RU2287576C1 - HUMAN MELANOMA CELLULAR LINE mel Ibr USED IN PREPARING ANTITUMOR VACCINE - Google Patents

Abstract

FIELD: biotechnology, medicine, oncology.

SUBSTANCE: invention relates to a novel human melanoma cellular line mel Ibr possessing stable cultural and morphological indices that is stored in the Specialized collection of cellular cultures in Cytology institute RAS at number PKKK (P) 689D. The cellular line is characterized by expression of melanoma (differentiating) markers - CD-63, HMW and cancer-testicular marker MAGE-3, the presence of histo-compatibility antigens of the first and the second class. The cellular line can be used in creature of antitumor (all-cellular, genetic engineering) vaccines used in treatment of melanoma and other malignant neoplasms. Using this cellular line provides preparing the novel effective antitumor vaccine. Invention can be used in medicine for vaccine therapy of malignant neoplasms.

EFFECT: valuable medicinal properties of cellular line.

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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 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), differentiation antigens of melanocytes (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 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 (cytotoxic 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 more "complementary" selection of cell lines for the development of vaccines.

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

Autologous / syngeneic whole tumor cells contain almost all the 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 have shown that the development of an immune response of both cellular and humoral type to these antigens correlated with increased 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 Ibr 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) 688D.

The pedigree of the mel Ibr cell line

The cell line was obtained from the tumor sample of the patient I.A.U. with a diagnosis of disseminated skin melanoma. The material was obtained by removal of the subcutaneous metastatic node. Before taking the patient’s material, surgical, chemotherapeutic, and immunotherapeutic treatments were performed.

The pedigree of the mel Ibr cell line

The cell line was obtained from a tumor sample of metastasis of melanoma of the skin. The material was obtained by removing the patient T.A.A. two subcutaneous metastatic nodes. Before taking the patient’s material, he underwent surgical and chemotherapeutic treatment.

Obtaining mel Ibr cell line

Tumor tissue was obtained by surgery to remove 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 15.

Morphological characters mel mel Ibr

mel Ibr is characterized by low-grade melanoma cells that are mostly irregularly rounded in shape with nuclei polymorphic in various shapes and abundant light blue vacuolated without clear contours of the cytoplasm. Large 2-core and giant multinuclear cells, mitoses are observed.

Karyological Characteristics of mel Ibr

The number of chromosomes in cells 57, 78. Permanent markers from m1 to m9.

Cultural properties of mel Ibr

The mel P cell line is cultured in RPMI nutrient medium (80%) - fetal calf serum 20% containing antibiotics (penicillin with 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 of 0.25% trypsin solution and 0.02% versene solution in a 1: 1 ratio. 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 nutrient medium (80%), fetal calf serum 20%, 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 fast 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 ² 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 Ibr cell line

Example 1. The cultivation of the cell line mel Ibr.

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 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 units / ml), streptomycin (100 μg / ml) and an amino acid and vitamin complex (Flow Lab.) in culture bottles (Costar). After passage 15, a stably growing cell line was obtained.

Example 2. The definition of antigens expressed on the cell line mel Ibr

The obtained mel Ibr cell line, which has stable cultural and morphological characteristics, was studied using immunofluorescence, immunohistochemistry, and PCR (polymerase chain reaction) analysis for expressed antigens (differentiating, tumor-associated, and histocompatibilities).

The differentiating melanoma markers determining the ratio of this line to melanoma were studied by us using monoclonal antibodies CD63, HMB45, MelanA, Tyrosinaza, HMW. Cancer-testicular marker-MAGE-3, which can be expressed on tumors of various histogenesis, was 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 Ibr cell line. Differentiation Antigens Cancer Testicular Antigens Histocompatibility antigens Cd63 will put MAGE-3 will put HLA (I class) will put HMB45 neg HLA-DR (II class) will put Melana neg Tyros neg Hmw will put

As follows from Table 1, this cell line is characterized by the expression of melanoma (differentiation) markers: CD63 and HMW, emphasizing the specificity of this cell line. The positive expression of the cancer-testicular marker MAGE-3 corresponds to the oncological profile and allows the wide use of this line to create an antitumor vaccine. A unique feature of this line is the presence of histocompatibility antigens of the first and second class, which leads to an increase in immunogenicity due to the presentation of tumor antigens directly to CD4 and CD8 cells.

Thus, this cell line of human skin melanoma mel Ibr has its own individual phenotype of tumor markers, consisting in the presence of differentiation antigens (CD63, HMW) and cancer-testicular - MAGE-3, as well as histocompatibility molecules of the first and second classes, which allows the use of the obtained cell line for the development of 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. 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 Ibr used to create antitumor vaccines is stored in the Specialized Collection of Vertebrate Cell Cultures of the Russian Cell Culture Collection under the number RACC (P) 689D.