CH678337A5 - - Google Patents

Description

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Description

The present invention relates to a process for the preparation and culture of a suspension of immune memory cells used for the prevention and / or treatment of an infectious disease or a malignant tumor such as cancer. The present invention also relates to a suspension of immune memory cells prepared using such a method.

A human or any animal, which has once had a bacterial or viral disease and which has reacted against a particular species of antigen, exhibits a remarkable defense reaction to the antigen when it is subjected to a second stimulation. due to the same type of antigen. This defense reaction thus provoked is the result of the individual's immunity to the particular disease and is usually called the secondary immune response or the body's anamnestic response to the antigen. Those skilled in the art know that an immune response results from an excessive multiplication of lymphocytes included in the body's reaction to stimulation by the antigen which contributed to the first manifestation of the disease. The lymphocytes thus developed in response to stimulation by an antigen are generally called immune memory cells.

The immune memory cells produced in an individual's body are a kind of lymphocyte found in the lymph and blood circulating in the vessels of the body's circulatory system. Lymphocytes from the circulatory system migrate into the thymus of the organism where they transform into T lymphocytes or T cells, which gives the organism an immune competence. As is known in the state of the art, these immunologically competent T cells, also sometimes called thymus-derived or thymus-dependent cells, are widely distributed in almost all of the peripheral tissues of the organism's circulatory system.

As with the immune memory cells forming part of the T cells thus developed from lymphocytes, the state of the art indicates that such cells are recognized simply conceptually as "immunological" memory cells. There have been no practical attempts to isolate or extract such cells or at least a fraction containing such cells from a tissue of the organism and to collect the resulting immune memory cells or the fraction containing the cells in a state usable in practice or in vitro. Likewise, no positive research for the use of immunological memory cells for the prevention and / or treatment of infectious disease or malignant tumor has been evoked or suggested on a serious basis, and no clinical or experimental approach aimed at using practice of immunological memory cells has not been seriously undertaken,

The present invention provides for carrying out a method of separating immune memory cells directly from a tissue of the organism, with a significantly high yield. Such a method according to the present invention depends for its effect on the various natures and properties of the immune memory cells which confer on the organism a cell type immunity. The present invention also provides for the establishment of a method of culturing the immune memory cells. for several generations to provide an established, continuous cell line that provides immune cells independent of tissue or an organism.

The present invention further provides a suspension of immune memory cells prepared from a tissue of an immune sensitized individual in accordance with the present invention or from any immune memory cells prepared from a tissue of an individual.

In accordance with one aspect of the present invention, there is presented a method for preparing a suspension of immune memory cells characterized in that it comprises the following steps:

- separation of cells consisting essentially of T cells from cells taken from an individual sensitized immune by immunization against a particular pathogen,

Addition to the separated cells of an antibody reacting to a specific histocompatibility antigen of a defined species of cells which form the antigen-antibody complexes with said antibodies and said separate cells having said histocompatibility antigens,

- addition of a blood serum to the cells containing said antigen-antibody complexes in order to cause in the blood serum the binding of the complement to said antigen-antibody complexes and therefore the destruction of the cells presenting said histocompatibility antigens and the survival of the remaining cells ,

- addition to persistent cells of an antibody reacting to a specific histocompatibility antigen of immune memory cells, and

- separation from the resulting cells, of those presented by specific histocompatibility antigens of immune memory cells.

According to another aspect of the present invention, there is provided a suspension of immune memory cells which comprises cells which are taken from an individual immune sensitized by immunization against a particular pathogen and which essentially consist of T cells, in which the cells representing about 98% by number of said T cells contain at least 103 immune memory cells.

Note that the term "pathogen" as used herein can be a cancerous or malignant tumor or any pathogenic microbe such as a bacteria or virus which is known to be the cause of disease or response toxic in a human or other animal.

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A suspension of immune memory cells prepared according to the method of the present invention can be separated, purified and cultured from any tissue of an organism which has been immunologically sensitized against an infectious disease or a malignant tumor. This means that immunological prevention and / or treatment of a disease can be carried out against any infectious disease of viral or bacterial origin or against any malignant tumor insofar as an immunologically sensitized organism, which has been cured of such a disease or tumor, is available. Typical diseases which can be faced by such immunological prevention and / or such treatment are those caused by pathogens including cancer cells, Salmonella, Corvnebacterium. Pseudo-mas. Pasteurella. Streptococcus, ectromelia virus and Sendai virus (Parainfluenza virus type I of 10 mice).

It should also be noted that the immune mechanism elicited by a suspension of immune memory cells differs fundamentally from humoral immunity which depends on the antibody-antigen reaction represented by vaccination. None of the adverse effects which are the inevitable result of vaccination should be caused by the prevention and / or treatment of a disease by the use of a suspension of immune memory cells.

When the immune memory cells provided by the present invention are cultured from the cells obtained in the form of a suspension of immune memory cells or first stage and then successively subcultured for several generations from a suspension of immune memory cells in the second stage, a continuous line of immune memory cells can be established to make possible the production of cells stably and economically on a quantitative basis.

To prepare a suspension of immune memory cells according to the present invention, the cells containing the lymphocytes are first removed from an individual who is known to have been cured and consequently immunized against any infectious disease of viral or bacterial origin. or 25 a malignant or cancerous tumor. These cells containing lymphocytes can, for example, be extracted from peripheral blood drawn from the heart or from a vein of the immune-sensitized individual. Alternatively, cells containing lymphocytes can be isolated from a sample of the spleen tissue of an immune-sensitized individual.

The T cells are then separated from the cells containing the lymphocytes. For this purpose, the cells containing the lymphocytes are treated by column affinity chromatography using an appropriate solid adsorber allowing selective passage of the T cells. B cells are also contained in the cells made up of the lymphocytes which confer on the cell-type immunity. The majority of these B cells will adhere to the solid adsorbent and therefore a cell suspension consisting essentially of T cells is eluted from the column fractionally. Preferably used in the chromatographic process is a nylon wool packaged in a tube.

A solid nylon wool adsorbent not only absorbs B cells but also unwanted cells such as particularly macrophages and leukocytes, as is known in the art, and for this reason chromatography affinity using a single so-40 nylon wool adsorbent cannot provide satisfactory high adsorption efficiency for B cells. According to a method of the present invention, it is preferable that the step of affinity chromatography on nylon wool is carried out, after another column chromatography step using another solid adsorbent such as glass wool. As is known in the state of the art, both T cells and B cells adhere strongly to glass wool which is however capable of stopping macrophages and leukocytes with significant high degrees of affinity. . By using a two-step affinity chromatographic method using glass wool first and then nylon wool, the B cells contained in the initial suspension of lymphocytes can be separated with satisfactory high efficiency from of the cell suspension freed from macrophages and leukocytes.

50 In substitution for such a two-step affinity chromatographic method, a T cell separation method can be used which depends, in principle, on the difference between the surface antigens (histocompatibility antigens) specific for T cells and B respectively. When a mouse is used as an immune sensitized individual, the Thy 1 surface antigen can be used to identify T cells in such a T cell separation process.

55 The cells, contained in the cell suspension resulting from the two-stage column affinity chromatography process, are largely made up of T cells but still contain a small quantity of unwanted species such as typically immature lymphocytes lacking immune capacity . Consequently, a method must be applied subsequently which removes the undesirable immature cells by causing an antigen-antibody reaction using the cells of the cell suspension as the antigen.

For this purpose, an antibody which reacts specifically with a particular histocompatibility antigen is added to the cell suspension which originates from the two-stage column affinity chromatography process. The preparation of antibodies to be added to the cell suspension is chosen so as to be able to react with cells which have histocompatibility antigens specific for the unwanted lymphocytes contained in the cell suspension.

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As a result of the antigen-antibody reaction thus provoked between the preparation of antibodies and the cells having these histocompatibility antigens, antigen-antibody complexes are formed by the antibody molecules linked with the cells presenting the histocompatibility antigens. To the antigen-antibody complexes is added an appropriate blood serum such as, typically, rabbit serum so that the complement contained in rabbit serum binds to the antigen-antibody complexes and destroys the cells presenting the histocompatibility antigens specific for unwanted lymphocytes. The result is that only cells with negative surface antigenic activity towards histocompatibility antigens can survive in the resulting cell suspension. It will be evident that these cells are essentially immune memory cells.

When a mouse is used as an immune-sensitized individual, the antigen la associated with region 1) can be used as the histocompatibility antigen in the reaction for the production of antigen-antibody complexes. In this case, the monoclonal anti-antigen la is added to the cell suspension to form the antigen-antibody complexes for binding of the antigen molecules with cells having positive antigenic activity. Appropriate blood serum is added to these antigen-antibody complexes so that the complement of the serum binds to the antigen-antibody complexes. Cells with positive antigenic activity are thereby destroyed and therefore only cells with negative antigenic activity can persist in the resulting cell suspension. It can be noted that the mouse la antigen corresponds to the HLA-D (or HLA-DR) histocompatibility antigen in humans.

The cell suspension obtained at this stage consists of cells which exhibit negative surface antigenic activity against the histocompatibility antigens specific for unwanted lymphocytes and which consist essentially of immune memory cells. To this cell suspension is then added an antibody preparation typically containing a rat antibody reacting with specific surface antigens of immune memory cells. Layers of rat antibodies are formed on surface antigen molecules to produce antigen-antibody complexes with rat antibody raised against specific antigen molecules in immune memory cells.

To these immune memory cells taken by means of the antigen-antibody reactions and of complement fixation, an antibody is then added, such as for example the rat antibody, which is particularly reactive with the surface antigens specific for immune memory cells. A layer of rat antibodies is therefore formed on the surface antigen of each immune memory cell.

Separately from the suspension of immune memory cells thus obtained, a mixture of anti-rat IgG antibodies (gamma immunoglobins) is prepared from an appropriate anti-rat IgG serum. The anti-rat IgG antibody preparation is applied to the internal surface of a glass container into which the cell suspension containing the immune memory cells is introduced in order to obtain the reaction with the anti-rat IgG antibody attached to the container. . The anti-rat IgG antibody reacts with the rat antibody enveloping the surface antigen present on each immune memory cell and, therefore, plays the role of the antigen in the antigen-antibody reaction which occurs between the anti-rat IgG antibody and rat antibody. it follows that each of the cells which has bound the rat antibody to its surface antigen will adhere to the internal surface of the container.

Cells that have not adhered to the inner surface of the container are apparently lacking in the ability to bind anti-rat IgG antibody on the surface of the container and are therefore considered to have no antigens. surface to form antigen-antibody complexes. This means that the cells that do not adhere to the surface of the container are essentially other cells than immune memory cells and can be eliminated from the container as unnecessary. invention, only cells fixed on the internal surface of the container and considered to be immune memory cells are taken as useful cells, The suspension of cells containing these useful cells is here called suspension of immune memory cells at the first stage. specific histocompatibility antigens of mouse immune memory cells include Thy 1, Lyt1, Lyt2, and Lyt3 antigens.

Experiments have shown that cells in the early stage immune memory cell suspension represent approximately 0.1% of the cells in the cell suspension from two-step column affinity chromatography. The results of these experiments further indicate that the immune memory of these cells in the first stage immune memory cell suspension is transferred almost perfectly to an unimmunized individual when the cell suspension contains at least 103 immune memory cells.

According to a second remarkable aspect of the present invention, immune memory cells are cultured efficiently from the cells obtained in the form of a suspension of immune memory cells at the first stage. Immune memory cells can therefore be cultured and subcultured successively for several generations to establish a continuous cell line which results in “first generation” immune memory cells from an immune-sensitized individual.

To grow "second generation" immune cells or memory cells from first generation immune memory cells, normal diploid cells are first iso4

CH 678 337 A5

linked from a mouse syngeneic mouse used as an immune-sensitized individual. These normal diploid cells are, after multiplication, exposed to radioactive irradiation such as, for example, gamma rays from cobalt 60 inhibiting the cells' ability to divide and multiply but allow the cells to continue living. Normal diploid cells whose ability to divide and multiply is thus inhibited will be referred to herein as normal diploid cells with inhibited mitosis.

On the other hand, pathogenic cells of the type which have caused the disease contracted by the immune-sensitized individual, are inactivated to prepare a suspension of inactivated pathogenic antigens. These pathogenic cells are used as the antigen of the type memorized by immune memory cells supplied in the form of the suspension of early stage immune memory cells. For this purpose, these pathogenic cells are removed from an individual who is in the process of developing the disease caused by the pathogen in question and suspended in a culture medium; the suspension of resulting cells is subjected to electromagnetic or radioactive irradiation with, for example, ultraviolet rays or gamma rays from cobalt 60.

The normal diploid cells with inhibited mitosis are then added to a suspension of first stage immune memory cells. To this mixture is then added a suspension of inactivated pathogenic antigen. The resulting mixture is incubated to promote the multiplication of immune memory cells in the suspension so as to thereby give a suspension of second stage immune memory cells. It may be noted that this suspension of second stage immune memory cells is the product of the primary culture of first generation immune memory cells separated from the immune sensitized individual. Next generation immune memory cells can be easily subcultured when the second stage immune memory cell suspension is treated in a similar manner to that used for primary cell culture from the first stage immune memory cell suspension . Thus, third generation immune memory cells can be obtained using the resulting mixture incubated under conditions similar to those used for the culture of second generation immune memory cells. The immune memory cells according to the present invention can thus be easily cultured from one generation to another in order to establish a strain of immune memory cells with good efficiency by repeatedly following a procedure similar to that used for the culture of memory cells. second generation immune systems.

According to the results of the experiments carried out by the inventor, the immune memory cells according to the present invention multiply at a rate which, in terms of number of cells, increases approximately by a factor of three during five days of incubation since the start of the culture of the cells obtained by the primary culture.

Such a rate of cell multiplication ensures the creation of an established line of immune memory cells and the preparation of such cells in a stable and economical manner on a quantitative basis.

It may further be noted that the second generation immune memory cells generally appear globular in shape and are approximately three times larger than the first generation immune memory cells when viewed under the microscope. It may be added that the second generation immune memory cells have nuclei which are almost all larger than those of the first generation immune memory cells, and molecules of capsular antigens which are largely identical to those of the immune memory cells of first generation.

The method according to the present invention can be implemented in different ways while remaining within the scope of the present invention. A preferred embodiment among these various possibilities is described in the following example, but this does not limit the scope of the invention.

50 EXAMPLE

f1i Separation of immune memory cells from the body:

A mouse cured of and immunologically sensitized against salmonellosis is used as an individual of the immune sensitized. It will however be obvious that, if desired, a mouse immunized against any infectious bacterial disease or cured of an infectious viral disease or of cancer or any other malignant tumor can be used in place of the immunized sensitized individual that we chose to use here.

60 Preparation of the initial cell suspension

(1-1) Peripheral blood is taken from the heart of the mouse to be used as starting material. The blood collected is diluted in five times the same volume of Hanks solution so as to dissociate the aggregates into a suspension of isolated cells, then centrifuged at 200 rpm. for 15 minutes at 65 at a temperature of 4 ° C. We separate a superficial skin (known as a "buffy coat") formed

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by the supernatant, of your resulting preparation, of the rest of this preparation by delicately aspirating this supernatant.

(1-2) The fraction of superficial skin containing a large proportion of leukocytes is again diluted in five times its volume of Hanks solution. The suspension of resulting cells is centrifuged at 2000 rpm for 15 minutes, at a temperature of 9 ° C., and the supernatant is then removed. The cell suspension thus obtained is again centrifuged in two stages, each under conditions similar to those used for the centrifugation stage immediately preceding it.

The supernatant formed on the surface of the cell suspension, produced during each of the two centrifugation steps, is removed at the end of each centrifugation step. To these cells which have i

been washed during the three centrifugation stages, each followed by the removal of the supernatant, a Hanks solution is added in a proportion giving a cell density of 4 × 10 7 cells / ml and containing 5% by volume of fetal calf serum. The cell suspension obtained at this stage of the process will hereinafter be called "initial cell suspension".

(1-3) In place of peripheral blood, one can use, if desired, the tissue of the spleen of an immune-sensitized individual. When the tissue of the spleen of a mouse is used as starting material, a sample of the tissue of the spleen of the mouse is excised and it is pressed on a steel sieve having holes of the type, for example of the screen Tyler's standard format No. 100. The cells which pass through the holes of such a steel sieve are dispersed in an appropriate volume of Hanks solution and can then undergo the same treatment as that of the cell suspension prepared from peripheral mouse blood. Thus, the suspension of cells containing lymphocytes derived from the spleen can undergo the same treatment as the suspension of cells prepared from the peripheral blood of the mouse.

Separation of T cells

(1-4) The suspension of initial cells prepared from the peripheral blood of the mouse is placed on a glass wool column comprising 10 g of glass wool placed in a vertical glass tube (with an internal diameter of 20 mm and a length of 100 mm), then Hanks solution containing 5% by volume of fetal calf serum is added. The cell suspension in the column is then heated to a temperature of 37 ° C for 45 minutes for incubation of the cells in the suspension.

After the incubation, the cell suspension is allowed to drop dropwise through the column by continuously adding Hanks solution of the same composition and maintained at a temperature of 37 ° C. The suspension of cells eluted from the column is taken in a centrifuge tube,

(1-5) As has been understood in the foregoing, it is observed in fact that the cells contained in the cell suspension resulting from the affinity chromatography on a glass wool column consist essentially of T and B cells The cells of the type which are separated from T and B cells and retained in the column are macrophages and leukocytes.

(1-6) Experiments were carried out with the suspension of cells taken by the affinity chromatographic process on a glass wool column. The results of the experiments show that the T and B cells separated by chromatography represent approximately 30% of the total amount of cells which were contained in the initial suspension of cells injected into the column,

(1-7) Subsequent to the glass wool column affinity chromatography, the suspension of cells taken from the centrifuge tube is subjected to centrifugation at 2000 rpm for 15 minutes at a temperature of 4 ° C., then remove the supernatant from the resulting suspension, To the suspension of cells thus obtained, a Hanks solution is added in proportion giving 4 x 10 7 cells / ml and containing 5% by volume of fetal calf serum. The resulting preparation is injected into a column of nylon wool comprising 10 g of nylon wool (produced by Fenwal Laboratories) placed in a vertical glass tube (internal diameter 20 m, length 100 mm), then solution of Hanks containing 5% by volume of fetal calf serum. The cell suspension in the column is heated to a temperature of 37 ° C for 45 minutes for incubation of the cells contained in the suspension. After the incubation, the cell suspension is allowed to drop dropwise through the column, continuously adding Hanks solution of the same composition and maintained at a temperature of 3 ° C. The cell suspension eluted from the column is taken up in a centrifuge tube.

(1-8) Experiments were carried out with the suspension of cells taken from the outlet of the affinity chromatography column on nylon wool; the results show that the cells separated by affinity chromatography on a nylon wool column represent approximately 10% of the total amount of cells which were contained in the initial suspension of cells injected into the glass wool column. ^

(1-9) The cells contained in the cell suspension resulting from the * chromatographic process

two phases essentially contain T cells which, according to the results of the tests carried out, represent approximately 95% of the population of total cells contained in the suspension. Most cells, other than T cells which were contained in the initial cell suspension before two-phase chromatography are considered to be partially adsorbed by glass wool fibers and partially by nylon wool fibers. However, as noted above6

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Also, the cell suspension obtained by the two-phase chromatographic process still contains unwanted lymphocytes and other cells.

Purification of T cells

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(1-10) In order to eliminate these unwanted cells, the suspension of cells taken from the centrifuge tube after affinity chromatography on a column of nylon wool is subjected to centrifugation at 2000 rpm for 15 min. temperature of 4 ° C; then the supernatant is removed. Culture medium is added to the resulting cell suspension in a proportion which results in a cell density of 8 x 10 7 cells / ml. The culture medium is a mixture of "Medium 199" containing 2% by volume of fetal calf serum. The culture medium having such a composition will then be called “medium A”,

(1-11) Then added to one part by volume of the cell suspension thus obtained, two parts by volume of a preparation of monoclonal anti-antigen antibody la (manufactured by Cedarlane Laborato-15 ries Ltd) diluted in 30 times the same volume of "medium A" and part by volume of rabbit serum diluted in twice the same volume of "medium A". The mixture of these three preparations is heated to obtain their interaction at a temperature of 37 ° C for 30 minutes.

(1-12) It results from the reaction which occurs in this mixture the formation of antigen-antibody complexes, by binding of anti-la monoclonal antibodies to cells having an antigen-only positive activity. Rabbit serum is added to the complexes thus formed so that the rabbit serum supplement binds to the antigen-antibody complexes. Cells with positive antigenic activity are destroyed by rabbit serum and, therefore, only cells with negative antigenic activity can survive. This cell suspension, containing a concentrated population of immune memory cells, is centrifuged at 2000 rpm for 15 min at a temperature of 4 ° C. so as to take up the immune memory cells.

(1-13) The resulting suspension of cells is then centrifuged in three successive stages, each at 2000 rpm for 15 min at a temperature of 4 ° C in "medium A". The cell suspension obtained at the end of these three centrifugation steps is diluted in “medium A” in a proportion giving a cell density of 8 × 10 7 cells / ml.

(1-14) In addition to this suspension of cells, a suspension of rat anti-immune memory cell antibodies is prepared according to a protocol which will be described in chapter 3 and is diluted in 5 times the same volume of "medium A". An equal volume of the rat antibody suspension and the cell suspension, each prepared as described above, is mixed and the reaction is allowed to proceed at a temperature of 9 ° C for 60 minutes. As a result of this reaction, the rat antibody binds to molecules of specific antigens in immune memory cells. Antigen-antibody complexes form between the layers of rat antibodies formed on the specific antigen molecules of immune memory cells. The suspension of cells containing the immune memory cells carrying the antigen-antibody complexes thus formed is subjected to centrifugation at 2000 rpm for 15 min at a temperature of 4 ° C. in “A” medium. Centrifugation is continued on the resulting suspension of cel-40 Iules, in three successive stages, each under conditions similar to those used for the preceding stage of centrifugation. To these cells obtained after the centrifugation steps, the "medium A" is added in proportions such that the cell density is 8 × 10 6 cells / ml.

(1-15) In addition to this suspension of immune memory cells, a preparation of anti-rat goat 45 IgG antibodies is carried out. For this purpose, a solution containing 10 ml of anti-rat IgG goat serum is diluted with thirty-five times the same volume of "medium A" in a sterile plastic Petri dish (manufactured by Becton Dickinson Co., Ltd.) measuring 100 mm inside diameter and allowed to stand overnight at 4 ° C. The excess antiserum is then removed from the dish by washing and the goat IgG antibody, anti-rat, is left in the form of a thin liquid layer covering the internal surface of the dish.

(1—16) The cell suspension prepared as described above is poured into the dish having the liquid layer of IgG, goat, anti-rat antibodies. The box is then held horizontally and should not be moved for 60 minutes. Thereupon, the cells which have not adhered to the layer of antibodies covering the box are removed and eliminated from the box by applying slight vibrations to it. 55 As explained, cells that do not bind to the anti-rat goat IgG antibody layer do not have antigen-antibody complexes that would otherwise have formed between the rat antibody and the antigens specific to immune memory cells. Most of the cells floating above the goat anti-rat IgG antibody layer on the surface of the dish are considered not to be immune memory cells.

60 (1—17) The interior surface of the dish is then rinsed in three stages, each using a mixture of RPM11640 medium containing 10% by volume of fetal calf serum, and then this same medium is sprayed onto the interior surface of the box. The cells which have adhered to the box are detached from the box and are taken as a suspension of immune memory cells at the first stage according to the method of the present invention.

65 (1-18) The cells removed at this stage of the process represent approximately 0.1% of the cells which

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were contained in the cell suspension from two-step column affinity chromatography. Experiments with this suspension of first-stage immune memory cells show that the cells representing 98% of the cells contained in the suspension of first-stage immune memory cells:

(1) belong to the family of small lymphocytes;

(2) exhibit positive antigenic activity with respect to the specific antigen molecules of immune memory cells, and

(3) exhibit positive antigenic activity with respect to the Thy1, Lytt, Lyt2 and Lyt3 antigens and negative antigenic activity with respect to the la antigen.

(1-19) In addition, experiments have been carried out in which the immune memory cells in the form of the suspension of immune memory cells in the first stage are introduced into an unimmunized syngeneic individual of the mouse used as an immune sensitized individual. The results of the experiments show that the immune memory possessed by the immune memory cells is transferred perfectly to the receptor, that is to say with a probability of 100% when the suspension of immune memory cells contains at least 103 immune memory cells. Other experiments have been carried out with the suspension of immune memory cells at the first stage, experiments in which individuals are irradiated, in which immune memory cells have been introduced, with gamma rays of cobalt 60. The results of these experiments show that the immune memory, which each of these individuals had acquired, remains unaltered when the individual is exposed to the gamma rays of cobalt 60 at a dose of at least 600 roentgens, but is destroyed when it is subjected to an irradiation of more than 1100 roentgens. This demonstrates that the immune memory cells contained in the suspension of early stage immune memory cells belong to a family of cells which are significantly resistant to radioactive radiation.

(2) Culture of cells for cell line establishment

The cells are then grown from the immune memory cells obtained in the form of the suspension of immune memory cells in the first stage.

Culture of normal Svnoenian cells

(2-1) Normal diploid cells are separated in the form of a cell suspension from a syngeneic mouse from your mouse used as an immune-sensitized individual and cultured in a sterile petri dish. Approximately, when you realize that a single layer of cells has formed in the box, these cells are exposed to gamma rays of cobalt 60 at a dose of 2000 roentgens to inhibit the ability of cells to divide and form. multiply.

Preparation of inactivated pathogenic antigen

(2-2) Pathogenic cells of the type that caused the disease in the immune-sensitized individual are inactivated to produce a suspension of inactivated pathogenic antigen. For this purpose, bacterial Salmonella cells are added to a culture medium, in a proportion which gives a cell density of 107 cells / ml. The culture medium consists of a mixture of RPMI 1640 medium containing 10% by volume of fetal calf serum, 2 g / l of sodium hydrogencarbonate (NaHCOa), 0.11 g / l of sodium pyruvate (NaOOCCOCCHs), and 0.1% by volume of yeast oleate. A culture medium exhibiting this composition will be referred to below as “medium B”

(2-3) The suspension containing the Salmonella bacterial cells dispersed in “medium B” is irradiated with ultraviolet rays at an intensity of 3.6 × 10 3 erg / cm 2 for 5 min in order to inactivate the bacteria. The use of “medium B” in the inactivation process is simply an example and, in the same way, any other medium having a desired composition can be used in substitution for medium B.

(2-4) When an immune-sensitized individual after contracting a viral infectious disease is used for the preparation of immune memory cells, virus particles can be dispersed in “medium B” in a proportion which gives a density of 109 particles / ml.

(2.5) If one wishes to use tumor cells such as cancer cells, a tissue fragment comprising the tumor is excised from the body of an individual and cut into small pieces. The fragments of the tumor tissue are immersed in a physiological saline solution, buffered with phosphate, containing 0.02% by volume of ethylenediaminetetraacetic acid (EDTA) and the mixture is stirred for 60 minutes at a temperature of 4 ° C. The resulting tissue fragments can be pressed against a steel screen with holes according to, for example, the standard screen of Tyler No. 100. The cell suspension is forced to pass through the screen and then centrifuged at a speed from 700 to 800 rpm for 5 min at a temperature of 4 ° C. The tumor cells harvested in this way are dispersed in medium B in a proportion such that the cell density is 2 × 10 3 cells / ml; the resulting cell suspension is exposed to the gamma rays of cobalt 60 at a dose of 2000 roentgens so as to produce a suspension of inactivated pathogenic antigen equivalent to that used in this example. It seems obvious that when an inactivated pathogenic antigen suspension has to be prepared for

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from tumor cell tissue, any desired culture medium other than medium B can be used for the manipulation of cells extracted from the tissue.

Culture of immune memory cells.

(2.6) To the suspension of immune memory cells at the first stage which was prepared as described above, medium B is added in proportions giving a cell density of 8 × 10 5 cells / ml. The resulting suspension is poured slowly into the dish containing the inhibited mitosis cells isolated from a syngeneic mouse. The volume of the cell suspension thus added to the cells with inhibited mitosis can be chosen according to the size of the box used and is chosen from 5 ml for a box with an internal diameter of 100 mm. If a petri dish with another diameter, for example 60 mm, is to be used, it is a good idea to use 2.5 ml of the first stage immune memory cell suspension in the dish.

(2.7) Add an equal volume of the suspension of inactivated pathogenic antigen, prepared as indicated above, to the mixture of the suspension of immune memory cells and cells with inhibited mitosis fixed on the dish. The resulting assembly is incubated at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide to promote the multiplication of immune memory cells. The preparation resulting from this manipulation is the suspension of immune memory cells prepared according to the method of the present invention.

(2.8) When the suspension of immune memory cells in the second stage is carried out in a similar manner to that of the primary culture from the suspension of immune memory cells in the first stage, it is easy to subculture immune memory cells of a later generation. For this purpose, the suspension of immune memory cells in the second stage can be diluted in “medium B” in a proportion giving a cell density of 8 × 10 5 cells / ml. The resulting cell suspension is mixed with cells with inhibited mitosis of the syngeneic individual with the suspension of inactivated pathogenic antigen prepared as described above. Immune memory cells of the next generation, that is to say the third, can thus be obtained when the resulting mixture is incubated and centrifuged under the conditions specified above. As noted, the experiments carried out by the inventor have revealed that, during the culture of the immune memory cells derived from the cells obtained by the primary culture, the immune memory cells multiply at a rate which increases by a factor of about 3 for 5 days of incubation.

(3Ì Preventive effect of immune memory cells.

Experiments are carried out with suspensions of immune memory cells each prepared according to a method of the present invention to test the preventive effects of the cells on the disease caused by various pathogens. The cell suspensions used for this experiment include those constituted by the suspension of first-stage immune memory cells containing immune memory cells isolated from an immune-sensitized individual and those constituted by the suspension of second-stage immune memory cells containing cells which are derived from early stage immune memory cells. The cell suspensions used for the tests contain different amounts of immune memory cells in a range between 102 and 104. These cell suspensions are inoculated into unimmunized mice as receptor individuals, and then these receptors are contaminated with different pathogens to assess the percentage of preventive effects on the disease. Pathogens used in these experiments include cancer cells, Salmonella, Corynebacterum, Pseudmonas, Pasteurella, Streptococcus, and the Electromelia and Sendai viruses. These bacterial, viral or cellular cancer pathogens are introduced into individuals in amounts chosen to cause a disease incidence rate of 100% for each of the pathogens. The results of these experiments are presented in the following table:

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Board

Pathogens

No. of cells

Preventive effect

Caricidal cells

1 x 104

100%

1 x103

100%

6x102

70%

3x102

42%

1 X102

34%

Salmonella

1 x 104

100%

1 x103

100%

6X102

68%

3x102

34%

1 x 102

20%

Corynebacterium

1 x 104

100%

1 x103

100%

6 x 102

48%

3X102

26%

1 x 102

14%

Pseudmonas

1 x 104

100%

1 x103

100%

6x 102

41%

3 x 102

23%

1 x 102

11%

Pasteurella

1 x 104

100%

1 x103

100%

6x102

78%

3 x 102

53%

1 x 102

35%

Streptococcus

1 x 104

100%

1 x103

100%

6x102

44%

3x102

31%

1 xlO2

17%

ectromelia virus

1 x 104

100%

1 x103

100%

6x102

51%

3 X102

27%

1 x 102

13%

Sendai virus

1 x 104

100%

1 x103

100%

6x102

88%

3x102

63%

1 x 102

29%

As seen in the analysis of these results, immune memory is successfully transferred to the unimmunized individual, with a 100% probability when the cell suspension contains 103 or more immune memory cells. The results of these experiments further show that considerably high degrees of the preventive effect of the disease can be obtained when a cell suspension containing less than 103 immune memory cells is introduced into the recipient individual. By

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CH678 337 A5

example, when an immune memory cell suspension containing less than 6 x 102 immune memory cells is introduced into an unimmunized individual, the immune memory is transferred to the recipient individual with a probability of approximately 50% and, if a suspension of immune memory cells containing less than 3 x 102 immune memory cells is used, immune memory is transferred to the receptor with a probability of about 30%.

(41 Preparation of anti-memory cell rat antibodies.

A description will be made below of the process for preparing the suspension of rat anti-immune memory cell antibodies used in the process steps described in paragraph 1.14.

The suspension of immune memory cells, prepared as described in paragraph 1.13, is diluted in physiological saline, phosphate buffered, and the resulting preparation is centrifuged under suitable conditions. The cell suspension obtained after the three stages of centrifugation is diluted, after washing, again with an identical saline solution, in a proportion giving a cell density of 108 cells / ml. A 0.25 ml suspension of the resulting immune memory cells is injected into the subcutaneous tissue of the femoral region of a series of female rats (of the Wistor species). After ten and twenty days respectively, similar fractions of cells are reinjected into the femoral subcutaneous tissue of the same rats.

The cell suspension, diluted in physiological saline, phosphate buffered, and centrifuged in three successive stages as described above, is again diluted in the same physiological saline in a proportion this time chosen to obtain a cell density of 109 cells / ml. A fraction of 0.25 ml is injected into the abdominal cavity of each female rat 40 days after the first injection of cells. The total volume of the blood of each of the rats is then mixed 50 to 60 days after the first injection of cells into the rats. Five to six ml of blood serum is obtained from each rat.

On the other hand, a sample of spleen tissue is excised from a newborn mouse syngeneic mouse from which immune memory cells were taken. The removal of spleen tissue is forced through a steel sieve and the spleen cells passed through the sieve are dispersed in physiological phosphate buffered saline. The suspension of cells thus prepared is centrifuged in three successive stages and then approximately 1.5 parts by volume of the rat serum prepared as described above is added to the resulting population of cells, and the mixture is stirred thoroughly. The preparation thus obtained is left to stand in ice water for 60 min, with stirring every 15 minutes, then the preparation is centrifuged under appropriate conditions.

The supernatant is removed from the resulting preparation and diluted in physiological phosphate buffered saline, and then shaken as the suspension of cells derived from spleen tissue. The supernatant is then removed from the preparation finally obtained in this way and is used as the preparation of rat antibodies against immune memory cells in the steps described above in paragraph 1-4.

Claims (1)

Claims 1. Method for preparing a suspension of immune memory cells, characterized in that it comprises the following steps: - separation of cells consisting essentially of T cells from cells taken from an individual sensitized by an immunization against a particular pathogen, addition to the separated cells of an antibody reacting to a histocompatibility antigen specific for a defined species of cells to form the antigen-antibody complexes with said antibodies and said separate cells having said histocompatibility antigens, - addition of a blood serum to the cells containing said antigen-antibody complexes to cause the binding of the complement present in the blood serum to said antigen-antibody complexes and thereby the destruction of the cells presenting said histocompatibility antigens and the survival of the cells remaining, - addition to persistent cells of an antibody reacting to a specific histocompatibility antigen of immune memory cells, and - separation from the resulting cells, of the cells which present the specific histocompatibility antigens of the immune memory cells. 2. The method of claim 1, wherein the cells which exhibit said specific histocompatibility antigen of immune memory cells are separated as a suspension of first stage immune memory cells from cells which have resulted from the adding said antibody to said persistent cells, characterized in that it also comprises the following steps: - preparation of normal syngeneic cells from the cells taken from said immune-sensitized individual, - exposure of said normal cells to radioactive irradiation to inhibit their ability to divide, 11 5 10 15 20 25 30 35 40 45 50 55 60 RR CH 678 337 A5 - preparation of pathogenic cells from a pathogenic agent of type identical to said particular pathogen, - inactivation of the separated cells from the pathogen identical to said particular pathogen, Preparation of a mixture of said suspension of immune memory cells at the first stage of normal cells whose division capacity is inhibited and of inactivated cells, and - Incubating said mixture to allow the cells of said suspension of immune memory cells at the first stage to multiply and to form a suspension of immune memory cells at the second stage. 3. Method according to one of claims 1 and 2 characterized in that the cells having your specific histocompatibility antigens of immune memory cells are separated by Spreading an anti-IgG antibody preparation on a surface to form a layer of the anti-IgG antibody preparation on said surface, The application to said layer of antibodies of cells which have resulted from the addition of said antibody molecules to said persistent cells so that the cells exhibiting said specific histocompatibility antigens of immune memory cells adhere to said surface, and - the removal of said surface from the cells which have adhered thereto. 4. Method according to one of claims 1, 2 or 3, characterized in that said cells, consisting essentially of T cells, are separated from the cells removed from said individual sensitized by the following steps: Treatment of the cells essentially consisting of T cells by a first step of column affinity chromatography using a solid glass wool adsorbent and - then treatment of the resulting cells by a second stage of column affinity chromatography using a solid nylon wool adsorbent. 5. Suspension of immune memory cells characterized in that it comprises cells which are removed dear from an individual sensitized by immunization by immunization against a particular pathogen and which consist essentially of T cells, in which the cells representing approximately 98% by number of said T cells contain at least 1 x 103 immune memory cells. 12