NO821233L - HIGHLY DETAILED INFORMATIONAL RIBONUCLEIC ACID (I-RNA), PROCEDURE FOR PREPARING IT AND USING THEREOF - Google Patents

Description

The object of the invention is i-RNS ("informative"', previously also called "immune" ribonucleic acid) which is specific against antigens...,, and which is extracted in the cell-free system and is so highly purified that it is tolerable in humans and animals by parenteral, preferably intravenous administration. Furthermore, the invention relates to methods for. production of that kind of high-purity

.i-RNS, as well as its application.

i-RNS is normally formed by various immune-competent cells in the body after contact with an antigen. It contains and transmits information for the synthesis' of antibody is,. regulator-prbtein and cell-bound immunity. i-RNS is_so always to

find in the peripheral blood and in lymphatic organs, when the macro-organism comes out of it with an antigen. With any blood transfusion, a large number of different i-RNS types are transferred, without knowing the antigens that react with those in

each case synthesized antibodies. The concentration of the individual i-RNS varieties is, however, far too small to be detected.

Prerequisite for the extraction of a particular i-RNS is

hence the experimental delineation of antigen recognition from antibody synthesis. This is most successful in cell-free bio-synthetic systems, which can either only recognize the selected antigen, or which can only synthesize antibodies.

From the work of Jachertz and others, it is known that the first concrete product from antigen recognition in the cell-free system is i-RNS. Cf. Jachertz "Zeitschrift fiir medizin-is.che Mikrobiologie und Immunologie" 154 (1968), p. 245,

"Annals of the New York Academy of Science" 207 (1973), p. 122, "Zeitschrift fiir Immunitatsforschung und experimentalle Therapie" 144 (1972), p. 260, as well as "Journal of Immuno-Genetics "• 1 (1974 ), pp. 355-36.2. The preparations of i-RNS described by Jachertz have still been highly contaminated and therefore unsuitable for administration to humans. An improvement of the purification was published by Jachertz in "Mole.cular and cellular Biochemistry".

.24 (1979) 93. Here too, however, as in the other works, was the experimental part. so ■ incomplete that working according to this several times was unsuccessful, and therefore the results in their entirety were called into question. This led to neither resul-

the interpretation or interpretation of these results has been accepted by those skilled in the art.

Attempts were also made to produce i-RNS in vivo, for. then extracting it from the spleen and lymph nodes of an animal;

cf. Liu Shi-Shan et al. "The Lancet", January 23, 1982, p". 197.. Apart from the troublesome presentation (horses were used,

■ which in each case had to be slaughtered), no complete purification succeeded here either.

The invention "has the task of extracting highly purified i-RNS in the cell-free system, which is specific against antigens and tolerable in humans by administration, especially by injection. This has surprisingly succeeded, whereby the i-RNS thus obtained is tolerable in humans and animals by parenteral, particularly • intravenous, administration, whereby warm-blooded animals and mammals are particularly contemplated. The method according to the invention for the production of i-RNS which is extracted in the cell-free system, is highly purified , specific against antigens and tolerable in humans by parenteral, in particular, intravenous,' administration, by conversion of antigen, DNS (including i-DNS), nucleotides and an enzyme system which is extracted from leukocytes, lymphocytes, other types of • cells or cell components, extraction with an aqueous solution of phenol and sodium dodecyl sulfate and further purification is characterized by centrifuging for purification over a density gradient, namely preferably over a density gradient of cesium chloride, sucrose and tris buffer: at approx. 130,000 G. This centrifugation lasts for approx. 4 hours. Preferably, a further centrifugation over cesium sulphate is also carried out.

It is particularly advantageous if one uses an enzyme system which is extracted from leukocytes or lymphocytes from a spleen homogenate by centrifugation over a density gradient. For this purpose, a density gradient of sucrose in tris buffers of 30 - 60% sucrose at approx. 100,000 G proved favorable. A prior stimulation of the immune system in the animal whose spleen homogenate is used for the recovery of the cell-free system,

is beneficial.. This stimulation can usually take place with Freund's adjuvant, BCG, but also particularly with Pirtd-Avi (radiation-inactivated chicken pox virus).

A further method for gentle purification is best.year in affinity chromatography'. 'The matrix can'f. e.g. consist of poly(u)-sepharose 4B or oligo-deoxythymidine.

The highly purified i-RNS obtained according to the invention in the cell-free system, which is specific against antigens and tolerable in humans and animals by parenteral, especially intravenous, administration, is actually suitable for use in direct therapy. It actually leads to the synthesis of - antibodies, regulatory protein and the cell membrane. immunity. You can therefore do without

further use it for active immunization as well as for the extraction of antisera and antibodies in vivo and in vitro. They thus

recovered antisera and antibodies can in turn also be used for targeted passive immunization. They can also be used for analytical purposes, as they are highly specific to the relevant antigens. An i-RNS according to the invention is thus a key substance with which it is extremely simple and elegant and thereby possible without after-effects to solve

the most diverse problems or to solve them better.. Of course, one can also administer them together with other tolerable substances, e.g. as an addition to transfusions of whole blood, whole blood preserves, plasma, plasma fractions, plasma-expanding agents, etc. ■

Typical antigens are e.g. bacteria, viruses, viroids, fungi, tumors and parasites. However, there can also be higher molecular chemical substances, e.g. steroids, plant and animal poisons, as well as allergens. Particularly advantageous are radiation-inactivated viruses, as they are no longer pathogenic, but have still retained their antigenicity.

It has been found that an i-RNS according to the invention is also capable of synthesizing specific antibodies in the cell-free system. Of greater importance is the finding that i-RNS in cells induces the synthesis of a regulatory protein which induces the new synthesis of the cell's own i-RNS in the recipient cell. The injected

i-RNS and the newly formed i-RNS are in turn capable of

induce the formation of antibodies. i-RNS is thus eminently suitable, with a view to specific therapy, to be administered parenterally, especially intravenously, and thereby to mobilize the body's own defense forces quickly and purposefully. If the body - still does not contain the antigen, then an active immunization takes place which proceeds faster and more complication-free than the usual vaccination with antigen.

Since a certain time after the injection of i-RNS according to the invention they also form significant amounts of antibodies, the serum can be used some time later for passive immunization. or extraction of antibodies,..• These kinds of sera and antibodies are of course also excellent for specific diagnostics and recognition of antigens.

Production according to the invention of i-RNS takes place

. without any manipulations of the genetic material whatsoever

in the sense of a recombination. Despite the synthesis in cell-free systems, according to the invention, i-RNS is to be considered a normal component in the peripheral blood. Criteria on

the safety of an application of i-RNS in humans can

. therefore orient yourself to■the criteria and experiences from blood transfusions. The injections of i-RNS that have been carried out in humans so far have not shown the slightest side effects in any case. However, the 'desired biological effect was

registered in almost all cases. From this it can be concluded that the i-RNS which is formed in the cell-free system by immunocompetent cells after stimulation with antigen, actually exhibits all the necessary information for the synthesis of antibodies and for expression of cell-bound immunity. Furthermore, i-RNS is free of antigens and antigen fragments as well as other types of contamination, such as proteins, DNS and RNS different information content. This can, for example, be demonstrated by agarose gel electrophoresis or thin-layer chromatography.

For carrying out the direct therapy, it is usually sufficient to inject the highly purified i-RNS according to the invention once. Only in some cases has it been shown to be recommendable to repeat the injection after e.g. 10 days'.

In the following examples, the invention is explained in more detail:

Oak sample 1

a) Production of the enzyme system For the production of the suitable enzyme system, leukocytes and lymphocytes can be used. It is particularly easy to proceed, the position based on the spleen. For this, 1-2 g of fresh, deep-frozen spleen (e.g. pig spleen) is pounded into powder in a mortar with dry ice and liquid nitrogen. and the suspension is frozen at -70°C. This process is repeated approx. 10x. The engulfed cells are then centrifuged for 30 minutes in a Beckman ultracentrifuge, Rotor SW 50.1 at 30,000 G. After the centrifugation,

1-1.5 ml of the red, clear, supernatant of and brought to a discontinuous sucrose gradient. This gradient consists of 1 ml of 60% sucrose in tris buffer A, above which 2.75 ml of 30% sucrose solution is in tris buffer A.

In a Beckman ultracentrifuge, Rotor SW-50.1' is centrifuged at 100,000 G, 0°C. After 4 hours, the desired fraction is obtained as a reddish, clear layer above the 30% sucrose. The layer is removed with a pre-chilled pipette and diluted with cold ■ .tris buffer. A in the ratio 1:1. The solution can be stored in liquid nitrogen. - ..

b) DNS (deoxyribonucleic acid) including i-DNS

(informational deoxyribonucleic acid)

1-2 g of deep-frozen spleen that has been stored in nitrogen is slowly thawed in 5 ml of Tris-buffer A and homogenized in the homogenizer at approx. 30 flask movements. ■ The suspension is added dropwise to an Erlenmeyer flask with 20 ml of an emulsion of 2% aqueous sodium dodecyl sulphate solution. with freshly distilled'

phenol (ratio 1:1), whereby the flask is shaken strongly to achieve homogeneous distribution. If the mixture is too viscous, 3% sodium chloride solution can be added. After .24 hours, the milky emulsion is centrifuged in a centrifuge for 15 minutes at 2000 G, whereby two phases emerge. The same volume (+ lo%) of anhydrous ethanol is added to the aqueous upper phase, so that DNS precipitates out. The DNS is transferred with a cast platinum loop to 10 ml of sterile 0.85% sodium chloride solution. 5 ml .of. 1% sodium dodecyl sulfate solution, 5 ml of 0.05 mol/l. tris-A .and 0.05 mol/l

EDTA buffer pH 7.6 and 2 mg of proteinase K are added, and everything is mixed. The resolution of DNS takes approx. 1 day, and. after this it can again be precipitated with alcohol. Resolution for renewed precipitation is repeated 3 to 7 times, until the ratio between E260' and ^ 2S0 has reached a value between 1.9 and 2 and the hyperchromic effect amounts to at least 20%. extinction increase at 260 nm. An absorption of DNS at -2 60 nm on lo.D. 'equivalent to approximately one. concentration of..40 ug/ml.. Using this relation

•the DNS solution is diluted so that you get a concentration of

1 ug/ml. This solution for use is frozen in portions of 0.5 ml<p>g and stored at -20°C.

c) Synthesis of i-RNS (informative ribonucleic acid)

In an ice bath, the purified enzyme system and the antigen are added

in tris-buffer A at pH. 8.0 and a mixture of the nucleotides ATP, GTP, CTP and UTP is added. For control, a small batch is made -, in it. same quantity ratio, as. however, tritium-labeled UTP is added... Pipetting is always done with chilled pipettes.^

The optimal concentrations of the individual components are obtained

by several attempts. Each of the samples is incubated in a water bath at 37°C and an aqueous solution of 1% sodium dodecyl sulfate and 1% aqueous phenol is added already after 3-6 minutes. The optimal incubation time also depends somewhat on the concentrations and the antigen in question and can be calculated by some manual experiments in advance. It is cooled in an ice bath and centrifuged at 2,000 G. The above contains the synthesized crude i-RNS.

d.) Purification of i-RNS

From the above, i-RNS is isolated over a discontinuous cesium chloride/sucrose gradient. Thus, in the centrifuge, the cellulose acetate tube is in each case 1 ml of cesium chloride solution with a density of 1.9 g/ml overlaid with 0.75 ml of a 30% sucrose solution in tris- buffer A pH 8.0. Every .2 ml of the samples is placed on the gradient, and the tubes are closed with sterile paraffin. After the centrifuge has run at 18°C, 130,000 G,

for 4 hours in Beckman R<p>tor SW 50.1, the cesium chloride layer is removed. Thus, the ice centrifuge tubes are pricked with an annealed needle at the bottom, and the entire caesium chloride solution is transferred to the sucrose layer in tubes. The solution thus obtained is diluted with -

sterile water 1:2 and can be stored frozen at -20°C. Further purification takes place by sedimentation over a cesium sulphate gradient. Thus, 4.8 ml of cesium sulphate solution with a density of 1.5 g/ ml in centrifuge tubes of cellulose acetate

.(Rotor Beckman SW 50.1). Layer 0.3 ml of the sample over this and centrifuge for at least 4 hours (preferably 12-19 hours) at 18°C and 130,000 G. After the centrifugation, the tubes at the bottom are pierced, and 13-14 fractions, each of 9 drops

, (= -0.4 ml) is divided into fractions. 0.1. ml of each fraction is used for determining the radioactivity, and the remaining. 0„ 3 ml is diluted with the same volume of water and. stored at -20°C. After it is determined in which fractions the highest radioactivity is contained, the corresponding fractions of the main batch are also selected. They contain the highly purified i-RNS.

The fractions, if desired, can be immediately injected without further purification steps.

The. is. possible to detect i-RNS electro-optically, whereby molecular lengths of between 1,500 and 2,000 nm have been observed. The degree of purity can be demonstrated, for example, by agarose gel electrophoresis.

Another demonstration of i-RNS consists in synthesizing antibodies in the cell-free system. For this purpose, a sample of i-RNS, highly purified ribosomes, the above-mentioned enzyme system and leucine and valine are brought together in tris-buffer A pH 8.0. The samples are incubated for 45 sec. at 7°C, and then protein synthesis is stopped by freezing at -20°C. The antibodies can be separated and purified by affinity chromatography on immuno-adsorption columns and detected analytically.

The highest concentrations of antibodies are elicited by the addition of carrier 10 i-RNS. This shows that a molecule of i-RNS in the cell-free system is capable of synthesizing at least 10<4> molecules of antibodies.

It is furthermore possible, with an RNS replicase, to increase the amount of i-RNS by a factor of 10 4 to 10 5 . By repeating these steps several times, a cloning takes place.

Since the thus obtained i-RNS also still has the same properties with regard to antibody synthesis as the one originally extracted, the conclusion can be drawn that antibodies are still produced by entrained antigen residues.

The analysis of i-RNS showed that the molecular weight is approx. 1-2 x 10 6. As antigens, Aujeszky virus, Herpes sim<p>lex virus, irifluenza virus, FMD virus, Varicella-. virus, Measles virus, HBs antigen, E.coli 0 antigen, sheep erythrocytes, ADH, tetanus toxin, as well as systems with tumor cells, such as LC^ from guinea pig, p815 from DBA2<->mus., L' 1210 from DBA2_mouse as well as the polyomavirus-induced tumor in Lewis rats. The first successful results■were further achieved with the attenuated Masernia virus "Moraten" and HBS antigen. Corresponding experiments showed that no cross-reactions occur. Thus, highly specific i-RNS arises in dependence on it. in each case antigen used.

Example 2

A specific i-RNS against hepatitis B was produced,

and this was tried on over 200 patients with regard to tolerability and activity. The dose of i-RNS was 10 mol/ml. In most cases, the patients received 1-2 ml of this solution once, but in rare, serious cases, another injection was made. The curative rate, which can be seen at the moment, is close to 100%.

Example 3 BALB/c mice were injected three times at weekly intervals, each time with 100 µl complete Freund's adjuvant. The complete Freund's adjuvant which is commercially available was mixed: 1:1 with ■ physiological saline solution. After the last injection waited

rest 4 weeks. Then the spleens of the animals were removed due to the recovery work for the recovery of the cell-free system.

The yields of i-RNS obtained with this cell-free system were a factor 10 higher.

Claims (11)

1. Highly purified i-RNS, extracted in the cell-free system, specific against antigens, tolerated by .humans and .animals when administered. 2. i-RNS as specified in requirements. 1,, characterized in that it is tolerable by parenteral, especially intravenous, administration. 3.. Process for the production of i-RNS which is extracted in the cell-free system, is highly purified, specific against antigens., tolerated by humans and animals by parenteral., especially intravenous,, administration, by turnover, of antigen, DNS ( including i-DNS), nucleotides by an enzyme system which is extracted from leukocytes, lymphocytes, other types of cells or cell components, extraction with an aqueous solution of phenol and sodium dodecyl sulfate and further purification, characterized by purification by centrifugation over a density gradient. 4. Method as stated in claim 3, characterized by centrifugation over a density gradient of cesium chloride/sucrose and tris buffer at approx. 130,000 G. 5. Method as stated in claim 4, characterized in that i-RNS is then purified by means of affinity chromatography. 6. Method as stated in claim .4., characterized by - in addition centrifuging over cesium sulphate. 7.. Method as stated in one ■ of the claims 3.-6, characterized by the use of an enzyme system which is extracted by cleaning a homogenise of tissue from the reticulo-endothelial system (RES), preferably spleen by centrifugation over “a density gradient. 8.. Method as stated in claim 7, characterized in that the immune system from animals, which is used for extraction. of the homogenise from tissue in the reticulo-endothelial system, is previously stimulated, preferably with BCG, Freund's adjuvant and Pind-Avi. 9. Procedure as stated - in claim 7, character - s e r t .by centrifuging over a density gradient of sucrose in Tris-buffer at approx. 100,000 G. ; 10. Application of i-RNS obtained in the cell-free system, highly purified, specific against antigens, tolerated by humans and animals by parenteral, preferably intravenous, administration, for direct therapy, for active immunization or. extraction of antisera and antibodies in vivo and in. vitro.. 11. Application according to claim 10, whereby the administration ■ takes place together with other tolerable substances.