WO1992002249A1

WO1992002249A1 - PROCESS FOR INHIBITING THE FUNCTION OF PROTECTED EXTRA-CELLULAR mRNAs OF CANCEROUS ORIGIN TO PROVIDE PROPHYLAXY AGAINST MALIGNANT DISEASES AND THEIR RELAPSES

Info

Publication number: WO1992002249A1
Application number: PCT/DE1991/000632
Authority: WO
Inventors: Viktor Balazs; Margit BALAZS-FRÖHLICH
Original assignee: Viktor Balazs
Priority date: 1990-08-08
Filing date: 1991-08-06
Publication date: 1992-02-20

Abstract

Prophylactic process against malignant diseases and their relapses by preventing the extra-cellular mRNAs of cancerous origin protected by protective complexes from acting as transmitters for affecting the host organism by cancer and the interaction between malignant cells in which the protective complex is isolated from an acellular (extra-cellular) liquid (like a serum of malignoma carriers, cancer cell breeding fluid) by ultra-high-speed centrifuging and using it as an immunising agent containing at least one antigen for active and passive immunisation and immuno-adsorption, immuno-assay, immunotherapy, immunocytotherapy and other similar purposes. Processes like haemofiltration and immunohaemadsorption are described for eliminating the protective complexes with their mRNA content from the bloodstream.

Description

Name: Method to prevent the function of the protected extracellular mRNAs from cancer cell origin for prophylaxis of malignant diseases or their relapse

The invention relates to a method for preventing or eliminating the extracellular protected mRNAs of cancer cell origin for the prophylaxis of cancer diseases or their relapse in accordance with the preamble of claim 1.

There are two phases in malignant processes in which the survival of a relatively small cancer cell mass is fatally crucial for the host, i.e. the patient:

1. Initial (initial) phase

2. Residual phase after therapy.

The outcome depends on the bilateral interaction between the malignant cells and the host, which has existed continuously since the emergence of the first malignantly transformed cells.

Any effect that strengthens the host's defense mechanisms and / or reduces the chances of survival of the cancer cells can have decisive effects in these two phases, meaning health or illness.

The interaction between host and cancer must first be analyzed.

This mutual interaction is a very complicated, multifactorial and versatile mechanism in which the rnRNA of cancer cell origin can play an important role, as detailed below. is described. 1. The blood plasma RNA fraction from malignancy carriers functions as a messenger in the protein-synthesizing cell-free system and ^{substantially} prevents the translation activity of other mRNAs when they are in competition with it.

2. This was in RNA transfer (experiments) bestä to normal cells ^¬ taken of: The translation product of plasma RNA from Malignomträgern was produced in the Rezipientzellen, while the production of the corresponding normal protein was dramatically inhibited.

This phenomenon is actually the functional mimicry on molecu ^¬ lar biological and cellular level of the main features of bösar¬ term process itself: promotion of its own existence on the cost of the host.

3. Poly (A) ⁺ RNA is secreted from malignant cells, protected in a lipoprotein shell or bound to other substances. As can be demonstrated in patients with malignant diseases, it can be isolated from the blood plasma.

4. Messenger RNA more than one substance from cancer cell origin can occur in the circulation of malignancy carriers.

5. A large spectrum of normal and malignant cells is able to take up mRNA from its environment if it is presented protected by lipid, lipoprotein or other substances. The RNA then exerts its messenger activity in the recipient cells. As a result, the translation product of the mRNA taken up is produced by these cells until the replenishment from the environment reaches through the circulation.

The recipient cell has acquired a new phenotypic quality, a new ability to produce, which it either does not have or cannot express genetically.

This new biological phenomenon and its meaning was first recognized by the inventors and is referred to as "phenotype borrowing". Phenotype lending is one of the most important mechanisms by which the malignant cells achieve their main goals:

a. Promoting their own existence, b. Hindrance (inhibition) of host functions, even c. the malignant cells force the host cells to produce what is advantageous for the malignant process. (Forced collaboration between the host and the malignant cells).

The influencing messages essentially go in two directions:

1. From the malignant cell population to the host cells.

2. From a subpopulation of malignant cells to other subpopulations either

a. within the tumor or b. from the mother tumor cells to distant metastatic tumor cell colonies, and c. vice versa.

Phenotype borrowing is a reversible, multi-step process that has the following important stages and characteristics:

1. Elimination of the protected mRNAs by malignant cells.

2. Transport of the mRNAs in this form in the extracellular liquid to the recipient cells. Direct cell-to-cell contact is not necessary.

3. Uptake of the mRNA by the recipient cells using endocytosis.

4. Acceptance of the command at least over a part of the system that synthesizes the system in the recipient cell:

a. Production of the translation product encoded in the guest mRNA, b. possible inhibition of the synthesis of one or some products of the recipient cell reversibly without impairing their viability.

5. Emergence of the effect of the phenotype loan product.

The product is either the end effector substance itself, which is directly useful for the cancer cells, or serves as an activator (inducer), the further, even several substances in parallel, or successively through an activation cascade in the same cell or in several other cells (cancer and Host cells) induced effector substances that are useful for the cancer cells. In this way, the invasiveness and the metastasis potential of the cancer cells or one of their subpopulations can be increased, either directly or indirectly, by means of intermediate cells (host cells). As a multi-activator u. a. the c-fos and the transforming growth factor (TGF) are mentioned here.

6. After interruption of the replenishment or the uptake of the guest mRNA, the original normal state in the recipient cell is produced within a few hours without gaps.

Therefore, it is very desirable to prevent the malfunctioning of the mRNAs of cancer cell origin in malignant diseases by interrupting the phenotype loan.

There are a number of published clinical and experimentally ^¬ len observations phenomena in malignancy describe where the above-mentioned exchange of information Phänotypausleih with mRNA can be truly operational:

A. Interaction between host cells and cancer cells.

1. The enzyme profile of the otherwise normal liver of malignancy carriers is more similar to that of malignant cells than that of normal people.

2. Normal lymphocytes in the overwhelming majority of myeloma patients have the myeloma protein as their surface immunoglobulin. 3. Normal leukocytes of the malignant carriers produce large amounts of enzymes with basement membrane-destroying activity, this is in contrast to the normal state.

4. The in vitro resistance of a tumor that developed complete resistance during chemotherapy in vivo was little or no increase. The resistance to chemotherapy developed in vivo can arise from the fact that the liver (liver again as in AI.) And partially kidney cells rapidly absorb larger quantities of chemotherapeutic agents from the circulation and excrete them. As a result, the chemotherapy concentration in the circulation is adjusted to a level which is still tolerable for the tumor. In this case may be conceivable that the rapid and increased Ausfiltrierung deε chemotherapeutic agent activated by Phänotypeausleih and was maintained ^¬ was present until the tumor in the host.

5. Recently, a new enzyme has become known which can break up the intercellular matrix and is only present in particularly invasive breast cancer cells. Nevertheless, the presence and the Pro¬ was production of this enzyme in the normal cells of the host in the tumor environment, even some of them will continue to demonstrate, but not rer to normal host cells in the area ande ^¬ tumors.

B. Interaction between different subpopulations of cancer cells

1. A subpopulation of malignant cells that is unable to metatatize when it s.c. is injected, but will metastatize after a subpopulation with metastatic potential of the same Timor IV in the same animal. was injected.

2. A subpopulation of malignant cells became resistant to a drug that they had never come into contact with before. is if only humoral contacts between this and another. Subpopulation that is resistant to this drug has been established. The list is not complete.

It should be noted that transcripts or their fragments of cellular oncogenes, which are activated by transferring units transcription in malignant medical ^¬, can be detected from the blood plasma of Malignomträgern by in vitro hybridization. Phenotype borrowing from these transcripts can cause phenomena similar to AI. , 5, and Bl. Bösar cause in ^¬ term Krankeiten, or manufacturing capabilities Wachs¬ tums-factors, enzymes, inter alia, intercellular matrix fracturing enzymes, or other substances into host cells that are removed from the tumor transmitted. Thus, in concert with other mRNAs of cancer cell origin, the surroundings of these host cells could become attractive for the incoming metastatic cancer cells and then feed them with growth factors and other substances. The inhibition of phenotypes by mRNA of malignant origin simultaneously prevents their own functions in the recipient cells of the host, which can lead to a reduction in the local and systematic defense mechanisms of the host. Small fragments of transcripts from cancer cell origin also have inhibitory activity.

Metastasis is what kills cancer. In order to be able to metastasize the cancer cells, they first have to leave the circulation (extravasation) and gain a foothold in a new place. The extravasation is considerably facilitated by the fact that the basement membrane-degrading enzymes were not only produced by the cancer cells but also by the Wirtzel¬ len because of phenotype loan (A. 3).

The newly arriving metastatic cancer cells need all the help and support from the cancer cells in the established colonies by phenotype borrowing all the more because they do not secrete any mRNA in the lipoprotein shell in the new environment for several hours (5 to 7) and so they do not Practice practicing loan type. Only then, after this period has elapsed, can you develop a protective and supportive microenvironment yourself through inhibition and forced collaboration of the surrounding host cells.

The occurrence of information exchange through the circulating mRNA was examined and proven in myeloma patients (A2 see list).

We have succeeded in the production of the Multiple Myeloma PROTEM m normal human lymphocytes with plasma RNA of myeloma patients to transfer what has caused the decrease in the production of its own top ^¬ surface-immunoglobin. The RNase-treated sample of the same plasma RNA was ineffective.

iαRNA transfer in the lipoprotein shell without RNase inhibitor is considerably (several hundred times) more efficient than transfer with isolated mRNA plus RNase inhibitor, all, prior incubation with anti-protective complex antibody prevents the transfer and the phenotype loan.

However, the RNA transfer method is a very complicated, arduous method and is not sensitive enough to give positive results in the early stages of malignancy.

The specific mRNA sequence of the H and L chains, corresponding to the chain combination of the myeloma protein, was detected from the blood plasma of the myeloma patients. The presence of the mRNA in the bloodstream or in the extracellular fluid is the most important requirement for borrowing the phenotype.

Phenotype lending has several advantages for cancer cells.

a) They save energy, components and production capacity, which they can then use for their own purposes, such as growth, multiplication, etc., because they only provide the encoded massage for the phenotype loan, the production of the translation product is carried out in the recipient cells. b) be extended at the time of its effectiveness: From a short-lived mRNA (a few hours) is a long-lasting (several days) Product c) increase the quantity of effector substances: A mRNA enables the handling of several molecules of Translationspro ^¬ domestic product. d) It also extends the spectrum of the effector substances (the active substances) by activator or by activation cascade. e) expand their sphere of influence to the whole Wirtorganis ^¬ mus.

It is believed that the inhibition of the effect by cancer is related to polypeptide, proein hormone-like substances in the serum, because proteolytic enzymes prevent this inhibition of the serum from malignancy carriers, but RNase is ineffective. The products of the phenotype loan (effector or activator and its products) are mostly proteins or polypeptides, which are also sensitive to proteolytic enzymes.

Phenotype lending is consistent with these because when the liporote shell is intact, the mRNA protects against the ubiquitous RNase. Proteolytic enzymes break down the protein component in the lipoprotein shell and thus release the mRNA to RNase activity, which is also highly active in serum. In another opinion, the involvement of the host is caused by the cancer with the presence of a large amount of abnormal lipids. Since the Liporoteinhülle large amount of abnormal, unknown under normal conditions in serum lipids ent ^¬ holds, the Phänotypausleih with this opinion also in line.

By preventing the Phänotypausleihε result εich prophylactic ^¬ cal and therapeutic facilities, to mention just a few:

a. In plasmacytoma preventing the transmission, the Myelomaproteinproduktionsfähigkeit to the normal Lymphozy ^¬ th of the host in the early stages of malignancy, the Inhi¬ bition of Immunεystems so far prevented so that the immune system can eliminate the relatively small Tumorzellπtasse without further chemotherapy when the malignancy frühzei ¬ tig recognized and treated according to the possibility mentioned above.

b. By preventing the endothelial basement membrane-destroying enzymes, the tumor cells present in the bloodstream cannot leave the circulation and thus cannot metastatize. This is particularly important during and after surgery when a large number of cancer cells can enter the bloodstream c. With the Phenotypausleih, the lower (20-25%) Er¬ success rate of the various forms of active immunotherapy will be explained at least in part because, therefore, the host ^¬ cells can not cooperate satisfactorily, or the increased in vitro and activated cells shortly after their Verabrei ¬ chung in the malignant carrier host in their function inhibited, ineffective. Hyperactivated cell functions can be less sensitive to inhibition by phenotype lending than the normal one.

d. Even preventing certain forms of chemotherapy resistance from developing in vivo would be possible as in the case of A4.

But here is the most important question of how to prevent phenotype borrowing. There are two ways to do this:

1. Immune defense induced by vaccination or

2. by removing the mRNA in its protective cover.

To achieve this is the object of this invention.

There is no method for general prophylaxis against malignant diseases.

Tests for immunization with killed tumor cells of the respective patient for therapeutic purposes are known. Similar experiments were carried out with cancer cells grown in vitro. In these cases, however, the success rate was very low and mostly temporary. In order to be able to achieve general prophylactic immunization, a vaccine must be prepared against every type of cancer and thus immunized, which is not technically feasible. Furthermore, a large number of antigen substances are used in fairly large quantities. administered, which is very disadvantageous on the effectiveness of the immunization. A sufficient immune response to the actual antigens with a protective effect is also not guaranteed. In addition, the protective antibodies must be directed against the surface antigens. and that they have to achieve in order to bind, so we have ^¬ fectively. What but when these antigenic substances leave the cell surface in large quantities often the case, dieεe inhibito act ^¬ driven into the extracellular Flüεεigkeit came antigens because they absorb large amounts of antibodies by binding, so the antibodies do not or only limited Konzentra ^¬ tion reach the cell surface and bind there. Can also be, and the surface antigens by the binding of the antibodies abspal ^¬ th and so prevent their action.

The dynamic heterogeneity of the cancer cell population in terms of surface antigenic substances is also an important source of the low success rate of this method.

Preventing an important function of cancer cells, which is necessary for their survival and occurs in every malignancy, would be better for general universal prevention.

One such mechanism is the Phänotypausleih, as is also evident from the up ^¬ take precedence versions.

By preventing the Phänotypausleihε the host can use ^¬ defense mechanisms from his. Important reinforcement options between different subpopulations are interrupted.

Induced active or passive immune response against the protective complexes of the mRNAs of cancer cell origin could prevent the mRNA from reaching its targets from the extracellular fluid, thus preventing the phenotype loan with all its consequences.

Previous observations have shown that the protective shell, ie the protective complexes of the mRNAs of cancer cell origin, has antigenic abilities and can thus trigger an immune response.

The abnormal lipid component of the protective complexes exert a certain inhibitory influence on the antigenic effect of their protein component. Thus, among other things, the absence of the "spontaneous" immune response to the protective complexes (the mRNAs of cancer cell origin) at their origin. presented by the malignant process in the host organism.

This inhibitory effect can be broken through in the case of artificial immunization with other forms of presentation (such as im, sc, etc.), particularly in combination of adjuvants and oil suspension. Similarly, the elimination of the abnormal lipid component in combination with Adju vant can ^¬ or the protein genetically introduced into microorganisms (genetically recombined) such as vaccinia virus, Mycrobakterium bovis BCG. , attenuated S.typhi.etc. Act.

Investigations in this regard and observations with liposomes, in particular with those which artificially contain mRNA, point to the following:

1. The surface properties, components and loading (batch) play an essential role in

a. the duration of circulation, b. Suεpenεion stability of the corpuscles (vesicles), in c. their ability to navigate (hike) to the target recipient cell, in d. their uptake into the recipient cell by endocytosis in their e. Leak resistance.

2. Antibodies affect all of these abilities

a. Change in surface charge, component. b. By reducing leakage resistance, the mRNA becomes accessible to RNase enzymes and is degraded. Simultaneous participation of antibodies and serum factors such as complement can cause perforation of the shell or its disruption (Vesiculolysiε, Particellysis), as with some types of RNA virus particles, where the fresh human serum disrupts the protective shell and the virus RNA is degraded by Seruir.- RNase. c. The antibodies change the suspension stability of the body none, d. cause aggregation. e. Opsonization promotes phagocytosis of the corpuscles f. Shorten the circulation time of the particles with the help of a. , cd and e .. g. Corpuscles with antibodies on their surface are prevented by endocytosis when they are absorbed into the recipient cell.

Which of these mechanisms will be effective in the given case depends on several factors, in particular on the properties and the quantity of the antibodies present and on the concentration of the corpuscles.

These mechanisms or a combination of these in the extracellular liquid in the immune state against the antigen substance (s) of the protective envelope of the mRNAs of cancer cell origin prevent the mRNAs from reaching their target recipient cell and being taken up by them. The chain of phenotype lending is broken (derailed) extracellularly. Cancer thus loses one of its most important survival mechanisms, the host is freed from forced collaboration with him and from the inhibition of his own functions, and thus strengthened in a defensive manner.

Similar effects can be achieved by administration of immune serum or its immunoglobulins, which are obtained by immunization with the specified vaccine (antigen substances of the protective cover of the mRNAs). The effect here is only temporary.

The second way of preventing the phenotype loan is to remove the protective sheath with its mRNA content from the extracellular fluid. There are two ways to do this:

1. Hemofiltration, the desired substances being removed according to their size by selecting the pore size of the filter. The previous devices are not suitable for removing the protective cover with the mRNAs because the pore size is not suitable for this. A major disadvantage of this method is its unselectivity, it removes everything harmful and useful the selected range of molecular size.

2. The second option is hemadsorptio. The distance is essentially ge according to the composition or the resulting properties. Three methods can be used here:

a. Through the lipid content. The previous methods do not seem to be suitable for removing the lipid envelope with mRNAs. It is also not selective.

b. Adsorption by immobilized protein A: suitable for removing all substances which contain IgG (immunoglobulin G) (IgG, immune complexes etc.) but not suitable for removing the protective cover. This method is also not selective, removes all harmful and useful IgG content.

c. Specific immunoadsorption. Here the patient flows through a porous matrix in which immunoglobulins containing antibodies are immobilized. The protective cover with its mRNA content is specifically removed. It must be mentioned here that the removal from the entire extracellular fluid is never complete. With this, only a decrease in the concentration in the circulation can be obtained. The reduction is less in the vicinity of tumor cells. This method can be used with other methods, e.g. B. 2.a. or 2.b. or combined with other forms of therapy.

The object of the invention is to provide a vaccine which contains the antigen substance (s) of the protective complexes or protective cover of the mRNA of cancer cell origin and is suitable for active and passive immunization so that phenotype lending is prevented and thus cancer or relapse is prevented. An additional goal is to develop a supplementary method for eliminating, removing the protective cover with its mRNAs.

This object is achieved by the features of the claims. The invention is explained in more detail below.

Blood is taken from untreated patients with active, malignant diseases and the serum is separated. The serum is subjected to KBr denity gradient flotation in an ultracentrifuge in order to isolate the lipoprotein protective envelope of the mRNAs from cancer cell origin. A third party (animal, volunteer) is thus immunized by repeated parenteral (i.m.) administration.

In unseru ge ^¬ is gained by blood collection and separation of Serumε. The immunoglobulin fraction with the antibody activity against the protective cover is separated from this by affinity chromatography with the aid of the immobilized protein A.

The Immunglobilinfraktion thus obtained with anti-Cases Antikör ^¬ per-content is bound to Sepharose active, immobilized to the protective sheath in their antigenic substance (s) to adsorb specifically at physiolo¬ Gischer ionic strength and pH. In this case, sera from patients with active malignant diseases are passed through in a column with the Sepharose prepared above, in order to be subjected to a specific immunoadsorption. The desorption is carried out after washing with solutions of low pH values and / or at higher ionic strength. The protective sheath fraction is dialyzed and lyophilized.

The above-mentioned method can be carried out with the cell-free supernatant liquid of cancer cell cultures after appropriate concentration.

The immunoadsorption method for isolating the protective sheath from patient sera can also be used in a corresponding scale-up form for therapeutic purposes. Here, however, the adsorbed protective sleeve is not desorbed, but removed with the Sepharose and by flowing serum after appropriate treatment in the Pa ^¬ tienten recycled.

The IgG isolated from the immune sera with an anti-protective Activity, especially after enzymatic removal of the Fc portion, can be used for passive immunization and treatment of those patients who are unable to be actively immunized. This can only be a temporary objective and may be linked to another therapy or measure.

Immunoadsorption the protective shell of patient sera can provides preconstruction ^¬ rend or practiced development of chemotherapy, radiation or surgical Behand¬. Passive immunization would be better used according to these forms of therapy if the active immunization with protective cover cannot be recommended for any reason.

The removal of the protective sheaths from the patient sera by immunoadsorption can also be combined with the removal of other undesirable substances such as immunoglobulin IgG and immune complexes (with IG content) by adsorption of immobilized protein A or according to their size.

Finally, it must be mentioned that the protective covers can be removed from the circulation due to their high lipid content by means of affinity chromatography. This method works non-discriminatory, non-selective and thus removes useful and harmful substances if they have lipid components in an accessible form.

A special form of immunotherapy is immunocytotherapy. Cells of the antibodies or other important bioactive substances are produced or tissue parts (pieces of tissue) with similar production capabilities are implanted in malignant carriers. Description of this form of therapy is given as part of this invention.

To be able to assess the immune status after immunization, a sensitive detection method of the antibodies is required. Such a method for the detection of the antibodies against the protective complexes from body fluids (such as serum etc.) is described.

The detection of the presence of the protective complex antigen substance in body fluids (such as serum) is also important, all the more because the presence of this antigen substance is a reliable early sign of malicious processes. A sensitive method for this will be from the body fluids (such as serum) prior tration without any concentrator ^¬ the antigenic substance or isolation of the lipoprotein is familiar.

The mRNA detection method can make even more sensitive when integrated isolated before extraction of RNA lipoprotein in constant property ^¬ RNase inhibitor by immunoadsorption and the RNA extraction performed it and identifies the target Sequencen with SENS ^¬ service methods.

The following example serves to further explain the invention without restricting it.

Example

The serum is obtained from 20 ml of blood in EDTA solution (final concentration: 4 mM, pH 7.4) from patients with malignant diseases (active malignant processes) on an empty stomach and separated by centrifugation at 4 ° C.

A sample (3 ml) of this sera is mixed with 0.3517 g / ml KBr and placed under an intermittent density gradient of KBr (1.006-1.221 g / ml) in an ultracentrifuge tube and flotation in the ultracentrifuge at 4 ° C with 105 000 xg for 16 h under ^¬ . The protective sheath Frank ion with the mRNAs will be visible as a band between opalisie ^¬ rendes VDL and HDL lipoprotein fraction and is collected by suction. Then this fraction is logical to physio ^¬ buffer solution with several changes of the dialysis buffer dialyzed and then lyophilized. Before further consumption, it is then dissolved or suspended in an appropriate buffer solution.

This method is used to isolate the protective envelope with its mRNAs from the cell-free culture fluid of the cancer cell cultures. In this case, however, the culture liquid after 24 hours Züch¬ processing time is made free of cells by centrifugation and at least five times ^¬ concentrated. Then the protective fraction by flotation separated by means of density gradient ultracentrifugation as indicated above and treated accordingly.

Cell cultures of various malignant cells are commercially available such as: HL 60 Leukemia Zeil-Line: Heia Krebszell-Line, Jlll Monozyten Leukemia Zeil-Line; RD reticulosarcoma etc. (etc.) (Flow Laboratories and Seromed).

The protective shell fractions from several patients or from several cell cultures are treated with gamma rays and treated with preservative. Active immunization is achieved by repeated administration (i.m.) of the vaccine, 1 to 3 ml per vaccination per animal (mouse, rat, rabbit). When vaccinating people, the vaccine is first mixed with immunostimulating substances (adjuvants) in oil droplet suspension and i.m. administered. The level of the developed immune status can be followed by immunoassay. Thereafter, the immune serum is obtained either by drawing blood (in humans) or by bleeding (in animals).

The IgG (mouse, rat, human) is isolated from the immune sera by affinity chromatography as follows: The IgG fraction with the antibody content is first isolated from the immune sera. For this purpose, an affinity chromatography method with a protein A Sepharose column (Pharmacia, Uppsala, Sweden) is used in accordance with the manufacturer's instructions.

The IgG fraction of the immune sera is adsorbed by normal human sera: 1 mg protein is bound to CnBr activated Sepharose 2B (3 ml of swollen activated Sepharose), with which a column is filled, washed and loaded with the IgG fraction. After flowing through, the unbound IgG fraction is collected and used as an anti-protective cover anti-body.

This IgG fraction is immobilized to CnBr-activated Sepharose 6 MB (Pharmacia, Uppsala, Sweden) and thus a column is filled, washed, equilibrated and with serum from patients with active malignant diseases or with concentrated sepa- rant of culture fluid from various cancer cell cultures (according to 24 h culture) and washed. Initial buffer for equilibration, sampling and washing: Physiological phosphate buffer saline, pH 7.5. Buffer for the desorption (elution) of the adsorbed protective covers is buffer B (150 M citrate buffer pH 2.5-2.8 and physiological NaCl solution). The protective fraction is collected, dialyzed and lyophilized.

This process can be carried out for the industrial production of larger quantities in scaled-up form (process scale), e.g. B .: A process glass column BRG 100/500 filled with 400 ml gel is suitable for 5 1 starting material (sera or concentrated culture liquid from cancer cell cultures), so large amounts of the protective cover can be isolated specifically and quickly (fast-flow).

The isolated and lyophilized Cases faction is suspended in phyεiolo- GisChem saline buffer and then with various biolo gical ^¬, microbiological Immunεtimulanzien in oil Tropfenεuspension mixed in the following composition:

1. Detoxified bacterial endotin (detox) produced for vaccination 10-400 μg, preferably 10-50 μg per dose, (commercially available).

2. Cell wall skeleton made from mycobacteria such as M.bovis BCG ^¬ according to Azuma et al. (Journal of National Cancer Institute, vol. 52, pages 95-101, 1974) or commercially, 180-300 μg per dose.

3. Trehalosedimycolate, which are prepared from M.boviε BCG or Corynebacteriu diphteriae as above, are commercially available, 120-180 μg per dose.

4. Corynebacterium parvum pyridine extract, 500 - 2500 μm per dose.

5. Oil droplet suspension in a final concentration of 0.5 to 2.5% by volume, prepared from light mineral oil, 7-n-heyloctadecane or Conocoperil etc. A detergent in 0.10 to 0.20% by volume can be used to stabilize the oil suspension. be mixed in saline. The above-mentioned mixture is mixed together with the isolated protective cover fraction and used for the active immunization. A dose is 1.0 to 10.0 mg of protective cover and is repeated at intervals of 1 to 6 weeks either sc or preferably im administered. Vaccination after several years is recommended.

In cases in which active immunization cannot be carried out or is not recommended, the IgG fraction isolated from immune serum by the stated method can be administered parenterally in order to protect the protective sheath with its mRNA in the circulation, but also in the extracellular liquid To prevent or eliminate tissue from cancer patients. It is particularly suitable, NEN the Fc portion of IgG antibody molecules by enzymatic treatment with pepsin to entfer ^¬, which is carried out with methods according to the prior art, with the Fc-free IgG antibodies fraction is interrupted chain of Phänotypaus ^¬ Leihs.

The total dose is several hundred mg of non-FC fragments of the IgG with an protective protective antibody activity in distributed portions at 1 to 6 day intervals, administered parenterally. The effect is according to the life of the antibody molecules or their Fragmen ^¬ te limited.

The protective sheath with its mRNAs of cancer cell origin can be removed from the blood or plasma of the cancer patients using the immunoadsorption method described above for the isolation of the protective sheath or its process scale form, specifically from the blood or plasma obtained. Or the immunoadsorption column can be connected to an extracorporeal circulation system, in accordance with the technical details of the method used for the removal of factor IX antibodies from the circulation of the hemophilia patients with protein A Sepharose column (Blood Vol. 58, p 38-44, 1981).

Corresponding apparatuses for extracorporeal circulation systems, to which the Sepharose absorption column bound with an antibody protective cover can be connected, are commercially available. In contrast to the insulation of the protective cover, the adsorbed material (Protective cover) is not desorbed, but instead the plasma which has ^flowed through is returned to the patient's circulation according to the selected extracorporeal system.

Thus, the concentration of the mRNAs of the cancer cell originating in the zir ^¬ can cause it to the cancer patients significantly reduced and thus the phenotype ^¬ borrowing be reduced through circulation. This condition can adsorption by repeated immune ^¬ be maintained. However, this method will not be very effective in tissues in the immediate vicinity of tumor cells, where the "first defense line" or "front line" is located.

It is therefore advisable to continue this treatment immediately afterwards with the administration of anti-protective IgG antibodies (one of its non-Fc fragments such as Fab, F (ab) ₂ or Fab ') or to combine it with other forms of therapy .

The protective sheath with its mRNAs can also be removed by hemofiltration. The devices for hemofiltration, which are commercially available, are not suitable for these purposes because the pore size of their filters is not large enough to allow the protective cover to pass through. By enlarging the pores it can be achieved that these devices are suitable for removing the protective cover from the circulation of the cancer patients. However, this method is not discriminatory, i.e. not selective, in contrast to the specific hemadsorption method described above. Although hemofiltration can significantly reduce the concentration of the protective sheath with its mRNAs in the circulation, it works much less effectively in tissues that are in the immediate vicinity of cancer cells. So it is also recommended here to continue the treatment with administration of anti-protective cover anti-bodies and to combine it with other forms of therapy.

Numerous modifications are possible, some of which should be mentioned:

The main protective antigen (s) can be isolated. After determining its amino acid sequence, its gene or its genes (DNA) are produced and introduced into a microorganism with a corresponding vector. This recombined microorganism is then either used for the immunization, or the introduced gene product is produced by in vitro cultivation and, after its isolation, is used for the immunization. These methods are now state of the art and can be implemented accordingly (Nature, London 302: 490-495, 1983; Proceeding of the National Academy of Sciences of the USA 82: 1359-1363, 1983). In this way, an infectious vaccinia virus, which expresses the antigen substance of the protective cover, can be produced and thus, as at that time, actively immunized against Variola. The same can be done with genetically recombined Mycobacterium boviε BCG or with attenuated S.typhi.

It is also possible to produce monoclonal antibodies against the protective shell antigen substance by mouse hybridαm method according to the prior art (Nature, London 256: 495-497, 1975). These technical principles are also used for human hybridoma technology.

The composition of the adjuvant mixture can be changed or supplemented. It is also possible, even advisable, to supplement the active immunization with simultaneous administration of other biological substances which can also stimulate the weakened immune system or increase its reaction, such as interleukins and substances with a similar effect.

A special form of immunotherapy is immunocytotherapy. In this form, human cells or human hybridoma cells which produce antibodies against the protective complexes are suspended in a corresponding culture medium and, under sterile circumstances, are implanted in the subcutaneous malignancy carriers in a space closed by porous material. For this, a closed bag, e.g. B. from Milipore membrane or material with appropriate pore size, or other similar porous material. The porous material is permeable to antibodies, proteins, nutrients, etc., but not to cells, which is why rejection of the implanted cells cannot occur and so human hybridoma cells or cells from genetically non-identical donors can be implanted . Similarly, one can perform pieces such immunotherapy with very small tissue ^¬, which consist only of a few cells but ur ^¬ nal cell-cell-contacts-still exist between the cooperating cells.

The antibodies from the Einplantate prevent the inhibition and the forced collaboration of the host organism by the Phänotypausleih and other cells and tissue particles that produce ge for Malignomträgern wichti ^¬ or missing bioactive substances (interleukins, etc.), can be miteinplantiert without in their functions to be inhibited or influenced in the malignancy carrier.

A great advantage of this therapy is that the implant can be completely removed at any time and a new one can be started as required.

Detection method for antibodies against the protective complex antigen substance from body fluids (such as serum etc.):

Isolated lipoprotein shell or its antigen substance is bound and washed on a water-insoluble substrate such as "Affi-Gel 702 Beads" (Bio-Rad) in accordance with the manufacturer's instructions. 0.2 ml of this gel is mixed in a tube with 0.2 ml of patient serum, then incubated either at 37 ° C for 4 h or at 4 ° C for 16, the gel is washed and 0.2 ml of anti-human immunoglobulin serum or its immunoglobulin Fraction which is conjugated with enzyme (peroxidase, alkaline phosphatase, Amersham) mixed in the prescribed dilution and incubated for 2 h at 37 ° C. After washing, the corresponding enzyme substrate is mixed in and the color reaction is evaluated in comparison with the negative control. For dilution and for washing (2 g / 1) ge ^¬ either the manufacturer's buffer or physiological NaCl-phosphate buffer (PBS) solution containing bovine serum albumin utilized.

Detection method for the protective complex antigen substance from body fluids (such as serum, etc.) without any prior isolation of the lipoprotein shell. Rabbit or rat sera or their Ig fraction with antibody activity against the protective complex antigen is bound to form "Affi-Gel 702 Beads" (Bio-Rad). From this gel, 0.2 ml with 0.2 ml of body fluid, such as serum, is incubated in a tube either at 4 ° C for 16 h or at 37 ° C for 2 - 6 h, then the gel is washed and then with 0.2 ml of anti-protection nplex-mouse-ImmunserOm or its IgG component, which are conjugated with enzyme, mixed, incubated at 37 ^β C for 2 h, washed and after adding the appropriate enzyme substrate, the color reaction compared to the negative control was evaluated. Conjugation kit for antibodies is commercially available (Amersham).

Both immune methods can be carried out even more simply if antigen or antibody are bound to microtiter plates instead of to the gel (such as: J. Im unol. 109: 129-135, 1972) and the method is carried out as indicated above. Numerous other methods are known for these purposes, which can also be used.

The sensitivity of other detection methods, such as mRNA detection from serum, can be significantly increased by the combination with the immunoadsorption of the lipoprotein envelope described in this invention. The adsorption is carried out in the constant presence of RNase inhibitor. The adsorbed material is treated with a proteinase (Proteinase K) at 37 ° C for 3 - 5 h and with a detergent, then the RNA is extracted with phenol-chloroform at 60 ° C and after admixing tRNA as career RNA precipitated with alcohol. The RNA extract is examined using nucleic acid hybridization, PCR (or a combination of the two) or by scanning-tuning microscopy for specific sequences of the mRNA of cancer cell origin (such as oncogen mRNA etc.). In this way, the sensitivity of the mRNA detection method and thus the malignancy test can be increased by at least two orders of magnitude, and several thousand cancer cells can already be detected in the host organism.

Claims

Expectations

1. A method of preventing the function exercise of extrazellula ^¬ reindeer, protected by protecting complexes mRNAs of cancer cell origin as transmitter substances of influence (inhibition forced ^¬ collaboration) of the host by the cancer and the interaction Zvi ^¬ rule of malignant cells, in which a vaccine for active and passive immunization, immunoadsorption, immunoassay, immunotherapy and immunocytotherapy, which contains at least one antigen substance of the protective complexes.

2. The method according to claim 1, characterized in that the vaccine is mixed with biological, microbiological and chemical immune ^¬ stimulants, or administered simultaneously therewith.

3. The method according to claim 1, characterized in that the protective complexes of the mRNAs of cancer cell origin are isolated from the sera of patients with active malignant diseases by flotation in an ultracentrifuge.

4. The method according to claim 1, characterized in that the protective complexes of mRNAs of cancer cell origin are isolated from concentrated, cell-free culture fluid from various cancer cultures after cultivation by flotation in an ultracentrifuge.

5. The method according to claim 1, characterized in that one uses the protective complexes according to claims 3 and 4 for active immunization ^¬ nization to obtain specific Immunseirum and here ^¬ to isolate the IgG fraction.

6. The method according to claim 1, characterized in that the specific IgG according to claim 5 is immobilized with anti-protective complex Antibody activity to a substrate such as Sepharose in order to the appropriate protective complexes thereon by immunoadsorption

, adsorb that you wash and then desorb and isolate.

7. The method according to claim 6, characterized in that one uses the protective complexes from Sera of various patent patients with active malignant disease or from concentrated, cell-free culture liquid from several cancer cell cultures after the breeding period according to claim 6 for the isolation of large amounts, in order to prevent prophylactic immunization perform.

8. The method according to claim 6 or 7, characterized in that one carries out the immunoadsorption without desorption to remove the protective complexes ^¬ complexes with their mRNAs from the circulation of cancer patients under sterile and pyrogen-free conditions and via a connected extracorporeal system.

9. The method according to claim 1, characterized in that the protective complexes with their mRNAs are removed from the circulation of cancer patients according to their size by hemofiltration.

10. The method according to claim 5, characterized in that the Fc portion of the IgG specified in claim 5 with anti-protective complex antibody activity is removed and one of these non-Fc fragments is repeatedly administered parenterally for therapeutic purposes, in particular ^¬ special in connection with other procedures and combinations with other forms of therapy.

11. The method according to claim 1, characterized in that cells or tissue particles or human hybridoma cells, etc., which produce anti-protective complex antibodies, and / or those cells which produce useful bioactive substances for the malignant carriers, including cells , which originate from genetically non-identical donors, are implanted in the sunkutis in a space closed by porous walls, which is permeable to proteins, nutrients, antibodies, etc., is not permeable to cells.

12. The method according to claim 1, characterized in that the antigen of the protective complexes from the patient serum without any prior isolation or concentration with the corresponding antiserum or with immunoglobulins with antibody activity against the protective complexes by solid-phase immunoassay or ahn- the method proves.

13 .. A method according to claim 1, characterized gekennzeichent that one examines the antibodies against the Schutzkαmplexe in serum or in other Un ^¬ tersuchungsmaterial using the antigen of the protective complexes by solid-phase immunoassay, or similar methods of detection.

14. The method according to claim 1, 6 and 8, characterized in that the protective complexes adsorbed from the serum (lipoprotein shell with their mRNA) are degraded in the constant presence of RNase inhibitor by proteinase, the RNA is extracted and the product is present for the presence of the mRNAs Cancer cell origin investigated with nucleic acid hybridization, PCR (or by a combination of the two) or by scanning-tuning microscopy.