. through the column and the eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration, etc., and then dried by lyophilization or the like to obtain the carcinostatic agent TF-132a.

The TF-132a thus obtained has the properties shown in Table 2.

(ii) -2) The steps for collecting the fraction which has not eluted with 0.1 M sodium chloride-phosphate buffer but has eluted with 0.2 M sodium chloride-phosphate buffer in the ion exchange treatment are carried out as follows using the water-soluble fraction obtained in (3) above or above. the internal solution obtained in (4) to obtain TF-132b. This means that the column packed with ion exchanger li 68080 is equilibrated with 0.2-0.3 M sodium chloride-phosphate drop buffer, preferably at a pH of about 8, followed by the water-soluble fraction or internal solution component obtained in (3) above, which is obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, 0.2 to 0.3 M sodium chloride-phosphate buffer is passed through the column and the eluted solution is collected. If necessary, wash the column with the above-mentioned 0.2 to 0.3 M sodium chloride-phosphate buffer and combine the eluted solution and washings. The ion exchange treatment can be performed several times. The solution prepared by diluting the above eluted solution with phosphate buffer to a concentration of 0.1 M sodium chloride is passed through a column in which the ion exchanger is equilibrated with 0.1 M sodium chloride-phosphate buffer, preferably at a pH of about 8, and a fraction , eluted with the above-mentioned 0.1 M sodium chloride phosphate buffer, is removed, after which the above-mentioned 0.2 M sodium chloride-phosphate buffer is passed through the column to obtain the eluate. In the specific example of the ion exchange treatment described above, a fraction not adsorbed on the ion exchanger equilibrated with the above-mentioned 0.2 M sodium chloride-phosphate buffer is collected, adsorbed on an ion exchanger equilibrated with the above-mentioned 0.1 M sodium chloride-phosphate buffer, washed with this 0.1 M sodium chloride-phosphate buffer. phosphate buffer and then eluted with the above-mentioned 0.2 M sodium chloride phosphate buffer to obtain an eluate. It is also possible to use a procedure in which, on the contrary, a fraction adsorbed on an ion exchanger equilibrated with the above-mentioned 0.1 M sodium chloride-phosphate buffer is collected and then a step is carried out to separate this fraction from the fraction adsorbed on the above-mentioned ion exchanger. 0.2 M sodium chloride-phosphate buffer, and the fraction eluted with this 0.2 M sodium chloride-phosphate buffer is pooled.

The eluate obtained as described above is then optionally concentrated and desalted by dialysis, ultrafiltration, etc., and then dried by lyophilization or the like to obtain the carcinostatic agent TF-132b.

680 8 0 The TF-132b thus obtained has the properties shown in Table 2.

(iii) -1) The internal solution component obtained in (4) above is passed through a column with an ion exchanger equilibrated with a phosphate buffer, preferably at a pH of n.

8, and 0.2 M sodium chloride-phosphate buffer, preferably at a pH of about 8, is passed through the column to wash it, and then 0.3 M sodium chloride-phosphate buffer, preferably at a pH of about 8, is passed through the column, or alternatively the aforementioned 0 , 3 M sodium chloride-phosphate buffer is passed through the column treated in (ii) -1) above with the remaining adsorbed component remaining. The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration, etc., and then dried by lyophilization or the like to obtain the carcinostatic agent TF-133a.

The TF-133a thus obtained has the properties shown in Table 2.

(iii) -2) The fraction which did not elute with 0.2 M sodium chloride-phosphate buffer but has eluted with 0.3 M sodium chloride-phosphate buffer in the ion exchange treatment is collected as follows in the same manner as in (ii) to 2), to obtain TF-133b. That is, the column packed with an ion exchanger is equilibrated with 0.3 M sodium chloride-phosphate buffer, preferably having a pH of about 8, followed by 0.3 M sodium chloride-phosphate buffer of the water-soluble fraction obtained in (3) above or an internal solution component obtained by dialysis of the water-soluble fraction. or ultrafiltration, pass through a column and collect the eluted solution. If necessary, the column is washed with the above-mentioned 0.3 M sodium chloride-phosphate buffer and the eluted solution and washings are combined. The ion exchange treatment can be performed several times. The solution prepared by diluting the above eluted solution with phosphate buffer to a sodium chloride content of 0.2 M is passed through a column in which the ion exchanger is equilibrated with 0.2 M sodium chloride phosphate buffer, preferably having a pH of about 8, and a fraction, eluted with the above-mentioned 0.2 M sodium chloride phosphate buffer, 68080 is eluted, after which the above-mentioned 0.3 M sodium chloride-phosphate buffer 1 is passed through the column to obtain an eluate. In the specific example of the ion exchange treatment described above, a fraction which is not an adsorbed ion exchanger slurry equilibrated with 0.3 M sodium chloride-phosphate buffer is collected, adsorbed on an ion exchanger equilibrated with the above-mentioned 0.2 M sodium chloride-phosphate buffer 1a, washed with 0.2 M sodium chloride-phosphate buffer and then eluted with 0.3 M sodium chloride-phosphate buffer to obtain the eluate. Alternatively, a method can be used in which, on the contrary, a fraction adsorbed on an ion exchanger equilibrated with 0.2 M sodium chloride-phosphate buffer is collected and then a step is performed to separate this fraction from the fraction adsorbed on an ion exchanger equilibrated to 0. 3 M sodium chloride-phosphate buffer 11a, and the fraction eluted with the above-mentioned 0.3 M sodium chloride-phosphate buffer 11a is collected.

The eluate obtained as described above is then optionally concentrated and desalted by dialysis, ultrafiltration, etc., and then dried by lyophilization or the like to obtain the desired carcinostatic agent TF-133b.

The TF-133b thus obtained has the properties shown in Table 2.

(iv) The water-soluble fraction obtained in (3) above or the internal solution component obtained in (4) above is formed into a 0.3 M sodium chloride-phosphate buffer, preferably having a pH of about 8, and the obtained buffer is passed through an ion exchange column which has previously been equilibrated with 0.3 M sodium chloride-phosphate buffer, preferably at a pH of about 8. The eluted solution is adjusted by adding phosphate buffer to a concentration of 0.1 M sodium chloride and then passed through an ion exchange column previously equilibrated to 0, 1 M sodium chloride-phosphate buffer, preferably at a pH of about 8, and then the above-mentioned 0.3 M sodium chloride-phosphate buffer is passed through a column, after which the eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like and dried. then lyophilized or the like to obtain the carcinostatic agent TF-1323.

The TF-1323 thus obtained has the properties shown in Table 2.

(v) The internal solution component obtained in (4) above is passed through an ion exchange column equilibrated with a phosphate buffer, preferably having a pH of about 8, and a 0.5 M sodium chloride-phosphate buffer, preferably having a pH of about 8, is passed through the column. to wash it, and then a 0.6 M sodium chloride-phosphate buffer, preferably having a pH of about 8, is passed through the column, or alternatively the aforementioned 0.5 M sodium chloride-phosphate buffer is passed through the column discussed in (ii) -2 above). and still has the remaining adsorbed component, after which the above-mentioned 0.6 M sodium chloride-phosphate buffer is passed through this column. The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration, etc., and then dried by lyophilization or the like to obtain the carcinostatic agent TF-136.

The TF-136 thus obtained has the properties shown in Table 2.

(7) Assembly of carcinostatic agent TF-140

Barium ions are added to the culture or the supernatant obtained in (1) or (2) above or to the water-soluble fraction obtained in (3) above, or to the solution obtained by dissolving TF-100 in water to form a barium salt. , after which the precipitate is collected and then subjected to a barium removal step and the aqueous solution is collected and then subjected to dialysis or ultrafiltration to obtain TF-140. Explaining in detail, an aqueous barium hydroxide solution, preferably a 0.1 to 0.5 M aqueous barium hydroxide solution, is added to the culture obtained in (1) or (2) above or to the liquid or water-soluble fraction obtained in the above. (3) or an aqueous solution obtained by dissolving TF-100 in water in an amount of about 10 to 15 times the amount of TF-100, and the pH of the resulting mixture is preferably adjusted to between 10 and 13 to form a barium salt. Sediment

II

19 68080 precipitates by adding a barium hydroxide solution and the amount of barium ions added is preferably set so that the total concentration of barium ions in the final volume of the mixture is 0.005-0.1 M.

The mixture thus obtained is filtered and a step of separating barium from the barium salt obtained is carried out. The barium is preferably removed by adding the barium salt to sodium sulfate, preferably 5-15% aqueous sodium sulfate solution, stirring the resulting mixture and then filtering to obtain a solution. The separated and removed precipitate is washed again with aqueous sodium sulfate solution and the washings are combined with the above solution. The solution thus obtained is subjected to dialysis, ultrafiltration, etc. to remove excess sodium sulfate or smaller molecules, and the resulting solution is then dried by lyophilization or the like to obtain the carcinostatic agent TF-140.

The TF-140 thus obtained has the properties shown in Table 2.

(8) Assembly of carcinostatic agent TF-150

The water-soluble fraction obtained in (3) above or the internal solution obtained in (4) above is treated with a quaternary ammonium salt, and the precipitate formed is collected and then subjected to a dissociation process using a solution containing sodium chloride, followed by adding a hydrophilic organic solvent to the soluble fraction. the precipitate is collected and then dried to obtain TF-150. The quaternary ammonium salt which may be used herein may usually be a complex with a polysaccharide, and particularly preferred examples thereof are cetylpyridinium chloride, cetyltrimethylammonium bromide, etc. The treatment with the quaternary ammonium salt is preferably carried out in the presence of a buffer such as borate buffer, etc. A specific explanation of the above treatment with a quaternary ammonium salt and a dissociation step using a solvent containing sodium chloride is as follows. For example, an aqueous solution of the quaternary ammonium salt is added dropwise to the water-soluble fraction obtained in (3) above or to the internal solution obtained in (4) above while keeping the pH weakly basic, and the resulting mixture is stirred at 0 ° -50 ° C: in

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Table 2 used a Sephadex G-50® marked with an "x", a 50 mm x x 600 mm column and distilled water as eluent; Sephadex G: 20 (¾ lie (registered trademark of Pharmacia Co., Ltd) marked with an "x", a 44 mm0 x 500 mm column was used for TF-110, 120, 132a, 133a for 136, 140 and 140 and 21 mm0 x 400 mm columns for TF-1316, 132b, 133b, 1323 and 150, and 0.1 M was used as eluent for all of them. phosphate buffer, pH 7, and 7.9 mm0 x 600 mm x 2 TSK-GEL G300SW ^ · (trade name of Toyo Soda Co., Ltd) was used to obtain a high performance liquid chromatogram labeled "xxx". column and elution was performed at room temperature at a flow rate of 0.8 ml / min using 0.1 M phosphate buffer pH 7 as eluent.

The pharmacological effects of these carcinostatic agents TF-100, TF-110, TF-120, TF-1316, TF-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF-140 and TF-150 are as follows: : (I) Effect of RF substances on cell viability (assay of growth colony formation).

J. H. Chim.'in et al. method (Cancer Res. 25, 698 (1965)), HeLa S-3 cells were cultured in Eagle's MEM medium supplemented with 10% calf serum for 3-5 days at 37 ° C in a CO 2 incubator, after which the culture was removed and the PBS (-) solution (aqueous solution containing 8.0 g / l sodium chloride, 0.2 g / l potassium chloride, 1.15 g / l sodium phosphate, monobasic, and 0, 2 g / l potassium phosphate, dibasic) containing 0.01% by weight of ethylenediaminetetraacetic acid and 0.1% by weight of trypsin were added to the cells adhered to the glass. diluted with Eagle's MEM medium supplemented with 20% calf serum to 200 cells / ml After inoculating 1 ml of cell suspension into petri dishes and culturing in a CO 2 oven at 37 ° C for 24 hours, in Example 1 (1), shown below, obtained super -natant was added to the cell suspension to a concentration of 1/16 to 1/128. To each 1 ml aliquot of cell suspension cultured at 37 ° C for 24 hours, each test substance was added and HeLa cells were cultured for 7 days in each case. After culturing, the culture medium was removed, after which the culture dishes were washed with Hank's solution and the cells were immobilized with 70% by weight of ethanol and then washed with 100% by weight of ethanol. After removal of ethanol, cells were stained with Gimsa staining solution, after which growth colonies were counted and cell viability was counted.

The results are shown in Tables 3 and 4. Cell viability in the tables was calculated from the following equation. The cell viability of each test substance is presented as the average of five replicates.

Number of growth colonies

Cell viability - in the case of the treated group --- x 100 number of growth colonies in the case of the untreated group 68080

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Table 4 Effect of TFs on cell viability

Substance Concentration (yg / ml) S Un ^^ viability | _i_v%) _ 10 99.4 TF-100 50 97, d 100 95.9 10 94.5 TF-110 50 88.9 100 87.3 10 98.8 TF-120 50 91.2 100 83.3 ji I 10 96.7 TF-132a 50 97.4 100 95.9 and 10 96.9 TF-133a 50 95.4 100 88.8 and I 10 95.9

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j 100 95.4! __! _____________: 10 98.5 50 98.6 TF-140 100 98.6 200 96.8 500 93.4 39 68080

As can be seen from this experiment, the cytotoxic effect of the ΤΓ agents of this invention on HeLa cells is very low compared to the supernatant.

(II) Immunostimulatory effect Four ICR strain mice were used for each group. Each test substance was dissolved in 0.2 in physiological saline and then administered intraperitoneally to mice. 24 hours after administration, 0.2 ml of a carbon suspension prepared by mixing 1 ml of Pelikan Drawing Ink 17 Black (manufactured by Gunther-Wagner Co., Ltd.) and 2 ml of physiological saline containing 3% by weight of gelatin was administered intravenously. injection into the tail of the mouse and 1, 5, 10 and 15 minutes after injection 0.02 ml of blood was taken from the orbit using a heparin-coated blood cell volume capillary and immediately diluted and the red cells were dissolved in 1.6 ml of 0.1 wt%. % aqueous sodium carbonate solution. This suspension was subjected to colorimetry at 675 nm and the mammalian cell index, namely the K value, was determined according to the method of Halpern et al. from the equation.

Mice in the control group were given 0.2 ml of physiological saline.

K = i ° g c0 - log c 1 - h where CQ = concentration of carbon powder in the blood at time tQ, and C = concentration of carbon powder in the blood at time t.

The results are shown in Tables 5-7.

40 68080

Table 5

Substance Dose (mg / kg) Mean of K values TF_1Q0 10 0.0851 ± 0.0145 50 0.060 5 0.0 30 2 TF-110 5 0.0901 i 0.0213 20 0.1025 ± 0.0025 TF_120 5 0 .0718 ± 0.0152 20 0.0559 ± 0.0107 TF_13l6 5 0.0832 ± 0.0193 20 0.1001 ± 0.0296 TF_132a 5 0.1102 i 0.0203 20 0.0809 ± 0.0296

Tp_133a 5 0.0851 ± 0.0057 20 0.0769 ± 0.0179 tf_136 5 0.0959 ± 0.0087 20 0.1027 ί 0.0159

Comparison - 0.0300 ± 0.0024 68080

Table c

Substance Dose (mg / kg) Mean of K values ____________, _ i TF-132b 5 0,1190 ± 0,0051 20 0,1073 ± 0,0031 TF_133b 5 0,0550 ± 0.0020 20 0,1099 ± 0.0075

Tp_150 δ 0.1399 ± 0.0011 20 0.0577 ± 0.0098

Tp_1323 5 0.1 ^ 00 ± 0.0075 20 0.0685 ± 0.0126

Comparison - 0.0286 ± 0.0035

Table 7

Substance Dose (rag / kg) Mean of K-values 1 0.0891 ± 0.0156 TF-140 5 0.09 ^ 6 ± 0.0139 20 0.0815 ± 0.0200

Comparison ~ 0.0379 i 0.0048 J

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As shown in Tables 5-7, the groups administered each of the TF agents of this invention showed activation of reticuloendothelial macrophages compared to the control group and increased cellular immunity in normal mice.

(III) Carcinostatic effect (a) Antitumor effect against Ehrlich aquatic tumor

Ehrlich aquatic tumor cells were inoculated intraperitoneally! «68080 to the ICR strain in mice (6 weeks old females) at 5 1 x 10 cells / mouse. Thereafter, each of the test substances was dissolved in 0/2 ml of physiological saline, and then administered intraperitoneally to the mice once a day repeatedly for 7 days from the day after tumor cell inoculation or once a day on the first day and from the third to the seventh day for a total of 6 days. For the test compounds shown in Table 9, each of them was administered to mice once a day on the first day and from the third to the seventh day for a total of six days after inoculation of tumor cells. The control group was given 0.2 ml of physiological saline in the same manner as above.

The results are shown in Tables 8 and 9.

Number of survival days of mice in the m / c _ group treated with anesthesia --- X 100 (»)

Number of survival days of mice in the control group 43-68080 —i — I- ^ I I \ si

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As shown in Tables 8 and 9, the group given each of the TF agents of this invention experienced a life-prolonging effect compared to the control group.

(b) Antitumor effect against solid Ehrlich tumor (1) Ehrlich tumor cells were implanted subcutaneously in the axillary well of ICR strain mice (females 6 weeks old) at 4 x 10 cells / mouse. Thereafter, each of the test compounds was administered to the mice once a day repeatedly for 7 days from the first day after tumor cell implantation, or once a day from the first and third days to the seventh day for a total of six days. The control group was given 0.2 ml of physiological saline in the same manner as described above. Tumor weights were determined on day 11 or 14 after tumor cell implantation. Tumor weights were determined by measuring the main diameter (a) mm and the smallest diameter (b) mm using a micrometer and calculating from the following equation: a x b2

Tumor weight = _ (mg) 2

The results are shown in Table 10.

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«63080 (2) Ehrlich tumor cells were implanted subcutaneously into the axillary well of ICR strain mice (female, 6 weeks old) at 4 x 10 6 cells / mouse. Thereafter, each test compound was administered to mice divided into groups for intravenous, subcutaneous, and intramuscular administration at doses of 10 mg / kg or 20 mg / kg once daily on the first day and from the third to the seventh day for a total of 6 days after tumor cell implantation. . The control group was given 0.2 ml of physiological saline in the same manner as above. Tumor weights were determined on day 13 after implantation of tumor cells.

The results are shown in Table 11.

50 68080 ^ s.

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As shown in Tables 10 and 11, the tumor growth inhibitory effect is observed in the groups administered each of the TF agents of this invention.

(c) Antitumor activity against Sarcoma-180 cancer

Sarcoma-180 cancer was implanted subcutaneously in ICR strain mice (female, 6 weeks old) at 1 x 10 5 cells / mouse. Thereafter, each of the test compounds was administered intraperitoneally to mice at doses of 1 mg / kg, 5 mg / kg, or 10 mg / kg once daily from day 1 to day 7 repeatedly for a total of 7 days after tumor cell implantation, or once daily on day 1 and day 3 to day 7. for six days. The control group was given 0.2 ml of physiological saline in the same manner as above.

The results are shown in Table 12.

52 68080

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As shown in Table 12, the groups given each of the TF agents of this invention had antitumor effects compared to the control group.

(d) Antitumor activity against B-16 melanoma tumor (1) B-16 melanoma tumor cells were implanted subcutaneously into the axillary well of BDF1 strain mice (male, 7 weeks old) at 1 x 10 6 cells / mouse. Thereafter, TF-133a was administered intraperitoneally to mice at doses of 5 mg / kg or 10 mg / kg once daily on the first day and from the third to the seventh day for a total of 6 days after tumor cell implantation. In the same manner as above, the control group was administered 0.2 ml of physiological saline and 20 mg / kg to the 5-FU group for 5-FU administration.

The results are shown in Table 13.

Table 13

Substance Dose (mg / kg x administration-Average number of tumors - weight (g) 'TF-133a 5x6 4.07 70 10 x 6 2.52 43 5-FU 20 x 6 4.21 72

Comparison - 5.84 100

As shown in Table 13, the groups given TF-133a had a clear anti-tumor effect compared to the control group, and the groups given both 5 mg / kg and 10 mg / kg TF-133a had an equal and more significant anti-tumor effect. effect, respectively, compared to the group given 5-FU.

55 6 8 0 8 0 (2.) Implantation of B-16 melanoma tumor cells and administration of TF-i32a, TF-133a-nf 5-FU and physiological saline were performed in the same manner as in (1) above. On day 21, after implantation of tumor cells, mice were dissected and colonies of dark cell tumor cells in the lung were examined.

The results are shown in Table 14.

Table 14

Substance Dose (mg / kg administered - Number of Branch Colonies) Avg. number of inhibitors% TF-132a 5x6 16.0 48 10 x 6 9.86 69 TF-133a 5x6 9.3 70 10 x 6 6.7 78 5-FU 20 x 6 24.8 20

Comparison - 31.0 0

As shown in Table 14, in the groups given both TF-132a and TF-133a, the branch colonies of B-16 melanoma tumor cells were significantly inhibited compared to the group given 5-FU.

(IV) Acute toxicity to LD ^ q mice is shown in Table 15.

56 68080

Table 15

Substance Antotie LD50 TF-100 Intraperitoneally 500

„---------------------- - -—H

Intravenously> 250 1Ί Ω

Intraperitoneal> 1000

Intravenous> 250 TF-120 Intraperitoneal> 1000 mrn Intravenous 300 TF-132a

Intraperitoneal> 500 TF-133a Intravenous 300

Intraperitoneal> 500

Intravenously ^ 00 TF-136a 3

Intraperitoneal> 500 TF-132b Intravenous> 100 TF-133b Intravenous> 200 TF-1 ^ 0 Intravenous> 100 TF-150 Intravenous> 200 TF-1323 Intravenous> 200 TF-13-16 Intravenous> t

As can be seen from the above pharmacological experiments, TFs are useful as carcinostatic agents and can be expected to have an effect against various cancers and further, they can be expected to have a significant effect specifically against solid cancers. All TF materials have excellent properties, although TF-130, especially 132a, 132b, 133a, 133b and 1323, are preferred.

The TF agents prepared according to the invention can be converted into various pharmaceutical forms, such as oral drugs, injectables, suppositories, etc., although they are preferably used in an injectable pharmaceutical form. When used as oral drugs, these oral drugs may contain various excipients and may be formulated as capsules, tablets, powders, or granules. When used as injections, the injections may be subcutaneous injections, intramuscular injections and intravenous injections and are used in the form of a suspension, solution or powder which is dissolved during use. Injections may contain a local anesthetic.

The dose of TFs is appropriately selected depending on the condition of the patient, although it is generally desirable to administer them to an adult at doses ranging from 0.01 to 50 mg / kg once daily or more per day and preferably by subcutaneous, intramuscular or intravenous injection or injection into an injured person. parts.

The present invention will be described in more detail below by way of example and with reference to the accompanying figures, in which Figure 1 is a photomicrograph showing the form of Fusobacterium nucleatum TF-031 used in the present invention; Figure 2 shows the infrared absorption spectrum of the carcinostatic agent TF-100; Figure 3 shows the ultraviolet absorption spectrum of this substance; Figure 4 shows an elution pattern obtained when this material was subjected to gel filtration using Sephadex G-50; Figure 5 shows a high performance liquid chromatogram; Rö 68080 58 Figure 5 shows the infrared absorption spectrum of the carcinostatic agent TF-110; Figure 7 shows the ultraviolet absorption spectrum of this substance; Fig. 8 shows an elution pattern obtained when this (K) substance was subjected to gel filtration using Sephadex G-200; Fig. 9 shows a high performance liquid chromatogram of this substance; Figure 10 shows the infrared absorption spectrum of the carcinostatic agent TF-120; Figure 11 shows the ultraviolet absorption spectrum of this substance; Figure 12 shows an elution pattern obtained when this (R) substance was subjected to gel filtration using Sephadex G-200 Figure 13 shows a high performance liquid chromatogram of this substance? Figure 14 shows the infrared absorption spectrum of the carcinostatic agent TF-1316; Figure 15 shows the ultraviolet absorption spectrum of this substance; Figure 16 shows an elution pattern obtained when this material was subjected to gel filtration using Sephadex G-200®; Figure 17 shows a high performance liquid chromatogram of this substance; Figure 18 shows the infrared absorption spectrum of the carcinostatic agent TF-132a; Figure 19 shows the ultraviolet absorption spectrum of this substance; Figure 20 shows an elution pattern obtained when this material was subjected to gel filtration using Sephadex G-200 ®; Figure 21 shows a high resolution liquid chromatogram of this substance; Figure 22 shows the infrared absorption spectrum of the carcinostatic agent TF-132b; Figure 23 shows the ultraviolet absorption spectrum of this substance; Figure 24 shows an elution pattern obtained when the material was subjected to gel filtration using Sephadex G-200®; 59 68080 Figure 25 shows a high resolution liquid chromatogram of this substance; Figure 26 shows the infrared absorption spectrum of the carcinostatic agent TF-133a; Figure 27 shows the ultraviolet absorption spectrum of this substance; Fig. 28 shows an elution pattern obtained when this material was subjected to gel filtration using Sephadex G-ZOO '' - '; Figure 29 shows a high resolution liquid chromatogram of this substance; Figure 30 shows the infrared absorption spectrum of the carcinostatic agent TF-133b; Figure 31 shows the ultraviolet absorption spectrum of this substance; Fig. 32 shows an elution pattern obtained when this (r) material was subjected to gel filtration using Sephadex G-200v; Fig. 33 shows a high resolution liquid chromatogram of this substance; Figure 34 shows the infrared absorption spectrum of the carcinostatic agent TF-1323; Figure 35 shows the ultraviolet absorption spectrum of this substance; Fig. 36 shows an elution pattern obtained when this (R) material was subjected to gel filtration using Sephadex G-200w; Figure 37 shows a high resolution liquid chromatogram of this substance; Figure 38 shows the infrared absorption spectrum of the carcinostatic agent TF-136; Figure 39 shows the ultraviolet absorption spectrum of this substance; Fig. 40 shows an elution pattern obtained when this material was subjected to gel filtration using Sephadex G-200; Fig. 41 shows a high resolution liquid program of this substance; Figure 42 shows the infrared absorption spectrum of the carcinostatic agent TF-140; Figure 43 shows the ultraviolet absorption spectrum of this substance; Fig. 44 shows an elution pattern obtained when this (ίδ substance was subjected to gel filtration using Sephadex G-200 ^; Fig. 45 shows a high-resolution liquid chromatogram of this substance; Fig. 46 shows the infrared absorption spectrum of the carcinostatic substance TF-150; Fig. 47 shows the uitraviolet absorption spectrum of this substance, Fig. 48 shows the elution pattern obtained when this substance was subjected to gel filtration using Sephadex G-200®, and Fig. 43 shows a high-resolution liquid chromatogram of this substance.

Example 1 (1) To each of 15 15 l culture flasks, each equipped with a screw cap, was charged with 2 l of culture medium containing 34 g of trypticase peptone, 6 g of phyton peptone, 20 g of protease peptone, 70 g of brain-heart extract, 6 g of yeast extract, 15 g of sodium chloride, 12 g of glucose, 10 g of lactose, 0.5 g of L-cystine, 0.2 g of sodium sulfite, 1.0 g of sodium aglycolate and 1.4 g of agar and the culture medium were adjusted to pH 7. The culture medium was sterilized at 1.2 atm at 120 ° C for 15 minutes, heated in a boiling water bath for 20 minutes, and immediately cooled thereafter, followed by a preculture solution of Fusobacterium nucleatum TF-031 prepared by culturing it in a culture medium having the same composition as above was inoculated into the water-cooled culture medium obtained above under sterile conditions in an amount of 100 ml per culture flask and subjected to unchanged culture in a culture cabinet at 37 ° C for 48 hours. When the culture was completed, the culture was centrifuged to remove the organisms at 4000 rpm at 5 ° C for 20 minutes. The amount of supernatant obtained was about 27 l.

(2) To the supernatant obtained in (1) above, 40 l of ethanol was added with stirring at 5 ° C, and the resulting mixture was allowed to stand at a low temperature until the amorphous precipitate had completely settled. The mixture was then centrifuged at 6000 rpm at 5 ° C for 15 minutes, and the precipitate was collected, washed with ethanol and dried under reduced pressure to give 60 g of a crude powder.

(3) 20 g of the crude powder obtained in (2) above was dissolved in 120 ml of water, and the resulting water-insoluble matter was removed by centrifugation at 7500 rpm for 10 minutes, after which the water-soluble fraction thus obtained was combined with the washing water obtained by washing with water-insoluble matter. substances twice with 60 ml portions of water, and the resulting solution was dried under reduced pressure to give 14 g of an off-white-light brown TF-100 powder.

68080

Example 2

The solution described in Example 1- (3), which was prepared by combining a water-soluble fraction free of water-insoluble substances with washing waters obtained by washing water-insoluble substances, was subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane no: HT.-1). ., manufactured by Asahi Kasei Kogyo Kabushiki Kaisha), and the internal solution was lyophilized to give 10 g of an off-white to light brown TF-110 powder.

Example 3 10 g of the powder obtained in Example 2 was dissolved in a small amount of water and the resulting solution was applied to a column packed with diethylaminoethyl-Sephadex A-50® equilibrated with 0.025 M phosphate buffer pH 8, and the eluted solution was collected. Further, 2.5 L of the same phosphate buffer as above was passed through the column, and the resulting eluted solution was combined with the eluted solution collected above, after which the resulting solution was desalted using a hollow fiber type ultrafiltration system (membrane no: HI-1), concentrated under reduced pressure, and then lyophilized. 470 mg off-white to light brown TF-120 powder.

Example 4 2 g of the powder obtained in Example 1— (2) were dissolved in 24 ml of water, and water-insoluble matter was removed by centrifugation at 7500 rpm for 10 minutes. The water-soluble fraction was dialyzed against distilled water overnight at 5 ° C using a cellophane tube, and the internal solution was concentrated under reduced pressure. The concentrated solution was then applied to a column packed with 150 ml of diethylaminoethyl-Sephadex Α-5 (ί ^ previously equilibrated with 0.025 M phosphate buffer pH 8, and the column was washed by passing 600 ml of 0.025 M phosphate buffer, pH 8, followed by 0.025 M phosphate buffer, 0.6 M sodium chloride, pH 8, was passed through the column to obtain an eluate, which was concentrated under reduced pressure to about 100 ml, dialyzed against distilled water at 5 ° C. , reconcentrated under reduced pressure, desalted using a Sephadex G-25 column and then lyophilized to give 470 mg of off-white to light brown TF-1316.

“68080

Example 5

The solution described in Example 1- (3) prepared by combining a water-soluble fraction free of water-insoluble matter and the washing water obtained by washing water-insoluble matter was dialyzed against distilled water overnight at 5 ° C using a cellophane tube and an internal solution. was concentrated under reduced pressure. The concentrated solution was then applied to a column packed with 50 g of diethylaminoethyl-Sephadex A-50-y previously equilibrated with 0.025 M phosphate buffer pH 8 and 2 L 0.025 M phosphate buffer pH 8 and 2 , 5 L of 0.025 M phosphate buffer with 0.1 M sodium chloride and pH 8 was passed successively through the column, followed by 2.5 L of 0.025 M phosphate buffer with 0.2 M sodium chloride and pH 8, which was passed through the column with the active ingredient adsorbed on the column. to elute the substance, and the resulting eluted solution was desalted using a hollow fiber type ultrafiltration system (membrane no: HI-1) and then lyophilized to give 600 mg of an off-white to light brown TF-132a powder.

Example 6 44.2 g of the powder obtained in Example 1- (2) were dissolved in 1.2 l of 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and pH 8, and the suspended insolubles were removed by filtration using Hyflo Super Cel. after which the resulting filtrate was applied to a 33 cm diameter column packed with ml§) 500 ml of diethylaminoethyl-Sephadex A-50Vfy previously equilibrated with 0.025 M phosphate buffer containing 0.3 M sodium chloride and pH 8. , and the eluted solution was collected. The column was washed with 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8, and the washings were combined with the above eluted solution to give 1.67 L of solution. The solution was again applied to an 8 cm diameter column packed with 400 ml of diethylaminoethyl-Sephdadex A-50, previously equilibrated with 0.025 M phosphate buffer, 0.3 M sodium chloride and pH 8, to give 1.68 l of elution. solution. The column was washed with 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8 to give 1.64 L of wash water. The eluted solution and washings were combined and 0.025 M phosphate buffer, pH 8, was added to give 4.98 L of 0.025 M phosphate buffer, sodium 68080 63

chloride concentration was 0.2 M and pH 8. The hand-obtained buffer solution was applied to an 8 cm diameter column packed with 500 ml of diethylaminoetvyii-Sephadex A-50® previously equilibrated with 0.025 M phosphate buffer. sodium chloride concentration was 0.2 M and pH 8, and the column was washed with 2 L of 0.25 C25 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH 8.

8. To 7 L of the solution, which had been diluted by combining the washings with the eluted solution, was added 0.025 M phosphate buffer, pH 8, to give 14 L of 0.025 M phosphate buffer, 0.1 M sodium chloride and pH 8. The eluted thus obtained the solution was applied to an 8 cm diameter column packed with 500 ml of diethylaminoethyl Sephadex A-50 previously equilibrated with 0.025 M phosphate buffer 0.1 M sodium chloride and pH 8, and the eluted solution was removed. The column was washed with 2 L of 0.025 M phosphate buffer with 0.1 M sodium chloride and pH 8, followed by 2 L of 0.025 M phosphate buffer with 0.2 M sodium chloride and pH 8, passed through the column and the eluted solution eluted. were assembled. The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10), further desalted using a Sephadex D-25® column, decolorized with activated carbon and then lyophilized to give 880 mg of off-white-light brown TF-132b.

Example 7 10 g of the powder obtained in Example 2 was dissolved in a small amount of water and the resulting solution was applied to a column packed with diethylaminoethyl-Sephadex A-SO 2 previously equilibrated with 0.025 M phosphate buffer pH 8. Then 5 l of 0.025 M phosphate buffer with 0.2 M sodium chloride and pH 8 was passed through the column, followed by 2.5 L of 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8 to elute the active substance adsorbed on the column. , and the resulting eluted solution was concentrated under reduced pressure. The concentrated solution was desalted using a cellophane tube and desalted completely using 64 68080 (r)

Sephadex G-25 and lyophilized to give 50C mg of an off-white to light brown? F-133a powder.

Example 8 44.2 g of the powder obtained in Example 1- (2) were dissolved in 1.2 l of 0.025 M phosphate buffer having a sodium chloride content of 0.3 M and a pH of 8, and the suspended insolubles were removed by filtration using Hyflo Super Cel. , after which the resulting filtrate was applied to a 33 cm diameter column packed with 500 ml of diethylaminoethyl Sephadex A-50, previously equilibrated with 0.025 M phosphate buffer, 0.3 M sodium chloride and pH 8. , and the eluted solution was collected. The column was washed with 0.025 M phosphate buffer at 0.3 M sodium chloride and pH 8, and the washings were combined with the above eluted solution to give 1.67 L of solution. The solution was re-applied to an 8 cm diameter column packed with 40 ml of diethylaminoethyl Sephadex A-50 previously equilibrated with 0.025 M phosphate buffer 0.3 M sodium chloride and pH 8 to give 1.68 1 eluted solution. The column was eluted with 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8 to give 1.64 L of wash water. The eluted solution and washings were combined and 0.025 M phosphate buffer, pH 8, was added to give 4.98 L of 0.025 M phosphate buffer, 0.2 M sodium chloride, pH 8. The eluted solution thus obtained was applied to an 8 cm diameter column. , supplemented with 500 ml of diethylaminoethyl-Sephadex A-50 ^ previously equilibrated with 0.025 M phosphate buffer at 0.2 M sodium chloride and pH 8, and the column was washed with 0.025 M phosphate buffer at 0 M sodium chloride. , 2 M and pH 8, followed by passing 2 L of 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8 through the column and collecting the eluted solution.

The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10 additionally desalted using a Sephadex G-25 column, decolorized with activated carbon and then lyophilized to give 590 mg of off-white to light brown TF-133L.

68080

Example 9 (1) 20 g of the crude powder obtained in Example 1— (2) was dissolved in 120 ml of water, and the resulting insolubles were removed by centrifugation at 7500 rpm for 10 minutes, followed by the water-soluble fraction thus obtained and the washings obtained by washing with water. insolubles twice with 60 ml portions of water, were combined and then subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane no: HI-1), and the internal solution was lyophilized to give 10 g of an off-white to light brown powder.

(2) In 1 L of 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and a pH of 8, 25 g of the powder obtained in the above (1) was dissolved, and the resulting solution was applied to a 33 cm diameter column packed with 500 ml of diethyl (D) aminoethyl-Sephadex A-SO 2 previously equilibrated with 0.025 M phosphate buffer, 0.3 M sodium chloride and pH 8. The column was also washed with 0.025 M phosphate buffer, sodium chloride 0 , 3 M and pH 8, and the eluted solution and washings were combined to give 1.6 L of the mixture. The mixture was applied to an 8 cm diameter column packed with 400 ml of diethylaminoethyl Sephadex A-50 ^ - /, similarly equilibrated with 0.025 M phosphate buffer 0.3 M sodium chloride and pH 8 to give 1 .65 l of eluted solution. The eluted solution was combined with 1.7 L of wash water obtained by washing the column with 0.025 M phosphate buffer, 0.3 M sodium chloride, pH 8, and the resulting mixture was diluted with 0.025 M phosphate buffer, pH 8, 10.35 l to prepare 0.025 M phosphate buffer with a 0.1 M sodium chloride concentration and a pH of 8.

The buffer solution thus obtained was then applied to an 8 cm diameter column packed with 500 ml of diethylamino-ethyl-Sephadex A-50 previously equilibrated with 0.025 M phosphate buffer 0.1 M sodium chloride and pH 8, and the column was washed with 2 L of 0.025 M phosphate buffer with 0.1 M sodium chloride and pH 8, followed by 2 L of 0.025 M phosphate buffer with 0.3 M sodium chloride and pH 8. , and the eluted solutions were collected. The resulting 66,08080 eluent solution was desalted and concentrated using an ultrafiltration system (filter used: Tovo Ultrafilter UK-10®) and further desalted with Sephadex A-25®, decolorized with activated carbon and then lyophilized to give 1.65 g of an off-white to off-brown color. -1323-powder.

Example 1Q

10 g of the powder obtained in Example 2 was dissolved in a small amount of water and the resulting solution was applied to a column packed with (S) diethylaminoethyl-Sephadex A-SO 4 previously equilibrated with 0.025 M phosphate buffer at pH 3. Thereafter, the column was 6 l of 0.025 M phosphate buffer with 0.5 M sodium chloride and pH 8 was passed through, followed by 2.5 l of 0.025 M phosphate buffer with 0.6 M sodium chloride and pH 8 to elute the active substance adsorbed on the column , and the resulting permeate was desalted using a hollow fiber type ultrafiltration system (membrane no: HI-1), concentrated under reduced pressure, and then lyophilized to give 130 mg of an off-white to light brown TF-136 powder.

Example 11 15 g of the crude powder obtained in Example 1- (2) was dissolved in 200 ml of water, and water-insoluble matter was removed by centrifugation at 7500 rpm for 10 minutes, after which the water-soluble fraction thus obtained was adjusted to pH 10 by dropwise addition. , 2 M aqueous barium hydroxide solution. A white precipitate precipitated by the addition of aqueous barium hydroxide solution. Barium ions were added to give a total barium ion concentration of 0.01 M in the final volume, after which the solution was stirred for 30 minutes and the precipitate was collected by filtration. The barium salt precipitate thus obtained was suspended in 10 μl of a 10% by weight aqueous sodium sulfate solution, stirred sufficiently, and then filtered and the filtrate was collected. The precipitate was resuspended, stirred and then filtered in the same manner as before once with 10 ml of 10% strength by weight aqueous sodium sulphate solution and twice with 5 ml portions of this solution 67 680 80 and each filtrate was collected. In addition, the remaining precipitate was washed. twice with 10 ml portions of water and then filtered to obtain washings. The above filtrate was collected and combined with washings, dialyzed against distilled water, concentrated under reduced pressure, and then lyophilized to give 0.45 g of an off-white to light brown TF-140 powder.

Example 12 32 g of the powder obtained in Example 1- (2) were dissolved in 450 ml of water and the suspended insolubles were removed by filtration using Hyflo Super Cel, after which the resulting filtrate was dialyzed against running water overnight using a cellophane tube. To the dialyzed solution was added dropwise 80 ml of a 20% by weight aqueous cetylpyridinium chloride solution with stirring, while adding 200 ml of 10% by weight borate buffer (pH 9.0), and the resulting mixture was stirred at room temperature for 30 minutes, after which the precipitate was collected by filtration. The precipitate was suspended in 150 ml of 0.1% by weight borate buffer (pH 9.0) containing 0.1 M sodium chloride and 0.2% by weight of cetylpyridinium chloride, and the resulting suspension was stirred for 30 minutes. The precipitate was then collected from the suspension by filtration and resuspended in 150 ml of borate buffer (pH 9.0) having the above composition, and the resulting suspension was stirred for 30 minutes, after which the precipitate was collected by filtration. The collected precipitate was suspended in 150 ml of 0.1% by weight borate buffer (pH 9.0) containing 0.5 M sodium chloride and 0.2% cetylpyridinium chloride, and the resulting suspension was stirred and dissociated for 30 minutes. The precipitate was then separated from the suspension by filtration to give a soluble fraction. The precipitate separated by filtration was then again subjected to the same dissociation process as above to give a soluble fraction. The two soluble fractions were combined and the resulting soluble fraction was adjusted to pH 3-4 with 10% hydrochloric acid, followed by the addition of ethanol to a final concentration of 80% by volume. The resulting solution was allowed to stand overnight at 5 ° C and the resulting precipitate was collected by filtration to give 5.6 g of an off-white to light brown TF-150 powder.

Claims (28)

68 63080 Patent ivar3.tiir.uk set A method for preparing a carcinostatic and immunostimulatory TF agent which is TF-100, TF-110, TF-120, TF-130, (TF-1316, TF-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF-136), TF-140 or TF-150, characterized in that the Tus-producing bacterium belonging to the genus Fuso is cultured under anaerobic conditions at pH 5-8.5 and at a temperature of 30-45 ° C 1-5 days in a culture medium containing nitrogen, carbon and vitamin sources as well as reducing agents and inorganic salts with or without agar; a precipitant consisting of a hydrophilic organic solvent is added to the produced culture or its supernatant after the pH of the culture or its supernatant is adjusted to 2-7 so that the concentration of the precipitant is 30-70% by volume, or a precipitant is added to the culture or the supernatant, consists of barium ions from a barium ion source such that the barium ion concentration is 0.005-0, 1M at pH 10-13; the precipitate formed is recovered; the precipitant is removed from the precipitate; The TF-containing fraction is separated as a water-soluble fraction from the precipitate; and recovering the TF from the aqueous solution and broth; wherein (A) TF-100 has the following properties; (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus and Ehrlich solid tumors and has an immunostimulatory effect, (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzoform, chloroform ethyl acetate and diethyl ether, (d) it has no clear melting point, it begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones near 3600-3200, 2960 -2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm (f) the UV spectrum of its aqueous solution shows strong absorption at the absorption limit and has an absorption peak near 256-260 nm, (g) when fractionated by gel filtration (column; 50 mm 4 x 600 mm, eluent: distilled water) using Sephadex 6-50® 68080 69 (Pharmacia Co., Ltd.) it has absorption zones with elution volumes close to the intermediate volume - 400, 430-530, 700-800 and 340-870 ml when measuring US absorbance a at 260 nir., and at non-extraction volumes close to the intermediate volume of -390 and 410-430 ml when measuring UV absorbance at 620 nm by the anthrone-sulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: 0.1 M phosphate buffer , pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GE1 G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm 6 x 600 mm x 2) has peaks near the solvent front, 38-60 and 65 min when measuring UV absorbance at 220 nm and 260 nm, (i) it is positive in the Molisch reaction, in the phenol-sulfuric acid reaction, in the anthrone-sulfuric acid reaction, in the indole-hydrochloric acid reaction, In the Lowry-Folin reaction and the ninhydrin reaction, (j) its Elemental Analysis is: C 30.6-35.7%, H 4.2-5.2%, N 4.2-5.2%, (k ) its saccharide content, measured by the phenol-sulfuric acid method, is about 40,0 to 46,0% by weight, expressed as glucose, and its protein content, as measured by the Lowry-Folin method, is about 20,0 to 23,0% by weight, expressed as calf serum albumin, (B) TF-110 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor and has an immunostimulatory effect, (c) it is soluble in water and insoluble in m ethanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) in its IR spectrum ( KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm (f) the UV spectrum of its aqueous solution shows a strong absorption at the absorption limit and has an absorption peak near 256 -260 nm, (g) when fractionated by gel formation (column 44 mm ό x 500 mm, eluent: 0.1 M phosphate buffer; pH 7) using 6 ° 68080 Saphadex 0-200 ^ (Pharmacia Co., Ltd.) it has absorption zones at elution volumes close to an intermediate volume of -380, 600-920 ml when measuring UV absorbance at 260 nn, at elution volumes close to an intermediate volume of -380, 420- 520 and 630-760 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, and at elution volumes close to the intermediate volume of -380, 420-520 and 620-760 ml when measuring UV absorbance at 620 nm by the anthrone-sulfuric acid method. (h) in its high performance liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Cc., Ltd ., column: 7.9 mm φ x 600 mm x 2) there are peaks near the solvent front, 38-60 and 65 min when measuring UV absorbance at 220 nm and 260 nm, (i) it is positive in the Molisch reaction, in the phenol-sulfuric acid reaction, the anthrone-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative ninhydrin reaction, (j) its Elemental Analysis is: C 35.9-41.0%, H 4.5- 5.2% N 4.2-5.2%, (k) its saccharide content measured by the phenolic-sulfuric acid method is about 30.0-69.0% by weight expressed as glucose, and its protein content measured by the Lowry-Folin method is about 18-22% by weight expressed as calf serum albumin, (C) TF ~ 120 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits mouse Ehrlich aquatic growth. growth and has an immunostimulatory effect, (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no clear melting point, it begins to decompose at about 110 ° C, and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1380-1360, 1140-1C00 and 820 cm *, (f ) the UV spectrum of its aqueous solution shows strong absorption at the absorption limit and has an absorption peak near 268-272 nm, il 7i 68080 (g) when fractionated by gel filtration (column: 44 mm ¢ 5 x 500 mm, eluent: 0-1M phosphate buffer, ph 7 ) using Sephadex G-200® (Pharmacia Co., Ltd.) it has absorption zones at elution volumes close to an intermediate volume of -325 and 775-875 ml, measuring UV absorbance at 260 nmr, at elution volumes close to an intermediate volume of -360, 360-480 and 510-760 ml when measuring the absorbance 490 n mr by the phenol-sulfuric acid method and elution volumes close to the intermediate volume of -380, 420-520 and 620-760 ml when measuring the absorbance at 620 nmr by the anthrone-sulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm φ x 600 mm x 2) has peaks near the solvent front, 38-60 min when measuring UV absorbance at 220 nmr and 260 nmr, (i) it is positive in the Molisch reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin reaction and negative in the ninhydrin reaction, (j) its Elemental Analysis is: C 35.1-40.2%, H 4.5-5.5% N 2.0-3.1%, (k) its saccharide content measured by the phenol-sulfuric acid method is about 56.0-73.0% by weight expressed as glucose, and its protein content as measured by the Lowry-Folin method is about 9.0-13.0% by weight expressed as calf serum albumin, (D) TF-130 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma and has an immunostimulatory effect, (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (c) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm-1! 72 680 8 0 (f) the UV spectrum of its aqueous solution shows strong absorption at the absorption limit and has an absorption peak near 256-280 nm, (g) it is positive in the Molisch reaction, in the phenol-sulfuric acid reaction, in the anthrone-sulfuric acid reaction, indo -chlorohydric acid reaction and Lowry-Folin reaction and negative ninhydrin reaction, (h) its Elemental Analysis is: C 27.5-39.8%, H 3.2-5.7% N 2.8-6, 3%, (i) it has a saccharide content, measured by the phenol-sulfuric acid method, of about 19.0 to 64.0% by weight, expressed as glucose, and a protein content, measured by the Lowry-Folin1 method, of about 6.0 to 28% by weight, expressed as calf seerunialbumiinina; (E) TF-1316 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether; (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm (f) the UV spectrum of its aqueous solution shows a strong absorption at the absorption limit and has an absorption peak near 256-260 nm, (g) when fractionated by gel filtration (column: 21 mm ¢ 5 x 400 mm, eluent 0.1 M phosphate buffer, pH 7) using Sephadex G-200 * (Pharmacia Co., Ltd.) has an absorption zone with elution volumes near the intermediate volume -160 ml when measuring UV absorbance at 260 nm, and absorption zone at elution volumes close to an intermediate volume of -160 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: 0.1 M phosphate buffer, pH 7; flow rate; 0.8 ml / min, room temperature) obtained using TSK-GEL G 3000 SVr ^ (Toyo Soda Co., Ltd., column: 7.9 mm <j> x 600 mm x 2) have peaks near the solvent front and 40-60 min when measuring UV absorbance at 220 nm, and close to the solvent front at 40-60 min when measuring UV absorbance at 260 nm, (i) it is positive in the Molisch reaction , in the phenol-sulfuric acid reaction, the anthrone-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative ninhydrin reaction, (j) it The elemental analysis is: C 30.0-34.0% f H 3.8 -4.4% N 4.9-5.7%, (k) its saccharide content measured by the phenol-sulfuric acid method is about 35.0-50.0% by weight expressed as glucose and its protein content measured by the Lowry-Folin method is about 10.0-23.0% by weight expressed as calf serum albumin-Nina; (F) TF-132a has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether; (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR septic tank (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm (f) the UV spectrum of its aqueous solution shows a strong absorption at the absorption limit and has an absorption peak near 270-280 nm, (g) when fractionated by gel filtration (column: 44 mm ώ x 500 mm, eluent 0.1 M phosphate buffer, pH 7) using Sephadex G-200® (Pharmacia Co., Ltd.). ) it has absorption zones with elution volumes near the intermediate volume -340, 600-700 and 720-880 ml when measuring UV absorbance at 260 nm, elution volumes close to intermediate volumes -340, 340-580 and 720-900 ml 74 68080 when measuring absorbance 4 at 90 nm by the phenol-sulfuric acid method, and at elution volumes close to an intermediate volume of -340 and 340-580 ml when measuring the absorbance at 620 nm by the anthrone-sulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: C, 1M phosphate buffer, pH 7; flow rate: 0.3 ml / min, at room temperature) obtained using TSK-GEL G 3300 S \ fö (Toyo Soda Co., Ltd., column: 7.9 mm φ x 6C0 mm x 2) has peaks near the solvent front , 30, 38-60 and 65 min when measuring UV absorbance at 220 nm, and near the solvent front, 62 and 65 min when measuring UV absorbance at 260 nm, (i) it is positive in the Moliseh reaction, fencli sulfur acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin reaction and negative ninhydrin reaction, (j) its Elemental Analysis is: C 34.8-39.8%, H 4.5-5.7 % N 2.8-3.6%, (k) its saccharide content, measured by the fencli-sulfuric acid method, is about 55.0-64.0% by weight, expressed as glucose, and its protein content, as measured by the Lowry-Folin method, is about 18 , 0-28.0% by weight expressed as calf serum albumin; (G) TF-132b has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether; (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm -1, (f) the UV spectrum of its aqueous solution shows strong absorption at the absorption limit and has a broad peak near 265-280 nm, 75 68080 (g) when fractionated by gel filtration (column: 21 mm i> x 400 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200® (Pharmacia Co., Ltd.) has a weak absorption zone at elution volumes of ca. with an intermediate volume of −150 ml when measuring UV absorbance at 260 nm, and an absorption zone with elution volumes close to an intermediate volume of −150 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) in its high-performance liquid chromatogram (eluent: 0.1 IM phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 sW® (Toyo Soda Co., Ltd., column: 7.9 mm <j> x 600 mm x 2) 1 solvent front, 36-37 and 48-50 min when measuring UV absorbance at 220 nm, and very low peaks near the solvent front, 32-39 and 45-52 min when measuring UV absorbance at 260 nm, (i) it is positive in the Molisch reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin reaction and negative in the ninhydrin reaction, (j) its Elemental analysis is: C 35.3-39.5%, H 4.5-5.6% N 2.8-5.4%, (k) its saccharide content as measured by the phenol-sulfuric acid method is about 23.6-45.5% by weight expressed as glucose, and its protein content as measured by the Lowry -Folin's method is about 15.5-28.0% by weight expressed as calf serum albumin; (H) TF-133a has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether; (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm 76 6 8 0 8 0 (f) its aqueous the UV spectrum of the solution shows strong absorption at the absorption limit and has an absorption peak close to 258-262 nm, (g) when fractionated by gel filtration (column: 44 mm φ x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using (R) Sephadex 0-200 ^ (Pharmacia Co., Ltd.) has absorption zones for elution volume near an intermediate volume of -300 and 630-340 ml when measuring UV absorbance at 260 nmr, absorption zone at elution volumes close to an intermediate volume of -420 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, and absorption zone near elution volumes at -4 ml when measuring UV absorbance at 620 nm by the anthronsulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: 0.1 M phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 sJ * (Toyo Soda Co., Ltd., column: 7.9 mm x 600 mm x 2) have peaks near the solvent front, 38 and 50 min when measuring UV absorbance at 220 nm, and near the solvent front 50-60 and 62 min when measuring UV absorbance at 260 nm, (i) it is positive in Molisch reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction , in the indole-hydrochloric acid reaction and in the Lowry-rFolin reaction and the negative ninhydrin reaction, (j) its Elemental Analysis is: C 28.0-36.6%, H 3.5-5.1% N 4.5- 6.3%, (k) its saccharide content as measured by the phenol-sulfuric acid method is about 26.0 to 35.0% by weight expressed as glucose, and its protein content as measured by the Lowry-Folin method is about 22.0 to 28.0 % by weight expressed as calf serum albumin; (l) TF-133b has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlicn solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, 77 6 8080 (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C (e) its IE septic tank (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm \ (f) the UV of its aqueous solution spectrum shows strong absorption at the absorption limit and has a broad peak near 270-280 nm, (g) when fractionated by gel filtration (column: 21 mm φ x 400 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-20 <β (Pharmacia Co., Ltd.) has an absorption zone with elution volumes close to an initial volume of -170 ml when measuring UV absorbance at 260 nmr, and an absorption zone with elution volumes close to an intermediate volume of -150 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) its high-resolution liquid chromatogram (eluent: 0.1 M phosphate, pH 7, phosphate ; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm φ x 600 mm x 2) has peaks near the solvent front 49 -50 min when measuring UV absorbance at 220 nm, and near the solvent front 38-39 and 52 min when measuring UV absorbance at 260 nm, (i) it is positive in Molisch reaction, phenol-sulfuric acid reaction, anthrone-sulfuric acid reaction, in the indole-chloro-hydrochloric acid reaction and the Lowry-Folin reaction and the negative in the ninhydrin reaction, (j) its Elemental Analysis is: C 31.1-38.5%, H 3.9-5.2% N 3.4-4 .7%, (k) its saccharide content, as measured by the phenol-sulfuric acid method, is about 19.0 to 24.5% by weight, expressed as glucose, and its protein content, as measured by the Lowry-Folin method, is about 12.9 to 22.9% by weight. -% expressed as calf serum albumin; (J) TF-1323 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma and has an immunostimulatory effect, 78 68080 (c) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C: (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm -1, (f) the UV of its aqueous solution spectrum shows strong absorption at the absorption limit and has a broad peak near 265-280 nm, (g) when fractionated by gel filtration (column: 21 mm © x 400 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex 0-200 ^ ( Pharmacia Co., Ltd.) has an absorption zone with elution volumes close to an initial volume of -160 ml when measuring UV absorbance at 260 nmr, and an absorption zone at elution volumes close to an intermediate volume of -150 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) its high-resolution liquid chromatogram (eluent: 0.1M phosphate buffer 7, phosphate buffer ; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm φ x 600 mm x 2) has peaks near the solvent front 36 and 48-50 min when measuring UV absorbance at 220 nm, and peaks near the solvent front at 36 and 50 min when measuring UV absorbance at 260 nm, (i) it is positive in the Molisch reaction, phenol-sulfuric acid reaction, anthronium in the sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative ninhydrin reaction, (j) its Elemental Analysis is: C 29.9-39.4%, II 3.9-5.6% N 2.8 -5.4%, (k) its saccharide content, as measured by the phenol-sulfuric acid method, is about 19.0-25.0% by weight, expressed as glucose, and its protein content, as measured by the Lowry-Folin method, is about 11.0-17, 0% by weight expressed as calf serum albumin; (K) TF-136 has the following properties: (a) a mixture of off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor, Ehrlich-68080 solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory effect, ( (e) it is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether; (d) it has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C , (e) its IR spectrum (KBr) has absorption bands close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm -1 (f) the UV of its aqueous solution spectrum shows strong absorption at the absorption limit and has an absorption peak near 256-260 nm, (g) when fractionated by gel filtration (column: 44 mm φ x 50 U mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200 ^ (Pharmacia Co., Ltd.) has absorption zones at elution volumes close to 540-930 ml when measuring UV absorbance at 260 nmr, and all fractions have a weak absorption zone when measuring UV absorbance at 490 nmr by the phenol-sulfuric acid method and when measuring UV absorbance at 620 nm by the anthrone-sulfuric acid method, (h) its in a high resolution liquid chromatogram (eluent: 0.1M phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SVP '' (Toyo Soda Co., Ltd., column: 7.9 mm φ x 600 mm x 2) has peaks near the solvent front , 28-40 and 42-60 min when measuring UV absorbance at 220 nmr and peaks near the solvent front and 42-60 min when measuring UV absorbance at 260 nm, (i) it is positive in the Molisch reaction, phenol-sulfuric acid reaction , in the anthrone-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative ninhydrin reaction, (j) its Elemental Analysis is: C 27.5-32.6%, H 3.2-4.0% N 5.0 to 6.1%, (k) its saccharide content, as measured by the phenolic-sulfuric acid method, is about 19.0 to 30.0% by weight, expressed as glucose, and its protein content, as measured by the Lowry-Folin method, is about 6.0 -12/0% by weight expressed as calf serum albumin; 80 68080 (L) TF-140 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor and has an immunostimulatory effect, (c) it is soluble in water and insoluble methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no clear melting point, begins to decompose at about 210 ° C and decomposes well above 280 ° C, (e) in its IR spectrum ( KBr) has absorption zones near 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm (f) the UV spectrum of its aqueous solution shows a strong absorption at the absorption limit and has an absorption peak near 255 -260 nm, (g) when fractionated by gel filtration (column: 44 mm x 500 mm, eluent: 0.1 M phosphate buffer, pH 7) using (6) Sephadex G-200'ty (Pharmacia Co., Ltd.) it has absorption zones at elution volumes near intermediate volume of -300, 500-900, and 900-1000 ml when measuring UV absorbance at 260 nm, and at elution volumes close to -500, 650-850 and 850-1000 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) in its high-resolution liquid chromatogram (eluent: Q , 1M phosphate buffer, pH 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm (6 x 600 mm x 2) at the peaks near the solvent front and 40-60 min when measuring UV absorbance at 220 nm and 260 nm, (i) it is positive in the Molisch reaction, the phenol-sulfuric acid reaction, the anthrone-sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry -Folin reaction and negative ninhydrin reaction, (j) its Elemental Analysis is: C 22.0-28.0%, H 3.0-3.5% N 5.0-6.5%, (k) its saccharide content measured by the phenol-sulfuric acid method is about 5,0 to 15,0% by weight expressed as glucose and has a protein content, as measured by the Lowry-Folin method, of about 23,0 to 32,0% by weight expressed as calf serum albumin; 81 68080 ( M) TF-150 has the following properties: (a) it is an off-white to light brown powder, (b) it inhibits the growth of mouse Ehrlich hydrocephalus tumor and has an immunostimulatory effect, (c) it is soluble in water and a is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) has no clear melting point, begins to decompose at about 110 ° C and decomposes well above 200 ° C, (e) its IR spectrum (KBr) has absorption zones close to 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm -1, (f) the UV spectrum of its aqueous solution shows strong absorption at the absorption limit and has an absorption peak near 255-260 nm, (g) when fractionated by gel filtration (column: 21 mm 0 x 400 mm, eluent: 0.1 M phosphate buffer, pH 7) using Sephadex G-200 ^ (Pharmacia Co., Ltd .) it has absorption zones at elution volumes close to an intermediate volume of -100 and 100-160 ml when measuring UV absorbance at 260 nm, and an absorption zone at elution volumes close to an intermediate volume of -150 ml when measuring UV absorbance at 490 nm by the phenol-sulfuric acid method, (h) its high performance liquid chromatograms a (eluent: 0.1M phosphate buffer, pH 7; flow rate; 0.8 ml / min, at room temperature) obtained using TSK-GEL G 3000 SW® (Toyo Soda Co., Ltd., column: 7.9 mm φ x 600 mm x 2) has peaks near the solvent front, 32 and 48-50 min when measuring UV absorbance at 220 nm, and near the solvent front, 32 and 48-50 min when measuring UV absorbance at 260 nm, (i) it is positive in the Molisch reaction, phenol-sulfuric acid reaction, anthronium in the sulfuric acid reaction, the indole-hydrochloric acid reaction and the Lowry-Folin reaction and the negative ninhydrin reaction, (j) its Elemental Analysis is: C 31.0-34.0%, H 4.0-4.4 S, N 2, 8-3,2%, (k) its saccharide content, measured by the phenol-sulfuric acid method, is about 40,0 to 55,0% by weight, expressed as glucose, 8 2 6 80 8 0 and its protein content, measured by the Lovry-Folin method, is about 7.0-14.0% by weight expressed as calf serum albumin. Process according to Claim 1, characterized in that a precipitating agent consisting of a hydrophilic organic solvent is added to form a precipitate. Process according to Claim 2, characterized in that the water-soluble fraction of the precipitate is separated from the precipitate and dried. Method according to Claim 2, characterized in that the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration; and the TF material is recovered from the internal solution. Process according to Claim 2, characterized in that the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, if necessary, and then treated with an ion exchanger; and the non-adsorbed fraction is recovered. Process according to Claim 2, characterized in that the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, if necessary, and then treated with an ion exchanger; and the TF material is recovered from the adsorbed fraction. Process according to Claim 6, characterized in that the absorbed fraction is treated with 0.6 M sodium chloride phosphate buffer; and the eluted fraction is collected. Process according to Claim 6, characterized in that the fraction which has not eluted with 0.1 M sodium chloride diphosphate buffer but has eluted with 0.2 M sodium chloride phosphate buffer is recovered from the adsorbed fraction. Process according to Claim 6, characterized in that the fraction which has not eluted with 0.2 M sodium chlorodiphosphate buffer but has eluted with 0.3 M sodium chlorodiphosphate buffer is recovered. Process according to Claim 6, characterized in that the fraction which has not eluted with 0.1 M sodium chloride-phosphate buffer but has eluted with 0.3 M sodium chloride-phosphate buffer is recovered. Process according to Claim 6, characterized in that the fraction which has not eluted with 0.5 M sodium chloride-83 68080 phosphate buffer but has eluted with 0.6 M sodium chloride-phosphate buffer is recovered. Method according to Claim 2, characterized in that the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, if necessary, 0.2-0.3 M sodium chloride-phosphate buffer is prepared from the water-soluble fraction of the precipitate, or 0.2-0.3 M sodium chloride-phosphate buffer from an internal solution component obtained by dialysis, or ultrafiltration, and then treated with an ion exchanger equilibrated with 0.2-0.3 M sodium chloride-phosphate buffer; the fraction which has passed through the ion exchange is recovered; this eluted solution is then adjusted to a concentration of 0.1 M sodium chloride and then treated with an ion exchanger equilibrated with 0.1 M sodium chloride-phosphate buffer to adsorb therein; and thereafter, the fraction that has not eluted with 0.1 H sodium chloride-phosphate buffer but has eluted with 0.2 M or 0.3 M sodium chloride-phosphate buffer is collected. A method according to claim 2, characterized in that the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, if necessary, 0.3 M sodium chloride-phosphate buffer is prepared from the water-soluble fraction of the precipitate or 0.3 M sodium chloride-phosphate buffer from an internal solution component obtained by dialysis or ultrafiltration, and then hand-filtered, , 3 M sodium chloride-phosphate buffer; the fraction which has passed through the ion exchanger is recovered; the sodium chloride concentration of this eluted solution is then adjusted to 0.2 M and the solution is then treated with an ion exchanger equilibrated with 0.2 M sodium chloride-phosphate buffer to adsorb therein; and thereafter, the fraction that has not eluted with 0.2 M sodium chloride-phosphate buffer but has eluted with 0.3 M sodium chloride-phosphate buffer is recovered. A method according to claim 1, characterized in that barium ions are added to the culture or its supernatant to form a barium salt, or a hydrophilic organic solvent is added to the culture 8J 68080 4 or its supernatant and the precipitate formed is recovered and then barium ions are added to the precipitate thus recovered. to a water-soluble fraction, or barium ions are added to a solution of a powder obtained by drying the water-soluble fraction of the precipitate to form a barium salt; the barium salt formed is recovered and then subjected to barium removal treatment; and the water-soluble fraction is collected and subjected to dialysis or ultrafiltration. A process according to claim 1, characterized in that a hydrophilic organic solvent is added to the culture or its supernatant; the precipitate formed is recovered; the water-soluble fraction of the precipitate or the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration is treated with a quaternary ammonium slurry; the precipitate formed is recovered and then subjected to a dissociation process using a solution containing sodium chloride; adding a hydrophilic organic solvent to the resulting soluble fraction to form a precipitate; and the precipitate thus formed is recovered. Method according to one of Claims 1 to 15, characterized in that the culture medium used for culturing the bacterium contains trypticase peptone, phytenipeptone, protease peptone, brain-heart extract (or heart extract), yeast extract, sodium chloride, glucose, lactose, L-cysteine, sodium sulphite and thioglycollate, or it contains them and agar. Process according to one of Claims 1 to 15, characterized in that the culture takes place at 36 ° to 38 ° C for 1 to 3 days in a culture medium adjusted to a pH of 7.2 to 8.2. Method according to one of Claims 1 to 15, characterized in that the bacterium belonging to the genus Fusobacterium is Fusobacterium nucleatum. Process according to one of Claims 2 to 15, characterized in that the hydrophilic organic solvent added to the culture or its supernatant is an alcohol. 85 68080 Process according to one of Claims 6 to 13, characterized in that the ion exchanger is a weakly basic ion exchanger or a weakly basic ion exchanger having molecular sieving ability. Process according to Claim 6, characterized in that 0.1 to 0.6 M sodium chloride-phosphate buffer is used to elute the fraction adsorbed on the ion exchanger. Process according to one of Claims 7 to 13 or 21, characterized in that the pH of the sodium chloride-containing phosphate buffer used for treating the adsorbed fraction is about 8. Process according to one of Claims 7 to 13 or 21, characterized in that the fraction which has been subjected to ion-exchange treatment is subjected to concentration, desalting and drying. Process according to Claim 15, characterized in that the quaternary ammonium salt is cetylpyridinium chloride. Process according to Claim 15, characterized in that the treatment with a quaternary ammonium filter is carried out in the presence of a borate buffer. Process according to Claim 15, characterized in that the sodium chloride-containing solution is a borate buffer containing sodium chloride and a quaternary ammonium salt. A method according to claim 6, characterized in that Fusobacterium nucleatuin is inoculated into a culture medium adjusted to pH 7.2-8.2 and containing trypticase peptone, phytonpeptone, proteose peptone, cerebral heart extract (or heart extract), yeast extract , sodium chloride, glucose, lactose, L-cystine, sodium sulfite or thioglycolate, or in a culture medium containing them and agar and then cultured under anaerobic Oc conditions at 36-38 ° C for 1-3 days: added to the culture or its supernatant alcohol to a concentration of 50-70% by volume; the precipitate formed is recovered; the aqueous solution of the precipitate is subjected to dialysis or ultrafiltration, if necessary, and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger having molecular sieving ability to remove the non-adsorbed fraction; the fraction eluted with 0.1 or 0.2 M sodium chloride-phosphate buffer is removed from the absorbed fraction; and then the fraction eluted with 0.3 M sodium chloride-phosphate buffer is collected, concentrated, deprotected and then dried. A method according to claim 6, characterized in that Fusobacterium nucleatum is inoculated into a culture medium adjusted to pH 7.2-3.2 and containing trypticase peptone, phytonpeptone, proteose peptone or cerebral heart extract (or heart extract), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite or thioglycolate, or a culture medium containing them and agar, and then cultured under anaerobic conditions at 36 ° -38 ° C for 1-3 days; alcohol is added to the culture or its supernatant at a concentration of 50 to 70% by volume; the precipitate formed is recovered; the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration, if necessary, and then 0.2-0.3 M sodium chloride-phosphate buffer of the aqueous solution of the precipitate or a 0.2-0.3 M sodium chloride-phosphate buffer of the internal solution component obtained from dialysis or ultrafiltration is treated with a weakly basic a basic ion exchanger having molecular sieving ability equilibrated with 0.2-0.3 M sodium chloride-phosphate drop buffer; the fraction which has passed through the ion exchanger is recovered; this eluted solution is adjusted to a sodium chloride concentration of 0.1-0.2 M and then treated with a low Kity basic ion exchanger or a weakly basic ion exchanger having a molecular sieve capacity and equilibrated with 0.1-0.2 M with sodium chloride-phosphate buffer, to adsorb to it; and the fraction eluted with 0.2-0.3 M sodium chloride phosphate buffer is recovered from the adsorbed fraction, concentrated, desalted and then dried. 87 68060