DD259139A1 - METHOD FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES FOR IDENTIFYING NON-T, NON-B ACIDS OF LYMPHOBLASTIC LEUKAEMIA - Google Patents

Abstract

The process for producing monoclonal antibodies against a surface antigen of non-T, non-B acute lymphoblastic leukemia (CALLA) has the goal of producing in an easily controllable manner the cost-effective production of larger amounts of specific antibodies for CALLA (mol 1 polypeptide chain) of non-T, non-B cells. The invention has for its object to provide a comprehensible method, in the course of which initially a hybridoma is produced, which is easy and inexpensive to handle and its cultivation is technically and economically advantageous manner possible. Using this hybridoma, monoclonal antibodies which react with the CALLA of Non-T, Non-B acute lymphoblastic leukemia are to be generated in subsequent process steps. The object is achieved by the production of the hybridoma H-BL-CALLA / 1 by means of cell fusion, its cultivation in vitro and in vivo as well as the separation of the monoclonal antibodies BL-CALLA / 1.

Description

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Field of application of the invention

The invention relates to a process for the preparation of monoclonal antibodies which react with an antigen which is responsible for the non-T neoplastic lymphocytes of Non-T, Non-B Acute Lymphoblastic Leukemia (referred to as Common Acute Lymphoblastic Leukemia Associated Antigen [CALLA]) and for the characterization of this Type of acute lymphoblastic leukemias, including the quantitative determination of the proportion of leukemic blasts, can be used, which has significance for the type of therapeutic measures to be initiated. Also, a therapeutic use of the monoclonal antibody itself is to be considered.

Characteristic of the known technical solutions

It is already known to produce monoclonal antibodies. For example, KÖHLER and MILSTEIN (Nature 256, [1975], 495, by fusing antibody-forming cells with a permanently growing plasmocytoma cell line, generated hybrid cells that grow permanently and stably secrete antibodies, leading to the production of various hybridoma lines that produce antibodies of defined specificity.

Thus, there are a number of monoclonal antibodies that serve to further characterize non-T, non-B acute lymphoblastic leukemias, which account for the majority of all types of acute lymphoblastic leukemia in humans. The majority of non-T, non-B acute lymphoblastic leukemias (approximately 80%) express a characteristic surface antigen called the Common Acute Lymphoblastic Leukemia-associated antigen (CALLA). This antigen is also expressed in about 40% of chronic myeloid leukemias (CML) at the stage of blast crisis.

Since then I. International leukocyte workshop, all monoclonal antibodies that react with CALLA are assigned to the CD 10 (nT, gp1OO). (Bernard A., Boumsell, L., Hill, C: Joint Report of the First International Workshop on Human Leucocyte Differentiation Antigens by the Investigators of the participating Laboratories.: Bernard A., Boumsell L., Dausset J., Milstein C Schlossmann SF Eds. Leucocyte typing: human leucocyte differentiation antigens detected by monoclonal antibodies Springer Verlag Berlin 1984, S. 9)

The best known monoclonal antibody against the CALLA of Non-T, Non-B acute lymphoblastic leukemia is the antibody J 5 (Ritz J., Pesando J.M., Notis-McConarty J., Lazarus H., Schlossmann S.F .: A monoclonal antibody to human acute

lymphoblastic leukemia antigen. Nature 283 [1980], 583). This monoclonal antibody is widely used by Coulter Electronics, Ine. distributed. The antigen recognized by J 5 is a 95-100000Da molecular weight glycoprotein (Newman RA, Sutherland R., Greaves MF: The biochemical characterization of a cell surface antigen associated with acute lymphoblastic leukemia and lymphocyte precursors, J. Immunol. [1981], 2024] .This surface antigen is not a leukemia-specific antigen, but is also expressed in a small population (<5%) of bone marrow cells of non-leukemic children and young adults.

Presumably, possession of this immunological marker is a characteristic of normal bone marrow cell precursors which are then involved in leukemic transformation (Janossy, C, Bollum, F., Bradstock K., McMichael, A., Rapson, N., Greaves, MF: Terminal transferase-positive human bone marrow cells exhibit the antigenic phenotype of common acute lymphoblastic leukemia, J. Immunol., 123, [1979], 1525). Distribution of CALLA on various non-hematopoietic tissues has also been known for some time (Metzgar, RS, Borowitz, MJ, Jones, NH, Dowell, BL: Distribution of cammon acute lymphoblastic leukemia antigen in nonhematopoietic tissues, J.Exp. Med. 154 , [1981], 1 249). From the different degree of glycosylation of the antigen in the various tissues, the fluctuation range of the molecular weight results.

In addition to the known reaction of J 5 with various human non-T, non-B cell lines (eg REH, Nalm-6 and the like), reactivity with isolated granulocytes and selected human cell lines is also evident the expression of CALLA has not been otherwise demonstrated (Braun, MP, Martin, PJ, Ledbetter, JA, Hansen, JA: Granulocytesand cultured human fibroblasts express common acute lymphoblastic leukemia-associated antigens, Blood 61, [19831, 718]. Also, cross reactions with cell surface antigens from various established T cell lines were observed with the monoclonal antibody J5.

From the Imminent Importance of CD 10 Monoclonal Antibodies for a Rapid Diagnosis of Non-T, Non-B Acute Lymphoblastic Leukemias Owned by CALLA, as Well as Prognostic Statements (Greaves, MF, Janossy, G., Peto, J. Kay, H .: Immunolögically defined subclasses of acute lymphoblastic leukemia in children: their relationship to presentation features and prognosis., Br. J. Haematol. 48, [1981], 179), the need for adequate availability of these monoclonal antibodies in the clinical setting is growing Practice.

Availability of the monoclonal antibodies in larger quantities is also from the point of view of potential use in serotherapy (Ritz, J., Pesando, JM, Salam, SE, Clavell, LA, Notis-Mc Conarty, J., Rosenthal, P.A. , Schlossman, SF: Serotherapy of acute lymphoblastic leukemia with monoclonal antibody, Blood 58, [1981] 141) or other therapy variants, such as ζ. As the cleaning of the bone marrow of leukemia in autologous bone marrow transplantation, absolutely necessary.

Object of the invention

The invention aims to provide a method for producing monoclonal antibodies, which in an easily controllable manner, the cost-effective production of large amounts of specific antibodies against the CALLA (mol wt. About 100000 Da) of non-T, non-B acute lymphoblastic leukemias allowed. It should be guaranteed a consistent quality. The anti-CALLA antibodies are said to have a better specificity than the known CD10 antibodies, especially side reactivity with T cells should be excluded. The antibodies should be capable of coupling with fluorescent dyes and other labeling substances with complete retention of activity. The monoclonal antibodies should be stable and therefore storable and suitable for use in a simple detection assay for CALLA-positive non-T, non-B acute lymphoblastic leukemias. The affinity of the antibodies should be higher than the known, so that shortened reaction times significantly streamline the detection. "

By providing such monoclonal antibodies, the diagnosis of non-T, non-B acute lymphoblastic leukemias should be qualified and a faster and more effective therapeutic intervention should be made possible.

Explanation of the essence of the invention

The invention has for its object to provide a comprehensible method, in the course of which hybridomas are first generated, which are easy and inexpensive to handle and their cultivation is possible in a technically and economically advantageous manner. Using these hybridomas, monoclonal antibodies are to be generated in subsequent process steps which react with most of the non-T, non-B acute lymphoblastic leukemias.

The object is achieved by isolating lymphoblasts from the peripheral blood of a patient with CALLA-positive non-B acute lymphoblastic leukemia in accordance with the known methods of density gradient centrifugation.

The leukemia loads thus obtained are used to immunize female BALB / c mice 6-8 weeks old. The immunization is carried out by repeated application of the antigen. From the spleens of such hyperimmunized mice, the lymphocytes are separated in a conventional manner. The B lymphocytes of this cell mixture are fused or hybridized with mouse B myeloma cells.

The appropriate mouse B myeloma cells P3-X63-Ag 8.653 (KEARNEY et al., J. Immunol. 123, [1979], 1548) are cultured in RPM1-1640 with 10% fetal calf serum (FCS). In a culture medium containing hypoxanthine, aminopterin and thymidine (LITTLEFIELD, Science 145, [1964], 709), the hybrid cells formed are separated from the unfused myeloma cells and, according to the invention, cell clones are selected which secrete antibodies of the desired specificity. The selection is made by dividing the cells into a plurality of separate 200 / ml cultures immediately after fusion.

The supernatants of the cultures containing the growing hybrid cells are checked for the presence of antibodies which react only with the non-T CALLA non-B cells.

For this purpose, first all culture supernatants are tested with cells of the human non-T, non-B cell line REH, and in parallel with human blood lymphocytes. Only those cultures are still used whose antibodies do not react with the human peripheral blood lymphocytes but with the majority of the REH cells. The testing of the culture supernatants with the

corresponding cells by means of indirect immunofluorescence. With the help of this technique, the selected crops are continuously checked. According to the invention, among the hybridomas preselected in the manner described, those whose antibodies are also capable of reacting with granulocytes and the human T cell line CEM are now being sought. In the third selection step, among the hybridomas selected in accordance with the invention, those are sought whose antibodies are capable of reacting with the cell surface protein of the non-T, non-B cells of about 100,000 Da.

For this purpose, the surface proteins of REH cells are radioiodinated according to the Lactoperoxidasemethode. The nonidet P-40 lysates of such labeled REH cells are incubated with the hybridoma culture supernatants to be tested, and then the mouse monoclonal antibodies are precipitated by adding anti-mouse immunoglobulin serums. The precipitate freed of unspecifically bound components are dissolved in sodium dodecyl sulfate buffer and subjected to NDS electrophoresis on polyacrylamide gels (5-15%).

By reference proteins, the apparent molecular weight of the radioiodinated, specifically separated by the monoclonal antibody cell surface antigen of non-T, non-B cells is determined. There are only such hybridomas. which react with a cell surface antigen having an app. molecular weight of about 10000 Da in the unreduced and reduced state.

To test the suitability of the thus selected hybridomas for the characterization of non-T, non-B acute lymphoblastic leukemia, tests are performed on peripheral blood leukemia from patients with clinically proven non-T, non-B type acute lymphoblastic leukemia , The appropriate hybridomas remaining after all selection steps are recloned and highly productive subclones are selected which stably produce the desired antibodies. The hybridomas thus obtained are referred to as H-BL-CALLA. Particularly suitable is the cell clone H-BL-CALLA / 1. The production of the monoclonal antibodies against the CALLA of Non-T, Non-B acute lymphoblastic leukemia BL-CALLA / 1 is based on a procedure which is directed to the use of the hybridomas H-BL-CALLA / 1. The hybridoma H-BL-CALLA / 1 is being cultured at the Life Science Section of KMU Leipzig. It is cryopreserved and accessible.

The produced monoclonal antibody BL-CALLA / 1 has received the registration number DDR-0149 in the Central Institute of Molecular Biology of the AdW of the GDR. The hybridoma H-BL-CALLA / 1 produces up to 100μg / ml monoclonal antibody BL-CALLA / 1 when in vitro culture. It is an IgG antibody. To obtain larger amounts of antibody, the hybridoma H-BL-CALLA / 1 in histocompatible BALB / c mice i. p. injected. It is advantageous if the mice used were pretreated with a tumor stimulator such as Paraffinum subliquidum, Pristan or the like. After ascites formation, the ascitic fluid containing the monoclonal antibody BL-CALLA / 1 is removed by puncture. From the ascitic fluid, the cellular components are separated by centrifugation, which consist predominantly of cells of the hybridoma H-BL-CALLA / 1. These cells are washed in physiological saline and conveniently injected into new, treated mice. The cell-free supernatant containing the monoclonal antibody BL-CALLA / 1 is subjected to further purification either directly or in a suitable dilution. For this come Salzpräzipitationen, DEAE Ionenaustausch.chromatographie, protein A adsorption, affinity chromatography or the like. in question.

The ascites contains 5-1 Omg BL-CALLA / 1 per ml of liquid in the inventive process implementation. Enrichment leads to a product with an antibody content of approx. 20 mg / ml.

To produce a stable storage form which is also suitable as a commercial preparation, the immunoglobulin fraction of the cell-free ascitic fluid is precipitated, for. B. with 40-50% saturated (NH ₄ ) ₂ SO ₄ solution and taken up in buffered saline. Dialysis against NH ₄ HCO ₃ solution results in a product which is stable in lyophilized form at 4 ° C. In another embodiment of the invention, the hybridoma cells H-BL-CALLA / 1 capable of producing antibodies are cultivated in a culture medium supplemented with serum. After appropriate cultivation in CC> 2-containing atmosphere, the antibody-containing culture medium is treated with 40-50% saturated (NH ₄ J ₂ SO ₄ , thus precipitating a gamma globulin fraction Further purification of the antibody is carried out by methods known per se, such as gel filtration, ion exchange chromatography or the like possible.

It is convenient to use MEM Eagle, Dulbecco's MEM, RPM11640 or the like as a culture medium. apply. Is particularly advantageous to Verwendund of RPMI 1640 is ^"5 M mercaptoethanol and 100 mg / l gentamycin added in a preferred embodiment of the invention, with 1OmM HEPES, 5 χ 10th As a serum supplement dientfetals calf serum in a proportion of 5-20% in the culture medium. It is advantageous to work with a serum content of 10% The temperature of the culture medium is between 35 and 38 ° C. It is advantageous to cultivate at 37 ° C. The CO ₂ content of the atmosphere above the medium is, in the most favorable case, 5%. It is advantageous to have a high level of atmospheric humidity, which can reach saturation of the atmosphere.The hybridoma is cultivated over a customary period of several days, with a culture time of 3-4 days, after which the medium is changed and the cell count is repeated set an optimal output value.

The monoclonal antibody BL-CALLA / 1 prepared according to one of these process variants has the reaction pattern with human blood cells given in Table 1. He reacted with permanently growing human cell lines in the manner documented in Table 2.

The monoclonal antibody BL-CALLA / 1 has a surprisingly high affinity for human non-T, non-B cells. Figure 1 comparatively shows the reactivity of BL-CALLA / 1 and J 5 (CD 10) over the non-T, non-B cell line REH. It can be seen that BL-CALLA / 1 stains the same cells much more, which can be attributed to a higher affinity of the antibody. Isolated granulocytes are recognized by BL-CALLA / 1 as well as J 5.

It should be noted that the monoclonal antibody BL-CALLA / 1 reacts only with the cell line CEM within the tested human T-cell lines. In contrast to this reactivity, J 5 also displays a complex nonspecific labeling of the cell line MOLT-4, as shown in Fig. 2. · _Ct

The cell surface antigen bound by the monoclonal antibody BL-CALLA / 1 is a glycoprotein having a molecular weight of about 10000Da and corresponds to CALLA.

Table 1

Indirect immunofluorescence of monoclonal antibodies BL-CALLA / 1 with different blood cells

Cells% labeled cells

Erythrocytes 0

Platelets 0

Monocytes 0

Granulocytes> 80

Lymphocytes (peripheral blood) 0

Claims (4)

claims: 1. Method for producing monoclonal antibodies for the characterization of non-T, non-B acute lymphoblastic leukemias by immunizing mice with leukemia loads from patients with non-T, non-B acute lymphoblastic leukemia, fusion of B cells obtained from the spleen of the mice. Lymphocytes with murine myeloma cells, separation of formed hybrid cells from unfused myeloma cells, selection of hybridomas producing antibodies reactive only with the Non-T CALLA, non-B cells, characterized in that hybridomas whose antibodies are conjugated to a cell surface glycoprotein of 100,000Da specifically selected to specifically label surface proteins of human non-T, IMon B cell lines and leukemia loads from non-T, non-B acute lymphoblastic leukemia patients carrying nonidentate P-40. Lysates of such labeled non-T, non-B cells are incubated with appropriate hybridoma culture supernatants, the mouse monoclonal antibodies are precipitated, e.g. B. by addition of anti-mouse immunoglobin serum, the precipitates of nonspecifically bound components washed free and dissolved in an N DS Puff he be reduced at an aliquot the disulfide bridges of the proteins, both samples are subjected to NDS electrophoresis on polyacrylamide gels, the molecular weight of the cell surface antigens specifically separated by the monoclonal antibody is determined, those hybridomas are selected whose antibodies meet the requirement of reaction with a cell surface antigen having a molecular weight of 100,000Da in the reduced and unreduced state, the hybridoma H-BL-CALLA which has been found to be particularly suitable / 1 is cultured, in vitro and in vivo, the monoclonal antibody BL-CALLA / 1 is isolated. 2. The method according to claim {, characterized in that the labeling of the surface proteins of human non-T, non-B cells by radioiodination is carried out with the Laktoperoxidasemethode. Method according to claim 1, characterized in that the selected hybridoma is recloned and highly productive subclones are selected. 4. The method according to claim 1, characterized in that for the in vivo production of the monoclonal antibody histocompatible BALB / c mice are used.