RU2390556C1 - HUMAN MELANOMA CELL LINE mel Z USED FOR MAKING ANTI-TUMOUR VACCINES - Google Patents

Abstract

FIELD: chemistry; biochemistry. ^ SUBSTANCE: invention relates to biotechnology, specifically to making cell lines used for making anti-tumour vaccines. The human melanoma cell line mel Z has stable culture and morphological characteristics and is stored in the special collection of vertebrate cell cultures of the Russian collection of cell cultures of the Institute of Cytology of the Russian Academy of Sciences under number RKKK (P) 711D. ^ EFFECT: invention widens the range of human melanoma cell lines used to make anti-tumour vaccines, used for treating melanoma and other malignant growths. ^ 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 (MUC-1, CDK4, β-catenin gp100-in4, p15, N-acetylglucose aminotransferase 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, with the exception of testicular tissue, which is not accessible 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 differentiation antigens of melanocytes is low due to the immunological tolerance to these "their" antigens. However, an antigen such as Melan-A / MART-1 contains several epitopes for the recognition of CTLs (pitotoxic lymphocytes) and is capable of inducing the generation of melanoma-specific 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 vaccine) contains a wide range of tumor antigens and is used as an allogeneic one. A multivalent vaccine such as the Mortona et al. [3] vaccine 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 of both cellular and humoral type 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 life expectancy in the treatment of malignant neoplasms.

The problem is solved in that a new cell line mel Z 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) 711D.

Pedigree of the melZ cell line

The cell line was obtained from a tumor sample of a female patient Z.Z.S., 55 years old, and / b 03/12300, who was treated in 2003 with a diagnosis of disseminated melanoma of the skin of the back. The material was obtained by surgical removal of the metastatic node from the soft tissues of the interscapular region. Previous treatment: surgical, chemotherapy, immunotherapy.

Obtaining melZ cell line

Tumor tissue obtained by surgical removal of skin metastases. 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 mel Z

The cytogram of mel Z culture consists of spindle-shaped, elongated, elongated, process-shaped cells and a few round-shaped cells. The nuclei of cells are oval, round and irregular in shape, normo- and hyperchromic with a granular and coarse-structure chromatin. The nuclei contain one or more small nucleoli. The cytoplasm of the cells is not homogeneous, moderately basophilic with a pinkish tinge. There are two nuclear cells and a small number of giant multinucleated cells with 4-6 nuclei. Cell division patterns are often detected.

Karyological characteristics of the melZ cell line

Cell fixation date: 04/27/06

The culture is uniform in core diameters, color density and chromatin state. 43 metaphases were analyzed. The number of chromosomes ranges from 44 to 46. The modal number of chromosomes corresponds to the diploid set (2n). Trisomy is observed on chromosome 7; monosomy on chromosome 11; a derivative chromosome consisting of a short arm of chromosome 1, a long arm of chromosome 9 and a segment of chromosome 1 proximal to 1p31 transposed to the end of the short arm of chromosome 1; translocation of two long arms of chromosome 1 with the formation of an isochromosome; translocation of the short arm 6 and long arm 9 of the chromosomes; translocation of part of the long arm 6 of the chromosome to the end of the long arm 17 of the chromosome (a segment of chromosome 6 distal to 6q22 was translocated to chromosome 17 in the 17q24 region). Thus, the karyotype imbalance consists in partial monosomy on the short arm of chromosome 1, trisomy on the short arm of chromosome 6, trisomy on chromosome 7, nullisomy on the short arm of chromosome 9 and monosomy on chromosome 11.

Kapiotype - 44 ~ 46, XX, der (1; 1 ; 9) (1pter → 1p31 :: 1 p36 → 1 p10 :: 9q10 → 9qter),

i (1) (qter → q10 :: q10 → qter), der (6; 9) (6pter → 6p10 :: 9q10 → 9qter),

t (6; 17) (6pter → 6q22 :: 17q24 → 17qter; 17pter → 17q24 :: 6q22 → 6qter), + 7, -11

The cultural properties of the melZ cell line

The met Z cell line is cultured in RPMI nutrient medium (90%), fetal calf serum 10% containing antibiotics (penicillin with streptomycin at a concentration of 100 units / ml and 100 μg / ml, respectively). 1 × 10 ⁶ cells are seeded in culture vials of 25 cm ³ 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 (90%), fetal calf serum 10%, 10% DMSO. Freezing mode: liquid nitrogen, temperature decrease by 1 ° С per minute to minus 25 ° С, then quick freezing to 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 and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, and transferred to a 25 cm ³ 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 the use of the melZ cell line

Example 1. Cultivation of the met Z cell line

Tumor tissue obtained by surgery to remove metastases of skin melanoma 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 ³ 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 units / ml), streptomycin (100 μg / ml) and an amino acid and vitamin complex (Flow Lab.) in culture bottles (Costar). After passage 20, a stably growing cell line was obtained.

Example 2. Determination of antigens expressed on mel Z cell line

The obtained mel Z cell line, which has stable cultural and morphological characteristics, was studied using immunofluorescence methods for expressed antigens (differentiating, histocompatibility, cancer-testicular).

The differentiating melanoma marker, which determines the ratio of this line to melanoma, was studied by us using monoclonal antibodies CD63. Histocompatibility antigens are determined using monoclonal antibodies in an immunofluorescence reaction. Cancer-testicular markers that can be expressed on tumors of various histogenesis, were investigated in the PCR reaction. The data obtained are reflected in the table.

MelZ cell line antigen expression Differentiation Antigens Cancer Testicular Antigens Histocompatibility antigens Cd63 will put. MAGE Weakly put. HLA _{(I class)} will put. HLA-DR _{(II class)} neg.

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

This cell line is characterized by the expression of a melanoma differentiating marker CD63, a histocompatibility class 1 antigen, and cancer-testicular markers of the MAGE class (weakly positive). The histocompatibility antigen of the second class is absent.

Thus, this cell line of human skin melanoma has its own individual phenotype of tumor markers, consisting in the presence of the differentiation antigen CD63; histocompatibility molecules of the first class and weakly positive cancer testicular markers of the MAGE 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.

Literature

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. 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 human melanoma cell line mel Z used to produce antitumor vaccines is stored in the Specialized Collection of Vertebrate Cell Culture of the Russian Cell Culture Collection of the Institute of Cytology RAS under the number RKKK (P) 711D.