Cell line of human melanoma mel me, applied for obtaining antitumour vaccine

FIELD: medicine.

SUBSTANCE: cell line of human melanoma mel Me has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of vertebrates of Russian collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 712 Д".

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

1 tbl, 2 ex


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

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

The Central event in the process of T-cell immune response against tumor cells is to stimulate recognition of the T-receptor antigenic determinants, selectively expressed on tumor cells. Tumor antigens, as a rule, subjected to processing prior to their presentation in the context of histocompatibility molecules on the cell surface. Different categories of tumor-associated antigens can be divided into three main groups: cancer/testicular antigens (MAGE, BAGE, PRAME, NY-ESO-1, HOM-MEL-40), differencirovanie antigens of melanocytes (tyrosinase, Melan-A/MART-1, gp100, TRP-1, TRP-2) and mutated antigens (MUC-1, CDK4, β-catenin gp100-in4, p15, N-acetylglucosaminyltransferase V). With immunologically cancer/testicular antigens may be good targets for immunotherapy of tumors, because in normal tissues, this group antigens (MAGE and PRAME) is not expressed, except in the tissue of the testicles, which not is available for cells of the immune system due to the absence of their direct contact with immunocompetent cells [1] and lack of expression of HLA antigens class I [2]. Unlike cancer/testicular antigens, immunogenic differencirovannyh antigens of melanocytes low due to immunological tolerance to these "its" antigens. However, this antigen Melan-A/MART-1, contains several epitopes for recognition CTLs (cytotoxic lymphocytes) and is able to induce the generation of melanoma-specific CTLs.

Thus, expression of various tumor markers plays a key role in the induction of antitumor immunity. A variety of relevant antigens allows for more "complementary" to select cell lines to generate antitumor vaccines.

Vaccines prepared on the basis of tumor cells are whole cell vaccines and are living allogeneic or autologous tumor cells.

Autologous/syngeneic solid tumor cells comprise almost all of the antigens expressed by the tumor host, which reduces the risk of allergic reactions to alien neoprogressive antigens, and also reduces the risk of contamination with pathogenic viruses and intracellular parasites. A vaccine consisting of multiple cell lines (polyvalent vaccine), contains a broad spectrum of tumor antigens and is used as allogeneic. Such polyvalent vaccine, as the vaccine forti et al. [3], consists of three allogeneic melanoma cell lines with high expression of immunogenic surface Glyco - and lipoproteins and gangliosides. Clinical trials of this vaccine have shown that the development of the immune response as the cellular and humoral type to these antigens was correlated with increased survival of patients. Another vaccine, "Melacine" [4] (Corixa corp., Canada), consisting of lysate allogeneic melanoma cell lines, induces antitumor effect in 5-10% of patients with melanoma.

The present invention is to obtain a new tumor cell lines human melanoma carrying a particular set of antigens that can be used in the creation of antitumor vaccines.

The technical result to be obtained by the use of the invention is expressed in expanding Arsenal of cell lines used to generate antitumor vaccine (whole cell, genetically engineered), which gives the opportunity to increase the effectiveness of treatment and increase life expectancy in the treatment of malignant tumors.

The problem is solved in that the resulting new cell line mel Me from tumor sample disseminated melanoma human skin.

The obtained cell line has a stable cultural and morphological characteristics. XP is doubt in cell culture collection of the Institute of Cytology RAS number RCCC(P) D.

Lineage cell line mel Me

Cell line derived from a tumor sample of a patient MME, and/b 02/1902, were treated in 2002 with a diagnosis of disseminated melanoma skin back. Tumor material obtained by removing metastatic site skin back. Previous treatment: surgery.

Obtaining cell lines mel Me

Tumor tissue obtained during surgical removal of metastases of the skin. The obtained cell suspension was seeded into flasks and cultured in a long time. Steadily growing cell line was obtained on 20 passage.

Morphological signs of cell lines mel Me

The culture they mel Me consists predominantly of epitheliopathy cells small, medium and large sizes. Cells are groups of small dense clusters and scattered. There are a few spindle cells, process and elongated elongated shape. Cell nuclei polymorphic, round, oval, persteviecobu and irregular in shape, have different sizes, thick edges, grobalization and granular chromatin structure, increased one or more nucleoli. The cytoplasm of the cells inhomogeneous, painted in basophilic tones of varying intensity. Commonly identified mitoses (0-6 in the field of view). Meet dual core cell and a single giant multinucleated cells.

Karyological characterization of cell lines mel Me

Date of fixation of cells: 24.11.05

The culture is characterized by different diameters of the nuclei at the same density of color and the state of chromatin, characteristic of interphase nuclei.

Analyzed 88 metaphases. The number of chromosomes in 60 cells ranges from 47 to 50. The modal number of chromosomes in these cells corresponds to a diploid set (2n). In 28 cells detected tetraploid (4n) set (about 100 chromosomes). There trisomy for chromosomes 2 and 7, the ring chromosome 15. Found additional chromosomal material of unknown origin on both arms of chromosome 1.

The karyotype is 47~50<2n>,XY,der(1)add(1)(p36)add(1)(q43),+2,+7,r(15).

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

A2, A31(19); V(17), V(22).

Cultural properties mel Me

Cell line mel Me is cultivated in a nutrient medium RPMI (90%), fetal calf serum 10%, containing antibiotics (penicillin with streptomycin at a concentration of 100 u/ml and 100 µg/ml, respectively). In the culture flasks of 25 cm3in 5 ml of medium seeded with 1×106cells. The cultivation temperature of 37°C. the Monolayer of cells is formed in 3-4 days. When seeding concentration of 70-100 thousand/ml monolayer is formed at 2-3 days without changing the medium. Cells are removed using standard the solutions of 0.25% solution of trypsin and 0.02% solution of Versene in the ratio of 1:1. When planting a concentration of 500 thousand/ml index of cell proliferation after 48 hours of cultivation is 3.6-4.6.

Conditions of cryopreservation

For long-term storage of cells preserved by freezing in liquid nitrogen. Cells resuspended environment for freeze - nutrient medium RPMI (80%), fetal calf serum 20%, 10% DMSO. The mode of freezing: liquid nitrogen, the temperature reduction of 1°C per minute up to minus 25°C, then rapid freezing to minus 70°C. Storage in liquid nitrogen at minus 196°C. Defrost quickly, at 37°C. Cells diluted in 10 ml serum-free medium and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, and transferred into a culture flask of 25 cm3. Cell viability is assessed according to the inclusion Trypanosoma blue. Cell viability after thawing is 90%.


With long-term observation of bacteria and fungi in culture is not detected. Test for Mycoplasma negative.

Examples of the use of cell lines mel Me

Example 1. Cultivation of cell lines mel Me. Tumor tissue obtained during surgical removal of melanoma metastases were divided mechanically into fragments the size of 2-3 mm3in the medium RPMI-1640, then, using Cell dissociation sieve-tissue kit is (Sigma), received cell suspension. The number of viable cells was determined by a standard method in the camera Goryaeva, using 0.5% solution Trypanosoma blue in PBS. In the culture flasks of 25 cm3in 5 ml of medium were seeded at 1×106cells. The cultivation temperature of 37°C. Cells were cultured in medium RPMI-1640 containing 10% fetal calf serum, 2 mm L-glutamine, 1% HEPES, penicillin (100 u/ml), streptomycin (100 μg/ml) and a complex of amino acids and vitamins (Flow Lab.) in the culture flasks (Costar). After 20 passages received steadily growing cell line.

Example 2. The determination of antigens located on the cell line mel Me. The obtained cell line mel Me with a stable cultural and morphological characteristics was investigated using methods immunofluorescence, immunohistochemistry, PCR (polymerase chain reaction) analysis of expressed antigens (differencirovanie, tumor-associated and histocompatibility).

Differencirovanie melanoma markers that define this line to melanoma, we investigated using monoclonal antibodies CD63, NPS, MelanA, Tyrosinasa, HMW. Cancer-testicular markers of class MAGE, which can be expressed in tumors of different histogenesis, studied in the PCR reactions. The histocompatibility antigen definition is Lena using monoclonal antibodies in immunofluorescence reaction.

This cell line is characterized by the expression of melanoma (differencirovannyh) markers: CD63, MelanA, HMB45, emphasizing the specificity of this cell line. Tyrosinasa neg. A unique feature of this line is the presence of antigens of the major histocompatibility first and second class, which causes the increase immunogenicity by presenting tumor antigens directly CD4 and CD8 cells. Cancer-testicular markers of class MAGE (A6; A12; A2; A3 and a10 - absent) (table).

The expression of antigens on the cell line mel Me
Differencirovanie antigensCancer-testicular AntigensThe histocompatibility antigen
CD63I promise.MAGERef.HLA (class I)I promise.
NMWI promise.HLA-DR(class II)I promise.
MelanAI promise.

Thus, this cell line melanoma human skin Me mel has its own individual phenotype of tumor markers, implying the presence of differencirovannyh antigens (CD63, MelanA, HMB45) and positive expressii molecules of the major histocompatibility first and second class; in the absence of the expression of antigen Tyrosinasa and cancer-testicular markers, which allows to use the cell line to create an antitumor vaccine (whole cell, genetically engineered), used to treat melanoma and other malignant neoplasms.


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

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

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

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

Cell line human melanoma mel Me used to obtain anti-cancer vaccines, the storage is carried out in a Specialized collection of cell cultures vertebrate Russian cell culture collection of the Institute of Cytology RAS number RCCC (P) D.


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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.

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1 tbl, 2 ex