RU2402602C1 - CELL LINE OF HUMAN MELANOMA mel Rac, APPLIED FOR OBTAINING ANTITUMOUR VACCINE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: cell line of human melanoma mel Rac has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 710 Д".

EFFECT: invention allows to extend arsenal of cell lines of human melanoma, used for creation of antitumour vaccines, which are applied for treatment of melanoma and other malignant neoplasms.

1 tbl, 2 ex

Description

The invention relates to the field of medical biotechnology, in particular to the production of cell lines used to create antitumor vaccines.

Vaccine therapy is one of the immunological approaches in the treatment of cancer. The principle of this method is based on the induction of antitumor immunity after the introduction of a tumor antigen into the body.

A central event in the T-cell immune response against tumor cells is the stimulation of T-receptor recognition of antigenic determinants selectively expressed on tumor cells. Tumor antigens, as a rule, are processed before their presentation in the context of histocompatibility molecules on the cell surface. The various categories of tumor-associated antigens can be divided into three main groups: cancer / testicular antigens (MAGE, BAGE, PRAME, NY-ESO-1, HOM-MEL-40), differentiating melanocyte antigens (tyrosinase, Melan-A / MART-1, gp100 , TRP-1, TRP-2), and mutated antigens (MUM-1, CDK4, β-catenin gp100-in4, p15, N-acetylglucose amine transferase V). From an immunological point of view, cancer / testicular antigens can be good targets for tumor immunotherapy, since in normal tissues this group of antigens (MAGE and PRAME) is not expressed except for testicular tissue, which is inaccessible to cells of the immune system due to the lack of direct contact with immunocompetent cells [1] and the absence of HLA expression of class I antigens on them [2]. Unlike cancer / testicular antigens, the immunogenicity of the differentiation antigens of melanocytes is low due to immunological tolerance to these “their” antigens. However, such an antigen? as Melan-A / MART-1 contains several epitopes for the recognition of CTLs (cytotoxic lymphocytes) and is able to induce the generation of melanomaspecific CTLs.

Thus, the expression of various tumor markers plays a key role in the induction of antitumor immunity. A variety of appropriate antigens allows for a more “complementary” selection of cell lines to create antitumor vaccines.

Tumor-based vaccines are whole-cell vaccines and are live allogeneic or autologous tumor cells.

Autologous / syngeneic whole tumor cells contain almost all antigens expressed by the host tumor, which reduces the risk of allergic reactions to foreign non-tumor-specific antigens, as well as the risk of contamination by pathogenic viruses and intracellular parasites. A vaccine consisting of several cell lines (multivalent vaccines) contains a wide range of tumor antigens and is used as an allogeneic one. A multivalent vaccine such as Mortona et al. [3], consists of three allogeneic melanoma cell lines with high expression of surface immunogenic glyco- and lipoproteins and gangliosides. Clinical trials of such a vaccine showed that the development of an immune response, both cellular and humoral, to these antigens correlated with an increase in patient survival. Another vaccine, Melacine [4] (Corixa corp., Canada), consisting of a lysate of allogeneic melanoma cell lines, causes an antitumor effect in 5-10% of melanoma patients.

The objective of the present invention is to obtain a new tumor cell line of human melanoma carrying a specific set of antigens, which will allow its use in the creation of antitumor vaccines.

The technical result obtained by using the invention is expressed in expanding the arsenal of cell lines used to create antitumor vaccines (whole-cell, genetically engineered), which makes it possible to increase the effectiveness of treatment and increase the life expectancy of patients with malignant neoplasms.

The problem is solved in that a new cell line mel Rac was obtained from a tumor sample of disseminated human skin melanoma.

The resulting cell line has stable cultural and morphological characteristics. It is stored in the collection of cell cultures of the Institute of Cytology RAS under the number RKKK (P) 710D.

Pedigree of the mel Rac cell line

The cell line was obtained from a tumor sample of the patient R.M., and / b 95/5520, who was treated in 2002 with a diagnosis of disseminated melanoma of the skin of the right lower leg. The previous treatment is surgical.

Obtaining met Rac cell line

Tumor tissue was obtained surgically by removing a metastatic node from the soft tissues of the right thigh. A cell suspension was prepared from tumor tissue. The resulting cell suspension was seeded into vials and cultured for a long time. A stably growing cell line was obtained at passage 20.

Morphological characteristics of the mel Rac cell line

The cytogram of the mel Rac cell line is represented by a polymorphic cell composition. There are cells of round, spindle-shaped, elongated, elongated, multi-branch and irregular shapes. The nuclei of the cells are sharply hyperchromic with a granular and coarse-grained chromatin structure, often contain hypertrophic nucleoli. Fragmentation and budding of nuclei is observed. The cytoplasm of cells is not homogeneous, intensively basophilic.

Karyological characteristics of the mel Rac cell line

Cell fixation date: 11/17/05

The culture is uniform in core diameters, color density and chromatin state. Analyzed 18 metaphases. The number of chromosomes ranges from 60 to 80. The modal number of chromosomes corresponds to the triploid set (3n). An additional chromosome material of unknown origin was found on the short arm of chromosome 1. Marker chromosomes (from 1 to 3 pieces per cell) are found. In general, in culture, there are complex multiple rearrangements that cannot be identified without the use of molecular cytogenetic methods. In almost all cells, dicentric chromosomes are detected (from 1 to 5 pcs. In the cell).

Karyotype - 60 ~ 80 <3n>, add (1) (p36), + 1 ~ 3mar, inc

Immunogenetic study (determination of HLA phenotypes (I class))

A23 (9), A26 (10); B39 (16), B8.

The cultural properties of the mel Rac cell line

The mel Rac cell line is cultured in RPMI culture medium (90%), 10% fetal calf serum containing antibiotics (penicillin and streptomycin at a concentration of 100 u / ml and 100 μg / ml, respectively). 1 × 10 ⁶ cells are seeded in culture vials of 25 cm ² volume in 5 ml of medium. The cultivation temperature is 37 ° C. A monolayer of cells forms in 3-4 days. At a seed concentration of 70-100 thousand / ml, a monolayer is formed on 2-3 days without changing the medium. Cells are removed using standard solutions: 0.25% trypsin solution and 0.02% Versen solution in a ratio of 1: 1. At a seeding concentration of 500 thousand / ml, the proliferation index after 48 hours of cultivation is 3.6-4.6.

Cryopreservation Conditions

For long-term storage, cells are preserved by freezing in liquid nitrogen. Cells are resuspended in freezing medium — RPMI culture medium (80%), fetal calf serum (20%), DMSO (10%). Freezing mode: liquid nitrogen, lowering the temperature by 1 ° С per minute to minus 25 ° С, then quick freezing to a temperature of minus 70 ° С. Storage in liquid nitrogen at a temperature of minus 196 ° C. Defrosting is quick at 37 ° C. Cells are diluted in 10 ml of serum-free medium, precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, and transferred to a 25 cm culture vial. Cell viability is assessed by the inclusion of trypan blue. Cell viability after thawing is 90%.

Contamination

With prolonged observation, bacteria and fungi were not found in the culture. Mycoplasma test is negative.

Examples of use of the mel Rac cell line

Example 1. The cultivation of the cell line mel Rac. Surgical tumor tissue was mechanically divided into fragments of 2-3 mm ³ in RPMI-1640 medium, then using a Cell dissociation sieve-tissue kit (Sigma), a cell suspension was obtained. The number of viable cells was determined by the standard method in the Goryaev chamber using a 0.5% solution of trypan blue in PBS. 1 × 10 ⁶ cells were seeded in culture vials of 25 cm ² volume in 5 ml of medium. The cultivation temperature is 37 ° C. Cells were cultured in RPMI 1640 medium containing 20% fetal calf serum, 2 mM L-glutamine, 1% HEPES, penicillin (100 u / ml), streptomycin (100 μg / ml) and an amino acid and vitamin complex (Flow Lab.) culture bottles (Costar). After passage 20, a stably growing cell line was obtained.

Example 2. The definition of antigens expressed on the cell line mel Ras. The obtained mel Ras cell line possessing stable cultural and morphological characteristics, using immunofluorescence, immunohistochemistry, and PCR (polymerase chain reaction) analysis methods, was tested for expressed antigens (differentiating, tumor-associated and histocompatibilities). Differentiating melanoma markers determining the ratio of this line to melanoma were studied using monoclonal antibodies CD63, HMB45, MelanA, HMW, Tyrosinaza. Cancer-testicular markers of the MAGE class, which can be expressed on tumors of various histogenesis, were investigated in the PCR reaction. Histocompatibility antigens are determined using monoclonal antibodies in an immunofluorescence reaction. The data obtained are shown in table 1.

Table 1 Expression of antigens on the mel Rac cell line. Differentiation Antigens Cancer Testicular Antigens Histocompatibility antigens Cd63 will put MAGE will put HLA (I class) will put HMB45 neg HLA-DR (II class) will put Melana neg Tyrosinaza neg Hmw will put

As follows from table 1, this cell line is characterized by the expression of melanoma markers: CD63, HMW, emphasizing the specificity of this cell line. The positive expression of cancer-testicular markers of the MAGE family corresponds to the oncological profile and allows the wide use of this line to create an antitumor vaccine. Histocompatibility antigens are represented by a molecule of the first and second class (HLA).

Thus, this cell line of melanoma of human skin mel Rac has its own individual phenotype of tumor markers, consisting in the presence of differentiating antigens HMW, CD63 and cancer-testicular markers of the MAGE family; the absence of antigens MelanA, HMB45, Tyrosinaza, as well as the presence of histocompatibility molecules of the first and second class, which allows the use of the obtained cell line to create antitumor vaccines (whole cell, genetically engineered) used to treat melanoma and other malignant neoplasms.

Bibliography

1. Barker C.F. et al. Immunologically privileged sites. ADV. Immunol. 1977; 25: 1-54.

2. Tomita Y. et al. Immunohistochemical detection of intracellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex class I antigens in seminoma J. Urol. 1993; 149: 659-663.

3. Morton DL et al. Ann N Y Acad Sci 1993; 690: 120.

4. Sondak V.K. Sosman J.A. Results of clinical trials with an allogenic melanoma tumor cell lysate vaccine: Melacine / Semin cancer Biol. 2003. Dec. 13 (6): 409-15.

Claims (1)

The mel Rac human melanoma cell line used to create antitumor vaccines is stored in the Specialized Collection of Vertebrate Cell Culture of the Russian Cell Culture Collection under the number RACC (P) 710 D.