UA62927C2 - Immunomodulating pharmaceutical composition containing peptides and adjuvants - Google Patents

Abstract

The invention concerns a pharmaceutical composition containing at least one immunomodulating peptide or a protein (fragment) together with an adjuvant. The peptide is derived from a pathogen or a tumour antigen. The adjuvant has the ability to increase the binding of the peptide to the cells of the individual to be treated or increase the absorption of the peptide by the cells and intensify the immunomodulating effect of the peptide. Preferred adjuvants are basic polyamino acids, such as polyarginine or polylysine, which are optionally conjugated with a cellular ligand, for example a carbohydrate group or transferrin. The composition is used in particular as a vaccine, for example a tumour vaccine.

Description

The invention relates to the field of immunomodulation and represents further developments in the field of therapeutic vaccines based on tumor cells, which are mainly based on the following premises: the existence of a qualitative and quantitative difference between tumor and normal cells; on the fact that the immune system has the fundamental property of recognizing this difference; on the fact that the immune system can be stimulated (by immunization with vaccines) to recognize tumor cells based on this difference.

In order to achieve an antitumor reaction, at least two conditions must be met. First, it is necessary to express an antigen against tumor cells, which is not found in normal cells or is found in a limited way, so that a qualitative difference between normal tissue and tumor tissue becomes possible. Secondly, it is necessary to activate the immune system accordingly so that it responds to this antigen. A significant difficulty in the immunotherapy of tumors lies in the low immunogenicity, primarily of humans. In the early stages, tumor-associated and tumor-specific antigens are identified, which are neo-epitopes and can be potential targets for the immune system to attack. If the immune system fails to eliminate tumors that express these neo-epitopes, then this is due to a failure of the immune response to the neo-antigen, rather than a deficiency of the neo-epitopes.

In cell-based cancer immunotherapy, two general strategies are being developed: on the one hand, adaptive immunotherapy, which uses the development of immunoreactive tumor-reactive T-lymphocytes and their repeated administration to patients; on the other hand, active immunotherapy uses tumor cells in anticipation of eliciting either a new or enhanced immune response that will lead to a systemic tumor response.

Antitumor vaccines based on active immunotherapy are obtained in different ways; an example is irradiated tumor cells that are replaced by adjuvant immune-stimulating agents such as

Sogupirakiegrit raguit or Vasiish5 SaItene ("zcehip (VS) to cause an immune reaction to the tumor antigen (Oeydep and Oi, 1991).

Recently, genetically modified tumor cells have been used in active immunotherapy against cancer. An overview of various attempts to eliminate tumor cells by enhancing immunogenicity due to the introduction of various genes is given in 2ayoika!i et al., 1993. One of the previously known directions involves the use of tumor cells that are genetically modified for cytokine production.

Identification and isolation of tumor antigens and tumor-associated antigens (Ta5), as well as peptides released from them (for example, described in Muogye! et al., 1994a and 19946; Saitei et al. 1993, Heptap et al., 1989 , Tir-reiv et al. 1993, or described in various published international applications MO 92/20356, MO 94/05304 UUO 94/23031, MO 95/00159) is a prerequisite for the development of the further direction of the use of tumor antigens as immunogens for vaccines against tumors , both in the form of proteins and peptides. From the works of Voop et al., 1992; Voop et al., 1994; Voop et al., 1995; maps of Reei and others. 1995; Map deg Eupaye, 1995 it is known that malignant melanoma is represented by peptides released from tumor antigens, in relation to MNO-1. An antitumor vaccine that is a tumor antigen per se has not yet achieved immunogenicity sufficient to elicit the cellular immune response required to kill tumor antigen-containing tumor cells (Magspapa et al., 1995). In order to achieve the fact that the antigen-presenting cells ("Apiideprgesepiipd seiIIv", "APS85") carried a certain peptide antigen on their surface, it was proposed to "add a pulse" to the peptides, which as a result leads to the filling of the cells with peptides (TukKosip5kKi, etc., 1996). It has also been noted that co-administration of adjuvants only relatively enhances the immune response (Oeidep and Oi, 1991).

The third direction in active immunotherapy, which aims to increase the effectiveness of anticancer vaccines, is based on xenogenized (alienated) tumor cells. This concept is based on the premise that the immune system responds to tumor cells that express a foreign protein, and in connection with this response, an immune response is elicited against those antigens that are delivered by the tumor cells of the vaccine.

The central role in the regulation of the specific immune response is played by a trimolecular complex consisting of components: the antigen-receptor of the T-cell, the MHC (Ma|og-Nisiosotraiariiu Sotriech) molecule and its ligands, and is a peptide fragment isolated from the protein.

MNO-molecules (and the corresponding NO-Av molecules) are peptide-receptors, which, with the required specificity, ensure the binding of many different ligands. The prerequisite for this is allele-specific peptide motifs, which reveal the following specificity criteria: peptides have a certain length, depending on

MNO-haplotype, for MNO-1-haplotype, as a rule, from eight to ten amino acid residues.

Usually, two of the positions of amino acid residues form a so-called "anchor", which is surrounded by closely related side chains through one amino acid or through an amino acid residue.

The exact position of the anchor amino acids in the peptide and the requirements for their properties vary depending on

MNeO haplotypes. The C-terminus of peptide ligands is often an aliphatic or charged residue.

Such MNO-peptide-ligand components are known so far in relation to H-2KU, Ke, KK, Kkt, r, NI А-А"0201,

A"0205 and B"2705 haplotypes.

As part of the protein metabolism inside the cell, non-degenerate, degenerate and foreign gene components, for example, viral proteins or tumor antigens, are broken down into small peptides, some of which are potential ligands for MNO molecules. This creates a prerequisite for their presentation through

MNO-molecules, the result of which is a cellular immune response, and, in particular, it is not yet fully understood how the peptides are produced in the cell as MNeO-ligands. Foreign or extraneous proteins and their fragments can be recognized, bound and eliminated by means of immunoglobulin, which represents an immune response. This also applies to all tumor antigens: on the example of tumor-associated antigens MOS1, CEA and HER2/novi, it has been proven that immunoglobulins that express themselves specifically for these proteins can recognize and kill cells containing the protein. In order to cause a specific tumor-antigen immune response, various forms of MISI and CEA were tested as immunogens (for example, in recombinant pox vectors; Vgopie et al., Journal of Ittipoi. 154: 5282, 1995) in animal experiments and in preliminary tests.

As part of the proposed invention, the considerations used in the development of cellular antitumor vaccines were continued: at the time when non-malignant, normal cells of the body are allowed by the immune system, the body reacts to the normal cell, if it, for example, on the basis of a viral infection, synthesizes proteins foreign to the body with immune protection . The reason for this is that MNO molecules represent foreign peptides, which belong to proteins foreign to the body. As a result, the immune system notes that something unwanted, foreign is happening to this cell. APC5 (these include macrophages, dendritic cells, Langerhan cells, B cells, and possibly recently discovered biphenotypic cells that exhibit properties of both B cells and macrophages; Tukosip5ki et al., 1996) are activated, generate new specific immunity and eliminate the cell.

Tumor cells contain appropriate tumor-specific tumor antigens, which are not sufficient vaccines because they are ignored by the immune system due to their low immunogenicity. If a tumor cell is loaded not with a foreign protein, but with a foreign peptide, then in addition to foreign peptides, the tumor's own antigens of this cell are also perceived as foreign. As a result of such alienation with the help of a peptide, it is possible to achieve that the cellular immune response caused by foreign peptides will be directed against tumor antigens.

The reason for the insignificant immunogenicity of tumor cells can be not only a qualitative, but also a quantitative problem. For a peptide derived from a tumor antigen, this may mean that it is presented by INO molecules, but at a concentration that is insufficient to elicit a tumor-specific cellular immune response.

An increase in the number of tumor-specific peptides on the tumor cell should promote the rejection of the tumor cell, leading to a cellular immune response. It has been proposed to present the tumor antigen or a peptide derived from it on the cell surface so that it is transmitted by means of DNA encoding the corresponding protein or peptide, as described in international applications UUO 92/20356, UUO 94/05303, MO 94/23031 and UMO 95 /00159.

In the German patent application P 19543649.0, a cellular vaccine is presented, containing as an active component tumor cells loaded with one or more peptides in such a way that the tumor cells together with the peptides are recognized by the individual's immune system as foreign, because they can be on one side " "foreign peptides" or "xenopeptides" means that they are different from peptides derived from proteins that are expressed by an individual's tumor cells. Another category of peptides is derived from tumor antigens that are expressed by an individual's cells. These peptides of another category contribute to increased immunogenicity due to the fact that they exist on tumor cells of the vaccine in a concentration that is higher than the concentration of the same peptides on tumor cells of the individual.

The basis of the proposed invention is the task of creating a new medicinal composition, in particular, a vaccine with an immunomodulatory effect.

In the development of the concept of a cellular vaccine, published in the German patent application P 19543649.0, within the framework of the proposed invention, a medicinal composition containing peptides with an immunomodulatory effect not on cells, but in combination with an adjuvant to cause or enhance cellular and/or humoral, is considered. mainly a systemic immune response, to pathogens or an antitumor reaction or to induce tolerance against proteins with an autoimmune effect.

It was unexpectedly found that certain excipients, for example, polycations, which became known in 1965, can facilitate the transfer of proteins in cells (Ruzeg et al., 1965; Kuzeg et al., 1978; Zpep et al., 1981), increase the immunogenicity of peptides.

The invention relates to a medicinal composition containing one or more peptides with an immunomodulatory effect, proteins and protein fragments in combination with an auxiliary agent. The composition is distinguished by the fact that the auxiliary agent has the ability to increase the degree of binding of the peptide, protein or protein fragment to the cells of the body of the individual to be treated or to increase the degree of penetration into the cells and contribute to the strengthening of the immunomodulatory effect of the peptides, proteins or protein fragments.

In the future, for brevity, peptides with immunomodulatory action, proteins or protein fragments will be referred to as "peptides". The term "peptide", if it does not refer directly to ligands, can temporarily denote fragments of proteins or proteins from which the peptide is derived, or is a product of cellular breakdown. "Immunomodulatory effect" means the occurrence or enhancement of a cellular and/or humoral, preferably systemic, immune response. In this form of real embodiment, the medicinal composition according to the invention acts as a vaccine. In a preferred embodiment, the peptides are ligands for at least one MNO molecule expressed by the body of the individual to be treated.

In accordance with international traditions, the MNOS-molecules of a person will be designated as "NO A" (Human Leukocyte Antigen) in the future.

The "cellular immune response" should be understood, first of all, as cytotoxic T-cell immunity, which, due to the generation of cytotoxic CP8-positive T-cells and CPA-positive helper T-cells, contributes to the destruction of tumor cells or cells affected by a pathogenic agent.

"Humoral immune response" should be understood as the production of immunoglobulins, which selectively recognize tumor cells or structures created by pathogens, and further, together with other systems such as complement, AOSS (antibody-dependent cytotoxicity) or phagocytosis, contribute destruction of these tumor cells, pathogens or cells affected by them.

The peptide contained in the vaccine is derived from an antigen or is, in the case of a protein, an antigen against which a cellular and/or humoral immune response should be elicited. This achieves the result that T-cells recognize, respectively, other cytotoxic effector cells containing the causative agent of the disease, tumor cells containing antigen, and/or generating antibodies.

For immunization against pathogens, such as bacteria, viruses, parasites, proteins or peptides are used, which are the protein of the corresponding pathogen or obtained from it. First of all, proteins that remain undamaged by high mutational clusters of these pathogens are suitable for this purpose.

For example, NRMU16/17 (Nytap Rarriuta Mite5; Reykatr et al., 1995); Neraiyiz V Migi5 Soge Apiidep (MibegPo et al., 1995), Riazto-diit Vegdpets (Uatanptp et al. 1992), Ipiiseplamigiz-MyKiIeorgoieip/ Nerashis S Migyv5.

A real embodiment of the invention is a protein that promotes the emergence of an antitumor reaction, a tumor antigen or a peptide derived from a tumor antigen; at the same time, a medicinal composition is used as an antitumor vaccine. In the case of therapeutic use of the vaccine, the peptide is obtained from the tumor antigen, which is expressed by the individual's tumor cells. These tumor antigens are, for example, antigens that are expressed by the individual's body in such a small concentration that the tumor cells are not recognized as foreign.

Antigens of an individual's tumor can be determined by standard methods in the course of establishing a diagnosis and developing therapeutic measures: tumor antigens can be detected in the simplest way - immunohistochemically using antibodies. If the tumor antigens are enzymes, such as tyrazinases, they can be detected using enzyme studies. The ET-RSV method can be used to determine tumor antigens with a known sequence (Voop, T., et al. 1994; Soshie, R., et al. 1994; Umeupapiv, R., et al., 1994). Further methods of detecting tumor antigens are studies based on STI 5 with specificity that corresponds to determined tumor antigens. Studies of this kind were described by Negip et al., 1987; Soshshie et al., 1993; Soh et al., 1994; Kimotsipi et al., 1995; Kazhakati et al., 1995, as well as in international application UMO 94/14459. Various tumor antigens or peptide epitopes obtained from them, which are applicable within the framework of the proposed invention, are borrowed from these sources. Examples of tumor antigens used are given in recently published review articles by Kozeprego, 1996 and Nepaegsop Ripp, 1996.

Regarding the tumor antigens used, this invention is not subject to limitation.

An antitumor vaccine containing a tumor antigen or a peptide derived from it can be used not only for therapeutic, but also for prophylactic purposes. For prophylactic use, it is advisable to prescribe a mixture of peptides obtained from representatives of frequently encountered tumor antigens. In the therapeutic use of an antitumor vaccine, one or more peptides are used, and they are believed to be present in an individual's tumor antigens. The antitumor vaccine according to the invention, compared to the cellular vaccine, has the advantage that it is useful for an individual at a relatively early stage (stage 1 and 11) of the disease, when it is not yet possible to obtain enough tumor cells for the production of a cellular vaccine.

In a preferred embodiment of the invention, the peptide will bind to the MNO-1I or MNO-1II subtypes of the individual being vaccinated, due to the occurrence of a cellular immune response. In this way, the peptide reveals or contains a sequence that ensures its binding to the MNO molecule.

Another real embodiment of the medicinal composition in the form of an antitumor vaccine contains, among other things, a polypeptide that has an immunostimulating effect, in particular, a cytokine. In the best form of the actual embodiment of the invention, interleukin 2 (II-2) or 5M-C5E is used as a cytokine, for example, in a dosage of about 1000 units; other examples for cytokines: 1-4, I/-12, IEM-o, IEM-D, TEM-u, IEM-o, TME-o, as well as their combination, for example, 1I--2-AIEM-u, 1 -2--111 -4, 1-24 TME-o or TME-o--TEM-u.

The medicinal composition in a real embodiment according to the invention serves the purpose of achieving stability in relation to proteins or fragments of proteins that cause autoimmune-induced diseases, that is, it is used for the therapy of autoimmune diseases. The peptides used in this actual embodiment of the invention are derived from proteins that cause autoimmune diseases.

In contrast to the use of the subject of the invention as an antitumor vaccine or as a vaccine against pathogenic agents, in which the peptides will combine with a region of the original protein (tumor antigen or protein of the pathogenic agent), in fact, in such a way that the peptide is recognized as the "original antigen", in the implementation of this invention, for the treatment of autoimmune diseases and other similar diseases, peptides are used that show critical differences in the amino acid sequence of the original protein. These peptides, based on their anchor positions, are bound to the MNO molecule, but have mutations in their sequence that cause them to function as antagonists that again turn off activated specific T-cells (Kegsey and Aiep, 1996).

Both natural antagonists found in viruses (Vepoientsi et al., 1994) and antagonists that were found as a result of systematic research, for example, thanks to the screening of peptide libraries, are suitable as peptide antagonists. An example of peptide antagonists are peptides that are able to turn off T-cells specific for Mueip Vasis protein. They were tested for effectiveness in animal experiments (VhosKe, 1996).

The peptide, which must cause a cellular immune response, must be bound to the MNSO molecule.

In order to result in an immune response in an individual's body, the individual being treated must have the appropriate NIG A molecule in their body. Determination of the NI A-subtype of an individual is, in relation to the occurrence of a cellular immune response, one of the essential prerequisites for the effective use of the peptide for this individual.

A patient's NO A subtype can be determined by standard methods, such as the microlymphotoxicity test (Practical Immunology, 1989). This test is based on the principle in which lymphocytes isolated from the patient's blood are mixed with therapeutic serum or with a monoclonal antibody against a specific HGA molecule in the presence of rabbit complement (C). Positive cells dissolve and are able to take up the indicator dye, while undamaged cells remain unstained.

To determine the NHA-I or NG A-II haplotype of an individual, CT-RSA can also be used ("Nemegv

Tgapzsgirsase Roiutegase Spaip Keasiip") (Scgt. Chapters 2 and 15). To carry out this study, blood is taken from an individual and RNA is isolated from it. This RNA is subjected to reverse transformation, resulting in the cDNA of the individual. The cDNA serves as a template for the polymer chain reaction with the starting by pairs that specifically cause an increase in the OMA fragment corresponding to a certain NI A haplotype. If a DNA band appears after agarose gel electrophoresis, the individual's organism expresses the corresponding NI A molecule. If a band does not appear, the patient is negative in this respect.

The determination of the peptide used according to the invention by means of NI A-molecule takes place relative to its anchor amino acids and its length; established anchor positions and length ensure that the peptide fits into the peptide binding groove of the individual's corresponding NI A molecule. The consequence of this is the stimulation of the immune system and the occurrence of a cellular immune response, which, in the case of the use of a peptide obtained from a tumor antigen, is directed against the individual's tumor cells.

Peptides used in the framework of the proposed invention are present in a wide band. their sequence can be obtained from immunogenic proteins of natural origin or their products of cellular decay, for example from viral or bacterial peptides, from tumor antigens; they can also be antagonists of peptides derived from proteins induced in autoimmune diseases.

Appropriate peptides can be selected on the basis of peptide sequences known from the literature.

In the case of the occurrence of a cellular immune response, peptides can be determined, for example, on the basis of peptides obtained from immunogenic proteins of various origins (described in relation to various NI A-motifs by Kattepzwee et al., 1993, Kattepzee et al., 1995, RaikK et al. , 1991) and which fit into the connecting groove of molecules of the corresponding NI A-subtype.

For peptides that reveal a partial sequence of a protein with an immunogenic effect, on the basis of already known or, if necessary, still established polypeptide sequences, by means of sequence correction taking into account NI A-specific requirements, it can be established which peptides represent suitable candidates. Examples of suitable peptides can be found, for example, in Kattepzvsee et al., 1993, yak et al., 1991, and Kattepsee, 1995, as well as in international application MO 91/09869 (NIM-peptides); peptides obtained from tumor antigens, described in published international patent applications UUO 95/00159,

UMO 94/05304. References are given to bibliographic sources and articles cited in connection with the problem under consideration. The best candidates include peptides whose immunogenicity has already been revealed, that is, peptides derived from known immunogens, for example, viral or bacterial proteins.

Instead of using original peptides that fit the binding grooves of MNO-1I or MNO-1II molecules, i.e., peptides obtained unchanged from naturally occurring proteins using established minimum anchor position and length requirements based on the sequence of the original peptides, changes can be made, if as a result of these changes, the effective immunogenicity of the peptide, which is in its connecting chemical affinity with the MNO molecule and its ability to stimulate T-cell receptors, will be not only not reduced, but also enhanced. In this case, according to the invention, peptides of artificial origin are used, which are synthesized according to the requirements of their ability to bind to the MNO molecule. So, for example, starting from the H2-KU-ligand Geu Rpe Sim Aia Pe Si Siu Rpeye (I REAIIESREI) amino acids that are not anchor amino acids can be changed to obtain a peptide of the sequence

Rpe Re Na su Aa Hey si Sish Ne (ERISAGEE)Y); in addition, the anchor amino acid He at position 9 can be replaced by He. Detection of epitopes of MNO-I or MNO-II ligands or their variations can be achieved according to the method described in Kattepzvsee et al., 1995. The length of the peptide corresponds, in the case of its agreement with MNO-I | molecule, preferably a minimum sequence of 8 to 10 amino acids with the necessary anchor amino acids. The MNO-II binding motif, which extends to new amino acids, reveals an increased degree of degeneracy in the anchor positions. Recently, on the basis of X-ray structural analysis of MNO-II molecules, techniques have been developed that make it possible to accurately analyze MNO-II binding motifs and, based on this, variations in peptide sequences (Kattepsee et al., 1995 and the original bibliographic sources cited there) .

If necessary, the peptide can be extended on the C- and/or on the T-terminus, since such an extension does not impair the ability of the peptide to bind to the MHC molecule and since the extended peptide can respond to the minimal sequence at the cellular level.

In a real embodiment of the invention, the peptide is negatively charged. Due to this, the peptide can be elongated with negatively charged amino acids or negatively charged amino acids can be introduced into the peptide mainly in positions that are not necessary for recognition due to specific

StTI5, or as anchor amino acids to achieve electrostatic binding of the peptide with a polycationic auxiliary such as polylysine.

In a real embodiment of the invention, the antigen is not present in the form of a peptide, but in the form of a protein, a protein fragment or a mixture of proteins or protein fragments. Within the framework of the proposed invention, large fragments of proteins or whole proteins are used, which can be guaranteed to be expected after application

ARS" will be transformed by the patient into peptides that are suitable for the MNO molecule.

The protein is an antigen or a tumor antigen, from which fragments are made that are preserved after transformation. In this form of the actual embodiment of the invention, the adjuvant serves the purpose of enabling or enhancing the stress of cells, especially APCs such as dendritic cells or macrophages, with tumor antigens or fragments. Proteins or protein fragments perceived in this way will be transformed by cells and can, accordingly, be presented to the MNeO correspondence of immunoeffector cells and, thus, cause or enhance the immune response (Vgasiaie and Vgasiaie, 1991; Komaszomish VapKauvki and KosK, 1995; MogkK by Kosk, 1996).

The actual embodiment of the invention, in which proteins or large fragments of proteins are introduced as antigens, has the advantage that there is little dependence on the individual's A-type NI, because the protein is broken down into a large number of fragments and thus a wide range of variation is possible. suitable" forms of the peptide.

In the case of input of proteins or protein fragments, the identity of the converted end products can be confirmed by chemical analysis (Edman cleavage or mass spectrometry of the converted fragments; see the review article by Kattepvsee et al., 1995 and the bibliography cited therein, or biological tests (the ability of APC5 to stimulate T cells specific for the transformed fragments).

The selection of peptide candidates capable of inducing a cellular immune response is traditionally carried out in several stages. In general, candidates are tested using a test, first of all, for their ability to bind to the MHC molecule.

The research method used in this case is based on the ability of peptides to stabilize voids

MHC molecules, as described, for example, in ZshiShbeg et al., 1994, and Msipiuge et al., 1996. In this case, the peptide is applied to cells capable of expressing the corresponding MHC molecule, but not binding endogenous peptides due to a genetic defect in MNeO-compliance. Suitable cell lines of this type are

AMA-5 (mouse) and T2 (human) or transforming variants thereof. In this way, only MNO molecules stabilized by the corresponding peptide can be detected. This is achieved mainly with help

EAS5 analysis, which is based on flow cytometry (RIom/ Suioteigu, 1989; РАС5 Mapiade TM Ozege

Syside, 1994, Seyi Otsebzi TM Ozegz syside, 1994). Stable MNOS molecules are suitable anti-

MNe-antibody and a second (for example, polyclonal) antibody, which is labeled with a fluorescent dye, for example, RITS (Ripnogeseipizoipiosuapa). Individual cells are stimulated by a laser of a certain wavelength in the stream; measure the fluorescence emitted and which depends on the number of peptides bound to the cell.

Another method for determining the amount of peptides bound to the cell is 5saispaga-Rioi, as described in Beze et al., 1994. For this purpose, a peptide labeled, for example, 1725 is used. This peptide is incubated overnight with isolated or recombinantly obtained MNO molecules at at a temperature of 4"C at various defined concentrations of the peptide. To determine the non-specific interaction of the peptide in some tests, an excess of unlabeled peptides is added, which paralyzes the non-specific interaction of the labeled peptides. Then the non-specifically bound peptide is removed, for example, by means of helium chromatography. The amount of bound peptides are determined in a scintillation counter on the basis of emitted radioactivity.The processing of the obtained data is carried out by standard methods.

A review of methods for characterizing MHC/peptide interaction, MHC ligand analysis, and peptide binding assays performed within the framework of the proposed invention can be found in Kattepsee et al. 1995.

In the next step, peptide candidates with good coupling quality are tested for their immunogenicity.

The occurrence of a cellular immune response can be confirmed by determining the peptide-specific STI 5, for example, using the method described in Sigtepe Rgoiosoi!5 ip Ittipo!podu, chapter 3, or in Viotrbega et al., 1993. Another determination of the presence of a cellular immune response is given then , when in the absence of T cells in the experimental animal (which is achieved by treating the animal with antibodies that displace CO4- or CO8-cells) no immune response occurs (Siggepi Rgoiosoi5 ip Ittipo/odu. Chapter 3).

Cellular immune response can also be detected by determining the reaction of "delayed hypersensitivity" - OTN-reaction in immunized animals. To do this, peptides are injected into the soles of the paws of mice, after which the degree of swelling at the injection site is measured (Sgoptanp et al., 1995; Rissenyi et al., 1994).

The induction of a humoral immune response by peptides, which are foreign antigens that are expressed by the body to be treated in a small concentration, can be determined by detecting specific antibodies in the serum. Antibodies in serum are used to determine the titer

Eplute Hypkei Ittipoazzau (EGIZA). At the same time, specific antibodies are determined by their binding to the peptide used for immunization using a color reaction. An alternative method is

Umeziet Vioi. At the same time, the specific antibodies of the serum are bound to the peptide mobilized on the membrane.

Bound antibodies are finally confirmed again by a color reaction (references to both methods are given in Sigtepe Rgoiosoi5 ip Ittipiodu. Editor Soiidap et al., 1991).

First of all, after vaccination with foreign antigens, for example, of viral origin, the formation of antibodies should be expected. It is possible that specific antibodies may be formed instead of mutated or overexpressed peptides derived from cellular tumor antigens. Tumor destruction by such antibodies can occur after binding of antibodies to tumor cells with the help of other components of the immune system. As other components can be used, for example, antibody-dependent cytotoxicity (AOSS) or phagocytosis with the help of macrophages (Ko I.M., Vgo5ioi I., Maie O.K. Ittipoiiodu, Spigspii! Simipd-v(ope ).

The generation of a cellular immune response by peptides derived from proteins of unknown immunogenicity can be tested as described in Rimoyip et al., 1995, or in Kamzhakat et al., 1994a.

This requires T-cells that are able to recognize the desired peptide if it is presented by MNO molecules. In the case of presence of peptides that originate from tumor cells, the corresponding T-cells are obtained from tumor-infiltrated lymphocytes (TI 5), as described in Kaulakata et al., 19945; in the case of the presence of foreign peptides, such T-cells are similarly obtained from peripheral blood. T-cells are incubated with cell lines such as T2 (Alehapaeg et al., 1989) or EMA-5 (Kagte et al., 1986) mixed with the appropriate peptide and dissolved in the case of an immunogenic peptide.

Another opportunity to test MHC-related peptide candidates for their immunogenicity lies in the study of the combination of peptides with T2 cells. Such a test is based on the peculiarity of T-2 cells (Alekhapaeg et al., 1989) or KMA-5 cells (Kagge et al., 1986) that they manifest themselves defectively in the TAP-pestide transfer mechanism and stably present MNO molecules only when peptides presented in MNO correspondence are applied to them. For the test, for example, T-2 cells or EMA-5 cells are used, which are stably transferred using the NI A gene, for example, NI A-A1 and/or NI A-A2 genes. If the cells are mixed with peptides that are good Ni A ligands, while they are presented in a NI A conformation such that they can be recognized by the immune system as foreign, such peptides contribute to the presence of NI A molecules on the surface of the cell in a significant amount. Determination of NIH A5 on the cell surface, for example with the help of monoclonal antibodies, allows identification of the corresponding peptides (Maipaiyi; et al., 1995; Zukshem et al. 1994)3. In this case, it is advisable to use a standard peptide with good Ni A-binding ability.

Taking into account the wide applicability of the medicinal composition according to the invention, it is advisable to use a mixture of several peptides, each of which can be connected to another MNO molecule, preferably to one of the two or three most frequently occurring MNEO subtypes. With a vaccine based on a mixture of peptides that can be associated with these haplotypes, a wide range of patients can be covered.

In a real embodiment of the invention, the vaccine may contain large peptide sequences. The applied peptides may in this case differ on the one hand in that they are associated with different NI A subtypes.

This achieves coverage of most or all of the NO A subtypes in a single patient or a large group of patients.

A further, under known conditions, additional variability in the peptides used may be that the peptides bound to a particular NI A subtype differ in their (not for NI A compounds) defining sequence, whereas they originate from e.g. , from different proteins of the same pathogens or from different pathogens. Such variability can lead to increased stimulation of the immune response or immunization against various pathogens.

The number of active peptides in the composition according to the invention can vary in a wide range.

The number of peptides depends, in particular, on the type of application and the corresponding formulary. The amount of peptides introduced should be from the order of 1O0tsdg to the order of 5000ng per dose, in general - from 1.04g to the order of 1000ng, in particular, from the order of 10shg to the order of 500tsg. Administration can be carried out once or multiple times, with multiple applications it is advisable to limit to at least three administrations.

In particular, with therapeutic use, the application can be carried out at intervals (for example, from 1 x a week to 1 x a month) for any length of time that depends on the specific immune status of the patient or the course of the disease.

The medicinal composition according to the invention can also be used ex mimo: the principle of possible application is that APC5, for example dendritic cells, are cultured ex mimo, the cell culture is incubated with the composition according to the invention, and APC5, which represent a peptide in MNOo- compliance, administered to the patient undergoing treatment. For this variant of application, methods known from bibliographic sources are used, for example, as described in Rogdadog and Siiroa, 1995: Moshpa and Ipabre, 1996.

The excipient contained in the composition according to the invention has the properties of facilitating the entry of peptides into cells or binding of the peptide to the patient's cells and enhancing the immunogenicity of the peptide. An adjuvant can, for example, at least temporarily permeable the membranes of the cells into which the peptide must enter in order to promote its penetration into the cell. It may be an advantage, but not necessarily, that the peptide is linked to the auxiliary agent, for example, through an electrostatic interaction between the electropermeable peptide and the polycationic auxiliary agent. The introduction of the peptide into the cell can be caused by the fact that the peptide, based on its spatial proximity to the cell membrane, can penetrate through it as soon as the auxiliary means ensures its absorption. The action of the adjuvant can be based on the fact that it increases the critical concentration of the peptide on the cell surface for penetration into the cell, or it carries out phagocytosis or fluid movement of the peptide into the cell.

It is unexpected that the presence of an auxiliary agent enhances not only the penetration of the peptide into the cell, but also increases the immunomodulatory effect of the peptide, which can be reduced to the correct presentation of the peptide through MNO molecules.

The medicinal composition may contain excipients, as well as almost all membrane-permeable substances used for the transfer of nucleic acids into cells; in this connection, a reference is made to the publication of the international application UMO 93/19768, which lists substances of this kind.

In a preferred embodiment of the invention, the auxiliary agent is a basic polyamino acid or a mixture of basic polyamino acids.

The degree of polymerization of polyamino acids can vary in a wide range. It ranges from order 5 to order 1000, in particular, from order 15 to order 500.

Polyarginine is preferably used as an auxiliary within the framework of the proposed invention.

Another preferred adjuvant within the framework of the proposed invention is polylysine.

Examples of other suitable, in particular, polycationic organic compounds (basic polyamino acids) are polyornithine, histone, protamine, polyethyleneimine or mixtures thereof.

The auxiliary agent, if necessary, can be connected with cellular ligands, for example, with transfer, dr 120, GO. (low-density hypoprotein), x-fetuin, ESE (epidermal growth factor) - peptides or with a representative of other cellular ligands, which are described in the international application UUO 93/07283, for the transfer of DNA by means of receptor-mediated endocytosis, carbohydrate residues, for example, mannose or fucose (ligands for microphages) or antibodies (or their fragments) instead of proteins on the cell surface. If necessary, polycationic auxiliaries are used, for example, polyagrinin or polylysine, which under known conditions are bound to cellular ligands as a component of a complex with DNA, for example, in the form of plasmid-DNA.

The DNA may be free of functional protein coding sequences, in which case the DNA is an empty plasmid.

In a real embodiment of the invention, DNA contains sequences encoding immunomodulatory proteins, in particular, cytokine, for example, 1-2, interferon, OM-C5E.

In order to investigate the mechanism of peptide transfer using basic polyamino acids, the release of lactate dehydrogenase (ION) is measured within the framework of the proposed invention. While in the processed polyarginine samples the concentrations of isolated OH were practically not detected, after incubation with polylysine in cellular excess conditions, high concentrations of ОН were detected. These results allow us to conclude that the effect of polylysine can be reduced to the permeability of the cell membrane.

Without being limited by theory, it can be said that the effect of the medicinal composition according to the invention is that the peptide, with the help of an auxiliary agent, penetrates into the cells representing the target, or binds to the cells found in the endothermal layer of the skin. The target cells can be antigen-presenting cells, from which the peptide under known conditions after the process can be presented by B and/or T cells. Examples of target cells are macrophages, fibroblasts, keratinocytes, Langerhans cells, dendritic cells or β-cells.

Within the framework of the proposed invention, it was investigated whether small peptides in the presence of basic polyamino acids or glycosylated forms of polycations can be accepted in an increased volume by similar macrophages, which (present antigen cells (APC5). Based on the results of the study of sugar residues, it became known that they are accepted by macrophages through receptor-mediated endocytosis. Observation of APC5 revealed that they are an in vivo (in vivo) cell type that takes up peptides and presents them to other immune cells. Results of in vivo (outside the body) experiments showing that APC5 in the presence of the tested adjuvants endocytose an increased number of peptide antigens, is a reference to the fact that these adjuvants are also capable of enhancing the presentation of peptides against cytotoxic effector cells, as well as causing their activation, which leads to an increase in the immune response against targets contained in the vaccine.

As auxiliary means, components in the form of particles can also be used under known conditions in addition to the auxiliary means mentioned above. Materials used for the synthesis of peptides, for example, silica gel or artificial resin, are suitable for particles, as they are physiologically receptive and it is possible to obtain particles from them that are small enough to penetrate the cell.

With the help of auxiliary means in the form of particles, it is possible to obtain high local concentrations of the peptide, which facilitates its "acceptance" by the cell.

The type of adjuvant adopted, the feasibility of its modification by cellular ligands or addition of DNA, as well as the required amount of adjuvant relative to the peptide can be determined empirically, for example, an arbitrarily chosen ratio of peptide to adjuvant (which can fundamentally vary over a wide range) can be determined by titration.

The testing of auxiliaries can be carried out in principle by the same methods as the testing of peptides, under known conditions, in several stages.

The ability of the auxiliary agent to increase the binding and/or internalization of the peptide with APC5 can be measured, for example, at the first stage, when APC5 is incubated with fluorescently labeled peptides and the auxiliary agent. Adjuvant-induced increased uptake or binding can be determined by flow cytometry by comparison with cells that are mixed only with peptides.

At the second stage, the excipients being tested ip migo (outside the body) can be studied to determine whether and to what extent their presence in the presence of the peptide affects APC5, and the MNO concentration on cells can be measured using the methods described above for testing peptides.

The next opportunity to test the effectiveness of the aid is to use an IP migo simulation system. At the same time, АРС35 is incubated together with an adjuvant and a peptide and the relative activation of a T-cell clone that specifically recognizes the used peptide is measured (Soyidap 1991 Gore 1993).

The effectiveness of the formulation can also be proven through the cellular immune response by confirming the "delayed hypersensitivity" (VTN) reaction in immunized animals.

Finally, the immunomodulatory effectiveness of the formulation is determined in an experiment on animals.

For this purpose, tumor models with known peptide sequences recognized by immune cells can also be used. A vaccine containing a peptide and an adjuvant can be used in varying ratios of peptide to adjuvant and total amount. The degree of protection against tumor growth is a measure of the effectiveness of an anticancer vaccine.

The medicinal composition can be taken parenterally, topically, orally or locally. It is preferably administered parenterally, for example subcutaneously, intradermally or intramuscularly, preferably subcutaneously or intradermally, to primarily reach skin cells (keratinocytes, fibroblasts), dendritic cells, Langerhans cells or macrophages as target cells. As part of tumor therapy, the antitumor vaccine can also be used intratumorally.

The composition for parenteral use according to the invention is presented in the form of a solution or suspension of peptides and an auxiliary agent in a pharmaceutically suitable carrier, preferably an aqueous carrier. Water, buffer water, saline solution (0.490), glycine solution (0.390), hyaluronic acid and similar known carriers can be used as an aqueous carrier. In addition to aqueous media, such solvents as, for example, dimethylsulfoxide, propylene glycol, dimethylformamide and their mixtures can be used. The composition may, in addition, contain pharmaceutically suitable excipients, for example, buffer substances, as well as inorganic salts to achieve normal osmotic pressure and/or effective lyophilization. Examples of this type of additives are sodium and potassium salts, for example, chlorides and phosphates, sucrose, glucose, protein hydrolysates, dextran, polyvinylpyrrolidone or polyethylene glycol.

The compositions can be stabilized using conventional methods, for example, using sterile filtration. The composition in this form can be packaged directly or lyophilized and mixed with a sterile solution before use.

In a real embodiment, the pharmaceutical composition according to the invention is presented in a form for topical application, for example, for dermal or transdermal application. The medicinal composition can be presented, for example, in the form of a hydrogel based on polyacrylic acid or polyacrylamide (OiorepeF, MegsClIe), in the form of an ointment, for example, with polyethylene glycol (PES) as a receiving medium, such as, for example, standard CA B 8 ointment (5095 РЕС 300, 5095 РЕС 1500), or in the form of an emulsion, primarily a microemulsion on the basis of "water in oil" or "oil in water" water, if necessary, with an additive with liposomes. As a penetration accelerator, sulfoxide derivatives are suitable, for example,

dimethylsulfoxide or decylmethysulfoxide, as well as transcutol (diethylene glycol monoethyl ether) or cyclodextrins, pyrrolidones such as 2-pyrrolidone, M-methyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid or M-(2-phydroxyethyl)-2-pyrrolidone, biodegradable and their fatty acid esters, urea derivatives such as dodecylurea, 1,3-didodecylurea and 1,3-diphenylurea, terpenes such as O-dimones, menthone, carvol, citric oxide or 1,8 - cineol.

Other forms of actual embodiment are aerosols, for example, for use as a nasal spray or for inhalation.

The composition according to the invention can also be administered with the help of liposomes presented in the form of emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, layered layers and similar substances. These means by which the composition is administered are a receptive medium for targeted delivery of peptides to a specific tissue, such as lymphoid or tumor tissue, or to increase the half-life of the peptide.

In the case of a real embodiment of the composition according to the invention in a local form, it can also contain a UV absorber, which, when used prophylactically against melanoma, acts at the same time as a means of protection from the sun.

A specialist can select appropriate auxiliary materials and forms from "KeMipdiop'5 Rpagptaseciisa! 5siepsev" 1990.

LIST OF DRAWINGS

Fig. 1: Vaccination of OVA/2 mice against Rae15 mastocytoma using the KUOAMTTTI peptide.

Fig. 2: Vaccination of OVA/2 mice against RZ15 mastocytoma using ZUEREITNI peptide

Fig. 3: Vaccination of OVA/2 mice against P8Z15 mastocytoma using ZUEREITNI

Fig. A4: Vaccination of OVA/2 mice against M-3 melanoma with a mixture of peptides

Fig. 5: Vaccination of OVA/2 mice against melanoma M-3 by the topical method of drug application

Fig. 6: Vaccination of OVA/2 mice against melanoma M-3 metastases

Fig. 7: Treatment of mice with M-3 micrometastases by vaccination with a mixture of peptides

Fig. 8: Polylysine-mediated filling of cells with tyrosinase

Fig. 9: Activation of T-cells of the therapeutic model after vaccination (IRM-y-secretion from the spleen cells of vaccinated animals as a reaction to M-3 cells)

Fig. 10: Induction of antiviral immunity using the influenza nucleocapsid peptide AZMEMMETM and fucosylated polylysine as an adjuvant (STI activation)

Fig. 11: Enhancement of binding of peptides to APC5 with the help of basic polyamino acids

Fig. 12: Ensuring the permeability of the cell membrane with the help of basic polyamino acids (CON- release after treatment of cells with polylysine or polyarginine

Fig. 13: Introduction of polyarginine inside peptides

Fig. 14: Transfer of peptides from bone marrow to antigen-presenting cells

Fig. 15: Increasing the efficiency of moving peptides into antigen-presenting cells using polycationic polyamino acids and histones

Fig. 16: Transfer efficiency depending on the degree of polymerization of basic amino acids

Fig. 17: Movement of peptides with low molecular weight polyarginines

Fig. 18: Influence on the efficiency of movement depending on the charge of the peptide

In the following examples, if there are no special instructions, the following materials and methods were used:

A) Cells a) cell lines

Melanoma mouse cell lines Siotsatap 591 (Clone M-3; ATOSS Massey 53.1), mastocytoma cell lines

РВ15 (АТС MeТ18В 64) and monocyto-macrophage cell lines РЗВ8О1 (АТС T18 63) are obtained from ATOS.

Cells are cultured in OMEM medium with a high glucose content ("Nidpy Siisoze OM EM, She

Tesppoiodiez) with the addition of 10955", 2 MIG. - glutamine and 20 dg/ti of gentamicin. The cells are subjected to the usual tests for the absence of infection with mycoplasma (RSK - Musoriazta Oeiesiup Kiyi, bigagadepe).

Murine cell line "KMA/5 (mouse lymphoma) is described in Kagge et al., 1986 and in | |pddgep and others, 1990.

BYU) cells from the bone marrow, representing the antigen.

First, the femurs of OVA/2 mice are perfused. Bone marrow cells are cultured in OMEM medium with a high glucose content with the addition of 1095 EC5, 595 horse serum, 2tM 1 - glutaline and 20nm /t! gentalicin in the presence of 200 units/ml of OM-C5E mice (Gi et al., 1989; Sepgute

Saturgidde, MA, PERSON). Every 24 hours during the first five days, two-thirds of the medium is replaced to remove unbound granulocytes and B-cells (Ipaba et al., 1992). Both bound and free cells are collected in the interval between 8 and 10 days as a result of incubation with РВ5/5 tM EOTA and at a density of 3x107 cells per well are seeded on a slide of an 8-well microscope (Mips, Kozkiaie, bapetagKk). More than 9,095 cells show a positive reaction with the G4/80 antibody (Epdodep, Satogiad, MA, O5A).

B) Synthesis of peptides

Peptides are synthesized in a peptide synthesizer (Model 433A from Reedrask Kitopiog. Arriei Viosuzietv,

Eozieg Sypu, Kapada) when using Tepiase! 5 RNV (Carr, Tibripdep) as a rigid phase according to the EGtos method (NVTO activation Razitosm, scale 0.25 mm). Peptides are dissolved in 1 M TEAA, pH 7.3 and subjected to purification by reverse chromatography in a Mudas C 18 column. Sequences confirmed by mass spectrometry on MAT GI azeghtai (Rigtipd, Zap dove, Kapada).

C) List of used peptides peptide | sequence | 7/0 | 0 Amino acid Z | MNO-haplotype designation! 77771711 | White numbers -:( F

OKRERI43 | VUABOMEE ///|/777777777yryai HER 11naKka

KRERI45 | RMIEOABVI 0000 tyrosinase.// | 77777777 | naKka OKRER'I46 | UMU5AOTY 0710000 tirovinlaza.// | 77777771 | naKka

CRERIBO | MUZMKKTE 77777777 вшрЯИГ///// 17771711 naka 11111111 ABZMEMMETM 0/0 influevntsna-.7//7/ | 77777771 nako 11111110 nucleocapsidgleptiad | //:////// HER. 77777711

Peptide mixtures

Peptide mixture 1 for M-3 anti-melanoma vaccine: crer143, crer145, crer146, crer150.

Peptide mixture from anti-mastocytoma RZ15 vaccine: crer117, crer118, crer162, crerib3, crer164

O) Production of the vaccine p1) Single-peptide vaccines a) For the production of a single-peptide control vaccine without an adjuvant, the peptide is taken at a concentration of 1mg/ml in PB5. The incubation period before injection is 4 hours at room temperature. p) Single-peptide vaccines with polylysine (if nothing else is specified further, polylysine with a chain length of 200 is used) as an adjuvant is prepared by mixing the peptide and polylysine in the indicated amounts in HB5. The incubation period before injection is 4 hours at room temperature. i) To obtain a vaccine containing the Tibdg effective peptide, mix 11.8 mg of polylysine with 160 mg of Creri17 peptide in a total volume of 1 ml HB5. iii) To obtain a vaccine containing 100 dg of the effective peptide, mix 74 dg of polylysine with 1 mg of the crer117 peptide in a total volume of 1 ml of HB5. c) A single-peptide control vaccine with Ipsotriei Egespaz5 Idiimap5 (adjuvant) (IBA) is prepared as a result of emulsifying the peptide and IRA in the specified quantities. The incubation period until the moment of injection is ЗОхв. at room temperature. j) For the control vaccine containing 1 bdg of the effective peptide, 192ng of Creri 17 peptide are emulsified in bOobnl HB5 with boOdl IRA. i) For the control vaccine containing 100 ng of the effective peptide, 1.2 mg of Creri 17 peptide is emulsified in HB5 bodl with IRA bodl. 02) Peptide mixtures as a vaccine a) Peptide mixture | as a control vaccine without an adjuvant containing 250 ng of each peptide Creri 43, Creri 45, Creri 46, Creri 50 in a total volume of 1 ml of PB5.

B) Peptide mixture I as a control vaccine without an adjuvant containing 250 μg of each peptide creri 17, creri 18, creri 62, creri 63, crerib4 in a total volume of 1 ml of PB5. c) A peptide mixture as a vaccine with polylysine as an adjuvant is prepared by mixing 1 mg of peptide mixture 1 (containing 250 ng of each peptide) with 74 dg of polylysine in HB5.

The incubation period before injection is 4 hours at room temperature. a) Peptide mixture II as a vaccine with polylysine used as an adjuvant is prepared by mixing 1.25 g of peptide mixture 111 (containing 250 dg of each peptide) with 9 g of polylysine with

NVZ5B. The incubation period before injection is 4 hours at room temperature. g) Peptide mixture | as a control vaccine with an adjuvant is prepared as a result of emulsification of 1.2 mg of peptide mixture I in bbodl HB5Z (containing Zbbig of each peptide) with bOOdl IBA.

The incubation period before injection is 30 minutes at room temperature.

Peptide mixture I as a control vaccine with an adjuvant is prepared as a result of emulsification of 1.5 mg of peptide mixture IP in bbodl HB5 (containing ZbOng of each peptide) with bOOdl IRA.

The incubation period until the moment of injection is 30 minutes at room temperature 9) For topical application, 1 mg of the peptide mixture is incubated with polylysine as an auxiliary agent

(containing 250 dg of each peptide) with 74 g of polylysine for 4 hours in a total volume of 400 dL HB5.

The resulting mixture is added to 1.6 OO OMEME (Megekie) hydrogels. n) For topical use of the control vaccine without an adjuvant, 1 mg of peptide mixture I (containing 250 ng of each peptide) in a total volume of 200 dl HB5 is added to 1.8 g of OVPOGOMEME hydrogel (MegekKie). i) The production of fucose-linked polylysine (chain length: 240 or 220) is carried out according to the method described in MasVgoogle et al. 1992, and a substitution of about 4095 is achieved (the starting materials p-fucopyranosylphenyl isothiocyanate and polylysine are obtained from BIOMA) .

C) If transferrin/polylysine conjugates are used (the manufacturing method is described in the international application UMO 93/07283), the amount is set in such a way that the absolute amount of polylysine is 75 µg of the peptide. If the plasmid DNA (empty plasmid reRb5, I Po-free, Voephipdeg Magtpeigp) is integrated in a complex, the ratio is 37.5 ug OMA/75 ng polylysine/1 mg of peptide. In the case of using 160 dg instead of 1 mg of peptide, the number of other components is reduced by the same factor (6.25).

E) Vaccine injection

Before subcutaneous injection, mice in groups of up to 8 animals are anesthetized in an isolated air chamber. After 3.5 minutes of treatment with halothane (4 95 in O»2, percentage of allocation 4), the mice are under anesthesia for about 1 minute; at this time, the vaccine is injected subcutaneously.

Intra-abdominal injection is performed without prior anesthesia. The injection volume of each vaccine is 100 dl per animal, which corresponds to 100 dl of a single peptide or peptide mixture | on the animal In the case of using a peptide mixture of III, each mouse is injected with 125 mg of the total peptide amount.

F) Topical application of the vaccine

200 mg of ointment containing 100 ng of peptide or peptide mixture is taken per mouse | or 125 mg of peptide mixture I. The ointment is rubbed into the skin of shaved animals, namely, the entire surface of the back and ears. The exact amount of ointment that is rubbed in is controlled with the help of scales. c) Use of a vaccine against tumor growth in mice

The protocol for testing the effectiveness of an anticancer vaccine for prophylactic or therapeutic purposes on mice corresponds, unless there were special instructions, to the principle described in the international application UMO 94/21808, and the OVA/2 model is used as a mouse model.

Example 1

Vaccination of YOVA/2 mice against mastocytoma P8Z15 16b0dg peptide sequence KUOAMTTTI. (krer118), obtained from the P815 tumor antigen described in I ee et al., 1992, the Hg-iC ligand is mixed with 11.8 dg of polylysine 300 in 50 bdl of HBV and incubated for 4 hours at room temperature. Then add 500 nl of EB55 (Eap5 buffered saline solution). 100 dl of the obtained mixture is injected subcutaneously into eight mice with a weekly interval. After this preliminary immunization, tumors appear a week later, and each mouse is contralaterally injected with 5x10" cells of the mastocytoma cell line PB15 (АТСС Mg

TIV 64; these cells express a tumor antigen from which peptide P8Z15) is obtained in t00nl EB55.

The result of this experiment is shown in Fig. 1 (colored squares).

In a parallel experiment, 200ng of the peptide was mixed with 5X0bdl of HB5 and then emulsified with 500dl of an auxiliary agent. Eight mice were subjected to preliminary immunization (per 100 dl of the obtained emulsion), which were then transplanted with RZ15 tumor cells, as described above (Fig. 1: colored circles).

To carry out the next parallel experiment, the cellular antitumor vaccine is prepared as follows: 16 bOng of the peptide krer118 is mixed with Zdg transferrin-polylysine (Tr), TO ri. and the big plasmid pp 65 (HRB-free) in Z0bil HB5O-buffer. After standing for 30 minutes at room temperature, the above-mentioned solution is added to a beaker with T 75-cell culture containing 1.5xX109 cells of the allogeneic fibroblast cell line MINNZTZ (ATS Mg SK 1658) in 200 l of OMEM medium (1095 RS5, 20tM glucose) and incubated at 37"С. After three hours, the cells are mixed with 15 ml of fresh medium and incubated overnight at 37"С and 595 СО". 4 hours before use, the cells are irradiated with 200u. Preparation of the vaccine is carried out according to the method described in the international application UMO 94/21808. Preliminary immunization with this vaccine is carried out at weekly intervals in a volume of 105 cells. After a week, the appearance of the tumor is recorded, as described above (Fig. 1 g: colored triangles). A vaccine containing a peptide with polylysine was found to best protect mice from tumor formation.

Example 2

Vaccination of OVA/2 mice against mastocytoma P815 using a single-peptide vaccine a) Three single-peptide vaccines containing either one creri!7 peptide in RVZ (Fig. 2a), or creri? peptide emulsified in IRA (Fig. 25), or peptide creri!/7 with polylysine (chain length 240) as an adjuvant (Fig. 2c) will be tested for their protective effect against the appearance of RZ15-tumor.

Vaccines are prepared according to the method described in the OO section. The injection volume is 100 dl each time; the injection is carried out subcutaneously (5s) or intraperitoneally (ir). Uninjected mice serve as a negative control, and a whole-cell vaccine consisting of PB15 cells secreting M-C5E serves as a positive control (P8B15-2M-C5E; 109 cells per 100 dl per mouse were injected subcutaneously). Each experimental group consists of eight animals, three vaccinations (5s) are carried out with an interval of 7 days. A week after the last vaccination, the animals receive a contralateral tumor challenge from 5x107 P815 cells. The animals are examined daily, the appearance of tumors is monitored at weekly intervals.

Crer1117 peptide with polylysine as an adjuvant shows a better antitumor effect when injected subcutaneously at 100 dg per animal (three out of eight animals were protected). This effect is also as good as the effect achieved with the whole-cell vaccine (four out of eight animals were protected). Injection of the 1bng peptide with polylysine per animal is less effective (two animals were protected), but it is clearly better than the injection of the 1О0dg peptide in РВ5 (Fig. 2a, no protective effect). Also, the peptide emulsified in IRA does not have the effect achieved in combination with polylysine (Fig. 2c).

B) In the next experiment on P8Z15-mastocytoma mice, two unmodified polylysines with different chain lengths are compared: a short one with 16 lysine groups (ri. 16) and a long one with 240 groups (ri. 240).

Animals from the control groups were injected with 100 μg of the peptide крер117 dissolved in РВ5 or emulsified in IRA. Two cellular control vaccines secreting ZM-C5E are used as a positive control (madI.a), and one vaccine is obtained from stably tolerant RZ15 cells, and the second vaccine is obtained using the rapid transfer method (AMET) described in Umadpeg et al., 1992. Both whole-cell vaccines provide protection in four to five out of eight animals. Vaccines based on peptides, consisting of a single peptide or a peptide emulsified in IRA, do not show a protective effect: in all animals, soon after the appearance of a tumor, the latter begins to develop. If, however, a peptide with polylysine is used, the vaccine protects the animals against tumor development: two out of eight animals are protected if the long polylysine (ri 240) is used and four out of eight animals are protected if the short polylysine is used. These results, presented in Figure 3, show that the single peptide in combination with polylysine as an adjuvant induces effective antitumor protection comparable to that provided by an effective whole-cell cytokine-releasing vaccine, which appears in the literature as a standard for antitumor vaccination (Ogapoi et al., 1993; Zsptiaii. and others 1995).

Example C

Vaccination of OVA?2 mice against P8Z15 mastocytoma using an antitumor vaccine containing PO15 single peptides or mixtures of ReO15 peptides. The following peptides are used to prepare the vaccine: Creri 18 (100 ng per injection)

Peptide mixture PI (Crer!I7, Crer118, Crer162, Crer163, Crer164). This peptide mixture contains all PB15 peptides known to date; ng of each peptide is administered per injection.

As a positive control, P815 cells secreting CM-SOE are used.

In the process of preliminary testing, Creri 17 shows itself as a peptide with a better protective effect against the appearance of RZB15-tumor, if 100 dg of the peptide is used together with polylysine (7.5 dg of polylysine /10 Orng of peptide in accordance with the standard ratio; polylysine: chain length - 200). A small amount of (Ibig) Crer!17 is less effective. In this example, t00dg of Cr118 are injected per animal, namely once with polylysine alone (group B), once with transferrin-polylysine (group C), and once with transferrin-polylysine / DNA (group 0). Kreri 18 from IBHA is used as a control. In this experiment, kreria!8 alone does not show a protective effect against the appearance of a tumor.

In the experiments carried out in example 4, it is shown that the vaccine containing a peptide mixture of melanoma peptides shows a protective effect against melanoma. Hence, this example tested whether the peptide blend concept was suitable for P815.

Peptide mixture I is used once only with polylysine (group E), once with transferrin-polylysine (group P) and once with transferrinpolylysine / OMA (group c). Peptide mixture I in IRA is used as a control. Mice are used as a negative control; as a positive control - transferred ЗМ-С5Е РВ15 - cells (105 cells per mouse).

The experiments carried out in this example were developed atypically, compared to other experiments: in the positive control group (ZM-C5E secreting cells), all animals developed tumors shortly after their appearance, and a large part of these tumors also quickly disappeared, as well as appeared A possible explanation for this is that the tumor had been developing for some time before it was destroyed.

Another possible explanation is that the swelling diagnosed as a tumor did not arise from tumor growth, but was a strong immune cell infiltrate (granuloma). Because the animals were not necropsied, the cause was not definitively determined; in any case, the supposed tumors were eventually destroyed. Another interesting result was obtained in group C, in which animals were treated with a combination of peptide mixture III and polylysine. All animals developed tumors, but in two of all animals, the size of the swelling (or immune cell infiltrate) was relatively small, did not enlarge, and the mice did not appear unhealthy. Both of these animals were not euthanized and were under further observation. Unexpectedly, nine weeks after its appearance, the tumor was not detected: the result is still not observed. Two of the eight animals eventually destroyed their tumor burden. The destruction of tumors could also be explained by the content of creri!7 in the peptide mixture, which is similar to example 2, where two of eight animals were protected with 1 µg of creri 17, and three of eight animals - with the help of 100 µg of creri117.

The protective effect could, however, be caused by more than one peptide of the mixture.

Example 4

Protection of OVA/2 mice against M-3 melanoma by preimmunization with an antitumor vaccine containing a peptide mixture.

A prophylactic vaccine containing a mixture of melanoma peptides (peptide mixture I, section rg) is used.

The order of the preliminary immunization with the vaccine, as well as the appearance of the tumor corresponds to the order described in example 2, with the difference that the appearance of the tumor is caused by the use of MZ cells (105 cells per animal). As a control vaccine, a whole-cell vaccine from M-Z3 cells is used, which secretes the optimal amount of --2 (1000-2000 units per 105 cells) and is produced according to the method described in 5sptiai et al., 1995. Under the selected test conditions, this vaccine allows you to get the 10095th protection (Fig. 4). Four groups of experimental animals are administered vaccines with a peptide mixture. Two groups receive (either 5.c or ir) peptides emulsified in IRA. The other two groups receive (or 5.s. or i.r.) peptides together with polylysine (ri. 240).

Fig. A4 illustrates the protective effect of a peptide vaccine with polylysine (ri 240) as an adjuvant; 5095 treated mice were protected against M-3 tumor challenge compared to untreated animals, which developed significant tumors very quickly. This effect could be achieved only by subcutaneous injection of the peptide-polylysine vaccine or by applying the hydrogel to the skin (Fig. 5). In three other control groups of mice treated with i.p. peptide/polylysine vaccines. or using peptides in IRA, the vaccine was largely ineffective. Presumptive tumors were not rejected and developed, compared to tumors of untreated control animals, with a slight delay. These results show that a vaccine containing a peptide mixture can achieve an antitumor protective effect if it contains polylysine. Under the selected test conditions, this peptide vaccine was half as effective as the cellular I-2 vaccine, which, according to reports that have recently appeared (7aiyoika!i, 1993, 2ayoiKka!i, 1995), protects up to 100,905 animals against the appearance of a tumor due to the use of 105 live M3Z cells.

Example 5

Protection of OVA/2 mice from M-3 metastases a) Use a therapeutic vaccine containing a mixture of melanoma peptides (peptide mixture | described in section 02). Three vaccinations (5s) are carried out with a weekly interval. The first vaccination is carried out five days after the appearance of metastases; are vaccinated against five-day metastases. 1.2x107 M-3 cells are injected for the formation of metastases; use the technology described in the international application UMO 94/21808 and in Zsptiivy et al., 1996.

Peptide mixture I is used as a vaccine: without an adjuvant (rertich 1 РВ5), with IRA as an adjuvant (BA rertich 1) or with fucose-modified polylysine (yiri rertich 1).

Control groups of mice do not receive any vaccine (non-injected) or receive the II-2-producing M-3 whole-cell vaccine shown in example 4. It can be seen from Fig.6 that the best degree of protection was achieved in the group in which the peptide mixture 1 with fucose-modified polylysine was used as an auxiliary agent (Fig.ba). 5095 mice were able to reject metastases (four mice out of eight). This method of treatment was even more effective than the method of using a whole-cell vaccine, which allowed to protect only 3390 mice (three out of nine). The use of the peptide vaccine with IRA or without an adjuvant resulted exclusively in slowing the growth of tumor metastases (Fig. 6B).

B) In the following experiment, the protective effect of a vaccine containing peptide mixture 1 and administered subcutaneously is investigated on a therapeutic model. Control groups of mice receive a peptide mixture

РВ5 (without adjuvant) or with IRA. Unmodified polylysine 240 is used as an auxiliary agent. In addition, fucose-modified polylysine 200 (ri 200) is used as an auxiliary agent. As a control in this experiment, a group of animals with I-2 cell vaccine expressing. As shown in Fig. 7a, the peptide/polylysine vaccine was effective in the treatment of MZ metastases. The characteristic rate of healing in this experiment was achieved only when fucoso-modified polylysine was used. When treated in this way, 5,095 animals rejected metastases, which is a good result compared to the use of the II-2 vaccine, which allows to cure 7,095 animals in this case. c) In the next experiment, the therapeutic model is tested to see how changes and modifications affect the antitumor effect. At the same time, in addition to unmodified and fucose-modified polylysine 200, short unmodified polylysine ri is tested. 16, long polylysine ri. 450 and the next polycation, namely polyarginine (pAgd720). As a positive control, a cellular MZ-vaccine is used, which releases the optimal amount (210 pd 105 cells) of CM-C5E (Zsptidi, 1995). Also in this experiment, the control groups contain the peptide mixture in IRA or without any adjuvant. As in example 50), the best effect was achieved if fucosopolylysin was used as an auxiliary agent, fig. 7p. In these groups, 4,090 animals rejected metastases, compared with 30,905 in the short polylysine ri group. 16. In addition to the group that received the peptides together with polyarginine, in the animals of the other groups that were injected with the peptide vaccine, only a short delay in the occurrence of tumors was noted. Unmodified polyarginine was as effective as fucoso-modified polylysine in this experiment, leading to rejection of metastases in four out of ten animals.

Figure 7 illustrates the repetition of this polyarginine-induced effect in an independent experiment. Here, too, vaccination with a peptide mixture in combination with polyarginine made it possible to achieve an antitumor effect in four out of eight treated animals.

Example 6

Filling cells with tyrosinase when using polylysine as an adjuvant.

This example serves as an experiment that shows that polylysine as an adjuvant is suitable for filling cells with large protein fragments or whole proteins, and as cells were used

Replacement M-3 cells.

To fill the cells, 1 g of RITO-labeled tyrosinase (EC 1.14.18.1; Sigma) is mixed with Zyg polylysine (p/240) and incubated at room temperature for 3 hours. Then the resulting solution is placed in a beaker with cell culture 775 with a volume of 2x106 M-3 cells and incubated at a temperature of 37"С. Then the cells were washed twice with РВ5, separated with РВ5З/2 tM EOTA and mixed with 1 ml of РВБ/5 95 ES5 for GAS5 analysis Fig. 8 illustrates the filling of M-3 cells with tyrosinase (the left curve shows the control, the right - the degree of filling with tyrosinase).

Example 7

Determination of T-cell activation after immunization in a therapeutic model

After the peptide/polycation vaccine in a therapeutic mouse model (see example 5) clearly demonstrated its effectiveness, it was investigated whether this method of treatment leads to the activation of T-cells. To do this, cytokine secretion is performed as a marker after incubation of spleen cells of vaccinated animals with parenteral M3Z cells (Kamakati et al., 19940).

Prepare single-cell suspensions of the spleens of vaccinated and untreated animals, which occurred due to erythrocytosis with a hypotonic buffer (0.15 MNASI, It KNSO»z, 0.1 tM EOTA, р 7.4). Bound cells are removed, while 3x1059 spleen cells per ml of OMEM medium (1095 EC5) are incubated in petra dishes for 90 minutes at 372 C. Bound cells are cultured by careful selection with a pipette and co-culture with 1x103 parental cells in different quantitative ratios. Cells are cultured in 2000 l of OMEM medium (1095 EC5), 2 tM glutamine and 2 µg/ti gentamicin in 96-well flat-bottom tissue culture dishes. On the ninth day, 100 nl of the composition are collected and the corresponding IEM-(- content is measured using commercially purchased sets of EGISA devices (Epdodep, Satagidd, MA, O5A) according to the manufacturer's instructions. It turned out that after a nine-day incubation, large of the amount of IEM-"in the environment, only spleen cells of vaccinated animals were released, while in the co-culture of spleen cells of untreated animals and MZ cells, practically no IEM was detected. The result of this experiment is shown in Fig. 9.

Example 8

Induction of antiviral immunity with the help of the influenza nucleocapsid peptide AZMEMMETM and fucosylated polylysine as an adjuvant A vaccine containing 1 mg of the peptide is administered

ABMEMMETM 75yg ?rGuz. The vaccine is administered by a single injection, as indicated in the methodological section, and 100 ng of the peptide is administered to the animal // 7. Big Trpuz. For control, an injection of 100 ng of a single peptide (PB5) is performed and, accordingly, no injection is performed (no-injection control).

AMA-5 mouse lymphoma cells are incubated overnight in a serum-free environment at a temperature of 2607C with 10 dg/ml of the ABMEMMETM peptide.

10 days after vaccination, spleen cells of vaccinated animals are isolated, mixed with peptide-filled KMA-5 cells in a ratio of 5:1 and cultured for another five days (5shshbeg et al., 1994). Determine the number of surviving functional cells of the spleen in different cultures. Then, to determine STI activity using a standard 4-hour europium release assay (Viotregud et al., 1993), cells are mixed in various ratios with KMA-5 cells, which are previously diluted with AZMEMMETM peptide and europium intracomplex compound. As illustrated in Fig. 10, specific immunity in this experiment was achieved only by vaccination with the peptide and three uvs, but not when a single peptide was administered.

Example 9

Testing of various basic polyamino acids for their ability to enhance the internalization and/or binding of peptides to APC5

To carry out these tests, fluorescent studies are carried out: the model of the peptide antigen sequence I REAIESRI (MSN Ka - limited) is marked with a fluorescent dye

Einogezsetizoipiosuapai (RITS) according to the manufacturer's instructions (MoiIesshag Rgore5). Reception or binding of the labeled RITS peptide alone (pulsed) or together with different concentrations of basic amino acids (polylysine with chain length from 16 to 490, polyarginine with chain length from 15 to 720) via MHC

The Ca-limited monocyto-macrophage cell line RZ8801 is determined by flow cytometry.

For this, 1x1097 RZ8801 cells are incubated in a final volume of ml medium (OMEM/10 95 EC5) in centrifuge tubes with 5 g of RITO-labeled peptide alone or with a mixture of peptide and polyamino acids for 30 minutes at a temperature of 37"C, and then intensively washed, to remove unbound peptide.

Polyamino acids are added in concentrations of 50, 25, 12, 6 and Zdg per mol. medium containing 5 μg of RITS-labeled peptide. The relative fluorescence intensity of different tests is compared to assess the efficiency of peptide uptake and/or binding. The result of these tests is shown in Fig. 11; tests are carried out, respectively, with 25yg ri 450 or RAgd450. Under the selected conditions, polyarginine proved to be almost five times more effective than polylysine.

Example 10

Investigation of the mechanism by which APC5 accepts peptides Peptides can be accepted

APC5 by specific mechanisms, such as, for example, macropinocytosis or receptor-mediated endocytosis (I aplamesspia, 1996). An alternative mechanism is that polyamino acids can make the cell membrane permeable and thus facilitate the diffusion of peptides from the medium into the cytoplasm. a) Whether the cell membrane has become permeable is checked by measuring the secretion of the cytoplasmic enzyme lactate dehydrogenase (OH) after incubation of RZ38801 cells with polyamino acids (polylysine or polyarginine) under isotonic conditions using commercially available kits (Suioch 96,

Rgoteda, Madizop, UMizsopzip, OBA) according to the manufacturer's instructions. On the basis of the results shown in Fig. 12r, it can be assumed that the effect of riy5 is that it makes the cell membrane permeable, which is manifested in high concentrations of the cytoplasmic enzyme released under isotonic conditions. In contrast, after using pAhod (Fig. 125), SON is practically not detected. When comparing samples using only polyamino acids and samples using mixtures of polylysine or polyarginine with peptide, no differences were found in HON release. After incubation with the peptide alone, no measurable IOH activity was detected.

B) Whether penetration of labeled RITSO-peptides occurred in the presence or absence of basic polyamino acids is investigated on the basis of the principle published by Midoikh et al., 1993. From the cells, the particles that got inside are transferred to the endosomes. Compared to the cytoplasm or cell culture medium, the neutral pH values show that these organelles with a pH value of the order of 5 are acidic.

The emitted RITS fluorescence is highly dependent on pH. In an environment with pH conditions such as those present in endosomes, fluorescence is suppressed. Hence, labeled RITS peptides, which are transferred from cells by endosomes, show reduced fluorescence. When monensin is added, the low pH value of the endosomes is neutralized, which leads to measurable enhanced fluorescence of the RITS-labeled peptides that have entered inside.

Cells are incubated with a mixture of polyarginine (average molecular weight 100,000, chain length 490) and fluorescently labeled peptide at a temperature of 4"C or 37"C. The number of samples incubated at 377 is stored and treated with 500M monensin before flow cytometric analysis at a temperature of 4"C.

This allows us to find that incubated APC5 with certain basic amino acids, for example, polylysine (ri uz) and polyarginine (pAgo) enhances the reception or binding of the peptide to APC5.

As can be seen from Fig.13, in the cells treated only with the peptide or with monensin, a slight increase in fluorescence is observed. In contrast, fluorescent signals in samples treated with monensin and a mixture of polyarginine and peptide were greatly increased. Peptide uptake was not observed if the samples were incubated at 4°C. The characteristic increase in fluorescence after treatment with monensin indicates that the pAgd-induced filling leads to the accumulation of peptides in vesicles inside the cells (Midoikh et al., 1993; Fig. 13). and as expected, a slight increase in fluorescence was observed after monensin treatment of polylysine-loaded samples. Polylysine loading at 4-7C resulted in a measurable increase in fluorescence, further indicating that the effect of polylysine is mainly due to the permeability of cell membranes (Fig. 12bB). .

Example 11

Study of APC5 filling with short peptides

АРС»5, isolated from the bone marrow and preserved with the help of ZM-C5E, are examined by the combination of fluorescently labeled peptide with polylysine (ri 200; 5BIZMA) or only the peptide by the method of fluorescence microscopy. To detect the uptake of peptides using a microscope, ARCs are seeded on a glass slide and incubated with 40 ng of fluorescently labeled peptide I REAPESRII or with a mixture of 50 µg/ml polylysine (ri200) and 40 dg of the peptide GREAIESORII for 30 minutes at a temperature of 3770. After thorough washing, the cells are fixed at 4956 -th paraformaldehyde, covered with anti-discoloration (Oako, sSiovigir,

Rapetagk) and fluorescently microscopied (7ei55). Nuclei are stained in contrasting colors with the help of 4.6b-diamedino-2-phenylindole (YUARI, 5ZISMA). As shown in the fluorescent photomicrograph, Fig.14, cells incubated with peptide and polylysine (A), compared to cells treated only with peptide (B), show a clearly increased degree of peptide uptake. While the fluorescence of cells treated only with the peptide (pulse) appears selectively and in the form of particles, the intense fluorescence of cells treated in the presence of polylysine and peptide appears non-localized and more evenly distributed throughout the cell.

Example 12

Quantitative determination of cell filling with a peptide by flow cytometry a) After the results of the experiments carried out according to example 11, it was shown that APC3 is an excellent target cell for filling with small peptides, GAS5 tests are carried out in vitro (outside the body) with the aim of identify suitable peptide vaccine adjuvants. This study enables rapid quantitative testing of fluorescently labeled peptides; as a peptide model, the peptide of sequence I REAIIESRI is used. In these experiments, mouse cell lines RZ8801 are used as APC5. 1x1092 cells in a final volume in iml OMEM medium with high glucose content and 1095 PC5 are incubated for 30 minutes at 37"C with 5 dg of peptide with a fluorescent final concentration of 5 mmol/ml. Cells are treated either with peptides alone or with a combination of peptide and polycations or peptide with histones at an increasing concentration (from 3 to 50 dg/ml), as shown in Fig. 15.

The following compounds are used: A: polyornithine (average molecular weight range 110,000, chain length 580); B: histone saturated with arginine; C: histone saturated with lysine; 0: polyarginine (average molecular weight range 100,000, chain length 490); E: polylysine (average molecular weight range 94,000, chain length 450). In previous tests, it was found that an incubation period of 30 minutes revealed the maximum degree of peptide uptake. A longer treatment period (4-8 hours) did not reveal a characteristic amplification of the fluorescent signal. Before the analysis, the cells were washed 5x with a large volume of RV5 containing 0.290 VBA. Cells in ice-cold RB5/0.295 VBA cells were resuspended and studied by flow cytometry (GASZzsap; Vesiop Oiskp5op, Zap uose, SA, OBA).

Polyarginine and polylysine prove to be the most effective auxiliaries; polyornithine exhibits a cytotoxic effect under selected conditions. Enhancement of peptide uptake due to polyarginine and polylysine correlates with concentration (Fig. 15j, e); the degree of reception increases according to the length of the chain (Fig. 16).

Polyarginine with a 0.3-fold increase over the control size was found to exhibit a wider relevant concentration range than polylysine, which showed a maximal increase of less than 0.2-fold, and that polyarginine at all chain lengths used was more efficient at transporting proteins than polylysine (Fig. 16): polyarginine enables efficient transfer at concentrations as low as 10 ng/ml, while polylysine requires concentrations of 25 ng/ml to cause a characteristic increase in fluorescence (Fig. 15j, e).

In order to determine whether there is a lower limit to the chain length in the transfer of peptides, polyarginines of different chain lengths (10-30 groups) are synthesized and tested for their ability to increase the transfer of peptides at high concentrations of polycations (Fig. 17). For these experiments, a peptide is used

IGREAESRI, tested polyarginine polymers are introduced at a concentration of 100 dg/mol.

Even a slight increase in peptide transfer was detected even with the shortest tested polyarginine. c) Basic amino acids are positively charged molecules. Hence, it can be assumed that negatively charged peptides can bind to these polycations through electrostatic interaction, which may lead to enhanced peptide uptake. To test this hypothesis, the ability of cationically active polyamino acids to accept short peptides was compared depending on their charge in RZ 8 801 cells. The following table shows the used negatively charged peptides that meet all the conditions necessary for the MNO-1 compound (Katep-5ee et al., 1995). Peptide 1 was obtained from mouse TER (protein from the tyrosinase family), peptide 2 - from influenza hemagglutinin (Zsptiai et al., 1996), peptide C - from mouse tyrosinase, peptide 4 - from P 198 tumor antigen, peptide 5 - from betagalactosidase ( sSamip et al., 1993). (Mg refers to the range of molecular weight, "Yashog" - to fluorescein).

Table

Based on the methods used to label the peptide with fluorescein, two negative charges are introduced. It was found that after incubation with polyarginine, peptides (5 pmol per sample) with the highest number of negative charges are most efficiently transferred to RZ880O1 cells. This indicates that the ionic interaction between the peptide and polycation enhances the transport of peptides into cells (Fig. 18). However, in comparison with cells that were treated only with peptide, also in the case of using neutral peptides in the presence of polycations, large amounts were taken up. Treatment with only the peptide made it possible to detect almost identical fluorescent signals in all tested peptides; on the basis of what is shown in Fig. 18 as "only peptide", the fluorescent signal is shown as a substitute, obtained with the peptide GREAESRI.

LITERATURE

AiIehapaeg, u. et al., 1989, Ittipodepeiisz5 29, p. 380

Aiighei, O.S. et al., 1992, Siip magazine. Opkoi 10 (4), pp. 599-605

Amgateav, A. And others, 1996, European Journal of Ittipoi). 26, pp. 394-400

Wentg, 9.R., 1994, Viosopind-Spet., September-October 5 (5), pp. 382-9

Vepoiei, A. and others, 1994, Mayige, 369, 407-410

Vioiodis Tpegaru oi Sapseg, editor: Oemiia M.T.Ug., NePtap, 5., Noseprego, 5.A., Mepad, 9.V. Iirripsoi

Sotrapu, Riade!rnia, Mem Moik, IHopaop, Nadegziomp

Viotrego, K. opa Oneteiyi, A.S., 1993, Ittipoi journal. Meipodz 160: pp. 27-34

Voop, T., 1992, Aam Sapseg Kez 58, pp. 177-210

Voop, T., 1993, Spectrum of Science (May), pp. 58-66

Voop, T. et al., 1994, App. Keum. And so on. 12, pp. 337-65

Voop, T. ipa map deg Vgyddep, R., 1996, Ehr tey journal 183, pp. 725-729

Vgasiaie, T9. opa Vgasiaie M.G., 1991, Ittipoi. Todau, pp. 124-129

Vgoske, 5, etc., 1996, Maige 379 (6563), pp. 343-346

Vgope, et al., 1995, Ittipoi journal. 154, p. 5282

Saitei, 5. ipa doppzop, u.R., 1993, Sstepi Oriop ip Opsoiodu 5, 383-389

Soyidap, 9U.E., et al., 1991, Maishge 351, pp. 290-296

Soiidap, 9U.E., et al., 1991, Sigtepi Rgoi. Ip Ittipoi., MMieu, Mem MogK

Sotzshiye, R.O. et al., 1992, Ipi. Sapseg magazine, 50, pp. 289-297

Society, R.O. et al., 1995, Rgos Mai! Asai Zsi OA 92, p., 7976-80

Society, R.O. et al., 1994, journal Ehr. Honey. 180, pp. 35-42

Soh, AH. et al., 1994, Zsiepse 264, 5159, p. 716-9

Sshitepi Rgoiosoi5 and MoiIesshiag Vioiodu, 1995, Negaizdebeg: A!izirei! E.M. dopp Umieu and 5opv, Ips.

Ogapoiy, Fr. et al., 1993, Rhos. May Asai. Zsi.O5A 90, pp. 3539-3543

Ohapai, co. ip Miiyidap, K.S., 1995 Admapsez ip Ittypo|odu 58, p. 417

Eakk, K. et al., 1991, Maishge 351, pp. 290-296

Eeidpeg, U.N. et al., 1994, journal OiOI.Spet. 269, pp. 2550-2561

EenKatr, M.S. et al. 1995, EEig. Ittipoi magazine. 25 (9), pp. 2638-2642

Eetop, K.O. et al. 1993, magazine Mai!. Sapseg Ipv5i. 85,16, cheese.1294-302

EIi5K, V. and others, 1995, journal Ehr. Mea. 1881, pp. 2109-2117

Rioj Suioteigu, Asad, Rgezz, Mayo ip Sei! Vioiodu, 1989, Moi. 33, author!: Oaglupkiemisl, 2. Opa Stizvtap, M.A.

Seadaerani, T. 1992, Nit Ittipoi. 33,4, 266-74

Stoptapp, O. 1995, Eig. And so on. 25, 2797-2802 cSvmaypi, A. 1995, SuyukKipez5 apa MoIesshag Tpegaru 1, 57064

Nap, H.K. 1995, RMAB5 92,9747-9752

Opposite: GAS5 Mapiade "m OOzeg5 scide, Argii 1994, Vesiop biskipsop

Naparisy: SES Ocevi "m Boimage Ozegz spcide, dipe 1994, Vesiop biskipsop

Nepaegzop, V.A., and Ripp, O.9., 1996 Admapsez ip Ittypoiodu 62, 217-256

Netip M. et al., 1987, Ipi. magazine Sapseg, 39, p. 390

Nosk, N. et al., 1993, Sapseg Kezeagsi 53, pp. 714-716

Neubieg", 9.0. etc. 993, Yeig. journal Ittipoi 23, pp. 2072-7

Nyapo, A.M.S., ipa Ragao!. OHM. (1996). Rgos. Mai! Aside. All together. OBA 93, pp. 9730-5

Ipabva, K., et al., 1992, Urnal Ehr. Mea. 176, pp. 1693-1702

Uypa, 5. and others, 1991, journal Ehr. May 173, 1,273-6

Kazhakati, MU. et al., 1994, Rhos. Mai)!. Aside. 5 OBA 91, pp. 6458-62

Kazmakati, U. and others, 1994a, Rgos. Mai)!. Aside. 5 O5A 91, 9, pp. 3515-9

KamzhakKati, U. and others, 1994, journal Ehr.Med. 180,1, pp. 347-52

Kazhakati, MU. et al., 1995, Ittipoi magazine. 154, pp. 3961-3968

Kaite, K. and others, 1986, Mashge 319,20. Rer., p. 675

Kegzp, 0... and others, 1996, Mayishge 380 (6574), pp. 495-498

Komaszomisv Vapkomuzki, M. Opa Nosk, K.G., 1995, 5siepse 267, pp. 243-246

Gapgamespia, A., 1996, Sit. Orip. Ittypo).. 8, pp. 348-354

Heptapp, -.M. et al., 1989, Rhos. Mai). Asai. from OBA 86, pp. 9891-9895

Goethe, V. And others, 1992, Yeig. Ittipoi magazine. 22, pp. 2283-2286

Ii, N. and others, 1989, journal Ehr. Honey. 169, pp. 973-986

Shi, M.G., Teueiiia, M.., Nepagiskwap, M/.2kh., ipa Temeipia, 5.5. (1992). magazine Ehr. Mea. 176, pp. 449-57

Tsuyepodgep, N.O., et al., 1990, Maishge 346, pp. 476-480

Y oenieg, 9.-R. et al., 1993, Meiypod5 Epgutoi. 217, pp. 599-618

Gorel, 9.A. et al., 1993, Yeig. Ittipoi magazine. 23, pp. 217-223

MasVgoot, S.M. et al., 1972, May. Epgutoi! 28, pp. 212-219

Maskiem/isl, A. And others, 1995, Nytap Sepe TPpegaru 6, pp. 805-811

Maipaii, M.5. et al., 1995, 5th issue 267, pp. 1016-1018

Mapave!Boiga, 0. And others, 1994, Maishge 369, 5 tau, pp. 67-71

Mabrae!roit, 0. And others, 1995, Mayige Meisipe 1,11, pp. 1179-1183

Magespapa, M., et al., 1995, IPI journal Sapseg 63, pp. 883-5

Msipiuge, S.A., et al., 1996, Sapseg Ittipoi!. Ittipoipeg. 42, pp. 246-250

Midoikh, R., et al., 1993, МАТО AFBI Zegieh5 Nb7, pp. 49-64

Mogizpa, K., et al., 1993, Siip journal. Ipmevi 91,6, pp. 2580-5

Mabei!, 0.., and others, 1993, Rgos. Mai). Asad 5 OBA 90, pp. 11307-11311

Modispi, MU, et al., 1994, Rhos. May). Asad 5 UZA 91, pp. 3171-3175

Oendep, N.R. pa OIYA, S... 1991, Vioiodie Tpegaru oi Sapseg, editor: Oe Mpa, M.T.9Ug., NeiItap, 5.,

Kozepbrego, 5.A., Mepad, 9.V., Girripsoi Sotrapu, RpPadeirpia, Mem MogK, Gopaop, Nadegviou/p, pp. 87-119

Ozvigapa-Boseprego, 5., 1994, Sitepi Oripiop ip Ittipoiodo 6, pp. 722-727

Ragaoi!, O.M., 1993, Ittipoiiodu Todau 14,6, p. 310

Rgasiisa! Ittypo!odu, editor Geziie Nidzop apa Rhapk S Nau, ViasKueyi

Zsiepiyis Rybiisayopv, Ohioga, I opdop, Ediprygoi, Vosiop, MeIroigpe

Rease, 0.., et al., 1991, Ittipoi journal. 146,6, pp. 2059-65

Reoriev, O.B. et al., 1994, Ittipoi journal. 152,10, pp. 4993-9

Riashya, O.E. et al., 1993, Rhos. Mai). Asaid. 5si. OA 90, pp. 4645-4649

Rogdadog, A., Siiroa, E., 1995, journal Ehr. Honey. 182, pp. 255-260

Ryssetsi, R. and others, 1995, Yeig. Ittipoi magazine. 24, pp. 1446-1452

Kattepsee, N.O., et al., 1993, Appy.Kem Ittipoi 11, pp. 213-44

Kattepsee, N.O., et al., 1993, Sstepi Oriope ip Ittypo|odu 5, pp. 35-44

Kattepsee, N.O., et al., 1995, Sitepi Vioiodu 7, pp. 85-96

Wattepsee, N.O., et al., 1998, Sstepi Oriop ip IttipoiPodu 7, pp. 85-96

Kattepsee, N.O., et al., 1995, Ittipodepeiisz 41, pp. 178-228

Vetipdyup'5 Rpaptaseciysa! bsiepsez, 18, AshShade 1990, Musk Rybiizpipd Sotrapu, Eazyup, Rep. 1990

Ketu, .5. et al. 1994, Viosopitsd-Spet., Mou-Oes, 5 (6), pp. 647-54

Keppie, 9. ipa Kiziipo, K., 1996, zsiepiyis Ategisap Zerietreg, pp. 28-30

Kimopy, S. And others, 1995, Journal of Ittypoiodu 154, 5, pp. 2257-2265

Kobrbipv5, R.R., et al., 1994, Sapseg Kez 54, pp. 3124-6

Kobrbipv5, R.R., et al., 1995, journal Ittipoi 154, pp. 5944-50

Kobrbrip5, R.E. opa Kozeprego, 5.A., 1996, journal Ehr. Honey. 183, pp. 1185-92

Kobrbip5, R.E. opa Kazhakati, U., 1996, Sigtg Orip Ittipoi 8, pp. 628-638

Voii I.M., Vgovioi .., Maiye O.K. Ittypoiodu, Spigepi Pimipuviope

Vozeprego, 5.A., 1996, Appitsa! Kemiemu5 oi Meadisipe, 47, pp. 481-491

Kuzeg, N.u. ipa Napsosk, K., 1965, 5siepse 150, pp. 501-503

Kuzeg, N.9. ipa zpip, MM.S., 1978, Rhos. Mai! Aside. 5 OBA 75, pp. 3867-3870 with Zspitidi, MU., et al., 1995, Rhos. May). Asai. 5 OBA, 92, pp. 4711-4714 with Zspitidi, MU., et al., 1996, Rhosz. May! Asai.zsi. O5A, 93, pp. 9759-63 Zetse, A., et al. 1994, Moi. And so on. 31 (11), pp. 813-822 zpep, UM.S. tspa Kuzeg, N.., 1978, Rhos. Mai). Asad from the U.S. OA, 75, pp. 1872-1876

Zpep, MU... ipa Vuzeg, N.)., 1989, Moi. Rpagtasoi 16, pp. 614-622 zpep, UM.S. tspa Kuzeg, N.U., 1981, Rhos. May). Asai. zsi OA, 78, pp. 7589-7593 zkKkirreg, u., ipa ziaivz5, N.., 1993, journal Ehr. Honey. 177, 5, pp. 1493-8

Ziipdiny, S... et al., 1994, Sctepi Oriop ip Ittypo!odu 6, pp. 73-740 ep, O. et al., 1994, journal EMVO, 13.6, pp. 1331-40 ziyireg, o ., and others, 1994, Yeig. journal Ittipoi 24, pp. 765-768 zukshem, U. et al., 1994, Ittipiu 1, pp. 15-52

Tpeoraiia, M., Hemipe, A.9., pa zpepgtap G.A. (1995) RMAZ 92, pp. 11993-7

Tirregv, H.M., et al., 1993, Sapseg, Uap. 15, Khoi.71,2, pp. 315-321

TukKosip5ki, MH., and others, 1996, At. magazine Raipoi. 148, pp. 1-16

Map deg Vgaddep, R., et al., 1994, EEig. Ittipoi magazine. 24, 9, pp. 2134-40 Ivvp: 0014-2980

Map deg Eupaee, V. ipa Viisnaga, M.S, 1995, Sitepi Oripiop Ittipo). 7, pp. 674-81

Map Rei, A. and Voop, T., 1982, Rgos.Matsy.Asai.5si. OBA 79, pp. 4718-4722

Map Rei, A, et al., 1995, Ittypo!odisa! Kemiemu5 145, pp. 229-250 mishepo, A., et al., 1995, Siip journal. Ipm 95,1, pp. 341-349

Umadpeg, E., and others, 1990, Rhos. Mai! Asad from O5A 87, pp. 341--4

Uadpeg, E, and others, 1992, Rgos. Mai! Asad 5 OBA 89, pp. 6099-1003

Umapad, K.R., et al., 1995, journal Ehr. Mea. 181, pp. 799-804

Uueupapiv, R. and others, 1994, Ipi. journal Sapseg 56, pp. 826-829

Umatapy, S. and others, 1992, Ittipoi journal. Me(yoaz 155 (1), pp. 95-99 um!ei, T. et al., 1994a), Ipi. magazine Sapseg 57, pp. 413-418 o! Gay, T. and others, 1994B), Yeig. Ittypo)y magazine. 24, pp. 759-764

MOgK, GA. ipa Kosk, K.G., 1996, App. Kemum. And so on! 14, pp. 369-396

Khozpipo, I. and others, 1994 a), journal of Ittipoi. 152, 5, pp. 2393-400

Hovzpipo, I. and others, 1994 B), Sapseg Kev., 54, 13, pp. 3387-90

Moipa, UM., Ipaba, K., 1996, journal Ehr. Mea., 183, pp. 7-11 2ganoikai, K. et al. 1993, SsSepe 135, pp. 199-20 2ganoikai, K. et al. 1995, journal Ittyp. 154, pp. 3406-3419

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Fig. 18

Claims (26)

1. A pharmaceutical composition for immunomodulation, which contains at least one peptide of immunomodulatory action in combination with an adjuvant, which is characterized by the fact that the peptide is a ligand for at least one MNO or NI A molecule expressed by the body of an individual subject to exposure , and the auxiliary means increases the degree of binding of peptides to the cells of the individual's body and increases the penetration into cells, as well as increases the immunomodulatory effect of the peptides. 2. Pharmaceutical composition according to claim 1, which differs in that the peptide is a ligand for the MNO-1 molecule. 3. Pharmaceutical composition according to claim 1, which differs in that the peptide is a ligand for the MNO-II molecule. 4. Pharmaceutical composition according to claim 1, which is characterized by the fact that the peptide is a ligand for NI A-A2-molecule. 5. Pharmaceutical composition according to any of items 1-4, which is characterized by the fact that it contains a peptide isolated from a protein of a pathogenic agent. 6. Pharmaceutical composition according to claim 5, which differs in that the peptide is isolated from a bacterial protein. 7. Pharmaceutical composition according to claim 5, which differs in that the peptide is isolated from a viral protein. 8. Pharmaceutical composition according to one of claims 1-4, which differs in that it contains a peptide isolated from tumor antigens. 9. Pharmaceutical composition according to claim 8, which is characterized by the fact that the tumor antigens are isolated from tumor antigens that are expressed from the organism of the subject subject to exposure. 10. Pharmaceutical composition according to claim 8, which is characterized by the fact that tumor antigens are isolated from antigens that are often encountered. 11. Pharmaceutical composition according to one of claims 8-10, which differs in that the tumor antigens are melanoma antigens. 12. A pharmaceutical composition according to one of claims 1-11, which is characterized by the fact that it contains several peptides, which are characterized by the fact that they bind different types of MHC from the organisms of individuals subject to exposure. 13. A pharmaceutical composition according to one of claims 1-11, which is characterized by the fact that it contains one or more peptides that are isolated from an immunogenic protein, or a tumor protein, or a product of their decay, having a natural origin. 14. Pharmaceutical composition according to one of claims 1-11, which is characterized by the fact that it contains one or more peptides other than peptides originating from immunogenic protein (proteins) or tumor antigen (antigens) of natural origin or product (products) their decay. 15. Pharmaceutical composition according to claim 1, which is characterized by the fact that the peptide is an antagonist of a peptide that is isolated from a protein that causes autoimmune diseases. 16. Pharmaceutical composition according to one of claims 1-15, which is characterized in that the excipient is an organic polycation or a mixture of organic polycations. 17. Pharmaceutical composition according to claim 16, which differs in that the peptide has a negative charge. 18. Pharmaceutical composition according to claim 16 or 17, which differs in that it contains basic polyamino acids or their mixture as an auxiliary agent. 19. Pharmaceutical composition according to claim 18, which is characterized by the fact that the excipient is polyarginine. 20. Pharmaceutical composition according to claim 18, which is characterized by the fact that the excipient is polylysine. 21. Pharmaceutical composition according to one of claims 16-20, which is characterized by the fact that the excipient is conjugated with cellular ligands. 22. pharmaceutical composition according to claim 21, which is characterized by the fact that the ligand is a carbohydrate residue. 23. Pharmaceutical composition according to claim 22, which differs in that the ligand is fucose. 24. pharmaceutical composition according to claim 21, which is characterized by the fact that the ligand is transferrin. 25. Pharmaceutical composition according to one of claims 1-24, which differs in that for parenteral administration it is a solution or suspension of peptides and an auxiliary agent in a pharmaceutically acceptable carrier. 26. Pharmaceutical composition according to one of claims 1-24, which differs in that it is a hydrogel for local use.