BE897470A - PROCESS FOR PRODUCING ANTI-TUMOR GLYCOPROTEINS - Google Patents

Abstract

Method for producing an anti-tumor glycoprotein of molecular weight lower than the molecular weight of the starting anti-tumor glycoprotein. An antitumor glycoprotein from a culture supernatant or an extract of reticuloendothelial cells, leukemia, lymphoblasts or fibroblasts of warm-blooded animals is subjected to a degradation treatment by reaction on a reducing agent and the desired glycoporotein of the reaction mixture.

Description

       

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 subject: Process for the production of anti-tumor glycoproteins Qualification proposed: PATENT OF INVENTION Priority of a patent application filed in Japan on August 4, 1982 under the number Sho 57-135852

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 EMI2.1
 The present invention relates to a method for producing an antitumour glycoprotein having a lower molecular weight than that of the starting antitumor glycoprotein, characterized in that the molecular weight of an antitumor glycoprotein having a molecular weight is reduced
 EMI2.2
 high, by subjecting it to a treatment with a reducing agent and then recovering the anti-tumor glycoproteins having reduced molecular weights from the reaction mixture.
 EMI2.3
 



  Despite the fact that a number of researchers around the world have so far developed many therapeutic agents for the treatment of tumors, the rate of tumor recovery by these therapeutic agents is still very low and research for the development of ever better therapeutic agents is being actively pursued.
 EMI2.4
 



  The present invention therefore relates to a process for the production of anti-tumor glycoproteins.



   The present invention also relates to a process for producing anti-tumor glycoproteins having lower molecular weights from an anti-tumor glycoprotein.
 EMI2.5
 moral with a higher molecular weight. The present invention also relates to a process for obtaining a CLx (CL = carcinolyser) with a high yield.



  The invention also relates to a process for obtaining a CLx2 with a high yield.



  Yet another object of the invention lies in a process for obtaining a CLx3 with a higher yield.



   The subject of the invention is a process for producing one or more anti-tumor glycoproteins having lower molecular weights than those of a glycoprotein.

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 EMI3.1
 starting antitumor, characterized in that an antitumour glycoprotein having a higher molecular weight is reacted with a reducing agent and in that the antitumor glycoproteins having a reduced molecular weight are collected thereafter from the reaction mixture.



   The implementation of the process according to the present invention makes it possible to obtain a carcinolyser (CL) with anti-glycoprotein.
 EMI3.2
 moral of a desired molecular weight with a high yield.



  The present applicants, realizing that reticuloendothelial cells playing an important role in the biophylactic mechanism would eventually be able to generate a substance capable of curing tumors, assigned the task of its researchers to continually- and extensively pursue studies relating to this subject .

   Most recently, the researchers concerned discovered four types of anti-tumor glycoproteins with different molecular weights, from a culture supernatant or an extract of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts warm-blooded animals
 EMI3.3
 and these researchers called them carcinolyser X (molecular weight 12,000-17,000), X1 (molecular weight 70,000-90,000), X2 (molecular weight 40,000-50,000) and X3 (molecular weight 7,000-9,000) (see Japanese patent applications NO 28993/1982, 87674/1982, 87675/1982, 10846/1982, and United States patent application NO 467. 840 of February 18 1983;

   these glycoproteins will be called in the remainder of this specification CLy, respectively and, if they are referred to collectively, they will be called
28992/1982, CL).



   The aforementioned Japanese applications correspond to Belgian patent NO 896. 027 filed on February 25, 1983.
 EMI3.4
 



  Since the CL., - CLy are present as a mixture in a culture supernatant or an extract of reticuloendothelial cells, lymphoblasts, leukemia cells

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 EMI4.1
 or fibroblasts from warm-blooded animals, it has been extremely difficult to obtain, in an efficient and exclusive manner, a carcinolyser having a desired molecular weight, that is to say CLX or CLX3 ′ in a large quantity.



  The Applicant's researchers have intensively studied the development of an efficient process for obtaining and purifying CLX3, CLX2 and / or CLx and have discovered, as a result of this research, that only by a degradation treatment of an anti-tumor glycoprotein (i.e. a carcinolyser) obtained from a culture supernatant or an extract of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts from blood animals hot, by a reducing agent, it was possible to obtain from the above-mentioned liquid reaction mixture, an anti-tumor glycoprotein having a molecular weight of 40,000-50. 000, that is to say CLX2, an anti-tumor glycoprotein having a molecular weight of 12,000-17. 000, ie CLX and / or an anti-tumor glycoprotein having a molecular weight of 7,000-9.

   000, that is to say CLx3 ′ in an easy manner and with a high yield, the objective of the present invention being achieved thereby.



  The abbreviations CL. ,,, and CLx3'telles as used throughout the present specification, are defined as designating antitumor glycoproteins which constitute the carcinolysers of which it was question in the preceding paragraph and which have the following properties: Cl (1 ) molecular weight: 12,000-17. 000;

   (2) coloring reactions: presents a coloring which indicates the presence of proteins during the Lowry reaction, presents a coloring which the presence of peptide bonds and amino acids or amino acids during the reaction with ninhydrin after hydrolysis to hydrochloric acid and has a color which indicates the presence of

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CLysugars during the reaction with phenol-sulfuric acid, during the reaction with anthrone-sulfuric acid, during the reaction with indole-sulfuric acid and during the reaction with tryptophan-sulfuric acid;
 EMI5.1
 (3) appearance and solubility: it is a white powder soluble in water, aqueous sodium chloride and a phosphate buffer and poorly soluble in benzene, hexane and chloroform;

   (4) sugar content: it is 27 to 33%, the composition of these sugars being 17 to 20% hexose, 5 to 7% hexosamines and 5 to 6 6 sialic acids; (5) isoelectric point: 4, 3-7, 3; (6) adsorbability: the substance is adsorbable on Sephadex conjugated with agglutinin from Ulex europeus in a 0.01 M phosphate buffer (pH 7.2);
 EMI5.2
 (7) stability: stable in an aqueous solution with a pH of 2.0, a pH of 7.0 or a pH of 11.0 to 40C for 24 hours and more and in an aqueous solution of a pH of 7.0 to 600C for 3 hours or more;

   (8) cytotoxicity: selectively damages tumor cells without causing appreciable harm to normal cells; and (9) differentiation: induces differentiation of tumor cells, that is to say it causes the recovery of tumor cells into normal cells.



   CLX1: (1) molecular weight: 70,000-90. 000; (2) staining reactions: shows staining
 EMI5.3
 which indicates the presence of proteins during the Lowry reaction, has a coloration which indicates the presence of peptide bonds and amino acids or amino acids during reaction to there ninhydrin after hydrolysis with hydrochloric acid and has a coloring which indicates the pre-
 EMI5.4
 sence of sugars during the reaction to phenol-sulfuric acid, during the reaction to anthrone-sulfuric acid,

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 EMI6.1
 during the reaction with indole-sulfuric acid and during the reaction with triptophan-sulfuric acid;

   (3) appearance and solubility: it is a white powder soluble in water, aqueous sodium chloride and a phosphate buffer and poorly soluble in benzene, hexane and chloroform; (4) sugar content: 35 to 45%, the composition of these sugars being 23-28% hexose, 8-11% hexosamine and 4 to 6% sialic acids; (5) isoelectric point: 4, 3-6, 2; (6) adsorbability: adsorbable on Sephadex conjugated with agglutinin from Ulex europeus in a 0.01 M phosphate buffer (pH 7.2); (7) stability: stable in an aqueous solution with a pH of 2.0, a pH of 7.0 or a pH of 11.0 to 40C for 24 hours and more and in an aqueous solution of a pH of 7.0 at 600C for 3 hours and more; and (8) cytotoxicity: selectively damages tumor cells without causing appreciable harm to normal cells.



  CL (1) molecular weight: 40,000-50. 000; (2) staining reactions: has a staining which indicates the presence of proteins during the Lorry reaction, has a staining which indicates the presence of peptide bonds and amino acids or amino acids during the reaction to ninhydrin after hydrolysis with hydrochloric acid and has a color which the presence of sugars during the reaction with phenol-sulfuric acid, during the reaction with anthrone-sulfuric acid, during the reaction with indole -sulfuric acid and during the reaction with triptophan-sulfuric acid; (3) appearance and solubility: it is a white powder soluble in water, aqueous sodium chloride and a buffer

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 EMI7.1
 phosphate and poorly soluble in benzene hexane and chloroform;

   (4) sugar content: 30-37%, the composition of these sugars being 20 to 23% hexoses, 6 to 8% hexosamine and 4 to 6% acids (5) iso- point electric: 4, 2-7, 3; (6) adsorbability: adsorbable on Sephadex conjugated with agglutinin from Ulex europeus in a 0.01 M phosphate buffer (pH 7.2); (7) stability: stable in an aqueous solution with a pH of 2.0, a pH of 7.0, or a pH of 11.0 to 40C for 24 hours and more and in an aqueous solution of pH from 7.0 to 600C for 3 hours and more; and (8) cytotoxicity: selectively damages tumor cells without causing appreciable harm to normal cells. r * T CLX3 (1) molecular weight: 7.000-9. 000;

   (2) coloring reactions: has a coloring which indicates the presence of proteins during the Lowry reaction, has a coloring which indicates the presence of peptide bonds and amino or amino acids during the reaction to ninhydrin after hydrolysis with hydrochloric acid and has a color which indicates the presence of sugars during the reaction with phenol-sulfuric acid, during the reaction with anthrone-sulfuric acid, during the reaction with indole-sulfuric acid and during the reaction to tryptophan-sulfuric acid; (3) appearance and solubility: it is a white powder soluble in water, aqueous sodium chloride and a phosphate buffer and poorly soluble in benzene, hexane and chloroform;

   (4) sugar content: 8-15 S composition of these sugars being 6 to 10% hexoses, 1 to 2% hexosamines

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 EMI8.1
 and from 1 to 3% of sialic acids; (5) adsorbability: adsorbable on the carboxymethylcellulose during an ion exchange chromatography, using carboxymethylcellulose in a 0.05 M phosphate buffer (pH 6.4); (6) stability: stable in an aqueous solution with a pH of 2.0, a pH of 7.0 or a pH of 11.0 to 40C for 24 hours and more and in an aqueous solution of a pH of 7, at 600c for 3 hours and more; (7) cytotoxicity: selectively damages tumor cells without causing appreciable harm to normal cells; and (8) the amino acid sequence of the N terminal of the protein part is alanine-alanine-.



  The present invention is generally implemented in the following manner: an anti-tumor glycoprotein CL having a molecular weight greater than the desired molecular weight is dissolved, which has been separated and purified from a supernatant of a culture or d a cell extract, at an appropriate concentration of, for example 10 to 10,000 g / ml and then subjected to a degradation treatment by reaction with an appropriate amount of a reducing agent. The reaction mixture is dialyzed, the dialysate is subjected to salification and the precipitates formed are separated by filtration.



  Then, these precipitates are dissolved in a small amount of physiological saline solution or a born solution and, after dialysis, the solution is subjected to gel filtration, using Sephadex G-50 to obtain an anti-tumor glycoprotein having the desired molecular weight.



  The expression `` degradation treatment '' as used in the present specification designates a treatment intended to convert a glycoprotein having a higher molecular weight into a glycoprotein having a weight

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 : lower molecular level by chemically causing the breakdown of intramolecular bonds, such as disulfide bonds.
 EMI9.1
 



  The starting material which is used for the implementation of the present invention is, suitably, CLX1 or CLx2 ′ although CLx can also be used when CLOY constitutes the desired product. Although as starting material, preferably a solution of CLy .. and / or CLx2 purified in a buffer, physiological saline solution, distilled water, etc., it is also possible to use a culture supernatant or cell extract containing CL per se.



   CL can be obtained from a culture supernatant or an extract of meticuloendothelial cells, lympho-
 EMI9.2
 basts, leukemia cells or fibroblasts from warm-blooded animals. The details of the production process and the anti-tumor effect of CL are described in the patent application of the United States of America NO 840 filed on February 18, 1983, corresponding to the Belgian patent NO 027 filed on February 25, 4671983, cited above, of which descriptions are incorporated herein for reference.
 EMI9.3
 



  As examples of the reducing agent which can be used for the purposes of the present invention, mention may be made of thiol-type compounds, such as 2-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acid, l monothiophosphoric acid, etc., hydrogenated metal complexes, such as sodium borohydride, tertiary amines, such as tri-n-butylphosphine, tris-diethylaminoethylphosphine, etc., sulfites, such as sodium sulfite, salts of cynic acid, such as cyanogen bromide, metal ions, such as silver ions, etc.

   These compounds are those generally employed for the decomposition and the elimination of protein contaminants with high molecular weights, for the degradation of a structure of a protein of a higher dimension and for the analysis of the fundamental structure or

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 EMI10.1
 basis of said protein, etc. The proteins treated with these compounds frequently lose their biological activity, but it is surprising that the degradation treatment in accordance with the invention is possible without causing loss of anti-tumor activity.



  The conditions for implementing the present invention, such as the concentration of the reducing agent, the treatment temperature, the treatment time, the treatment pH, etc., can be appropriately chosen depending on the purpose, for example, CLx or CLx3 must be obtained in isolation or that these two substances must be obtained simultaneously, etc. The reducing agent can be used in a concentration of 10-1 preferably, for example, 10-5 is 2-mercaptoethanol, preferably - for example, 10 when it is dithioerythritol , when it is monothiophosphoric acid, it is sodium borohydride, or when it is thioglycolic acid.

   The treatment temperature is not particularly limited, provided that the glycoprotein does not undergo denaturation and it is convenient to use a temperature from 0 to 370C, preferably the ambient temperature around 15-250C. The reaction time can be chosen appropriately so as to obtain a CL carcinolyser having a desired molecular weight, this time generally varying from 10 minutes to 2 hours.

   Preferably, the pH is chosen from the range from weakly acidic to neutral pHs, that is to say between approximately 5, 5 and 7. In addition, during the implementation of the degradation treatment, a surfactant -active or surfactant, for example, an ionic surfactant, such as sodium dodecyl sulfate (DSS), a nonionic surfactant, such as Triton X, etc., may preferably be present in the reaction mixture, in order to 'increase yield. The optimal concentration of surfactant fluctuates depending on the starting material used

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 when to carry out the degradation treatment, this concentration
 EMI11.1
 tion fluctuating in the range of 0.001 to 10%. The ionic surfactants or surfactants include those of the anionic, cationic and amphoteric type.

   As examples of suitable anionic surfactants, mention may be made of DSS, a salt of colic acid, etc. As examples of suitable cationic surfactants, mention may be made of dodecylammonium bromide, etc. and examples of suitable amphoteric surfactants are deoxylysolecithin, etc. As examples of suitable nonionic surfactants, mention may be made of Triton,
 EMI11.2
 those of the Tween type, those of the Span type, etc. Surfactants can be used individually or in mixtures.



  The present invention will now be described in more detail with the aid of the following nonlimiting examples.



    EXAMPLE 1
 EMI11.3
 a) Preparation of starting materials.



   CLs of higher molecular weight (i.e. CLX1, CLX2 and CLX) were prepared, used as starting materials in Examples 1-7.
 EMI11.4
 



  1 D Human lymphocytes (2 x cells) were suspended in 4.000 ml of Eagle medium containing 10% calf serum and, after the addition of phytohemaglutinin (Difco Co.) to a final concentration 50 g / ml, the culture was carried out at 370C for 48 hours in an atmosphere consisting of 5% CO 2 and 95% air.



  Then, the culture medium supernatant was dialyzed against a 0.01 M phosphate buffer (pH 7.2) and the fraction was salified with ammonium sulfate to 40-80% from the dialysate. This fraction was redialysed against the aforementioned phosphate buffer and was then subjected to gel filtration using Sephadex G-100 (Pharmacia Co.).

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 EMI12.1
 



  We obtained the CLy, crude in fractions having molecular weights of 70,000-90. 000, 40. 000-50. 000 and 12. 000-17. 000, respectively. The crude CLy. Was adsorbed on Sephadex conjugated with agglutinin from Ulex europeus (Maruzen Oil Co.) and it was eluted with a 0.01 M phosphate buffer containing 0.5 M of fucose. After elimination of the fucose by dialysis, the CLX1 was again adsorbed on Sephadex conjugated with agglutinin europeus, then it was eluted by a method of teeth, using a phosphate buffer (pH 7, 2), so as to elute the purified CLX1. Purified CLX1 was obtained with a specific activity of 50,000 units / mg, in a total amount of 5,000 units in one operation.



  The crude CLx2 was purified in the same manner and the purified CLOY2 was obtained with a specific activity of 20,800 units / mg, in a total amount of 5,200 units in one operation.



  The crude CLx was also purified in the same manner and the purified CLx was obtained with a specific activity of 10,000 units / mg in a total amount of 10,000 units in one operation. The purified CLX was also obtained with a specific activity of 7,500 units / mg, in a total amount of 150 units, starting from the same number of human lymphocytes and the same method was then used except that the medium of culture did not contain dephytohemaglutinin.



  Activity is expressed throughout this specification based on the concentration which inhibits 50% of the proliferation of KB cell cells serving as a unit. b) Degradation treatment.



  We dissolved 200,000 CLy units <(molecular weight 70,000-90,000) in saline and reacted for 1 hour at room temperature in the presence of 5 x 10-7M 2-mercaptoethanol. After the reaction,

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 EMI13.1
 the reaction mixture was dialyzed and salified with 80% ammonium sulfate and the precipitated fraction was separated by filtration. This fraction was dissolved in physiological saline and thus subjected to gel filtration using Sephadex G-50. The fractions corresponding to the molecular weights of 40,000-50 were collected. 000, 12. 000-17. 000 and 7. 000-9. 000 and they were called fraction A, fraction B and fraction C respectively.

   The procedures described above were also repeated in the presence of 0.05% DSS so as to obtain the same fractions.



  The physicochemical properties of these fractions A, B and C were examined and they were identified as being based on CLX2'CLx and CLx3 respectively. The results obtained are collated in Table I.

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  TABLE l - Physicochemical properties of fractions A-C
 EMI14.1
 
 <tb>
 <tb> Fraction <SEP> A <SEP> Fraction <SEP> B <SEP> Fraction <SEP> C
 <tb> (PM <SEP> 40. <SEP> 000-50. <SEP> 000) <SEP> (PM <SEP> 12. <SEP> 000-17. <SEP> 000) <SEP> (PM <SEP> 7. <SEP> 000-9. <SEP> 000) <SEP>
 <tb> Identification <SEP> CLX2 <SEP> CLX <SEP> CLX3,
 <tb> Appearance <SEP> and <SEP> solubility <SEP> Powder <SEP> white <SEP> soluble <SEP> in <SEP> water, <SEP> on <SEP> chloride <SEP> from <SEP> sodium <SEP> aqueous
 <tb> and <SEP> a <SEP> buffer <SEP> to <SEP> phosphate <SEP> and <SEP> poorly <SEP> soluble <SEP> in <SEP> benzene,
 <tb> hexane <SEP> and <SEP> chloroform.
 <tb>



  Reactions <SEP> from <SEP> coloring <SEP>
 <tb> Lowry <SEP> Blue <SEP> (links <SEP> peptides)
 <tb> Ninhydrin <SEP> Blue <SEP> purple <SEP> (amino acids)
 <tb> Phenol-acid
 <tb> sulfuricê <SEP> Brown <SEP> greyish <SEP> (sugars)
 <tb> Anthroneacide <SEP> sulfuricê) <SEP> Blue <SEP> greenish <SEP> (sugars)
 <tb> &alpha;

  -Naphtholacid <SEP> sulfuric2) <SEP> Purple <SEP>
 <tb> Indoleacide <SEP> sulfuric2) <SEP> Brown <SEP> greyish <SEP> (sugars)
 <tb> Tryptophan-2)
 <tb> acid <SEP> sulfuric <SEP> Purple <SEP> greyish <SEP> (sugars) <SEP>
 <tb> Holff3) <SEP> Colorless <SEP> (not <SEP> from <SEP> lipids)
 <tb>
 

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 EMI15.1
 
 <tb>
 <tb> Fraction <SEP> A <SEP> Fraction <SEP> B <SEP> Fraction <SEP> C
 <tb> (PM <SEP> 40. <SEP> 000-50,000) <SEP> (PM <SEP> 12,000-17,000) <SEP> (PM <SEP> 7.

    <SEP> 000-9.000)
 <tb> Content <SEP> in <SEP> sugar4) <SEP> 30 <SEP> - <SEP> 37 <SEP>% <SEP> 27 <SEP> - <SEP> 33 <SEP>% <SEP> 8 <SEP> - <SEP> 15 <SEP>%
 <tb> Hexose <SEP> 20-23 <SEP>% <SEP> 17-20 <SEP>% <SEP> 6-10 <SEP>%
 <tb> Hexosamine <SEP> 6 <SEP> - <SEP> 8 <SEP>% <SEP> 5 <SEP> - <SEP> 7 <SEP>% <SEP> 1 <SEP> - <SEP> 2 <SEP>%
 <tb> Acid <SEP> sialic <SEP> 4 <SEP> - <SEP> 6 <SEP> 01 <SEP> 5 <SEP> - <SEP> 6 <SEP>% <SEP> 1-3 <SEP>% <SEP>
 <tb> Adsorbable <SEP> on <SEP> Sephadex <SEP> conjugate <SEP> to <SEP> from <SEP> Adsorbable <SEP> on <SEP> carboxyAdsorbability <SEP> agglutinin USE <SEP> <SEP> europeus <SEP> in <SEP> buffer <SEP> methylcellulose <SEP> to <SEP> course <SEP>
 <tb> of a <SEP>
 <tb> at <SEP> phosphate <SEP> 0.01 <SEP> M <SEP> (pH <SEP> 7, <SEP> 2)

    <SEP> exchange <SEP> of ions <SEP> in <SEP> using <SEP> from <SEP> the <SEP> carboxymethylcellulose <SEP> in <SEP> buffer
 <tb> at <SEP> phosphate <SEP> 0.05 <SEP> M
 <tb> (pH <SEP> 6, <SEP> 4 <SEP>
 <tb> Point <SEP> isoelectric5) <SEP> 4.2 <SEP> - <SEP> 7.3 <SEP> 4.2 <SEP> - <SEP> 7.3 <SEP> 6.3 <SEP> - <SEP> 7.8 <SEP>
 <tb> Stability <SEP> Stable <SEP> in <SEP> solution <SEP> aqueous <SEP> of a <SEP> pH <SEP> from <SEP> 2, <SEP> 0, <SEP> of a <SEP> pH <SEP> from <SEP> 7, <SEP> 0 <SEP> or <SEP> of a
 <tb> pH <SEP> from <SEP> 11.0 <SEP> to <SEP> 40C <SEP> during <SEP> 24 <SEP> hours <SEP> and <SEP> more <SEP> and <SEP> in <SEP> solution <SEP> aqueous
 <tb> of a <SEP> pH <SEP> from <SEP> 7.0 <SEP> to <SEP> 600C <SEP> during <SEP> 3 <SEP> hours <SEP> and <SEP> more.
 <tb>



  Cytotoxicity <SEP> Damage <SEP> selectively <SEP> them <SEP> cells <SEP> tumor <SEP> without <SEP> to wear <SEP> from
 <tb> harm <SEP> sensitive <SEP> to <SEP> cells <SEP> normal
 <tb>
 

  <Desc / Clms Page number 16>

 
 EMI16.1
 1) the process of Seikagaku Jikken Koza, Vol. 1 (Tanpakushitsu Teiryohou), 1971 2) the process of Seikagaku Jikken Koza, Vol. 4 (Toshitsu Teiryohou), 1971 3) Following the method of Seikagaku Jikken Koza, 3 (Shishitsu Teiryohou), 1971 4) Spiro, J. A., Methods in Enzymology, Vol. 8, p. 3 1966.



  E \ n 'using Ampholine (marketed by LKB-Produkter. AB, Sweden).



  6 5 'The cells were cultured in 1 ml of Eagle medium containing 10% calf serum and the culture of each fraction at 370C for 48 hours in an atmosphere consisting of 5% CO2 and 95% of air, then counted the number of viable cells not stained with Trypan blue and the concentration of the fraction at which 50% of the cells was killed was taken as a measure of cytotoxicity.



  The recovery of the fraction activity in Example 1 is presented in Table II.



  TABLE II
 EMI16.2
 
 <tb>
 <tb> NextTreatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 200. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 10. <SEP> 000 <SEP> 160. <SEP> 000 <SEP> 15. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 5. <SEP> 000 <SEP> 150. <SEP> 000 <SEP> 25. <SEP> 000
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
 

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 EMI17.1
 EXAMPLE 2 200,000 CLX2 units were dissolved in physiological saline and reacted for 1 hour at room temperature in the presence of 2-mercaptoethanol.

   After the reaction, dialysis and ammonium sulfate salification were carried out to obtain a fraction which was subjected to gel filtration using Sephadex G-50. Fractions with a molecular weight of 12,000-17 were collected. 000 (fraction B) and 7,000-9,000 (fraction C). The roof modes described above were also repeated in the presence of 0.05% DSS. The fractions were determined to be based on CLx and CLx3 'in the same manner as that described in Example 1.

   Table III presents the results obtained with regard to the recovery of CLx and CLx3 'TABLE III
 EMI17.2
 
 <tb>
 <tb> 10 <SEP> 7ItTreatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 200,000 <SEP> - <SEP> Treatment <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 170. <SEP> 000 <SEP> 20. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 140. <SEP> 000 <SEP> 37. <SEP> 000
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
 
 EMI17.3
 EXAMPLE 3 200,000 CLx units were treated with 5 x 10-7M of 2-mercaptoethanol and, in a similar manner to that described in Example 1, a fraction having a molecular weight of 7,000 was obtained. -9. 000 (fraction C). This fraction was identified as being based on CLx3 as described in Example 1.

   Table IV presents the recovery of CLX3

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   TABLE IV
 EMI18.1
 
 <tb>
 <tb> Processing <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 200. <SEP> 000 <SEP> - <SEP>
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol <SEP> 110. <SEP> 000 <SEP> 58. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 90. <SEP> 000 <SEP> 73. <SEP> 000 <SEP>
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
   EXAMPLE 4
 EMI18.2
 A mixture of 100,000 units of CLX1 and 100,000 units of par 5 x was treated in a similar manner to that described in Example 1. The results of the recovery of CLx2'de and CLX3 are presented in Table V.



  TABLE V
 EMI18.3
 
 <tb>
 <tb> CLX2Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 100. <SEP> 000 <SEP> 100. <SEP> 000 <SEP> - <SEP> Treatment <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 20,000 <SEP> 150,000 <SEP> 20,000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 3. <SEP> 000 <SEP> 140. <SEP> 000 <SEP> 42. <SEP> 000
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
   EXAMPLE 5
 EMI18.4
 A mixture of 100,000 CLX1 units and 100,000 CLx units was treated with 5 x 10-7M of 2-mercaptoethanol, in a similar manner to that described in Example 1.

   The results of the recovery of CLx2'de and Cl, are presented in Table VI.

  <Desc / Clms Page number 19>

   TABLE VI
 EMI19.1
 
 <tb>
 <tb> Processing <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 100. <SEP> 000 <SEP> - <SEP> 100. <SEP> 000 <SEP> - <SEP>
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 19,000 <SEP> 130,000 <SEP> 40,000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 7. <SEP> 000 <SEP> 120. <SEP> 000 <SEP> 57. <SEP> 000
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
   EXAMPLE 6
 EMI19.2
 A mixture of 100,000 CLX units and 100,000 CLx2 units was treated with 5 x 10-7M 2-mercaptoethanol in a similar manner to that described in Example 1.

   The results of the recovery of CLx and CLx3 are presented in Table VII.



  TABLE VII
 EMI19.3
 
 <tb>
 <tb> Processing <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 100. <SEP> 000 <SEP> 100. <SEP> 000 <SEP> 0
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 120. <SEP> 000 <SEP> 65. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 90. <SEP> 000 <SEP> 77. <SEP> 000
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
   EXAMPLE 7
 EMI19.4
 A mixture of 100,000 CLx1 units of 100,000 CLX2 units and 100,000 CLX units was treated with 5 x 10-7M 2-mercaptoethanol in a similar manner to that described in example 1. The results of recovery

  <Desc / Clms Page number 20>

 
 EMI20.1
 CLX2'de and CLx3 are shown in Table VIII.



  TABLE VIII
 EMI20.2
 
 <tb>
 <tb> CLxTreatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 100. <SEP> 000 <SEP> 100. <SEP> 000 <SEP> 100. <SEP> 000 <SEP> - <SEP>
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol <SEP> 0 <SEP> 23. <SEP> 000 <SEP> 240. <SEP> 000 <SEP> 22. <SEP> 000
 <tb> Processing <SEP> to <SEP> 2mercaptoethanol
 <tb> + <SEP> 0 <SEP> 8. <SEP> 000 <SEP> 240. <SEP> 000 <SEP> 33. <SEP> 000 <SEP>
 <tb> Processing <SEP> to <SEP> DSS
 <tb>
   EXAMPLE 8
 EMI20.3
 A mixture containing 100,000 units of CLy, 100,000 units of CLX2 and 100,000 units of CLx was treated with each of the reducing agents presented in Table IX, according to a process similar to that described in the example. 1.

   The recovery results of the CLx2'CLX and CLX3 obtained are shown in Table IX.

  <Desc / Clms Page number 21>

 
 EMI21.1
 



  TABLE IX
 EMI21.2
 
 <tb>
 <tb> Agent <SEP> reducer <SEP> Concen-Activity <SEP> (Units)
 <tb> tration
 <tb> CBX1 <SEP> CBX2 <SEP> CBX <SEP> CBX3
 <tb> Before <SEP> treatment <SEP> - <SEP> 100,000 <SEP> 100,000 <SEP> 100,000 <SEP> Acid <SEP> thioglycolic <SEP> 10-6 <SEP> M <SEP> 0 <SEP> 26,000 <SEP> 240,000.

    <SEP> 25,000
 <tb> Acid <SEP> monothio
 <tb> phosphoric <SEP> 10-5 <SEP> M <SEP> 0 <SEP> 27,000 <SEP> 260,000 <SEP> 11,000
 <tb> Tri-n-butylphosphine <SEP> 10-6 <SEP> M <SEP> 0 <SEP> 23,000 <SEP> 230,000 <SEP> 32,000
 <tb> Tris-diethylamino-10-6 <SEP> M <SEP> 0 <SEP> 21,000 <SEP> 230,000 <SEP> 27,000
 <tb> Sulfite <SEP> from
 <tb> sodium <SEP> 10-7 <SEP> M <SEP> 0 <SEP> 25,000 <SEP> 220,000 <SEP> 29,000
 <tb> Bromide <SEP> from <SEP> cyanogen <SEP> 10-7M <SEP> 0 <SEP> 24,000 <SEP> 240,000 <SEP> 28,000
 <tb> Ions <SEP> money <SEP> 10-6 <SEP> M <SEP> 0 <SEP> 23,000 <SEP> 240,000 <SEP> 25,000
 <tb>
 
 EMI21.3
 EXAMPLE EXEI @ LE 1 x 1011 cells of human BALL-1 cells (Miyoshi, Nature, Vol. 267, p.

   843-844, 1977) which had proliferated subcutaneously in nude mice were put into Zion in 20 l of Eagle medium containing 10% calf serum and, after the addition of 1 x 108 pfu (plaque-forming units ) of the Sendai virus, cultivated at 370C in an atmosphere consisting of 5 CO 2 and 95% air, for 48 hours.

  <Desc / Clms Page number 22>

 
 EMI22.1
 



  The culture supernatant was used as the starting material. This culture supernatant contained a mixture of CLyj, Lyp, and CLX3. The treatment was carried out on this culture supernatant by the addition of 10-6M of 2-mercaptoethanol and 0.1% of DSS at room temperature for 1 hour. The reaction mixture was dialyzed and salified with ammonium sulfate to obtain a fraction which was subjected to gel filtration using Sephadex G-50 to obtain CLX2'du and CLX3. The results obtained appear in Table X.



  PAINTINGS
 EMI22.2
 
 <tb>
 <tb> 9Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 47. <SEP> 000 <SEP> 49. <SEP> 000 <SEP> 98. <SEP> 000 <SEP> 500
 <tb> After <SEP> treatment <SEP> 0 <SEP> 1. <SEP> 000 <SEP> 150. <SEP> 000 <SEP> 37. <SEP> 000
 <tb>
 
 EMI22.3
 EXAMPLE The procedure described in Example IX was repeated, except that 5 × 2-mercaptoethanol was used as the reducing agent and that each compound indicated in the table was used. XI in a given concentration.

   The results also presented in Table XI.

  <Desc / Clms Page number 23>

 
 EMI23.1
 TABLE XI
 EMI23.2
 
 <tb>
 <tb> 10Agent <SEP> reduc-Activity <SEP> (Units)
 <tb> tor <SEP> Surfactant
 <tb> (Concentration) <SEP> (Concentration)
 <tb> CBX1 <SEP> CBX2 <SEP> CBX <SEP> CBX3
 <tb> Before <SEP> treatment <SEP> 48,000 <SEP> 51,000 <SEP> 99,000 <SEP> 400
 <tb> tcide <SEP> cholic. <SEP>
 <tb>



  (0.01%) <SEP> 0 <SEP> 2,000 <SEP> 154,000 <SEP> 36,000
 <tb> Bromide <SEP> from <SEP> dodecyltrimethyllammonium <SEP> 0 <SEP> 1,000 <SEP> 149,000 <SEP> 39,000
 <tb> 2-Mercapto- <SEP> (0.01%)
 <tb> ethanol <SEP> Deoxylysolecithin <SEP> 0 <SEP> 3,000 <SEP> 160,000 <SEP> 31,000
 <tb> (0.01%) <SEP>
 <tb> (5 <SEP> x <SEP> 10-7 <SEP> M)
 <tb> Tween <SEP> 20 <SEP> 0 <SEP> 2,000 <SEP> 159,000 <SEP> 29,000
 <tb> (0.01%) <SEP>
 <tb> Span <SEP> 60 <SEP> 0 <SEP> 1.000 <SEP> 158,000 <SEP> 31,000
 <tb>
 
 EMI23.3
 EXAMPLE 11 5 × bovine peripheral lymphocyte cells were suspended in 1,000 ml of Eagle medium containing 10% calf serum and they were cultured at 370C in an atmosphere consisting of 5% COp and 95% air, for 48 hours. The culture supernatant was used and obtained as a starting material.

   This culture giant contained a mixture of CLX1'CLX2'CLX and L) -A to CLX3. The degradation treatment was carried out on this culture supernatant AO by the addition of 10-3M of dithiothreitol at room temperature for 2 hours, then procedures similar to those described in Example 9 were used. ,

  <Desc / Clms Page number 24>

 
 EMI24.1
 to obtain CLx2'CLx and CLx3. The results obtained appear in Table XII.



  TABLE XII
 EMI24.2
 
 <tb>
 <tb> 109Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 13. <SEP> 000 <SEP> 12. <SEP> 000 <SEP> 23. <SEP> 000 <SEP> 200
 <tb> After <SEP> treatment <SEP> 0 <SEP> 1. <SEP> 000 <SEP> 32. <SEP> 000 <SEP> 11. <SEP> 000
 <tb>
 
 EMI24.3
 EXAMPLE 10 3 x Flow 7,000 human fibroblast cells (Flow Co.) were suspended in 6,000 ml of Eagle medium containing 10% calf serum and, after addition of P1yrohemagglutinin to obtain a final concentration of 500 g / ml, they were subjected to culture at 370C in an atmosphere consisting of 5 CO 2 and 95% air for 48 hours. The culture supernatant obtained was used as the starting material. This culture supernatant contained a mixture of CLx1'CLx2'CLx and CLx3.

   A degradation treatment was implemented on this culture supernatant in the presence of 0.111 sodium borohydride, at room temperature, for 2 hours, this treatment being followed by the implementation of methods similar to those described. in Example 9, with a view to obtaining CLyp, the results obtained are presented in Table XIII.



  TABLE XIII
 EMI24.4
 
 <tb>
 <tb> 12Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 16. <SEP> 000 <SEP> 1 <SEP> 8. <SEP> 000 <SEP> 35. <SEP> 000 <SEP> 600
 <tb> After <SEP> treatment <SEP> 0 <SEP> 1. <SEP> 700 <SEP> 52. <SEP> 000 <SEP> 17. <SEP> 000
 <tb>
 

  <Desc / Clms Page number 25>

 
 EMI25.1
 2 x 10 human peripheral lymphocytes were suspended in 4,000 ml of Eagle's medium containing 10 5 calf serum and, after addition of phytothemaglutinin to obtain a final concentration of 50 g / ml, they were subjected to culture at 370C in an atmosphere consisting of 5% COp and 95% air, for 48 hours. The culture supernatant which was obtained was used as the starting material.

   This culture supernatant contained a mixture of CLx1'CLx2'CLX'CLx3. A degradation treatment was implemented on this culture supernatant by the addition of 5 x dithioerythritol at room temperature for 1 hour, this treatment being followed by the implementation of methods to those described in Example 9 , to obtain CLx2'CLx and CLX3'The results are presented in Table XIV.



  TABLE XIV 1. 1
 EMI25.2
 
 <tb>
 <tb> EXEl'lPLE <SEP> 13Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb> Before <SEP> treatment <SEP> 23. <SEP> 000 <SEP> 25. <SEP> 000 <SEP> 51. <SEP> 000 <SEP> 800
 <tb> After <SEP> treatment <SEP> 0 <SEP> 2. <SEP> 000 <SEP> 83. <SEP> 000 <SEP> 13. <SEP> 000
 <tb>
 
 EMI25.3
 .



  EXAMPLE 14 1 x human Ball-1 cells which had been previously proliferated by culture on tissue, were placed in suspension in 2,000 ml of Eagle medium containing 10% calf serum and subjected to culture at 370C in an atmosphere consisting of 5% COp and 95% air, for 48 hours. The culture supernatant was used and obtained as a starting material. The culture supernatant in question contained a mixture of CLX1'de and CLX3. We implemented a degradation treatment

  <Desc / Clms Page number 26>

 
 EMI26.1
 , on this culture supernatant in the presence of 5 × 10 2-mercaptoethanol, at ambient temperature, either for 2 hours or for 5 minutes, this treatment being followed by the implementation of methods similar to those described in example 9, in order to obtain CLx2'CLX and CLy ,.

   The results recorded are presented in Table XV.



  TABLE XV
 EMI26.2
 
 <tb>
 <tb> 1010Treatment <SEP> from <SEP> Activity <SEP> (units)
 <tb> degradation
 <tb> CLX1 <SEP> CLX2 <SEP> CLX <SEP> CLX3
 <tb>. <SEP>
 <tb>



  Before <SEP> treatment <SEP> 5. <SEP> 200 <SEP> 4. <SEP> 900 <SEP> 11. <SEP> 000 <SEP> 110
 <tb> After <SEP> treatment <SEP> 0 <SEP> 300 <SEP> 13. <SEP> 000 <SEP> 7. <SEP> 100
 <tb> (2 <SEP> h)
 <tb> After <SEP> treatment <SEP> 3. <SEP> 600 <SEP> 5. <SEP> 400 <SEP> 12. <SEP> 000 <SEP> 120
 <tb> (5 <SEP> mn)
 <tb>
 
 EMI26.3
 When the degradation treatment was carried out for 5 min, CLX2 and CLX could be obtained with high yields, while the degradation treatment carried out for 2 hours produced CLX and CLX3 with high yields.



  EXAMPLE Purification of CLx2'CLX and CLx3.



  If desired, the crude CLx2 'CLX obtained can be further purified by implementing the methods described in the previous examples, conventionally used to purify biological substances.



  The CLX2 and Clay were purified, for example, by implementing the method described in Example 1 a) above for the preparation of starting materials using Sephadex conjugated with agglutinin from Urex europeus. We got. CLX2 and CLX purified with specific activities of 2,400 units / mg and 2,500 units / mg respectively.

  <Desc / Clms Page number 27>

 
 EMI27.1
 



  The CLx3 ′ was purified, for example, by adsorption on sephalosis conjugated with concanavalin A, this operation being followed by elution with a 0.01 M phosphate buffer (pH 7.2) containing 0.5 M a-methyl-D-mannoside. elimination of K-methyl-D-mannoside by dialysis, the solution was applied to balanced carboxymethylcellulose using a 0.05 M phosphate buffer (pH 6.0), this being followed by elution using a 0.05 M phosphate buffer (pH 7.8). This operation produced a purified preparation with a specific activity of 2,100 units / mg.



  Efficacy, toxicity, instructions for use and dosage of CL according to the present invention.



  EXPERIENCE 1 Selectivity of the Cytotoxic effect.



  In 1 ml of Eagle's medium containing 10 calf serum and each substance tested, the culture was carried out, at 370C, for 48 hours and under an atmosphere consisting of 5 iso of CO 2 and 95% air, samples of 105 selected from KB cells encompassing multiple tumor cell lines (nasopharyngeal cancer), MX-1 cells (breast cancer), provided by Dr. Shigeru Tsukagoshi, Cancer Institute), HEp-2 cells (cancer throat) and HEL cells (hepatoma, Flow Co.) and normal cell lines including intestine 407 cells, Girardi heart cells, liver cells, Chang cells, Vero cells (monkey kidney) ) and MDCK cells (dog kidney (Flow Co.),

   cells which were all respectively subjected to a 24 hour preculture and cells chosen from P388 and L1210 cells (leukemia, supplied by Dr Shigeru Tsukagoshi, Cancer Institute), which were used immediately. We then counted the

  <Desc / Clms Page number 28>

 
 EMI28.1
 viable cells not stained with Trypan blue under a light microscope and the concentration of the test substance for which 50% of the cells were killed was calculated, in comparison with a control taken per 100. As test substances, the CLx2'CLX'CLX3 obtained in Example 15 were used, the a-, ss- by implementing a known method (Granger GA et al., Cellular Immunology, Vol. 38, p. 388-402 (1978)) and Mitomycin C.



  A unit of lymphotoxins is expressed by a conventional index based on cytotoxicity on L mouse cells (Eds., Bloom, BR and Grade, PR "In Methods in Cell-mediated Immunity", Press, 1979). results are shown in Table 16.

  <Desc / Clms Page number 29>

 
 EMI29.1
 



  TABLE-XVI r

  <Desc / Clms Page number 30>

 
 EMI30.1
 As is evident from the above results, CL selectively damages tumor cells, substantially without causing any harm to normal cells. On the contrary, both the a-, ss- and y-Iymphotoxins as well as mitomycin C showed non-selective cytotoxicity with respect to normal cells and tumor cells.



    EXPERIENCE 2 Influence on mice to which we had transplanted the
 EMI30.2
 Sarcoma 180 or Ehrlich's tumor.



  3 x Sarcoma 180 or tumor cells were transplanted per animal to male mice (strain ddY) weighing 25 to 30 g, intraperitoneally and the survival period in days was observed. CLx, in Example 13, was administered intravenously to groups of 5 mice, administration being
10 daily from one day after transplant until death. The results, expressed as a percentage of the average of the days of survival of the animals of the groups tested compared to those of a control group, are presented in Table XVII.

  <Desc / Clms Page number 31>

 
 EMI31.1
 



  BOARD
 EMI31.2
 
 <tb>
 <tb> XVIIDays <SEP> from
 <tb> Substance <SEP> Dose <SEP> daily survival
 <tb> Tumor <SEP> tested <SEP> dienne <SEP> means <SEP> (%)
 <tb> 1,2 <SEP> ut / kg <SEP> 112
 <tb> CBX
 <tb> 4 <SEP> uts / kg <SEP> 132
 <tb> 12 <SEP> uts / kg <SEP> 165
 <tb> 1 <SEP> ut / kg <SEP> 114
 <tb> CBX2 <SEP> 3 <SEP> uts / kg <SEP> 134
 <tb> Sarcoma <SEP> 10 <SEP> uts / kg <SEP> 160
 <tb> l80
 <tb> 1 <SEP> ut / kg <SEP> l09
 <tb> CBX3 <SEP> 3 <SEP> uts / kg <SEP> 133
 <tb> 10 <SEP> uts / kg <SEP> 154
 <tb> Mitomycin <SEP> C <SEP> 0.5 <SEP> mg / kg <SEP> l41
 <tb> Cyc1ophosphamide <SEP> 20 <SEP> mg / kg <SEP> 170
 <tb> 1.

    <SEP> 2 <SEP> ut / kg <SEP> 140
 <tb> CBX <SEP> 4 <SEP> uts / kg <SEP> 160
 <tb> 12 <SEP> uts / kg <SEP> 186
 <tb> 1 <SEP> ut / kg <SEP> 136
 <tb> Tumor <SEP> CBX2 <SEP> 3 <SEP> uts / kg <SEP> 164
 <tb> ascites
 <tb> by Ehrlich <SEP> 10 <SEP> uts / kg <SEP> 187
 <tb> 1 <SEP> ut / kg <SEP> 140
 <tb> CBX3 <SEP> 3 <SEP> uts / kg <SEP> 155
 <tb> 10 <SEP> uts / kg <SEP> 181
 <tb> Mitomycin <SEP> C <SEP> 0.5 <SEP> mg / kg <SEP> 165
 <tb> Cyclophosphamide <SEP> 20 <SEP> mg / kg <SEP> 205
 <tb>
 

  <Desc / Clms Page number 32>

 
 EMI32.1
 EXPERIENCE 3 Influence on the days of survival of mice carrying pulmonary carcinoma.



  To male mice of the BDF1 'strain weighing 20 to 25 g, 2 × Lewis lung carcinoma cells were transplanted, intramuscularly, into the right thigh and the days of survival were observed. CLy, and CLX3 'obtained as described in Example 15, were administered intravenously to groups of 6 mice, the administration being from the day following transplantation to death. The results, expressed as percentages of the average of the days of survival of the animals of the groups subjected to the test compared to those of a control group are collated in Table XVIII.



  TABLE XVIII
 EMI32.2
 
 <tb>
 <tb> 106Substance <SEP> Dose <SEP> Days <SEP> from
 <tb> survival
 <tb> tested <SEP> daily <SEP> means <SEP> (%)
 <tb> 1, <SEP> 2 <SEP> ut / kg <SEP> 108
 <tb> CBX <SEP> 4 <SEP> uts / kg <SEP> 114
 <tb> 12 <SEP> uts / kg <SEP> 150
 <tb> 1 <SEP> ut / kg <SEP> 107
 <tb> CBX2 <SEP> 3 <SEP> uts / kg <SEP> 123
 <tb> 10 <SEP> uts / kg <SEP> 145
 <tb> 1 <SEP> ut / kg <SEP> 116
 <tb> CBX3 <SEP> 3 <SEP> uts / kg <SEP> 140
 <tb> 10 <SEP> uts / kg <SEP> 158
 <tb> Mitomycin <SEP> C <SEP> 0.5 <SEP> mg / kg <SEP> 120
 <tb> Cyclophosphamide <SEP> 20 <SEP> mg / kg <SEP> 162
 <tb>
 

  <Desc / Clms Page number 33>

 
 EMI33.1
 EXPERIENCE 4 Influence on lung metastases from cancer.



  A square fragment next to 2 mm of Levais lung cancer was transplanted into the backs of animals from groups of male mouse mice of the BDF1 strain weighing 20 to 30 g each, the groups consisting of 6 animals each. CLX'CLX2 and CLX3 'obtained in Example 15 were administered, by the intravenous route, once a day, for 12 days from the 9th day after transplantation. On the 21st day after transplantation, the mass of the primary tumor was isolated and weighed and the number of metastases in the lungs of the animals was calculated, according to the method of Wexler, H. (J. Natl. Cancer Institute, Vol 36, 641, 1966).



  The results obtained are presented in Table XIX.



  TABLE XIX
 EMI33.2
 
 <tb>
 <tb> fromSubstance <SEP> Dose <SEP> Weight <SEP> from <SEP> the <SEP> Number <SEP> this <SEP> nodules
 <tb> tested <SEP> daily <SEP> tumor <SEP> (g) <SEP> from <SEP> metastasis <SEP> in
 <tb> them <SEP> lungs
 <tb> Control <SEP> 8.8 <SEP> # <SEP> 0.8 <SEP> 30.1 <SEP> # <SEP> 5.6 <SEP>
 <tb> CBx <SEP> 3 <SEP> utsjkg <SEP> 4, <SEP> 4 <SEP> # <SEP> 1, <SEP> 4 * <SEP> 6, <SEP> 7 <SEP> # <SEP> 2, <SEP> 9 *
 <tb> 1 <SEP> 30 <SEP> uts / kg <SEP> 3, <SEP> 2 <SEP> # <SEP> 0, <SEP> 5 ** <SEP> 0, <SEP> 5 <SEP> # <SEP> 0, <SEP> 3 **
 <tb> CBX2 <SEP> 3 <SEP> uts / kg <SEP> 4.5 <SEP> # <SEP> 1, <SEP> 5 * <SEP> 6, <SEP> 9 <SEP> # <SEP> 2.1 *
 <tb> 30 <SEP> uts / kg <SEP> 2, <SEP> 3 <SEP> # <SEP> 0, <SEP> 4 ** <SEP> 0, <SEP> 7 <SEP> # <SEP> 0, <SEP> 4 **
 <tb> CBX3 <SEP> 3 <SEP> uts / kg <SEP> 4, <SEP> 1 <SEP> # <SEP> 1, <SEP> 3 * <SEP> 7.0 <SEP> # <SEP> 2,

    <SEP> 8 *
 <tb> 30 <SEP> uts / kg <SEP> 2, <SEP> 1 <SEP> i <SEP> 0, <SEP> 3 ** <SEP> 0 ') <SEP> 6 <SEP> # <SEP> 0, <SEP> 3 **
 <tb> Cyclophosphaminde <SEP> 20 <SEP> uts / kg <SEP> 3.6 <SEP> # <SEP> 0.7 ** <SEP> 0.7 <SEP> # <SEP> 0 <SEP> 4 **
 <tb>
 

  <Desc / Clms Page number 34>

 
 EMI34.1
 Notes) The results which appear in the tables are expressed as mean standard error.



  * Domains statistically different from the control group at a significance level of p <5%.



  ** Statistically different data from the control group at a significance level of p <1%.



  As the above results clearly show, CLX'CLX2 and CLX3 have successfully suppressed primary lung cancer and its lung metastases.



  EXPERIENCE 5 Toxicity test (single administration).



  10,000 units / kg of Cl'10 were administered intravenously, respectively. 000 units / kg of CL or 100. 000 A A <1 units / kg of CLX3 to animals of 3 groups of male mice of the BDF1 strain each weighing 20 to 25 g, the groups being formed of 10 animals each and the animals were observed for 7 days. The animals of the respective groups survived without showing any change in body weight and general condition, despite the respective administration of its high glycoprotein units.



  EXPERIENCE 6 Toxicity test (continuous administration for 30 days).



  CLX'CLX2 or CLX3 were administered for 30 days to male mice of the BDF1 'strain each weighing 20 to 25 g, each group comprising 10 animals and the number of dead animals, the change in body weight and the 'condition. The body weight was taken between 9 and 10 hours before noon and the general condition was observed on the 10th, 20th and 30th days, according to the method of Arvien (Science, Vol. 36, p. 123 (1962)). As a result, no dead animals were observed during these 30 days when 1,000 units / kg / day of CLX or

  <Desc / Clms Page number 35>

 
 EMI35.1
 Cl2 10,000 units / kg / day CLx3 and the weight gain curve was approximately the same as that of the animals in the control group.

   In addition, it was found that the general conditions were normal as in the case of those of the control group animals.



  As can be seen from the experiments described above, CLX'CLX2 and CLX3 selectively suppress the growth of tumor cells and, moreover, they not only remarkably suppress cancer metastases, but are also extremely effective against various tumors and are always d very safe use, even at doses higher than the dose at which the pharmaceutical effect is manifested. Therefore, CLy, and CLx3 are extremely useful for the therapeutic treatment of various tumors, such as stomach cancer, lung cancer, hepatoma, colon cancer, breast cancer, uterine cancer, leukemia, etc. .

   Furthermore, relatively low molecular weight forms of CL (CLX'CLx2'CLx3) can be derived from relatively high molecular weight forms of CL (CLX'CLX1'CLX2) by treatment with a reducing agent, without prejudice their effectiveness in treating tumors.


    

Claims (1)

 EMI36.1   CLAIMS  EMI36.2  1. Method for producing an anti-tumor glycoprotein of reduced molecular weight, characterized in that a degradation treatment is implemented on at least one starting anti-tumor glycoprotein, obtained from a culture supernatant or an extract of reticuloendothelialos cells, lymphoblasts, leukemia cells or fibroblasts of warm-blooded animals, by reacting the abovementioned starting antitumor glycoprotein with a reducing agent and then at least one glycoprotein is recovered reduced molecular weight antitumor. 2. Manufacturing process 1, characterized in that the starting antitumor glycoprotein is the glycoprotein CLOY1 and the antitumor glycoprotein of reduced molecular weight is at least one chosen from the group formed by the antitumor glycoproteins CLx2'CLX and CLX3. 3. Method according to claim 2, characterized in that said CLX1 glycoprotein is obtained from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts of warm-blooded animals, by proceeding salifying the supernatant and fractionating a precipitate thereof by gel filtration so as to obtain a fraction having a molecular weight of 70,000-90. 000 and in that said fraction is then subjected to adsorption and elution on Sephadex conjugated with agglutinin from Ulex europeus. 4. Method according to claim 1, characterized in that said starting anti-tumor glycoprotein is CLX2 and in that the anti-tumor glycoprotein of reduced molecular weight is at least one chosen from the group formed by CLX and CLx3. 5. Method according to claim 4, characterized in that said starting antitumor glycoprotein CLX2 is obtained naked from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or  <Desc / Clms Page number 37>    EMI37.1  fibroblasts of warm-blooded animals, by salifying the supernatant and fractionating a precipitate thereof by gel filtration, so as to obtain a fraction having a molecular weight of 40,000 to 50,000 and whatever 'said fraction is then subjected to adsorption and elution on Sephadex conjugated with agglutinin from Ulex europeus.  EMI37.2   6. Method according to claim 1, characterized in that said starting antitumor glycoprotein is CLx and in that the reduced molecular weight antitumour glycoprotein is 7. Method according to claim 6, characterized in that the starting antitumor CLx glycoprotein is obtained only from a culture supernatant of reticuloendothelial cells, lymphoblasts, leukemia cells or fibroblasts of warm-blooded animals by salifying  EMI37.3  cation of the supernatant and by fractionating a precipitate thereof by filtration through gel so as to obtain a fraction having a molecular weight 12,000-17. 000 and in that said fraction is then subjected to adsorption and elution on Sephadex conjugated with agglutinin from Ulex europeus.  8. Method according to claim 1, characterized in that the reducing agent is at least one substance chosen from  EMI37.4  the group formed by a thiol compound, a metal hydride complex, a tertiary phosphine, a sulfite, a cyanate and a metal ion.    9. Method according to claim 2, characterized in that the reducing agent is at least one substance chosen from  EMI37.5  the group formed by a thiol type compound, a metal hydride complex, a tertiary phosphine, a sulfite, a cyanate and a metal ion.    10. Method according to claim 4, characterized in that the reducing agent is at least one substance chosen from  EMI37.6  the group formed by a compound of the thiol type, a complex  <Desc / Clms Page number 38>  of metal hydride, a tertiary phosphine, a sulfite, a cyanate and a metal ion.    11. Method according to claim 6, characterized in that the reducing agent is at least one substance chosen from  EMI38.1  the group formed by a thiol type compound, a metal hydride complex, a tertiary phosphine, a sulfite, a cyanate and a metal ion.  EMI38.2   12. Method according to claim 8, characterized in that the thiol type compound is chosen from the group formed by 3-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic and monothiophosphoric acid. 13. Method according to claim 9, characterized in that the thiol type compound is chosen from the group formed by 3-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic and monothiophosphoric acid. 14. Method according to claim 10, characterized in that the thiol type compound is chosen from the group formed by 3-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic and monothiophosphoric acid. 15. The method of claim 11, characterized in that the thiol type compound is selected from the group formed by 3-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic and monothiophosphoric acid. acid 16. Method according to claim 1, characterized in that the degradation treatment is carried out in the presence of a surfactant or surfactant.    17. The method of claim 8, characterized in that the degradation treatment is carried out in the presence of a surfactant or surfactant.    18. The method of claim 9, characterized in that the degradation treatment is carried out in the presence of a surfactant or surfactant.    19. Method according to claim 10, characterized in that the degradation treatment is carried out in the presence of a surfactant or surfactant.  <Desc / Clms Page number 39>      20. The method of claim 11, characterized in that the degradation treatment is carried out in the presence of a surfactant or surfactant.    21. The method of claim 16, characterized in that the surfactant or surfactant is chosen from the group formed by sodium dodecylsulfate, sodium cholate, dodecyltrimethylammonium bromide, deoxylysolecithin, TritonR X, TweenR 20 and the SpanR 60.    22. Method according to claim 17, characterized in that  EMI39.1  that the surfactant or surfactant is chosen from the group formed by sodium dodecyl sulphate, sodium cholate, dodecyltrimethylammonium bromide, deoxylysolecithin, TritonR X, TweenR 20 and SpanR 60. 23. The method of claim 8, characterized in that the degradation treatment is carried out at a temperature which varies from 0 to 370C, in a pH range weakly acid to neutral. 24. The method of claim 17, characterized in that the degradation treatment is carried out at a temperature varies from 0 to 370C, in a pH range weakly acid to neutral.    25. The method of claim 23, characterized in that the temperature varies from 15 to 250C.    26. The method of claim 24, characterized in that the temperature varies from 15 to 250C.