DD272473B3 - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODY AGAINST THE EPSILON CHAIN OF CD 3 ANTIGEN HUMAN T-LYMPHOCYTES - Google Patents

Description

Field of application of the invention

The invention relates to a process for the production of monoclonal antibodies which react with a cell membrane antigen (p207) of the CD3 complex of human T-lymphocytes. The monoclonal antibodies can be used for medical diagnostics and for the elimination of normal and neoplastic human T-lymphocytes.

Characterization of the known technical solutions

It is already known to produce monoclonal antibodies. The principle solution of the hybrid dome production was described by KÖHLER and MILSTEIN (Nature 256, (1975), p. 495).

Monoclonal antibodies are known which react with different cell surface antigens of human T-lymphocytes (P.CKUNG, G.GOLDSTEIN: Human T-lymphocyte differentiation antigens detected by monoclonal antibodies, Research Monographs in Immunology, Vol.3, p.5-15, Editor: TURK, Elsevier / North Holland Biomedical Press, Amsterdam, New York, Oxford 1981 and JALEDBETTER, AEFRANKEL, LAHERZENBERG: Human Leu T-cell differentiation antigens: quantitative expression of normal lymphoid cells and cell-lines, ibid, p. 16 -22). Among the most common monoclonal antibodies that react with all human T cells are OKT3 (U.S. Patent 4,515,893), OKT1 (U.S. Patent 4,515,894), Anti-T12 (EP 0097518), and OKT11 (U.S. Patent 4,614,720). , In addition, other monoclonal antibodies have become known which also react T cell-specific (OD 238168A 3) and even bind to the antigen-specific receptor complex of human T cells (DD-PS 250333).

The monoclonal antibodies against human leukocytes are of great importance for medical diagnostics, so that within the framework of three previous workshops on leukocyte antigens (Paris 1982, Boston 1984, Oxford 1986) an international standardization has been started, which is part of the CD nomenclature ("Cluster The different monoclonal antibodies of the CD groups 2, 3, 5, 6 and 7 react with all or at least the great majority of the human T cells (Leukocyte Typing II. Vol , Ed .: EL REINHERZ et al .: Springer-Verlag, New York, Berlin, Heidelberg, Tokyo 1986).

Freckle the Nachwels the totality of human T cells is often the monoclonal antibody OKT3 (US-PS 4615893) used. One equivalent is Anti-Leu 4 from Becton / Dicklnson. Both monoclonal antibodies belong to CD3.

The special importance of the antibody of the CD3 group lies in the close association of this CD3 antigen complex with the antigen-specific T cell receptor. Both the major alpha / beta receptor and the relatively rare Qamm / Qamma and Gam.na / Delta receptor are characterized by a relatively strong noncovalent binding to the CD3 complex on the mature T lymphocyte cell membrane connected. The rearrangement of T cell receptor gene fragments, their synthesis, and cell surface expression are the determining events in the ontogenesis of T lymphocytes. It is certain that the T cell receptor requires cooperation with the CD3 complex for the realization of its signal transduction function. There is still no hope that the T cell receptor on the cell membrane without the CD3 complex occurs or could be functionally effective without him. This structural and functional association of the antigen-specific T cell receptor and the CD3 complex makes monoclonal antibodies against the CD3 targets such highly specific T lymphocyte detection reagents.

The CD3 complex consists of at least three different chains. The gamma chain is a glycoprotein with a molecular weight of 25 kDa, but due to the different glycosylation but also components in the entire range of 25 to 29kDa can occur. The delta chain is also a glycoprotein and has an M ₁ of 2OkDa. After deglycosylation, two polypeptides with molecular weights of 14 and 16 kDa are produced (BORST, J., et al., Nature 312, (1984), pp. 455-458). The epsilon chain also has an M ₁ of 20 kDa, but unlike the other two chains is not glycosylated (BORST et al., Eur. J. Immunol., 13: 576-580 (1983)).

As far as is known, most described monoclonal CD3-Antlkörper react either with the gamma or delta chain, which due to the close association in different proportions, the other chains (epcilon, but also alpha and beta) can be mitisoliert (Leucocyte Typing III, Ed .: AJ McMICHAEL et al., Oxford University Press, Oxford, New York, Tokyo, 1987). Only two monoclonal antibodies (3P6 and SP10) have been reported to be reactive with the epsilon chain (PESSANO et al., EMBO J.4, (1985), pp.337-344). These monoclonal antibodies were generated by immunization with the isolated denatured epsilon chain and also react with NDS-denatured epitopes of this chain in immunoblotting experiments. So far no monoclonal antibodies have been selected with native molecules of the CD3 complex that react with the cell-containing epsilon chain.

Object of the invention The aim of the invention is to provide a process for the preparation of monoclonal antibodies which is easily controllable Consider cost-effective production of larger amounts of specific antibodies to the epsilon chain of the CD3 antigen (protein

2OkDa) of the human T-lymphocytes.

The monoclonal antibodies are said to have high affinity and an epitope of the epsilon chain of the CD3 antigen

They are said to be expressed on both quiescent and activated T lymphocytes and are intended to be used for qualitative and quantitative flow cytometric analysis for the immunohistological detection of T cells in T cells

Cryostat tissue sections and for the elimination of normal and neoplastic T cells from lymphocyte preparations

suitable.

The monoclonal antibodies should be stable and storable. They should also be used for direct coupling with Fluorescent dyes are suitable. By providing such monoclonal antibodies to the medical diagnosis of human T lymphocytes

be qualified.

Explanation of the essence of the invention The invention has for its object to provide a comprehensible method, in the course of which first hybridomas

be produced, which are easy and inexpensive to handle and their cultivation in technically and economically advantageous

Catfish is possible. Using these hybridomas, monoclonal antibodies should be used in subsequent steps

which are in association with the epsilon chain of the CD3 antigen, a 2OkDa protein, of human T cells.

The object is achieved in that first lymphocytes according to the known methods of density gradient centrifugation

isolated from the peripheral blood of healthy donors. By passage over nylon wool or e-rosette formation, T lymphocytes are obtained therefrom.

With human T lymphocytes obtained in the manner previously described or otherwise obtained Mice immunized. Preferably, 6-8 week old female BALB / c mice are used. The immunization takes place

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 with mouse B myeloma cells. The appropriate mouse myeloma cells P3-X63 Ag 8,653 (KEARNEY et al., J. Immunol., 123, (1979), p. 1548) are administered in RPMM640 with 10% fetal calf serum (FCS).

held.

In a culture medium containing hypoxanthine, aminopterin and thymidine (LITTLEFIELD, Science 145, (1964), p.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 and of the other properties mentioned in the target position.

The selection of the hybridomas is carried out in such a way that the cells immediately after the fusion into a plurality of separate

200 ^ l cultures are divided. The supernatants of the cultures containing the growing hybrid cells are applied to the

Presence of antibodies that react only with Zetloberflächenantigen of human T-lymphocytes. For this

For example, any culture supernatant that reacts with human blood lymphocytes will be treated with B lymphocytes by elimination of the

Τ-ΖβΙΙβη be obtained by E-rosette and / or Nylonwolloaeparatlon tested. Only those cultures are used which antibodies do not react with B lymphocytes, but with the vast majority of blood lymphocytes and all separated T lymphocytes. Detection of culture supernatants with the appropriate cells can be carried out both by indirect radio-binding technique and by indirect immunofluorescence.

However, cultures selected in this way must be checked in any case with the indirect immunofluorescence technique. This excludes the possibility of further growth of hybridomas whose antibodies react with B-lymphocyton, monocytes, granulocytes, platelets and erythrocytes.

According to the invention, those clones are now being sought among the cybridomes preselected in this manner whose antibodies do not double-label cells after binding to lymphocytes and development with FITC-labeled anti-mouse immunoglobulin and subsequent addition of TRITC-labeled Antl-Pan-B cell antigens , As Pan B cell Antlkörper can be used: BL-B 22 or BL-B 20.

In a further selection step, double-labeling with a TRITC-tagged Antl-Pan-T cell antibody verifies that all lymphocytes labeled by this Pan-T cell antibody are also labeled by the hybridoma supernatant. Pan-T-cell antibodies are especially TRITC-BL-T / Rec.

Among the hybridomas selected in this way, only those whose monoclonal antibodies co-modulate with an established CD3 antibody are then referred to.

In a subsequent selection step of all the hybridomas are further cultured, whose antibodies stain human T lymphocytes sufficiently strong after indirect labeling, so that the monoclonal antibodies for both qualitative and quantitative analysis> r. flow cytometric method, such. B. the analysis with a fluorescence activated cell sorter (FACS 440) can be used.

In a further selection step, the selected hybridomas are checked whether they are suitable for the labeling of antigen-carrying cells in cryostat-blocked lymphoid tissue and that they have no side reactions with other tissues.

In a further selection step, only hybridomas are then drawn on whose monoclonal antibodies isolate an antigen from the membranes of the T lymphocytes, which has an apparent molecular weight of about 20 kDa, even after deglycosylation by Endo-F.

To determine the molecular weight, the surface proteins of native human T-lymphocytes are radioiodinated (125-iodine) or otherwise labeled by the lactoperoxidase method. The Triton X-10O lysates of such labeled T cells are incubated with the hybridoma supernatant to be tested, and then these mouse antibodies are precipitated by addition of Antl mouse immunoglobulin serum.

The precipitates freed of unspecific components are dissolved in sodium dodecyl sulfate buffer and then subjected to NDS electrophoresis on polyacrylamide gels (5-10%). Using reference proteins, the apparent molecular weight of the radioiodinated, specifically separated by the corresponding monoclonal antibody T cell antigens is determined. There are selected those hybridomas whose monoclonal antibodies react with cell surface antigens of human T cells, which have an apparent molecular weight of 2OkDe in the reduced state. even after Endo-F treatment.

The hybridomas obtained by such selection are recloned and highly productive subclones are selected which stably secrete the desired monoclonal antibodies. The hybridomas thus obtained are referred to as H-BL-TP-3. The production of the monoclonal antibody BL-TP-3 is based on a method which is directed to the in vitro or in vivo culture of the hybridoma H-BL-TP-3. The hybridoma H-BL-TP-3 is cultivated at the Life Sciences Section of the Karl-Marx-Universität Leipzig or is cryopreserved and accessible to interested parties. The monoclonal antibody BL-TP-3 has received the registration number ZIM-0512 in the central institute for molecular biology of the AdW of the GDR. The monoclonal antibody BL-TP-3 obtained by in vitro or in vivo culture reacts with leukocytes, other blood cells, and lymphoid cells from certain organs in the manner shown in Table 1.

The reaction pattern of the monoclonal antibody with permanently growing cell lines is shown in Table 2. The monoclonal antibody BL-TP-3 can be coupled to FITC, TRITC, phycoerythrin and biotin by the usual methods known to those skilled in the art. These directly labeled preparations show strong labeling of the human T cells.

The most important properties of the monoclonal antibody BL-TP-3 are summarized in Table 3. The monoclonal antibody BL-TP-3 belongs to the (gG1 Subklassr.

Ausführungsbeisplel Selection of the hybridoma H-BL-TP-3 First, according to the known method of density gradient centrifugation, lymphocytes are extracted from the peripheral blood

healthy donor isolated. Passing through nylon wool and E-rosette formation, T lymphocytes are prepared therefrom.

8 weeks old female BALB / c mice are immunized with these human T lymphocytes. The immunization takes place

by repeated administration of the antigen.

Four days after the last immunization, the spleen is removed and a single cell suspension is prepared. The B lymphocytes of this cell mixture are fused with mouse B myeloma cells. The appropriate mouse myeloma cells P3-X63 Ag 8,653 (KEARNEY et al., J. Immunol., 123, (1979), p.1548) are administered in RPMI-1640 with 10% fetal calf serum (FCS).

held.

In a culture medium containing hypoxanthine, aminopterin and thymidine (LITTLEFIELD, Science 145, [1964], p. 709),

The hybrid cells formed are separated from the unfused myeloma cells.

The selection of the hybridomas is done in such a way that the cells immediately after the fusion in 576 separate iCD ^ cultures

be split. The supernatants of cultures containing the mature hybrid cells are assayed for the presence of

Antibodies that react only with cell surface antigens of human T-lymphocytes. This will be all Culture supernatants that react with human blood lymphocytes, with B lymphocytes, by eliminating the T cells by E rosette and / or Nylonwolleseparatlon be tested. Only those cultures become welter

whose antibodies are not with B lymphocytes, but with the vast majority of blood lymphocytes and all over

Nylon wool separated T lymphocytes respond. The culture supernatants are tested with the appropriate cells

by means of I; direct immunofluorescence.

However, cultures selected in this way must be checked in any case with the indirect immunofluorescence technique

become. The use of the indirect immunofluorescence technique precludes the onset of hybridomas whose antibodies react with B lymphocytes, monocytes, granulocytes, platelets and erythrocytes.

Among the pre-selected in this way hybridomas now such clones are sought, the antibody after binding to Lymphocytes and development with FITC-labeled anti-mutaguloglobulin and subsequent addition of TRITC

Labeled anti-pan B cell antibodies do not duplicate cells. As the Pan-B-cell Antlkörper can be used: BL-B 22 or BL-B 20.

In a further selection step, double labeling with a TRITC-labeled Antl-Pan-T cell antigen

Verify that all lymphocytes labeled by this Pan-T cell antibody are also labeled by the hybridoma supernatant. As a Pan-T-cell Antlkörper is special TRITC-BL-T / Rec.

Among the hybridomas selected in this way, only those whose monoclonal antibodies have a well-established Comodulate CD3 antibody. In a subsequent selection step, all of the hybridomas whose antibodies are human T cells are further cultured therefrom. Lymphocytes stain sufficiently strongly after indirect labeling, so that the monoclonal antibodies to both

qualitative as well as for quantitative analysis in flow cytometric methods, such. B. the analysis with a fluorescence activated cell sorter (FACS 440) can be used.

In a further selection step, the selected hybridomas are checked whether they are suitable for the labeling of

antigen-carrying cells in cryostat sections of lymphoid tissues (Tonsllle, lymph nodes and thymus) are suitable and that they have no side reaction with other tissues, especially skin and connective tissue.

In a further selection step, only hybridomas are then moved on whose monoclonal antibodies are removed from the Membranes of T lymphocytes isolate an antigen, which has an apparent molecular weight of about 20 kDa, even after Deglycosylation by Endo-F. To determine the molecular weight, the surface proteins of native human T-lymphocytes after the Lactoperoxidase method radiolated (125-iodine). The Triton X-100 lysates of such labeled T cells are used with the

incubated with hybridoma supernatants examined and then this Ma ¹ isantikörper by addition of anti-

Mouse immunoglobulin Serumpräzipitiert. The washed free of unspecific components Precipitate be dissolved in sodium dodecyl sulfate buffer and

subsequently subjected to NDS electrophoresis on polyacrylamide gels (10%). By reference proteins, the apparent molecular weight of the redioiodinated, specifically separated by the corresponding monoclonal antibody

T cell antigens determined. There are selected those hybridomas whose monoclonal antibodies with Cell surface antigens of human T cells, which in the reduced state an apparent molecular weight of

2OkDa, even after Endo-F treatment.

The hybridomas obtained by such selection are screened and highly productive subclones are selected which are the

stably secrete the desired monoclonal antibody. The hybridomas thus obtained are referred to as H-BL-TP-3.

The preparation of the monoclonal antibody BL-TP-3 is based on a method based on in vitro or in vivo culture

of the hybridoma H-BL-TP-3.

embodiment Recovery of Monoclonal Antibody BL-TP-3

2 × 10 cells of the liquid nitrogen-stored hybridoma H-BL-TP-3 are thawed and, after washing twice, taken up in 4 ml RPM11640 cell culture medium containing 10% fetal calf serum. Of these, four separate 1-ml Kultu. in

Tissue culture plates applied to polystyrene. After proliferation of the cells, 5 ml, 200 ml and 50 ml cultures are succesively placed in tissue culture flasks. The cell-free culture supernatants of the hybridoma cell culture cultured in sufficient density and time contain the

monoclonal antibody BL-TP-3. The monoclonal antibody can be isolated or enriched by a conventional method.

Table 1 Reaction pattern of the monoclonal antibody BL-TP-3 with human blood cells and cells from lymphoid organs

cell type Marked cells (%) PBL 73 +/- 6 T lymphocytes 100 B lymphocytes <3 LGL (NK) <5 monocytes 0 granulocytes 0 erythrocytes 0 l .ymphoblasts (PHA-stim.) > 90 Lymphoblasts (Con A-stim.) > 90 thymocytes 32 +/- 5 Tonsillenlymphozyten 41 +/- 9

Table 2 Binding pattern of the monoclonal antibody BL-TP-3 with permanently growing human cell lines

Zeil-line Reaction of the mAK BL-TP-3 IgQI with the cells ρ 20; Epeilon chain of the CD 3 T cells: Yes CEM + ever MOLT-3 - Yes MOLT-4 - JURKAT + very well HSB-2 _ very well SKW-3 - very well T cell clones (IL 2 dependent): AK-15 +++ very well KT-2 +++ very well B cells: very well DEER - Raji _ IM-09 - GM-607 - HMY-2 - Myeloid line: U-937 - Erythroid line: K-562 - Detection by indirect immunofluorescence: +++: 100% strong market ++: 100% weakly marked +: <100> 25% +/-: <25% -: unniarklert Table 3 Characteristics of Monoclonal Antibody BL-TP-3 isotype antigen Modulation of the CD 3 / TCR Comudulation with CD3 mAb Comodulation with BL-TRec / 1 Fitness for immunocytology flow cytometry immunohistochemistry Fitness to the mark FITC TRITC biotin

Claims (1)

Method for producing monoclonal antibodies against the epsilon chain of the CD3 antigen of human T-lymphocytes by immunization of BALB / c mice with enriched human T-lymphocytes, fusion of B-lymphocytes obtained from the spleens of such mice with murine myeloma cells, selection of Hybridomas secreting monoclonal antibodies with Pan-T cell reactivity, characterized that hybridomas which are monoclonal antibodies specifically react with a protein of 20 kDa, are selected in such a way, that T-lymphocyte-specific clones are selected first, subsequently those hybridomas whose monoclonal antibodies are detected by two consecutive selection steps carried out on the double-labeling of the lymphocytes with the antibodies contained in the culture supernatants, which are developed with FITC-labeled anti-mouse immunoglobulin, and TRITC-labeled Pan-B cell and pan T-cell antibodies that guarantee Pan-T cell specificity are selected, it is ensured in a fourth selection step that the antibodies are comodulating with CD3 antibodies, wherein in a fifth selection step only those monoclonal antibodies are taken into account that have a sufficiently high affinity for the stable labeling in the case of indirect immunofluorescence and flow cytometric evaluation, in a sixth step, the antibodies confirm their suitability to label T cells in cryostat tissue sections and do not undergo side reactions with other non-lymphoid tissues, in a further selection step hybridomas are selected, the monoclonal antibodies of which are selected with a radiolabeled T cell-solubilized, non-glycosylated cell membrane antigen having a molecular weight of 2OkDa, - That the thus determined hybridoma H-BL-TP3 (registration number ZIM-0152) is cultured in vitro and in vivo and the secreted monoclonal antibody BL-TP3 isolated, enriched and optionally coupled with markers or to solid phases.