FR2616069A1 - IN VIVO INHIBITOR FACTOR OF TUMOR PROLIFERATION AND MATERIALS CONTAINING SAME, PARTICULARLY CAPABLE OF OBTAINING BONE MARROW - Google Patents

Abstract

A substance which inhibits tumoral proliferation in vivo, obtainable from a supernatant of bone marrow, contains a heat-stable factor which is responsible for the said nonspecies-specific activity, is distinct from serum albumin and can be separated by gel filtration from this supernatant together with any serum albumin present.

Description

In vivo inhibitory factor of tumor proliferation and materials containing it, particularly susceptible to be obtained from bone marrow.

 The invention relates to an in vivo inhibitory factor for tumor proliferation and materials containing it, in particular capable of being obtained from bone marrow.

 It relates more particularly to an inhibitory factor of the tumor proliferation in vivo, nonspecific species, which is likely to be obtained from a bone marrow supernatant, and to be extracted from this supernatant at the same time as the serum albumin contained in this marrow supernatant and, where appropriate, at the same time as with protein substances contained in the marrow, whose molecular weights are in a range of about 60,000 to about 68,000 daltons.

 It should be noted that the term "protein substances" as used herein refers to peptides, polypeptides and proteins, with no need to distinguish between them.

 The term "marrow supernatant" refers to the acellular fluid in which the bone marrow normally bathes and which is separated from the cellular constituents, after the removal of the marrow from the bones which normally retain it. It can also be envisaged as constituting the natural environment of the bone marrow cells containing the excretion products of these cells at the moment when said separation, in particular by filtration and / or centrifugation, is carried out.

 The factor according to the invention comprises in particular one or more thermostable polypeptides or peptides (in that the inhibitory activity of the tumor growth is not affected, or even increased, by a heat treatment at a temperature of 80 ° C. 30 minutes), these polypeptides or peptides having apparent isoelectric points between 4.5 and 6.0, when these are determined by isoelectric focusing tests carried out with said factor on a gel comprising a pH gradient, in the presence of said serum albumin and, where appropriate, proteinaceous substances initially contained in the marrow supernatant in molecular weight ranges from about 60,000 to about 68,000 and separable by gel filtration from a bone marrow supernatant.

 When these focussing tests are carried out, for example under the conditions described below, they show at least one migration band, distinct from the migration band characteristic of serum albumin (isoelectric point of the order of 4.9%). ) and characteristic of the aforesaid factor (isoelectric point between 5 to 6.0).

 In particular, the following tests show three migration bands corresponding to isoelectric points contained in the region 5.0 to 6.0.

 The invention also relates to an in vivo tumor proliferation inhibiting material, obtainable from a bone marrow supernatant, this material being characterized: in that it contains a heat-stable factor responsible for the aforesaid non-specific activity of species, distinct from serum albumin, separable by gel-filtration from this supernatant, with the serum albumin that it may contain, - in that it consists essentially of one or more protein substances, in that it has an essentially anionic character, one or more isoelectric points in the region 4,5-6,0, in that it is essentially free of hexoses and gel-free protein substances filtration from a marrow supernatant and having molecular weights lower than 60,000 and greater than 68,000 and proteinaceous substances having isoelectric points greater than 6.0, in particular protein substances that meet this condition when the determination of these isoelectric points is performed by isoelectric focusing tests carried out on this material, when it contains other protein substances, including bone marrow serum albumin.

 This material may also contain serum albumin and gel-filtration separable protein substances from a marrow supernatant and contained in a molecular weight range of about 60,000 to about 68,000.

In other words, the invention also relates to a material, characterized in that all the protein substances it contains are contained in a range of apparent molecular weights ranging from about 60,000 to about 68,000.
It should be noted that the above-mentioned molecular weight values, however significant, can not be regarded as being necessarily limiting in nature - as regards the inhibitory factor that can be further purified, since the It can not be ruled out that in the purification steps which will be described later, this factor remains relatively closely associated with one of the distinct protein substances whose apparent molecular weight is contained in the above-mentioned range. the apparent molecular weight of the inhibitory factor could also be due to the sum of the molecular weights of the tumor proliferation inhibitory factor, in the isolated state, and of another protein constituent, possibly acting as a carrier with respect to this factor.

 The invention finally relates to a process for obtaining a material containing the inhibitory factor of the invention, this method being characterized in that, starting from a bone marrow previously taken from an animal, in particular beef or rabbit, the acellular medium in which the cells of the marrow are normally immersed and, if appropriate, after dilution in an appropriate physiological solution, is separated, in particular by gel-filtration and fractionation of the eluted fractions, protein substances having molecular weights less than about 60,000 and greater than about 68,000, as well as proteinaceous substances having isoelectric points greater than 6; to retain only those fractions consisting of protein substances having apparent molecular weights between about 60,000 and 68,000 and isoelectric points less than 6.0, particularly when the determination of these isoelectric points is performed by isoelectric focusing tests carried out over all of this fraction.

Additional features of the invention will become apparent from the following description of the conditions under which products according to the invention can be isolated and in which the properties mentioned above have been demonstrated, in particular by reference to drawings, in which - fig. 1 is a replica of the chxomatogram of the first gel filtration operation of a bone marrow supernatant, and fractionation of the eluate into several fractions having major molecular weight ranges respectively. decreasing - fig. 2 is a replica of the chromatogram of the second filtration operation, applied to one of the purified fractions at the end of the first filtration (fraction C), to obtain the "FIT fraction" (inhibitory factor for tumor proliferation) which will then be the purpose of the analyzes for the determination of the main characteristics of the substances and the factor
According to the invention - figs. 3A and 3B are respectively representative of the elution profiles (evaluated by the variations of the absorbances A measured at 280 nm as a function of the eluted volume) of the FIT fraction and, for comparison, of pure bovine serum albumin, on gel marketed under the designation SUPEROSE 12 (PHARMACIA brand) - fig. 4A and 4B are respectively representative of the electrophoretic migration profiles of a bovine serum albumin used for comparison and of the FIT fraction, on a cellulose acetate band; the percentages of each population separated by electrophoresis are also indicated on the abscissa axes - FIG. 5 is a replica of the comparative SDS-PAGE gel electrophoretic behaviors of the fraction
FIT and constituents used for comparison, and - fig. 6 is a replica of the electrophoretic behaviors, in a gel pH gradient, of the FIT fraction and the various substances used for comparison.

 The chromatograms or elution profiles of FIGS.

1, 2 and 3 show the variation of the absorbance A (measured at 280 nm on the ordinate axis) of the fractions collected as a function of the volumes eluted (in ml on the abscissa axis)
A. OBTAINING BONE MARROW FROM BONE OF A
MAMMAL - PARTICULARLY A RABBIT OR BEEF - COMING
To be sacrificed
1) Preparation of bone marrow from laPin
The following is the protocol for the preparation of a rabbit marrow extract from 10 "New Zealand" rabbits weighing between 1.5 kg and 2 kg.

 The following operations are performed as sterile as possible. They are all done at + 4-C.

 Rabbits are sacrificed by rupture in the medulla oblongata, then cut up.

 The bones of the animal: femur, tibia, fibula, humerus, radius, ulna are removed with as little flesh as possible. After extraction and "cleaning", the bone is immersed in a biological solution consisting of a sterile 0.15M-pH7.0 sodium chloride solution.

 The bones are washed three times, each time with 1.5 liters of biological solution. Bones thus washed are kept in a fresh biological solution until use.

 For the extraction of the bone marrow, bone epiphyses are cut above a container containing 150 to 200 ml of biological solution. The marrow is then extracted using a syringe, by injecting a little solution along the inner walls of the bones, in order to expel it out of the bones.

 The extracted marrow is then dispersed in the solution. To this end, the two marrow-biological solution phases are homogenized using the syringe by sucking and then expelling the mixture several times in succession.

 The dispersion obtained is centrifuged for 30 minutes at 10,000 rpm. The supernatant is filtered on sterile gauze to remove fat. The caps are set aside. The supernatant, filtered, is immediately frozen.

2) Preparation of a bone marrow of beef
The ribs of a freshly slaughtered yearling heifer weighing about 1000 kg were separated from the carcass. The soft tissue was separated and each rib was sawn into four parts. The bones were rinsed several times with a sterile isotonic solution at -4iC, before being mounted in a vice. The bone marrow was extracted from the spongy material with a hand drill and driven at low speed. The material obtained was immediately suspended in sterile isotonic saline solution and the molecules were dissociated by gentle stirring.

The suspension of the bone marrow cells in the isotonic saline solution was then subjected to a first centrifugation at 15,000 g, to remove particulates such as cells and bone fragments. The supernatant was filtered on natural gauze in order to retain any fat still present. The precipitate was set aside and the supernatant immediately frozen.

 4 liters of acellular bone marrow supernatant were obtained from all the ribs of a heifer. They were collected and frozen (in the form of "aliquots") at -30 ° C.

 All the above operations, starting with the above-mentioned rinsing of the ribs, were performed under sterile conditions and at + 4-C.

 This is the last supernatant hereinafter referred to as "bone marrow", which constituted the starting material for subsequent purifications. It is understood that the aforementioned rabbit supernatant could have been used under identical conditions.

B. PREPARATION AND PURIFICATION OF THE FACTOR INHIBITING THE
TUMOR GROWTH (FIT) FROM THE STRENGTH
BONE.

 1) Preparation of the surnaaeant.

 Sterile material and sterile solutions prepared from freshly distilled water are used and sterilized by autoclaving for 20 minutes at 120 ° C.

 Two vials each containing about 200 ml of bone marrow are thawed by incubation with stirring in a water bath previously equilibrated at 37-C. The thawed solution is distributed in 100 ml centrifuge cups and then centrifuged for 30 minutes at 38,000 g at + 4 ° C. The supernatant is recovered and then filtered on sterile gauze. Its volume is measured. In a typical experiment, this volume is about 400 ml. For practical reasons, this volume is divided into four identical fractions (volume = 100 ml) each corresponding to a sample intended to be chromatographed.

1 ml of a 2% (w / v) solution of sodium azide (NaN 3) in sterile distilled water is added to each fraction.

 Pending the subsequent chromatography, the fractions are stored at + 4-C.

 The casings used for dialysis are tested with freshly distilled water and then divided into groups of five in sterile "plasma" flasks containing about 100 ml of freshly distilled water. The flasks are then sterilized by autoclaving for 20 minutes at 120.degree.

2) First stage of purification: Obtaining a
active fraction Purified by filtration on ael.

 The gel used consists of a mixed matrix of a high resolution polyacrylamide and a stabilizing agarose in aqueous suspension, having a preferential zone of fractionation between about 20.0 and about 350,000. It is more particularly a polyacrylamide gel (3%) - agarose (4%), marketed under the designation ULTROGEL ACA34 (LOB brand), which was used in the test described below.

 Columns of ULTROGEL AcA34 (5 × 90 cm) previously equilibrated with about 5 liters of a 0.9% sodium chloride solution containing 0.02% NaN 3 are used, using a peristaltic pump.

 Part of the supernatant (100 ml) is deposited on the column (flow rate: 60 ml / h).

 Elution is carried out with a solution of sodium chloride 0.9% containing 0.02% NaN 3 (flow rate: 60 ml / h).

 Fractions of 10 ml are collected and the progress of the chromatography is followed, by the use of a U.V. detector (A = 280 nm) connected at the column outlet and coupled to a recorder.

 The fractions collected are left for a few hours at room temperature, then their absorption at 280 nm is measured with a spectrophotometer. After reading, the fractions are stored at + 4in.

 The absorption chromatogram (at 280 nm), as a function of the elution volume (in ml), is plotted (FIG. 1). The limits of each fraction are determined and the tubes are combined to provide six fractions, respectively designated A, B, C, D, E, F, which are then stored at + 4-C.

 The six fractions A, B, C, D, E and F respectively correspond to the elution volumes which are indicated in FIG. 1 (abscissae). We see that the initial distribution was carried out more particularly in order to collect each of the main elution peaks (fractions A, C, D and F) and the intermediate volumes (fractions B and E).

Eluted fractions are also characterized by apparent molecular weight (MW) ranges that are approximately as follows:
Fraction A: PM greater than 300-350,000,
Fraction B: about 300,000 PM> about 150,000,
Fraction C: about 68,000>MW> about 60,000,
Fraction D: about 50,000> PM about 25,000,
Fraction E: about 25,000> MW about 10,000,
Fraction F: PM less than <10,000.

 The same chromatographies are repeated three times under the same conditions, each time on 100 ml of supernatant.

 Between each chromatography, the column is washed with about 3 liters of 0.9% sodium chloride solution containing 0.2% NaN 3 (flow rate: 60 ml / h). After each chromatography, the fractions A, B, C, D, E and F obtained are combined, respectively with fractions A, B, C, D, E and F homologous to the preceding chromatographies.

 After the last chromatography, each fraction is divided into sterile dialysis casings (for example those marketed under the designation NOJAX) (except the fraction F for which SPECTRAPOR brand casings with a 1 x molecular cutoff threshold are used and then dialysed against several volumes. The end of the dialysis is determined by the absence of chloride ion (negative reaction in the presence of 0.1M silver nitrate) in the dialysis water.

The different dialyzed fractions (retentates)
A, B, C, D, E and F are recovered, frozen at -30 ° C and sterilized lyophilized for 48 hours. The lyophilizates are stored at +4 ° C.

In a typical experiment, using 400 ml, the respective weights of material in the fractions
A, B, C, D, E and F were as follows
Fraction A: 114 mg
Fraction B: 79 mg
Fraction C: 489 mg
Fraction D: 827 mg
Fraction E: 109 mg
Fraction F: 30 mg.

 The in vivo antitumor activity is associated with the fraction C molecular weight of between about 60,000 and about 68,000).

3) Second stage of Purification
The active fraction (fraction C) is subjected to a second gel filtration, using the same gel as in the first operation (recycling operation) but operating under the following conditions.

 The same column of ULTROGEL AcA34 (5x90 cm) was previously equilibrated with approximately 5 liters of PBS-pH7.40 buffer containing 0.02% NaN3 (flow rate 60 ml / h).

The sample is prepared under a laminar flow hood in sterile equipment as follows:
In a typical experiment, 470 mg of fraction C, exactly weighed, are dissolved in 20 ml of PBS buffer pH 7.40 containing 0.02% NaN 3. The solution obtained is subjected to magnetic stirring for 30 minutes, then centrifuged for 30 minutes at 38,000. g to + 4C.

The supernatant obtained is deposited on the column (flow rate: 60 ml / h) and the elution is carried out with buffer
PBS pH 7.40 containing 0.02% NaN 3 (flow rate: 60 ml / h).

Fractions of 10 ml are collected and the progress of the chromatography is followed, by the use of a UV detector (A = 280 nm) connected at the column outlet, and coupled to a recorder, as in the previous case.
The fractions collected are left for a few hours at room temperature, then their absorption at 280 nm is measured spectrophotometrically. After reading, the fractions are stored at + 4 ° C.

 The absorption chromatogram is plotted (at 280 nm according to the elution volume (in ml).

 The chromatogram obtained is shown in FIG. 2. The collected tubes are transferred under the laminar flow hood, divided into three groups A ', B and C', in order to separate the fraction corresponding to the absorption peak (fraction B '), anterior elution fractions (A' fraction) and posterior fractions (C 'fraction). The contents of the tubes in each of these groups are then combined.

 Fraction B 'again contains constituents whose apparent molecular weights range from about 60,000 to about 68,000, with proportions of molecular weight less than 60,000 and greater than 68,000 now become negligible.

 Each fraction is divided into sterile dialysis casings and dialysed against several volumes of freshly distilled water. As before, the end of the dialysis is determined by the absence of chloride ion (negative reaction in the presence of 0.1M silver nitrate) in the dialysis water.

 Each retentate (each corresponding to a fraction) is frozen at -30 ° C, and lyophilized sterile for 48 hours. The lyophilizate is stored at + 4in.

In a typical experiment, for 470 mg of committed product, the weight of each A ', B', C 'fraction is as follows:
Fraction A ': 47 mg
Fraction B ': 253 mg
Fraction C ': 61 mg.

 Antitumor activity, determined in vivo, is in fraction B '. It is this fraction that constitutes the "FIT fraction" that will be subject to the biological tests discussed below, and whose activity must be attributed to its FIT content.

C. ELEMENTS OF CHARACTERIZATION OF THE "FRACTION FIT".

1. U.V absorption spectrum

 This spectrum is characterized by a maximum absorption at 278 nm.

2. Protein assay.

 This assay is performed according to the method of Lowry (according to a modification of Miller, Anal Chemical Reference 1959, 31, page 964). This assay indicates a protein content of substantially 100 S for the active fraction.

3. Dosages of hexoses.

 This assay is carried out by an anthrone-sulfuric method (Methods Enzymol., 1957, III, 84).

The assay indicates the absence of hexoses (content less than 0.5%).

4. Study of the behavior of the active fraction (FIT) in
various chromatography systems.

4.1. Filtering tests on the gel marketed under
the brand "SUPEROSE 12" (PHARMACIA).

 This gel is constituted by an agarose gel, formed of particles calibrated with high precision, to allow a high resolution of separation of biological molecules according to their respective molecular weights, in liquid chromatography operations under high pressure and in the conditions recommended by its manufacturer, the Company PHARMACIA, and designated by it under the phrase "FAST PROTEIN LIQUID CHROMATOGRAPHY" (fast liquid chromatography of proteins).

The FIT fraction was chromatographed on this support under the following conditions:
100 μl of a solution containing 1.6 mg / ml of the fraction
FIT in 0.1 M Tris-HCl buffer pH 7.0 NaCl1M-NaN3 0.02 eo are injected into a column (1x30 cm) of SUPEROSE 12 equilibrated and eluted with the same buffer; elution rate 0.5 ml / minute; ambient temperature.

By way of comparison, 100 μl of a 1.2 mg / ml solution of bovine serum albumin of the same 0.1 M Tris-HCl pH 7.0-NaCl1M NaN3 0.02% buffer are chromatographed under identical conditions. The bovine serum albumin used was that marketed by SIGMA under the reference A4378. The profile obtained for the fraction
FIT is shown in Figure 3A.

 Two main peaks are respectively eluted at a volume of 11.69 ± 0.03 ml and 12.95 ± 0.01 ml.

 The profile obtained under identical experimental conditions for bovine serum albumin is shown in FIG. 3B. The two main peaks are eluted at 11.66 ml and 13.03 ml, respectively.

 Comparative examination of Figures 3A and 3B reveals the presence of significant proportions of a constituent that is eluted as serum albumin in the FIT fraction.

 The presence of the active factor according to the invention alters to a certain extent the spectrum due to serum albumin. The existence in the FIT fraction of constituents other than albumin is thus highlighted. The constituents are nonetheless found in very similar elution volumes. The apparent molecular weight of the main peak arising from this test for the FIT fraction is in the order of 65,000.

4.2. Chromatography on cation exchange resins
of the MONO-S type, HR 5/5.

Several tests have been carried out on various cationic resins, more particularly on the resins marketed under the designations - MONO-S, HR 5/5, which is a resin exchange agent.
cations with CH 2 SO 3, - CM SEPHADEX C-50, which is a
cations, carrying charged groups CH2COO
These tests were carried out using appropriate buffers at pH 7. The activity of inhibition of tumor growth, under the conditions described later, was found in the very first fractions of elution, attesting-thus the anionic character of the FIT fraction (absence of fixation, at pH 7, on a cationic resin).

5. Study of the electrophoretic behavior of the fraction
active (FIT).

 The electrophoreses - were carried out in two different systems - on cellulose acetate support, - in polyacrylamide gel in the presence of SDS (PAGE-SDS).

 5.1. Electrophoresis on cellulose acetate tape.

 Comparative electrophoresis of the FIT fraction and SIGMA bovine serum albumin were again carried out.

The tests were carried out under the following conditions on a cellulose acetate band, in the PHOROSLIDE-MILLIPORE system, under the conditions described by MILLIPORE: a) 1.2 μl of a solution of bovine serum albumin (SIGMA) ref A4-378) at 11 mg / ml in bidistilled water are separated for a period of 20 minutes under a voltage of 100 V in Veronal-pH 8.6 buffer. The band is revealed by incubation for 10 minutes in a dye solution
S-PONCEAU, rinsed with several baths of a solution of 5% acetic acid, then diaphanized for reading at densitomer at 520 nm.

b) 0.9 μl of a solution of the FIT fraction (10 mg / ml) in double distilled water are separated for a period of 30 minutes under a voltage of 100 V, in buffer
Veronal-pH 8.6. The band is revealed by incubation for 10 minutes in a PONCEAU-S dye solution, rinsed with several baths of a 5% acetic acid solution, and then diaphanized for reading at the densitometer at 520 nm.

 The electrophoretic profile of the FIT fraction (FIG 4B) has characteristics both common to that of the bovine serum albumin preparation (FIG 4A) and characters that distinguish the active constituents of the FIT fraction from the serum-serum albumin. albumin.

The results indicate that FIT contains a significant proportion (estimated at 40%) of a migrating constituent such as serum albumin. This confirms what was observed during the gel filtration experiments.
SUPEROSE 12, namely very similar elution volumes for albumin and for FIT.

 5.2. Electrophoresis on SDS-PAGE.

 The behavior of the FIT fraction in electrophoresis is compared with that of several reference molecules identified below.

The experiment is carried out in 0.1 M sodium phosphate buffer, pH 7.1, containing 1% of SDS, with a 10% acrylamide gel, under the conditions described by LKB (Operating Instructions 306). Each sample is dissolved in 0.1M pH 7.1 sodium phosphate buffer containing 10
SDS, 1% ss-mercaptoethanol and 6M urea, and then incubated in a boiling water bath (protein concentration between 2 and 11 mg / ml).

Duration of the experiment: 4.5 hours. Color: Blue
Coomassie. Deposit: 10 days.

A: Ovalbumin (Worthington), PM 43,000
B: Serum bovine serum albumin (SIGMA), MW 68,000
C: Lysozyme (Sigma), PM 14,000
D: Alcohol dehydrogenase subunit (Bohringer), PM
37,000
E: Catalase subunit (Serva), PM 60,000
F: Fraction B of first gel-filtration (fraction B,
Fig. 1)
G: Fraction FIT (fraction B of second gel-filtration
see fig. 2)
H: Fraction D of first gel filtration (fraction D,
Fig. 1).

 This test confirms, by comparing the migration distances (Fig. 5) of the FIT constituents with the migration distances of the known molecular weight substances, that the bulk of the constituents of the FIT fraction are contained within a molecular weight range. apparent 60,000-68,000, with a prevalence of about 65,000.

Isoelectric focusing on ael plates marketed by the company LKB under the brand AMPHOLINE (PAG).

 These thin plates, described in French Patent No. 1,554,045 or US Pat. No. 3,485,736, are formed of a thin-layer acrylamide-based gel containing ampholytes (mixtures of polyacids and polybases) which, when the constituents of the plate are subjected to the action of an electric field, they are distributed in a stationary pH gradient.

 The experiment is carried out with the deposits of the materials identified below, on a plate allowing the formation of a gradient of pH 3,5-9,5 under the conditions described by the manufacturer (LKB) in the instructions for use accompanying the PAG plates.

A: deposit of 15 μg serum albumin-bovine (SIGMA
ref. A4378).

B: 75 μg deposit of the FIT fraction.

C: deposit of 30 μg of trypsin inhibitor (SIGMA),
marker of pI = 4.55.

D: deposition of 30 μg of carbonic anhydrase B (SIGMA),
PI marker = 6.57.

E: deposit of 75 μg of fraction D obtained at the end
of the first purification on ULTROGEL AcA34
supernatant of the bone marrow
artwork.

 Fig. 6 shows the compared distances of migrations of these different substances in the pH gradient. The migration bands are revealed by staining with Coomassie blue.

 This experiment allows the following observations: Serum albumin is characterized by an isoelectric point (pI) of 4.9.

The migration distances of the known substances (C pI 4.55, A: pI 4.9, D: pI 6.57) make it possible by comparison to specify the isoelectric points of some of the substances contained in the fraction FIT.

The FIT fraction comprises a large band of pI 4.9, attributable to serum albumin, and three minor bands at pI values in a range of pI between 5 and 6.0.

The fraction FIT is essentially free of constituents clearly distinct from the band corresponding apparently to serum albumin and having isoelectric points between 7 and 8, which is found in the track E of FIG. 6 (corresponding to the fraction
D obtained after the first purification on
ULTROGEL AcA44). More generally, the FIT fraction is free from constituents having isoelectric points greater than 6.0.

BIOLOGICAL ACTIVITY OF FIT FRACTIONS.

 The tests described below were performed with batches of FIT fractions obtained from different bone marrow preparations.

1) In vivo activity.

 The active product IC 86 1773 (FIT) was tested for its antitumor activity in vivo on a reputable experimental model, using a carcinoma of colon CL 26 transplanted in mice (BALB: c mice).

 The in vivo inhibitory activity of tumor growth, which the FIT factor has, is appreciated by the reduction in the importance of the tumors induced in the animal, when the latter has been injected with FIT, compared to development of tumorization in control animals.

 The following experimental protocol has been implemented.

 Mice, BALB / c females receive at OJ days tumorigenic injection of 400,000 carcinoma cells in 0.2 ml of physiological solution, subcutaneously. The cells had been prepared shortly before under sterile conditions. Right after. the tumorizing injection, the mice received intraperitoneally, 109 μg per animal of the product to be tested in 0.5 ml of a phosphate buffer solution. The treatment is continued for 8 to 10 days. The animals are then sacrificed, the tumors removed and weighed.

 The results are expressed as a percentage of the weight of the tumors of the treated animals compared with the control animals which received in an equivalent volume 100 μg of bovine serum albumin.

 The results are reported in the following table which relates to 4 different lots, numbered from (1) to (4), of the fraction IC 86 1773.

TABLE 1
Results
Lot tested Controlled Treatment Inhibition p
(1) 664 + 197,111 t 174 38% 0.01
(2) 92 t 38 28 + 16 70% 0.01
(3) 125 + 65 39 t 19 69% 0.01
(4) 215 + 78 92 + 73 57 and 0.005
These results testify to the presence in the factor FIT of very significant properties of in vivo inhibition of tumor development since this is expressed by a reduction in the weight of the tumors, ranging from 38 to 70%, according to experiments with respect to the weight of tumors in control animals.

2) Absence of in vitro activity.

 The test used, deemed to be effective, consists in evaluating the in vitro influence on the development of tumor cells in culture, in contact with the substance studied introduced into the culture medium.

 Lot FIT 3 was tested on breast carcinoma cell lines, BR 3 and MCF 7 lines, using the experimental protocol that follows.

 The cells are incubated in petri dishes, at a rate of 104 cells per dish in a medium composed of methylcellulose in the medium of DUL BECCO modified with ISCOVE (IMDM) + 20% fetal calf serum + insulin. The products to be tested are introduced into the medium at the following dilutions: 0, 5, 25 and 100 μg / ml.

 The results are expressed in number of colonies after 7 days of culture. The readings were done in duplicate.

TABLE 2
TABLE 2
Lineage Dilution Number of colonies
BR 30 pg / ml 296 + 35
5 μg / ml 267 t 19
25 μg / ml 256 + 23
100 μg / ml 294 + 28
MCF 7 0 pg / ml 159 + 12
5 pg / ml 159 + 12
25 pg / ml 126 t 14
100 pg / ml 138 t 27
These results demonstrate the absence of significant in vitro activity induced by the factor FIT.

 A similar experiment was also carried out with lot (4), but with respect to a KB strain, a human cancer of the nasopharynx. Lot (4) was introduced into the culture at a rate of 200 μg / ml. On this very sensitive strain and at the very high dose tested, no cytotoxic activity was observed, confirming the results of the table above.

3. Activity of the product aHeating.

A batch (5) of IC 861773, which exhibited moderate activity, was heated for 30 minutes at 80 ° C. The product, after heating, showed a significant increase in its activity
The results are reported in the table below:
TABLE 3
BEFORE HEATING Inhibition AFTER HEATING Inhibition Controlled control treated control 206 + 81 141t108 32 224 + 102 93 + 42 58 4. Histohematological studies of control tumors and
control taken after administration to the animal of the
lot (3) of the product IC 86 1773 under the conditions described above. above.

 In control animals, the tumors are larger than in the treated animals. They have necrotic surfaces and the connective tissue is composed only of fibroblasts oriented to encapsulate the tumor and the capsule is thick. Many mitoses are visible in the tumor mass. Carcinoma is known to have a strong malignant character.

 In the treated animals, some tumors have massive necrotic surfaces infiltrated by inflammatory tissue marked by the presence of granulocytes, macrophages, lymphocytes. The tumors are completely surrounded by newly formed granulomatous tissue which has clearly replaced the tumor. In fact the capillaries are visible; they are oriented perpendicular to the tumor mass as if the granulomatous tissue had replaced the tumor surfaces after their necrosis. However, there is still some mitosis in the remaining tumor tissue.

 It follows from the above that the invention therefore provides an active ingredient capable of inhibiting proliferation or tumor growth. As such, the invention relates to any pharmaceutical preparation for use in human or veterinary therapy, in association with a pharmaceutically acceptable vehicle suitable for the chosen mode of administration, especially parenterally. It will be up to the clinician to determine each time, in the light of the state of progress of the tumor disease to be treated, the effective doses that will have to be administered to the patient or the animal, in order to inhibit the tumor process. course, for example in unit doses of 100 mg to 2,000 mg.

 It goes without saying and as it follows already from the foregoing, the invention is not limited to those of its modes of application and of realization which have been more especially envisaged; it encompasses all the variants, especially those consisting of compositions and tumor growth inhibitory factors that can be obtained under similar conditions from cell supernatants having in common with the bone marrow to contain lymphoid cells. for example from supernatants of spleen cells.

Claims (13)

 1. In vivo tumor proliferation inhibiting material, obtainable from a bone marrow supernatant, characterized in that it contains a thermostable factor responsible for the aforesaid non-specific species activity, distinct from the serum albumin, separable by gel-filtration from this supernatant with the serum albumin which it may contain, in that it consists essentially of one or more protein substances, in that it presents an essentially anionic character, one or more isoelectric points in the region 5-6.0, in that it is essentially free of hexoses and gel-filtration separable protein substances from a marrow supernatant and having molecular weights of less than 60,000 and greater than 68,000 and protein substances having isoelectric points greater than 6, in particular protein substances which This condition is when the determination of these isoelectric points is made by means of electrical focusing tests carried out on this material when it contains other protein substances.  2. Material according to claim 1, characterized by the absence of inhibitory activity of tumor proliferation in vitro, when this material also contains serum albumin and gel-filtration-separable molecular weight protein substances from a marrow supernatant and contained in a molecular weight range of about 60,000 to about 68,000.  Material according to claim 1 or claim 2, characterized in that all the protein substances of this material are contained in a range of apparent molecular weights ranging from about 60,000 to about 68,000.  4. The material of claim 1, characterized by an apparent molecular weight of the order of 65,000.  5. In vivo non-species specific tumor proliferation inhibitor, obtainable from a bone marrow supernatant, and being separated therefrom with the serum albumin contained in this supernatant of marrow.  6. The factor according to claim 5, characterized in that it is separable from said marrow supernatant with its content of protein substances, whose molecular weights are in a range of about 60,000 to about 68,000.  7. Factor according to any one of claims 5 and 6, characterized in that it comprises one or more thermostable peptides, having apparent isoelectric points between 5 and 6.0, when these are determined by tests of electrical focusing carried out with said gel factor comprising a pH gradient, in the presence of said serum albumin and, if appropriate, protein substances initially contained in the marrow supernatant in molecular weight ranges from about 69,000 to about 68,000 and separable by gel filtration from a bone marrow supernatant.  8. Factor according to claim 7, characterized in that the above focusing tests show at least one migration band, distinct from the migration band characteristic of serum albumin (isoelectric point of the order of 4.9). ) and characteristic of the aforesaid factor (isoelectric point between 5 and 6).  9. The factor of claim 8, characterized by three distinct migration bands in the region of isoelectric points of 5 to 6.  10. Factor according to any one of claims 5 to 9, characterized in that it consists of a peptide.  11. The factor according to any one of claims 5 to 10, characterized in that it can also be separated by gel-filtration, from supernatants of spleen cells.  A pharmaceutical or veterinary composition, characterized by combining an effective dose of the tumor proliferation inhibiting material in vivo according to any one of claims 1 to 4 with a pharmaceutically acceptable carrier.  13. A pharmaceutical or veterinary composition, characterized by combining an effective dose of the tumor proliferation inhibitory factor in vivo according to any one of claims 5 to 11, with a pharmaceutically acceptable carrier.