RU2333767C2 - Vaccine compositions, methods for application thereof in melanoma prevention and treatment, and genetic constructions for obtaining active substances of composition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns genetic engineering, biotechnology, immunology and medicine and can be applied in obtaining antitumoral vaccine and melanoma treatment. Vaccine compositions are based on heat shock proteins and antigen peptides for treatment of tumor disease, the said compositions containing hybrid protein consisting of heat shock HSP70 family protein and one of specific melanoma MAGE peptides (A1, A2, A3) or gp 100, as well as suitable pharmaceutical carrier. The invention also claims a method for obtaining the said hybrid proteins by gene expression in synthetic recombinant vectors in producer strains of E coli BL21 (DE3) cells.

EFFECT: improved immunising power.

3 cl, 4 ex, 1 tbl, 2 dwg

Description

The invention relates to the field of genetic engineering, biotechnology, immunology and medicine and can be used to obtain antitumor vaccines, preventive vaccination and treatment of melanoma.

The invention also includes methods of using antigen-presenting cells sensitized by hybrid proteins consisting of proteins of the HSP family and specific melanoma peptides.

Peptides of the MAGE group are cancer-specific markers, mainly expressed in melanoma cells, present in a healthy body only in testicles that are not accessible to cells of the immune system due to the lack of direct contact with immune cells and the lack of expression of proteins of the major histocompatibility complex of HLA class I on the surface of testicle cells. MAGE genes are represented by three subfamilies - MAGE-A, -B and -C - of which the products of MAGE-A expression are the most immunogenic. MAGE-A genes are represented by a family of 12 genes located on the long arm of chromosome X (Xq-28 region) [De Plaen E. et al., Immunogenetics 40: 360-369, 1994]. In most tumors studied, only the MAGE-A1, -A2, -A3, -A4, -A6, -A11 and -A12 genes are expressed. The epitopes recognized after presentation by cytotoxic lymphocytes are possessed only by the expression products of MAGE-A1 [van der Bruggen P. et al., Science 254: 1643-1647, 1991], MAGE-A2 [Viressen M.J. et al .. Int. J. Cancer 73: 125-130, 1997] and MAGE-A3 [Gaugler B. et al., J. Exp. Med. 179: 921-930, 1994]. The antigenic epitope MAGE-A1 presented by HLA-A1 (EADPTGHSY) has been identified [Yamashita N. et al., Hepatology 24: 1437-1440, 1996]. Two peptides (KWVELVHFL and YLQLVFGIEV) have been identified in the structure of the MAGE-A2 protein that are capable of binding to HLA-A2 and induce the formation of cytotoxic lymphocytes capable of killing cells expressing MAGE-A2 in transgenic mice.

The MAGE-A3 protein contains four known epitopes, the first of which (EVDPIGHLY [Gaugler B. et al, J. Exp. Med. 179: 921-930, 1994]) is presented by HLA-A1, the second (FLWGPRALV [van der Bruggen P. et al., Eur. J. Immunol. 24: 3038-3043, 1994]) - HLA-A2, third (IMPKAGLLI [Tanaka F. et al., Cancer Res. 57: 4465-4468, 1997]) - HLA- A24 and finally the fourth (MEVDPIGHLY [Herman J. et al., Immunogenetics 43: 377-383, 1996]) is presented by HLA-B44.

A particularly high level of expression of MAGE-A antigens is observed in melanoma cells (65% MAGE-A3 [De Plaen E. et al., Immunogenetics 40: 360-369, 1994]), breast carcinomas, head and neck cancers, lung carcinomas and bladder, as well as sarcoma [Van Pel. A. et al., Immunol. Rev. 145: 229-250, 1995]. A very high level of expression of MAGE-A1 (80%) is observed in liver cancer [Yamashita N. et al., Hepatology 24: 1437-1440, 1996]. There is also a correlation between the expression level of MAGE-A antigens and the degree of tumor progression [Brasseur F. et al., Int. J. Cancer 63: 375-380, 1995; Eura M. et al., Int. J. Cancer 64: 304-308, 1995; Katano M. et al., J. Surg. Oncol. 64: 195-201, 1997; Patard J.J. et al. Int. J. Cancer 64: 60-64, 1995]. The study of the ability of the peptide fragments of MAGE-A1 and MAGE-A3 to induce an anti-melanoma immune response in vivo showed that the peptide fragment (EVDPIGHLY) of MAGE-A3 showed the highest antitumor activity. The function of the proteins MAGE-A1 and MAGE-A3 is unknown, although it is known that they are localized in the cytoplasm of cells [Kocher T. et al. Cancer Res. 55: 2236-2239, 1995; Margolin L.F. et al., Proc. Natl. Acad. Sci. USA 91: 4509-4513, 1994] and are relatively small proteins: MAGE-A1 - 309 amino acids, MAGE-A2 and MAGE-A3 - 314 a.k., MAGE-A4 - 317 a.k. [Gamier J. et al., Methods Enzymol. 266: 540-553, 1996].

From an immunological point of view, antigens of the MAGE group are very good targets for immunotherapy. They are widely represented in a number of tumors and are absent in normal human tissues, with the exception of the testes, on which the cells of the immune system do not act due to the lack of direct contact between testicular cells and immune cells, as well as the absence of class I HLA on the surface of reproductive cells [Barker C.F. and Billingham R.E., Adv. Immunol. 25: 1-54, 1977; Tomita Y. et al., J. Urol. 149: 659-663, 1993]. MAGE proteins are relatively large proteins with a number of potential epitopes bound by Class I HLA. However, when the peptide fragments of these proteins are used in vivo, their immune response and antitumor activity remain very low.

One of the explanations for the low immunogenicity of MAGE antigens is the stability of their structure - like other highly organized proteins, MAGE have a high percentage of α-helices in the molecule, providing an optimal geometry for the maximum number and strength of hydrogen bonds. Inside the cell, for subsequent presentation of the antigen, the MAGE protein must be partially renatured, ubiquitated, and then subjected to proteolytic degradation. Obviously, a highly organized protein with a structure stabilized by a large number of bonds will be more resistant to proteosomal degradation, and therefore, it will be difficult to present on the cell surface as a part of type I HLA, which is observed in the case of MAGE melanoma proteins [Kirkin A.F. et al., APMIS 106: 665-679, 1998]. It is likely that tumor cells are characterized by an extremely low level of expression of the presented antigen from trace amounts of partially denatured tumor-specific MAGE proteins. Because of this, the limiting stage may be the training of “naive” lymphocytes, which requires approximately 1000 times higher concentrations (levels) of antigenic peptide presented on the cell surface than is required for the lysis of a cell carrying an antigen by already trained lymphocytes.

A significant increase in the immunogenicity of MAGE peptides can be achieved when they are used in vaccines in the form of heat shock proteins (stress proteins, HSP). Heat shock proteins have the ability to bind to a wide range of tumor-specific antigenic peptides. Such a complex has the ability to internalize into antigen-presenting cells, followed by effective presentation of the peptide on their surface as part of HLA class I. Presented accordingly, the antigen presented after activation of “naive” lymphocytes will cause the development of a cellular immune response directed to cellular proteins involved in the tumor process . An advantage of the approach proposed by the applicant is the possibility of creating complex anti-melanoma vaccines by mixing a set of hybrid proteins consisting of heat shock protein HSP70 and various specific antigenic melanoma peptides. Such vaccines will be distinguished by the simplicity of manufacture, safety and high effectiveness of the therapeutic effect of suppressing tumor growth.

Along with the MAGE peptides, one of the most promising for the induction of the cellular immune response of tumor antigens is the gp 100 gene expression product. The gp 100 protein is a membrane glycoprotein consisting of 661 amino acids. The gp 100 gene is expressed in most melanoma cell lines, as well as on cultured melanocytes. In non-melanoma cancer cell lines, gp 100 is not expressed. In normal tissues, gp 100 was found only in the retina [Adema G.J. et al., Am. J. Patol. 143: 1579-1585, 1993], where the protein is predominantly localized in the membrane and filament matrix of premelanosomes, where, apparently, is involved in the synthesis of melanin [Schaumburg-Lever G. et al., J. Cutan. Pathol. 18: 432-435, 1991; Vennegoor C. et al., Am. J. Patol. 130: 179-192, 1988]. The gp 100 protein is recognized by HLA-A2 restricted lymphocyte infiltrating tumors [Cox A.L. et al., Science 264: 716-719, 1994]. The sequence YLEPGPVTA recognized by cytotoxic lymphocytes was identified. Obviously, the use of the gp 100 protein or its fragments in the composition of HSP vaccines for prophylactic vaccination or treatment with melan has great prospects. A prerequisite for the therapeutic use of gp 100 is [Adema G.J. et al., Am. J. Patol. 143: 1579-1585, 1993], which demonstrated that the lymphocytes introduced into the patient with tumor melanoma and used to clone the gp 100 gene caused a regression of metastases in patients. The use of a hybrid protein consisting of HSP 70 and an antigenic fragment of gp 100 to activate antigen-presenting cells and subsequent induction of an immune response can make therapy much more predictable and effective.

Recently, a number of authors have carried out work on the creation of vaccines, which include heat shock proteins. Due to the low immunogenicity of synthetic antigenic peptides, several authors have attempted to increase it by creating in vitro non-covalent complexes of antigenic peptides and human or mycobacterial heat shock proteins [Barrios C. et al., Eur. J. Immmunol. 22: 1365-1372, 1992; Roman E. and Moreno C., Immunol. 88: 487-492, 1996]. These complexes have the ability to induce strong specific reactions to the antigenic peptides that make up their composition. Complexes of HSP proteins with antigenic peptides can be considered as readily available vaccine preparations that can be used without any additional adjuvants. Also, non-covalent complexes of HSP proteins with antigenic peptides can be used to activate ex vivo antigen-presenting dendritic cells taken from patients. After administration of activated dendritic cells to the patient, a sufficiently strong cellular immune response to the antigenic peptides that make up the complexes can be achieved.

Due to the high need for effective and safe vaccine preparations for the prevention and treatment of cancer, from which about 6 million people die every year in the world, work in the field of creating such drugs is extremely urgent. The result of work in this area was a series of inventions that formed the basis of a strategy for the development of antitumor vaccines, including heat shock proteins.

Application for invention of the Russian Federation No. 2003101965/14 describes a method for treating diseases associated with human papillomavirus (warts, cancer of the cervix and rectum, etc.) using a composition containing a fusion protein comprising mycobacterial heat shock protein (HSP65 or HSP70 from Mycobacterium bovis) and papilloma virus protein. Treatment can be carried out either by introducing a recombinant fusion protein to a subject, or by introducing a nucleic acid encoding a fusion protein contained in a viral vector. The disadvantages of the approach used by the authors of the application include the use of a viral vector, the consequences of which are not well understood and may pose a potential danger to the patient. It should also be noted that the use of the described invention is limited only to neoplasms associated with infection with the human papillomavirus.

U.S. Patent Application No. 20020216315 describes a method for inhibiting the growth of primary tumors and metastases by administering to a subject a recombinant fragment of a heat shock protein (HSP60, HSP70, HSP90) lacking a peptide binding domain. In this case, genes encoding these proteins from any organism can be used. The inventors consider a recombinant fragment of the heat shock protein as a means of stimulating the immune response in cancer and other diseases. According to the authors, this protein may also find application as an adjuvant to stimulate a specific immune response to exogenous antigens. Co-administration of a protein and an antigenic peptide to a subject should elicit a specific adaptive immune response directed against the antigen.

However, it should be noted that obtaining, as a recombinant product, not a whole native heat shock protein, but its fragment lacking a peptide-binding domain, is more advantageous from a technological point of view, since the process of obtaining protein is simpler, cheaper and takes less time. However, from the point of view of the practical use of such a protein, serious disadvantages are seen. Although the fragment retains the properties of an adjuvant, the absence of a peptide-binding domain in its composition does not allow the use of this protein for the creation of effective vaccines in vitro, in which antigenic peptides are in strong contact with the heat shock protein. Therefore, it seems more promising to create recombinant vaccines based on chimeric proteins consisting of an antigen associated with mycobacterial HSP70 or HSP65.

The present invention is based on the ability of heat shock proteins to stimulate immune responses. Moreover, heat shock proteins were previously used as adjuvants to enhance the immunogenicity of viral and bacterial proteins [RF patent No. 2229307]. When developing new vaccine and immunotherapeutic compositions, the applicant solved the problem of enhancing the immune response to peptides - fragments of specific protein melanoma antigens - using stress proteins (heat shock proteins). The hybrid proteins proposed by the applicant, consisting of the HSP70 protein and the specific melanoma peptides listed below, have not been previously used for vaccination against melanoma and its immunotherapy, and the composition and effectiveness of their action has been experimentally established by the applicant for the first time. Thus, the proposed technical solution meets the criteria of "inventive step" and "novelty."

An inventive task is to create new vaccine compositions for the prevention and treatment of human melanoma and methods for their use, as well as methods for using antigen-presenting cells sensitized by the components of these compositions.

The inventive task is solved by the fact that vaccine compositions are proposed for the prevention and treatment of melanoma containing an inert carrier, diluent and antigenic component in an effective amount, and characterized in that as the latter it contains individual hybrid proteins consisting of Mycobacterium tuberculosis HSP70 and various peptide fragments specific proteins of melanoma MAGE and gp 100, or mixtures thereof (MAGEA1-HSP70, MAGEA2-HSP70, MAGEA3-HSP70, PEPGP-HSP70).

A method is also proposed for producing said hybrid proteins by expressing their genes as part of synthetic recombinant vectors in producer strains of host cells.

Also provided is a method of immunotherapy and prophylactic vaccination, characterized in that the stimulation of immunity is carried out by introducing to the patient a composition containing these fusion proteins.

An immunotherapy and prophylactic vaccination method is also proposed, characterized in that the patient is stimulated with autologous antigen-presenting cells sensitized by hybrid proteins consisting of HSP70 Mycobacterium tuberculosis and various peptide fragments of specific proteins of the melange MAGE and gp 100.

The invention is illustrated by the following examples.

Example 1. Obtaining recombinant plasmid DNA encoding the synthesis of a hybrid protein MAGEA1-HSP 70

Synthesis of the DNA sequence encoding the C-terminal fragment of the protein HSP70 (DHSP70)

A cDNA library of M. tuberculosis genes was used as a source of the HSP 70 gene.

The M. tuberculosis HSP70 gene was isolated by amplification using the following primers:

where the selected fragment is the recognition site of the restriction enzyme XhoI, underlined - HindIII

Synthesis of the sequence required to create DNA encoding a fusion protein.

Sequence synthesis primers with the WatH I and XhoI restriction sites needed to create a fusion protein consisting of the MAGE-A1 peptide fused to HSP70 (MAGA1HSPD).

The underlined fragment is the BamHI restriction enzyme recognition site, isolated by XhoI

To synthesize the following sequence, the corresponding primer pairs were mixed and amplified, resulting in the following oligonucleotide was obtained:

где подчеркнутый фрагмент - сайт узнавания ферментом рестрикции BamHI, выделенный - XhoI

where the underlined fragment is the recognition site for the BamHI restriction enzyme isolated by XhoI

Synthesis of a sequence with restriction sites BamHI and HindIII encoding a fusion protein consisting of the MAGE-A1 peptide fused to HSP70 (MAGA1HSP)

To obtain MAGA1HSP, the DHSP70 fragment and the MAGA1HSPD oligonucleotide were treated with Xhol restriction enzyme and doped with T4 ligase.

The sequence encoding a hybrid protein consisting of a peptide

where the underlined fragment is the BamHI restriction enzyme recognition site, highlighted is HindIII, *** is the missing fragment of the DHSP70 sequence

The sequences encoding the synthesis of hybrid proteins, which include the other specific melanoma peptides listed in Table 1, were synthesized according to a similar scheme.

Creation of vectors for the expression of hybrid proteins

To create expression vectors, DNA fragments encoding the corresponding fusion proteins were treated with the BamHI and HindIII restriction enzymes and inserted at the BamHI and HindIII restriction sites into the pET vector (Novagen). The resulting vectors were called pEMAGA1HSPD, pEMAGA2HSPD, pEMAGA3HSPD, pEGPHSPD.

The general scheme of the obtained vectors is presented in figure 1.

The induction of the synthesis of the target hybrid proteins was evaluated in 10 ml of IPTG-induced cultures (growing to OD ₆₀₀ 0.5, introducing IPTG to 0.2 mM, then incubating for 3 hours at 37 ° C on a shaker). Protein production of the expected size was verified by SDS-PAGE by electrophoresis. For expression of recombinant proteins, E. coli strain BL21 (DE3) was used.

The products obtained as a result of these procedures are hybrid proteins consisting of Mycobacterium tuberculosis HSP70, to the C-terminus of which is attached one of the specific antigenic melanoma peptides listed in Table 1.

Table 1.
Amino acid structure of peptides - fragments of tumor-specific antigens. Antigen Amino acid sequence of the peptide fragment MAGE-A1 EADPTGHSY MAGE-A2 KMVELVHFL MAGE-A3 FLWGPRALV gp 100 YLEPGPVTA

Example 2. Induction of cytotoxic lymphocytes and their cytotoxic activity against tumor target cells.

Preparation of Dendritic Cells (DC)

DC was isolated from peripheral blood leukoconcentrate of healthy donors according to the method [Thumer B. et al., J. Immmunol. Meth. 223: 1-15, 1999]. Using gradient centrifugation (400 g, 30 min) through a ficoll-pack solution (Pharmacia, Sweden), a fraction of mononuclear leukocytes was obtained, which was then washed three times with Versen's solution (250 g, 175 g and 110 g no 15 min), resuspended in medium RPMI 1640 supplemented with 10% heat-inactivated bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin at a concentration of 50 million cells / ml, 10 ml were placed in plastic Petri dishes and incubated for 2 hours in a humidified atmosphere of 5% CO ₂ at 37 ° C. Then, non-adherent cells (mainly lymphocytes) were selected, centrifuged, frozen and stored in liquid nitrogen. 10 ml of fresh medium was added to the monolayer of adherent cells (mainly monocytes) and incubated under standard culture conditions until the next day, which was designated as "day 1". On days 1 and 3 of cultivation, GM CSF (800 units / ml) and IL-4 (1000 units / ml) were added to the incubation medium. On day 5, cells were harvested and transferred into 6-well plates at a density of 5 · 10 ⁵ cells per well in 3 ml of fresh medium with cytokines in the same concentration. On day 6, DC maturation inducers were added as a complex preparation of the first phase of the Leukinferon immune response cytokines and one of the hybrid proteins. On day 8, DCs were collected.

Induction of cytotoxic lymphocytes and determination of their cytotoxic activity (CTA)

Lymphocytes isolated from peripheral blood as described above were thawed and suspended in culture medium. To stimulate lymphocytes, DCs were washed with phosphate-buffered saline and placed in 24-well plates with lymphocytes at a DC: lymphocyte ratio of 1:20 in 1 ml of HyQ IMDM medium containing 10% inactivated bovine serum. The next day after the start of stimulation and then once every 3 days, 30 units / ml of human recombinant IL-2 were added to the culture. After 7 days, lymphocyte cultures were restimulated using thawed DCs in the same amount as for primary stimulation. After each stimulation, syngeneic lymphocytes inactivated by ultraviolet radiation (1 million per well) were introduced as feeders to the lymphocyte cultures. 5 days after the second stimulation, the determination of CTA lymphocytes was carried out in relation to the target tumor cells. For this purpose, a suspension of lymphocyte effectors in the amount of 12.5-100 thousand cells per well was introduced into the wells of a 96-well plate, and then tumor cells (5 thousand cells per well) pre-labeled with ³ H-thymidine so that the cell ratio target: cell effectors was 1: 2.5-1: 20. After 24 hours of incubation, the survival of tumor cells was determined and the CTA of effector lymphocytes was calculated by the formula: CTA = (1-P _{target + effector} / P _target ) -100%, where P _target and P _{target + effector} are the radioactivity of samples in which only incubated tumor target cells and the same cells together with effector cells, respectively.

The results of a study of the cytotoxic activity of stimulated lymphocytes against tumor cells are shown in figure 2. As target tumor cells, B16 melanoma cells were selected in which MAGE and g 100 antigens are expressed. As can be seen from Fig. 2, the cytotoxic lymphocytes obtained after co-cultivation with dendritic cells loaded with the corresponding fusion proteins had higher cytotoxic activity compared with control lymphocytes induced by intact dendritic cells, with all the studied target: effector ratios.

Example 3. Immunization of mice with vaccine compositions based on hybrid proteins.

For immunization, a vaccine composition containing the MAGEA1-HSP70 fusion protein (see Example 1) was injected intradermally into C57B1 / 6 mice in a dose range of 0.01 to 100 μg. 7 days after immunization, mice were injected subcutaneously with a suspension of tumor cells of the B16 line melanoma in an amount of 1 · 10 ⁶ cells / mouse. This number of grafted tumor cells is 10 times the dose commonly used in experiments to model tumors of this line in mice. The sizes of developing tumors were measured 3 times a week. Vaccination efficacy was evaluated by slowing the growth of tumors in immunized mice compared to control (intact) animals, as well as by increasing the average life expectancy of animals. At the time of death of the control animals, the average tumor volume in immunized mice was only 53% of the control, and the life expectancy of immunized animals was increased by 59%. Similar results were obtained by immunizing mice with vaccine compositions containing other hybrid proteins of Example. 1 (data not shown).

Example 4. Sensitization of vaccine compositions of antigen-presenting cells - macrophages and dendritic cells.

Dendritic cells are obtained from peripheral blood leukoconcentrate of healthy donors, as described in Example 3. Macrophages are obtained from mononuclear cells isolated from human peripheral blood by Ficoll gradient centrifugation. Cells are seeded on Petri culture dishes previously coated with human serum and incubated for 1 h at 37 ° C. Then, the non-attached cells are removed, and phosphate-buffered buffer containing 1 mM EDTA is added to the attached cells, cooled to + 4 ° C, and left for 10-15 minutes. Cells are harvested, washed and suspended in RPMI medium. Further cultivation and sensitization of cells is carried out as described in example 3.

Sensitized autologous antigen presenting cells can be intravenously administered to a patient with a malignant neoplasm. From 10 ⁶ to 10 ¹² sensitized macrophages can be simultaneously administered to one patient. Various immunomodulators, such as IFNα, IFNγ, TNFα, IL-2, -4, -6, etc. can also be used to enhance the effect.

Claims (3)

1. A vaccine composition for the treatment of melanoma based on a hybrid protein, which is an antigenic peptide and human heat shock protein HSP70, characterized in that it contains specific melanoma peptides selected from the group: MAGE (A1, A2 as an antigenic peptide in the composition of the hybrid protein) , A3) or gp 100 (PEPGP) with the sequences shown in table 1, as well as a suitable pharmaceutical carrier. 2. The method of producing the hybrid protein according to claim 1, comprising expressing the synthetic genes of these proteins in E. coli BL21 (DE3) as part of the recombinant vectors: MAGA1HSPD, MAGA2HSPD, MAGA3HSPD and PEPGPHSPD, described in Fig. 1. 3. The method of immunotherapy, characterized in that the stimulation of immunity is carried out by introducing the patient the vaccine composition according to claim 1.

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