DE2352662A1 - METHOD FOR PRODUCING A TRANSFER FACTOR - Google Patents

Description

PATENTANWÄLTE HENKEL —- KERN —FEILER —HÄNZEL — MÜLLER

DR. PHIL. DIPL.-ING. DR. RER. NAT. DIPL.-ING. DIPL.-ING.

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19 OCT. 1973

PIKTOR LIMITED, London, England

Method for producing a transfer factor

The invention relates to a method for producing Imunization material and in particular of transmission factors and interference substances.

Part of the immunization reaction in humans and animals is ensured by antibodies or polypeptides, which form part of the immunoglobulins produced by the lymph cells and circulating in the blood, which chemically combine with antigens in order to neutralize them. In many cases, however, the antibodies do not ensure the required level of immunization. Increasingly, the important role that lymphocytes play in ensuring cell immunity is therefore being investigated. The lymphocytes carry the "immunization memory", and they offer a natural defense against virus

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Infections as well as against the growth of neoplasms. While some disease states are characterized by a lack of cell immunity, treatment is through Lymphocyte transfusion is unsatisfactory for two reasons: first, a donor is required, and second, the transferred lymphocytes can provoke a transplant response in the recipient.

The Presence of "Transfer Factors" in Lymphocytes and Their Role as Carriers of "Cell Immunity Memory" was first used by H.S. Lawrence occupied. Lawrence H.S., "The transfer in humans of delayed skin sensitivity to streptococcal M substance and to tuberculin with distrupted leucocytes "J. Clin. Invest (1955) 34, 219-30; Lawrence H.S., Al Askari S., David J., "Transfer of immunological information in humans with dialysates of leucocyte extracts ", Trans. As. Amer. Physicians (1963) 76, 84-91; Lawrence H.S., "Transfer Factor and Cellular Immune Deficiency Diseases," New. Engl. J. Med. (1970) 283, 411-9; and Lawrence H.S., "Transfer Factor" Advances in Immunology (1969), 195-266. This substance, found in humans and other primates, can only have cell immunity responses, but does not transfer the ability to secrete antibodies. Contains after contact with an antigen lymphocyte memory transfer factors that currently considered to be specific to the class of antigens which is the antigen with which the lymphocyte was contacted, listened to. The transfer factors therefore seem to have a double specificity: On the one hand they are specific for a class of antigens and on the other hand they are specific for cell immunity. the However, lymphocytes of a normal adult contain transmission factors which are common to every virus, bacterial

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or the like antigen with which the relevant Person was contacted, so that the dialysate of Leucocyteextrakteii a multiply specific transfer factor mixture contain.

When the transfer factors are dialysed, they are divided into a dialyzable transfer factor and a non-dialyzable transfer factor. The former appears to be a small molecule, the approximate molecular weight of which is estimated to be around 7,000? In fact, however, it appears to be a mixture of molecules with different molecular weights, the majority of the molecules being close to a molecular weight of 7000, while the number of molecules with molecular weights significantly below 4,000 or significantly above 10,000 is negligible. The dialyzable transfer factor appears to be in the form of a ribonucleotide, but is not completely destroyed by ribonuclease, so that it appears to be a double-chain ribonucleic acid (RNA). This is stable for indefinite periods of time when lyophilized and stored at temperatures from -20 ⁰ C. The non-dialyzable transfer factor, on the other hand, is a larger molecule and has been characterized as RNA with a molecular weight of more than 50,000 (6 to 12 S). It is not species-specific, but specific for an antigen class and also specific for the transmission of cell immunity.

Since the dialyzable transfer factor is not antigenic Being natural, i.e. unable to induce an immune response, "its therapeutic administration is the lymphocyte transfusion preferred. A trial treatment

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In fact, transfer factor treatment has already been shown to be successful in overcoming cell immunity deficiencies and has been shown to provide clinical improvement in various diseases. For example is it already successful for the treatment of subacute sclerosis-panencephalitis, system,! scher candidiasis,

malignant melanoma, breast cancer, measles, and chickenpox been applied. In the previously known methods, which rely on donor blood as the starting material, is however, no satisfactory production of the transfer factors as therapeutic agents is possible. Much more , represents the manufacture of a pure substance from whole blood a laborious process that requires the prior separation of lymphoid cells from other blood fractions requires. None of these previously known processes is suitable for the large-scale production of transmission factors under standardized, reworkable conditions. There If donor blood is also used in these procedures, these procedures can only provide transfer factors which are specific to the type of antigens with which the donor has been contacted. But you can't be modified in such a way that they deliver a certain antigen-specific transfer factor at will, except through the appropriate choice of an appropriate blood donor.

It has now been found that transfer factors in lymphoid cells, specifically lymphocytes, grown in vitro were, are present and can be obtained from them, and especially that the formation of a certain antigen-specific transfer factor can be introduced into such a culture. On this basis is now with the invention the generation of

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Transfer factors are possible in large quantities according to a standardized, repeatable procedure, and the specific antigen-specific transfer factors can be produced at will. The invention also enables the extraction of interference substances from a lymphocyte culture.

Generally speaking, the invention consists in a method of generating a transfer factor (which is an analyzable and / or a nondialysable transfer factor), in which one or more cell strains, preferably of lymphoid origin, can be cultured in vitro, e.g. lymphocytes, which are the transmission factor and the transfer factor is extracted from it. The extract from the cells can according to known Procedure to be purified in order to obtain a pure preparation of the transfer factor.

Surprisingly, it turned out that all cells grown in an artificial culture can be stimulated to deliver the transfer factors, although of course certain cells are better providers of transmission factors than others. Generally lymphoid cells preferred. This term generally refers to lymphocytes (those obtained from the peripheral blood can be) as well as on lymphoblastoid rows of cells. In general, the lymphocytes themselves are not a continuous replicate when grown in vitro Cell cultures for long periods of time, as the lymphocytes usually within a few days die and, at best, cannot be expected to survive for more than two weeks. Since every-

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yet the lymphocytes are the best known suppliers for are the transfer factor, it is desirable to use them in the method according to the invention. This can be done by known modification of the lymphocytes for profit The so-called "lymphoblastoid cell series" happens. This modification can be achieved through repeated contacting of the cultured cells with phytohemagglutinin (P.H.A.) using methods such as those described, for example by G.E. Moore and K. Ultrich in J. Surg. Res. 5, 270-282 (1965) and G.E. Moore, R.E. Gerner and H. Addison-Franklin in J. Amer. Med. Assoc, 199, 87-92 (1967). Using these procedures will be the peripheral blood lymphocytes as permanent rows of cells (lymphoblastoid cell rows), which are expediently reduced to a concentration of 2 10 to 1 χ 10 in the culture Cells per ml are kept.

Various preferred methods according to the invention for generating the transfer factor are explained below. Generally speaking, however, the lymphoid cells and specifically the lymphocytes are either cultured alone, so that the transfer factors obtained are specific for the class of antigens with which the The donor has been contacted or the cells will be specific for a desired class of antigens Transfer factor incubated, in which case the recovery of transfer factor specific for this class of antigen is initiated.

In a cultivation process used in the method according to the invention, the cells are consistently with it incubated at a constant concentration of the transfer factor. For example, a 10, 10 ^, or 10 Cells equivalent amount of the transfer factor of a

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200 ml of culture with 4 × 10 cells / ml was added. If desired, a lower initial concentration of cells can be used, although the initial concentration should preferably not be lower than Kr cells / ml. The cells are then allowed to grow until they reach a concentration of 10 cells / ml, whereupon fresh culture medium containing the selected concentration of transfer factor is added and the cell concentration is diluted back to its initial value, i.e. 4 10 " ^5. This cultivation method continues until enough cells have been obtained to extract the transfer factor.

An optional cultivation method will be the lymphoid cells initially for a few hours incubated with a high concentration of transfer factor. The cells are then washed and combined in one cultivated from the transfer factor free medium. After about six days there are enough cells for the extraction of the transfer factor available. Another method is to cultivate the cells for a period of time of five days in a transfer factor-free medium after an initial induction or Operating time of 24 hours in one with the transfer factor

4th
medium added to an amount equivalent to 10 cells.

Another, optionally possible, cultivation method consists in incubating the lymphocytes or the like Lymphoid cells (e.g. lymphoblastoid cell rows) with. Transfer factor in an amount corresponding to 'KK or

10 cells. The cells will then last for about two days

let grow. Then it becomes a medium that does not have any transmission factor contains, to dilute the cells,

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ORIGINAL INSPECTED

whereupon the cells finally do about three days later be won.

According to a preferred embodiment of the invention, it is possible to produce a specific transfer factor to induce in the lymphoid cell culture. As mentioned, this introduction is achieved by im Cultivation medium during at least part of the cultivation period the relevant specific transmission factor is available. It has been found that lymphocytes originate from primates other than humans Delivery of the appropriate transfer factor for the Induction can be used just as effectively as the human transfer factor (cf. Paque R.E. and Dray S. "Monkey to human transfer of delayed hypersensitivity in vitro with RNA extracts" Fed. Proc. (1972) 792, Paragraph No. 3287). Thus, if the transfer factor is equivalent to 10 baboon lymphocytes for a period of 6 hours is incubated with 10 cultured lymphocytes (a lymphoblastoid cell line), it shows the ability to the latter lymphocytes show a cell immunity. speak to purified tuberculin protein derivatives as well to other antigens, e.g. lepromin antigen. The procedures outlined above can be used for the Generation of transfer factor can be used, which for numerous virus, bacterial or tumor antigens, specific is. The transmission factor specific for a rare antigen can be generated in the monkey, and will then used to induce transfer factor formation in a lymphoid cell culture. It can be monospecific or polyspecific transfer factors can be generated. In general, it is also possible to culture lymphoid cells to apply that have been obtained from animals other than humans, and these cultures, with the human

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to vaccinate the transfer factor. For example, cultured simian lymphoid cells may have a human transmission factor vaccinated to produce more transfer factor. Cultured cells from others can also be used Animals als.Primaten to obtain the transfer factor for humans and. Animals are used, including Animals other than the animals from which the cells were taken. For example, can carried out the known mouse neuroblastoma cell line (N / 41) Vaccination with the appropriate transmission factor for the Obtaining transfer factor for various others Animals are used.

Baboons were injected with a specific antigen to induce a corresponding cell immunity response. This response can be assessed by making comparative measurements of lymphocyte migration from immunized and non-immunized baboons in the presence of this antigen. A positive response (i.e., an emigration index below 0.60) is an indirect indication of the presence of for this Antigen specific transfer factor. (An explanation of the leucocyte used for this purpose Migration experiment can be found e.g. in Moulias R. Goust J.M., Deville-Chabrolle A., Buffet C, Muller-Berat CJ. "Le test de migration des leucocytes (TML), un nouveau test d'hypersensibilite retardee in vitro chez l'homme "press Med. (1970) 78, 2315-8). An antigen-specific transmission factor obtained from the lymphocytes of the immunized baboons is added to a human lymphocyte culture similar to those already described in vitro Conditions is grown in order to form the antigen-specific transfer factor.

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The same procedure can be used with other laboratory animals such as rats or guinea pigs.

The transfer factors can be set according to the usual procedures extracted from the cultured lymphocytes or other cells. The cells are repeated by Freezing and thawing are broken, and the product is subjected to centrifugation, and ultrafiltration if necessary subjected to gel chromatography. One

in the range of 256 mils absorbent fraction is collected and lyophilized. Instead of breaking open the cells by freezing and thawing them, you can use treated with a chemical agent that disrupts the cells, such as Triton-X 100, which is normally used used in an amount of 0.1 to 1% in a buffer will. The transfer factor is then separated off in the manner described above.

The invention is explained in more detail below with the aid of examples.

Beigdel 1

Starting from cultures of the strains F48, V2F, BF and BM / 5, all of which are lyraphoblastoid cell lines obtained in the usual way from lymphocytes, 5 × 10, 6.9 × 10, 7 × 10 ′ and 6, respectively 10 lymphocytes removed, lyophilized and ^{stored at -20 0} C in preparation for the extraction of the transfer factor. The lyophilized cells were resuspended in water for injection and disrupted by freezing them five times and then thawing them at 37.degree. Each lymphocyte culture was separately centrifuged for 20 to 30 minutes at 150 to 250 times gravity. The protruding

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Liquid was for 1 hr at 20,000 times gravity (G) and a temperature yon 4 ⁰ C in a Sorvall RC2B centrifuge centrifuged. The product obtained from each lymphocyte culture was placed in a high-throughput pressure ultrafiltration device (Amicon DC2), using successively "Diafiber" filter cartridges XM 50 (which retain substances with a molecular weight of over 50,000) and then those of the PM type (which Withholding substances with a molecular weight of over 10,000) were used. The dialysate, consisting of the supernatant fluid separated from the broken lymphocytes and diluted tenfold with 0.05 M physiological saline solution, was concentrated on an Amicon UM-2 membrane, which retained substances with a molecular weight of over 1000. This concentrate finally obtained, which in the four individual cases had a volume of 10 to 20 ml, was then lyophilized.

Example 2

Human peripheral blood lymphocytes were placed in a conventional nutrient solution at a concentration of about 10 cells / ml. About 10 ml of this medium was used. Then phytohemagglutinin (PHA) was added in an amount of. about 0.1 µg / ml (equivalent to 0.1 Wg / 10 cells) added. Every two days was the old nutrient medium removed and replaced with fresh medium containing P, HA (0.1 u g / ml). This process was continued until the lymphocytes had been modified to such an extent that they formed a lymphoblastoid cell line which could continuously be reproduced without external stimulation. This usually took three to six weeks.

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chen, the exact time required being determined empirically became.

The lymphoblastoid cell line thus formed was then diluted with fresh nutrient medium, so that 200 ml of the culture with 4 × 10 ** cells / ml were formed. One 10 * cells an equivalent amount of an antigen-specific transfer factor was then added to this culture, and the cells were grown until they reached a concentration of 10 / ml. Then was fresh Nutrient medium, which has the transfer factor in an amount

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corresponding to 10 cells was added in order to dilute ^{the cell concentration to 4 × 10 5 again.}

Thereafter, the cells were collected, lyophilized, and stored at -20 ⁰ C in preparation for the extraction of the transmission factor. The lyophilized cells were resuspended in water for injection and disrupted by treatment with Triton-X 100 at a concentration of 0.5 $ in a buffer for 5 minutes. The supernatant liquid was then ^{centrifuged for 1} hour at 20,000 G and 4 ° C. in a centrifuge of the Sorvall RC2B type. The supernatant from this centrifugation was placed in a high throughput pressure ultrafiltration device (Amicon DC2) containing successive "Diafiber" filter cartridges XM50 (which retain substances with molecular weights above 50,000) and PM 10 (which contain substances with molecular weights of hold back over 10,000). The dialysate, consisting of the supernatant fluid from the broken lymphoblastic cells and diluted tenfold with 0.05 M physiological saline solution, was concentrated on an Amicon UM 2 membrane , which sub-

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punches with a molecular weight of over 1000 holds back. This final concentrate having a volume of 10 to 20 ml was lyophilized.

Various series of tests can be carried out in order to prove that the cultivated in this way from the Lymphocyte extract contains transmission factor which has essentially the same properties like the transmission factor isolated from human blood. This can primarily be done in vitro will be demonstrated, with experiments measuring the MIF secretion are used, especially the previously mentioned leukocyte migration test or the lymphocyte conversion test, to prove that the transfer factor obtained by the method according to the invention in lymphocytes can induce an immunity reaction against the corresponding antigens. These results are through Studies carried out in vivo confirmed in which the obtained by the method according to the invention Transfer factor is injected into baboons, whose immunity response is then examined. Human Patients are against subacute sclerotic panencephalitis, systemic candidiasis, malignant melanoma, Breast cancer, measles, and chickenpox have been treated.

The results of these biological tests are confirmed by physical-chemical evaluation. To this The purpose is the transfer factor to Biogel PtO (which Substances with a molecular weight of 10,000 to 2000) is chromatographed, which was previously done with tris (hydroxymethyl) aminomethane Püffer has been adjusted to equilibrium. In the case of the " Chromatography shows the method according to the invention obtained transfer factor, as well as the one from

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obtained from human blood, an absorption band at 256 m U * This band is characteristic of the presence of nucleic acids. Both types of transfer factor show negative reactions to biphenylamine, which indicates the lack of DNS. A positive reaction, however, is obtained in Orein, eliminating the presence indicated by ribose and thus the RNA natural of the Product is proven.

In addition, the ultraviolet spectrum of the transmission factor isolated from lymphoblastoid cell lines is identical with that obtained from natural uncultured lymphocytes Transfer factor; both show a UV absorption minimum at 242 nm and a maximum at 258 nm. As a result it is proven that the product obtained by the method according to the invention is actually a transfer factor.

As mentioned, the generation or recovery of the transfer factor can be promoted by adding it to a culture of lymphoid cells. However, other methods for favoring the formation of the transfer factor are also possible. For example, those cells that do not form the transfer factor can be eliminated, and it has been shown that the brief incubation of cells with mitostatic agents, _{e.g. e} Velbe, or by brief cold incubation during the induction period, the amount of the transfer factor formed by selective killing the non, t-productive cells is increased, since apparently not all cells in the cultivated colonization are good suppliers of the transmission factors. Another alternative is to separate those cells that have proven to be good transmission factor suppliers.

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ben. Optionally, by synchronizing a cell culture, the formation of the transmission factors is improved by enabling induction during a specific, more precisely determined period of time in the cell cycle. In addition to humans and other primates, the animal species which can be treated with the transmission factor produced according to the invention and whose cells can be used in the culture IiUV, extraction of the transmission factors, include the following animal families: cattle, sheep, horses, cats, dogs and chickens.

It has also been shown that by adding a human Donor transfer factor to a lymphocyte culture the production of interference substance in the culture - is stimulated. Much higher concentrations of interference substance are formed than they normally are found in continuous series lymphocyte cultures. With a corresponding modification of the one described Extraction and purification processes can do that Method according to the invention for the production of interference substance in large quantities by induction with transfer factors can be used in lymphocyte cultures.

In general, the use of the lymphoblastoid Cell series preferred as they are very good suppliers of the Represent transfer factors and see continuously can reproduce. However, any cells can also be used may be used, although they should be able to continuously replicate themselves in vitro, and if the Cells do not naturally contain transfer factors, the formation of transfer factors can be caused by addition are induced or excited by transmission factors.

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Claims (6)

Patent claims ohe HU process for the production of transmission factor by extraction from cells containing the transfer factor, characterized in that that the cells are cultured in vitro prior to extraction. 2. The method according to claim 1, characterized in that to initiate the formation of a specific transmission factor by the cells of the transmission factor is added to the cells before or during their cultivation. 3. The method according to claim 1 or 2, characterized in that that lymphoid cells are used. 4. The method according to claim 3> characterized in that Lymphocyte cells can be used. 5 · The method according to claim 3 * characterized in that used a lymphoblastoid cell array as the cells will. 6. The method according to claim 1 or 2, characterized in that the cells from the mouse neuroblastoma cell line be won. 40981 8/1170