DD245902A1 - METHOD FOR OBTAINING BISPECIFIC MONOCLONAL ANTIBODIES - Google Patents

Abstract

The invention relates to a method for obtaining bispecific monoclonal antibodies. The aim of the invention is the production of monoclonal antibodies with two different binding sites against different antigens or antigenic determinants. The process according to the invention is characterized in that hybrid cells which produce different monoclonal antibodies are marked in the cell culture with different fluorescent dyes, then fused, the tetradomes formed are selected with a fluorescence-activated cell sorter which selects tetradomas which produce bispecific antibodies the cell culture and on mice for antibody production weitervermehrt. Fields of application of the invention are various fields of medicine, biology, veterinary medicine, agriculture and biotechnology.

Description

embodiment

1) Hybrid cells producing monoclonal antibodies to the tumor marker AFP (alpha-fetoprotein) were labeled in cell culture with the fluorescent dye FITC (fluorescein isothiocyanate) which gives green fluorescence. Other hybrid cells that produce monoclonal antibodies against the immunoassay enzyme POD (horseradish peroxidase) were labeled in the cell culture with the fluorescent dye TRITC (tetramethyl-rhodamine-isothiocyanate), which gives a red fluorescence.

The stained antibody-producing hybrid cells were fused in the cell culture using PEG (polyethylene glycol).

2) cells with double fluorescence (Tetradome), which must contain genetic material from both hybrid cells were selected using the fluorescence-activated cell sorter (FACS III, Becton Dickinson), cloned in cell culture and further increased.

3) All tetradoma clones were subsequently tested for their production of antibodies to AFP and POD. Those clones producing antibodies to both antigens were subsequently tested for production of bispecific antibodies. For this purpose AFP purified on a solid carrier, PVC (polyvinyl chloride) film was adsorbed and then incubated with the culture supernatants to be tested. Following a washing procedure, it was then incubated with POD and then the POD detection reaction was performed on the appropriate substrate, which was converted to a colored product by the enzyme reaction.

4) The tetradomas, which were positive in the double specificity assay, were frozen in liquid nitrogen in a known manner and further amplified in cell culture and inbred mice for antibody recovery. Tetradom clone T1-F2 / B1 was developed at the

deposited under the number in the RGW reference center for cell lines at the Friedrich-Löffler-Institut Insel Riems.

Claims (1)

Invention claim: Method for obtaining bispecific monoclonal antibodies, characterized in that two hybrid cell lines producing different monoclonal antibodies are labeled in the cell culture with different fluorescent dyes, the stained hybrid cells are fused in the cell culture, the tetradomes formed are selected with the aid of a fluorescence-activated cell sorter Tetradomes that produce bispecific antibodies, selected and cultivated in a known manner and transplanted to mice. Field of application of the invention The invention relates to a method for obtaining monoclonal antibodies with double specificity, ie with a binding site against an antigen or an antigenic determinant and a second binding site against a second antigen or a second antigenic determinant.
Field of application of the invention is medicine, veterinary medicine, agriculture, biology and biotechnology. Characteristic of the known technical solutions Antibodies with double specificity can be obtained biochemically by reductive cleavage of the bivalent antibody molecules into monovalent fragments and subsequent oxidative association of different monovalent fragments (A.NISONOFF and M.M.RIVERS, Arch. Biochem., Biophys., 93, 460, 1961). This method is also applicable to monoclonal antibodies. Dual specificity monoclonal antibodies were also obtained using hybridoma technology. Hybrid cells which produce a specific monoclonal antibody and which have been provided with an enzyme defect by cell culture manipulations are fused to spleen cells of immunized mice in cell culture. Due to the enzyme defect, the fused cells (tridomes) can be selected using a special medium. The tridomas producing bispecific antibodies were selected by specific immunological tests (CMILSTEIN, CCUELLO: Nature 305, 537, 1983; J. MARTINIS, GS DAVID, RM BARTHOLOMEUS, and R. WANG, p. 129 in Basic Biology on New Developments in Biotechnology). Ed. Holloender et al., Plenum Publ. Corp. 1983). Furthermore, two hybrid cell lines producing different monoclonal antibodies each provided with a different enzyme defect were fused in the cell culture. The fused cells (tetradomas) were selected with the appropriate two selection media and the tetradomes which produce bispecific antibodies were selected by immunological tests (KG BURNETT, JF KULL and J. MARTINIS, presented at the 5 ^th International Congress of Immunology, 21. 27.8.1983, Kyoto, Japan). The disadvantage of these methods is the lengthy selection procedure for the tridomes or tetradomes, since the induction of enzyme defects in the fusion partners used is associated with a considerable amount of work and time. Object of the invention The aim of the invention is to obtain bispecific antibodies from fused cell lines, whereby the cell fusion and the selection of the fused cells should be carried out by a more effective and faster route. Explanation of the essence of the invention The method according to the invention for producing bispecific monoclonal antibodies against 2 different antigens is characterized in that a hybrid cell line which produces a particular monoclonal antibody is labeled with a fluorescent dye and this with another hybrid cell line which produces a different monoclonal antibody and with one different fluorescent dye was labeled, fused in cell culture. Tetradomes (= hybrid hybridomas), which have double fluorescence, are selected via a fluorescence-activated cell sorter (FACS), cloned and propagated in the cell culture. The clones are tested for antibody production by immunological assays and those tetradoma clones producing antibodies with dual specificity are selected, propagated and then stored in liquid nitrogen, cultured in a known manner and transplanted to ascites form mice. The method according to the invention, the core of which is the fluorescent dye label, requires considerably less work than the previously known methods. It is applicable to a wide variety of antigens. With the antibodies with double specificity produced according to the invention, the previous immunological detection and purification methods can be qualitatively improved. They also offer opportunities for therapeutic use.