CS267400B1 - RG-12 hybridoma mouse lymphocytes - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing a monoclonal antibody against a tumor antigen associated with human malignant melanoma cells, which is stored in the hybridoma collection of the Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava under the designation RG-12. The monoclonal antibody of the hybridoma RG-12 is suitable for use in the diagnosis of malignant melanoma and monitoring the course of this malignant disease by determining the levels of circulating antitumor antibodies by the method of competitive radioimmunoassay and in the differential diagnosis of malignant melanomas, benign skin tumors and tumors of neuroectodermal origin by the method of immunohistochemical examination of paraffin tissue sections.

Description

The invention relates to a novel cell hybridoma constructed by a cell fusion of a mouse myeloma line Sp2 / 0 and a mouse saline lymphoid cell producing an antibody against the membrane antigen of cells of the human malignant melanoma line IIMB 2.

The introduction of hybridoma technologies has paved the way for the preparation of monoclonal antibodies directed against antigenic determinants of cells of lower and higher organisms, including tumor cells. Anti-tumor monoclonal antibodies have found wide application in the immunohistochemical, radioimmunological and localization diagnosis of primary tumors and their metastases and in the control of tumor progression after therapy. The vast majority of anti-tumor monoclonal antibodies prepared to date are directed against transplant-type antigens, cytoskeleton antigens, differentiation antigens, and antigens of embryonic origin. At present, there are only a few monoclonal antibodies in the world that react with neoantigens associated with tumors of the same histogenesis.

In recent years, monoclonal antibodies have been developed against antigens associated with human cutaneous malignant melanoma cells [Ziai, MR, Imberti, L., Nicotra, MR, Badaracco, G., Segatto, O., Natali, PG, Ferrone, S .: Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen. Cancer Research 47, (1987), 2474; Hotta H., Ross, AH, Huebner, K., Isobe, M., Wendeborn, S., Chao, A., V., Risciardi, RP, Tsujimoto, Y., Croce, CM, Koprowski, H .: Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Research, 48, (1988), 2955]. However, none of them is strictly specific for malignant melanoma cells. Their simultaneous reactivity with tissues and tumors of neuroectodermal origin as well as with tumors of other histogenesis has been demonstrated [Steplewski, Z., Herlyn, D., Clark, WH, Ross, AH, Blaszcyk, M., Pak, KY, Koprowski, H .: Production and characterization of monoclonal antibodies against human malignant melanoma. Cancer Investigation, 1, (1983), 215; Hotta et al., Cancer Research, 48, (1988), 2955). Most of the antimylanoma antibodies developed have reacted with antigens that are denatured with formol (a fixative routinely used to fix tissues for histological examination) and are therefore not suitable for immunohistochemical assays. For these assays, several anti-melanoma monoclonal antibodies with varying degrees of Specificity are commercially prepared by Dakopatts A. S. (Santa Barbara, California, USA) by Hybritech Company (San Diego, California, USA) and others. At present, antimelanoma monoclonal antibodies are not commercially available in kits for radioimmunodiagnostic testing of circulating antimelanoma antibodies in the sera of patients with malignant melanoma.

These shortcomings are largely eliminated by a hybridoma producing a monoclonal antibody against an antigen associated with human malignant melanoma cells, which is stored in the hybridoma collection of the Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. Čsl. Army 21, under the designation RG (2.

The RG-12 hybridoma was obtained by fusing a spleen cell of a Balb / c mouse, immunized with cells of the human maize melanoma line HMB-2, with a mouse plasma cell cell Sp2 / O according to the method described in the literature [Kohler, G., Milstein, C .: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, (1975), 495] and subsequent cloning of the hybrid cell set by standard techniques.

The resulting hybridoma produces a monoclonal antibody that specifically binds to a tumor antigen present in human malignant melanoma cells and in tumor cells of epithelial origin (carcinomas) but not in healthy tissue cells.

RG-12 hybridoma cells can be propagated in vitro in standard animal cell culture media, or in vivo in the peritoneal cavity of Balb / c mice. Viable hybridoma cells can be stored in vials at -180 ° C in liquid nitrogen. By thawing, viable cultures are obtained which are used as a source of monoclonal antibody.

Example

Hybridoma cells were propagated in vivo by applying 1 x 10 ⁷ cells intraperitoneally to Balb / c mice without prior premedication. On day 21 of growth of the hybridoma in ascites, the mouse was sacrificed and ascites fluid was collected by abdominal puncture. 5.6 ml of ascites fluid containing 6.9 mg / ml of immunoglobulin was obtained. This was isolated from the ascites fluid by precipitation in 2 M ammonium sulfate and purified by diethylaminoethyl cellulose column chromatography. Ascites fluid containing both the monoclonal antibody and the purified antibody showed specific binding to human malignant melanoma cells and carcinoma cells in an immunohistochemical assay on paraffin sections of tissues, but not to cells of healthy tissues and tumors of other histogenesis. In a competitive radioimmunoassay, a monoclonal antibody detects circulating anti-tumor antibodies in 85% of the sera of patients with malignant melanoma.

Under in vitro conditions, the RG-12 hybridoma grows as a suspension culture. The hybridoma cells have the size and shape characteristic of myeloma cells. They contain fused cell nuclei with an aneuploid karyotype. As a growth medium, at least essentially MEM medium (Institute of Sera and Vaccines, Prague) enriched with 10% fetal calf serum (Bioveta, Ivanovice na Hané), gentamicin and buffer solution of sodium bicarbonate is used. The cultures are maintained in a thermostat at 37 ° C in a humidified atmosphere in the presence of 5% CO ₂ .

CS 267 400 Bl

Under these conditions, the mean generation to hybridoma cells is 22.5 hours. The hybridoma produces an IgG class monoclonal immunoglobulin. '

The RG-12 hybridoma can be used industrially as a source of a monoclonal antibody against malignant melanoma. In a competitive radioimmunoassay using HMB-2 melanoma cell membrane proteins, a monoclonal antibody can be used for the quantitative and qualitative determination of circulating anti-tumor antibodies in patients with malignant melanoma in the diagnosis, differential diagnosis and monitoring of the course of the disease after radical treatment. In immunohistochemical examination of paraffin sections of tissues, the monoclonal antibody is used in the differential diagnosis of malignant melanomas and their metastases from benign pigmented skin tumors and other tumors of neuroectodermal origin. Potentially possible uses can be expected in the immunoscintigraphic localization] diagnosis of metastases of malignant melanoma in vivo.

Claims (1)

Mouse lymphocyte hybridoma RG-12, produced against a tumor antigen associated with a cell group IgG monoclonal antibody of human malignant melanoma.