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

FIELD: medicine.

SUBSTANCE: cell line of humans melanoma mel H 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) 715 Д".

EFFECT: invention allows to extend arsenal of human melanoma lines, which are used for creation of antitumour vaccine, 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, FRAME, 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 FRAME) is not expressed, except in the tissue of the testicles, which half 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 antigen immunogenicity 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 Mrtona 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, 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 N of the tumor sample disseminated melanoma human skin.

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

Lineage cell line mel H

Cell line derived from a tumor sample of a patient HNS, and/b 00/9865, were treated in 2001 with a diagnosis of disseminated melanoma skin of the left foot. Tumor material obtained by removal of metastatic inguinal lymph node. Previous treatment: surgery, immuno - and chemotherapy. Steadily growing cell line was obtained on 31 passage.

Morphological signs of cell lines mel H

Cell line mel H consists of relatively monomorphic cells of the small and medium size, round, oval and elongated without a clear cytoplasmic loops. The cytoplasm is abundant inhomogeneous, painted in basophilic tone with a pinkish tinge. Cell nuclei Hypo - and normocapnia with granular chromatin structure, sometimes contain nucleoli. Rare 2 nuclear and giant mononuclear and multinucleated cells. There are mitoses.

Karyological characterization of cell lines ml N

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 69 cells ranges from 54 to 65 chromosomes, reflecting triploid set of (3n). In 19 cells detected hexaploid (6n) set (about 120 km HRO is Osom). This cell with replicated chromosome sets in which there was no separation of the cytoplasm, i.e., the presence of increased polyploidization. Found additional chromosomal material of unknown origin on the long arm of the two chromosomes 19 and on the long arm of chromosome from the group D, the inversion of part of chromosome 16, translocation of chromosomes 13 and 15. In 32 cells observed chromosomal aberrations, presents dicentrine (47), tricentenary (1) and ring (3) chromosomes. Therefore, this culture is characterized by high chromosomal variability. Karyotype - 54~65<3n>,der(13;15)(q10;q10),add(Dq),inv(16)(p11q12),add(19)(q13.4).

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

A3, A30(19); B13, B7.

Cultural properties of the cell line ml N

Cell line mel H 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 culture flasks (25 cm2in 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 solutions of 0.25% solution of trypsin and 0.02% solution of Ve is sung in a 1:1 ratio. 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 with a volume of 25 see cell Viability is assessed according to the inclusion Trypanosoma blue. Cell viability after thawing is 90%.

Contamination

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

Examples of the use of cell lines ml N

Example 1. Cultivation of cell lines mel H. 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" (Sigma)is Uchali 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 culture flasks (25 cm in 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 passage 31 received steadily growing cell line.

Example 2. The determination of antigens located on the cell line ml N. The obtained cell line mel H, with a stable cultural and morphological characteristics, using methods immunofluorescence, immunohistochemistry, PCR (polymerase chain reaction) analysis was investigated on expressed genes (differencirovanie, tumor-associated and histocompatibility). Differencirovanie melanoma markers that define this line to melanoma, was investigated using monoclonal antibodies CD63, NPS, MelanA, Tyrosinaza, HMW. The histocompatibility antigen identified by using monoclonal antibodies in immunofluorescence reaction.

This cell line is characterized by the expression of melanoma (differencirovannyh) markers: HMW and CD63, antigene the histocompatibility first class emphasizing the specificity of this cell line. Negative experse differencirovannyh markers NMW, MelanA, Tyrosinaza. Positive expression of cancer-testicular genes MAGE corresponds cancer profile and can be used for this line to create cancer vaccines. The data obtained is displayed in table 1.

Table 1
The expression of antigens on the cell line mel N
Differencirovanie antigensCancer-testicular antigensThe histocompatibility antigen
CD63I promise.MAGEI promise.HLA (class I)I promise.
NMWRef.HLA-DR(class II)Ref.
MelanARef.
TyrosinazaRef.
HMWput

As follows from the table. 1, this cell line is characterized by the expression of melanoma markers: HMW and CD63, emphasizing the specificity of this cell line. Positive expression of cancer-testicular markers collection MAGE corresponds cancer profile and can be used for this line to create cancer vaccines. Antigens presented by the major histocompatibility molecule of the first class (HLA).

Thus, this cell line melanoma human skin mel N has its own individual phenotype of tumor markers, implying the presence of differencirovannyh antigens - HMW, CD63, cancer-testicular genes of the MAGE class, as well as molecules of the major histocompatibility first class and the absence of such differencirovannyh markers, as MelanA, HMB45, Tyrosinaza. This phenotype allows the use of the cell line to create an antitumor vaccine (whole cell, genetically the engineering), used to treat melanoma and other malignant neoplasms.

References

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. 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 H used to obtain anti-cancer vaccines, is stored 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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