DD272472A1 - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODY AGAINST A 50 KDA ANTIGEN HUMAN T-LYMPHOCYTES - Google Patents

Abstract

The invention relates to a method of producing monoclonal antibodies for a 50 kDa cell surface antigen of normal and neoplastic human T lymphocytes. The object of the invention is to provide a process for the preparation of corresponding monoclonal antibodies, which allows the production of relatively large quantities of these antibodies in an easily controllable manner. The monoclonal antibodies are said to have a high specificity and affinity allowing their use in flow cytometry and immunohistology. For this purpose, hybrid cells of B-lymphocytes of immunized BALB / c mice are generated in a known manner and selected hybridoma cells whose monoclonal antibodies have the desired properties. The selection method is characterized in that hybridomas are produced and cultured in a multistage selection system whose monoclonal antibodies have Pan-T cell specificity, reactivity with cortical and medullary thymocytes and binding to a cell surface protein of 50 kDa, sufficiently high affinity for use in flow cytometry and possess immunohistology. The monoclonal antibody BL-TP 2 of the hybridoma H-BL-TP 2 has the desired properties. The monoclonal antibody has the registration number ZIM-0156. The method of producing the monoclonal antibody is based on the in vitro or in vivo cultivation of the corresponding hybridoma H-BL-TP 2 and the separation of the monoclonal antibody. The field of application of the invention is medical diagnostics {monoclonal antibody, hybridoma, T lymphocytes, CD 2 antigen, human lymphocytes, leukocyte differentiation, leukemia diagnostics}

Description

$ 27,247

Title of the invention

Process for the preparation of monoclonal antibodies against a 50 kDa antigen of human T lymphocytes

Field of application of the invention

The invention relates to a process for the production of monoclonal antibodies which react with a cell surface antigen (gp 50) 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 hybridoma production has been described by KÖHLER and MILSTEIN (Nature 256, (1975), p. 495).

Monoclonal antibodies are known which react with different human T lymphocyte cell surface antigens (PCKUNG, G.GOLDSTEIN: Human T-lymphocyte differentiation antigens detected by monoclonal antibodies, Research Monographs in Immunology, Vol.3, p.5-15, eds TURK, Elsevier / North Holland Biomedical Press, Amsterdam, New York, Oxford 1981 as well

J.A.LEDBETTER, A.E.FRANKEL, L.A. HERZENBERG: Human Leu T-cell (differentiation antigens: quantitative expression of normal lymphoid cells and cell-lines, ibid., P.16-22). The most common monoclonal antibodies that react with all human T-cells include OKT3 (U.S. Patent 4,515,893), OKTI (U.S. Patent 4,515,894), Anti-T 12 (EP 0 097 518), and OKT 11 (U.S. Patent 4,614,720). In addition, other monoclonal antibodies have become known which also react T cell-specifically (DD 238 168 A3) and even bind to the anti-gene-specific receptor complex of the human T cells (DD-PS 250 333).

The monoclonal antibodies against human leukocytes are of great importance for medical diagnostics, so that in the context of

So far, three workshops on leukocyte antigens (Paris 1982, Boston 1984, Oxford 1986) have begun an international standardization that has led to the CD nomenclature ("Cluster of Differentiation") of the corresponding monoclonal antibodies. The monoclonal antibodies of CD groups 2,3,5,6 and 7 react with all or at least the vast majority of human T cells (Leukocyte Typing II. Vol. 1-3, ed. ELREINHERZ et al .: Springer Verlag, New York, Berlin, Heidelberg, Tokyo 1986).

for the detection of the totality of human T cells, the monoclonal antibody OKT 11 (US Pat. No. 4,614,720) is frequently used. One equivalent is Anti-Leu 5 from Becton / Dickinson. Both monoclonal antibodies belong to CD 2. Several monoclonal antibodies have become known for this group of antibodies (Leukocyte Typing 11 I.Hrsg .: A. J. M. MICHAFL et al., Oxford University Press, Oxford, New Prec, Tokyo 1987).

All these antibodies have in common that the determination of the T-lymphocytes is often associated with difficulties, since in flow cytometric measurement flowing transitions between the unlabelled and the labeled cells occur. This phenomenon is partly due to the nature of the CD 2 antigen, since it is not a pure differentiation antigen but has the character of an activation-dependent antigen. There are on the CD 2 antigen epitopes that appear only when the T lymphocytes are activated. Overall, however, the CD 2 antigen is also more strongly expressed after activated T lymphocytes (SCMEUER, THHUTTEROTH, KHMEYER zur BÜSCHENFELDE: Monoclonal antibodies as probes to define critical surface structures involved in cell activation.) In: Leukocyte Typing II, Vol. 1, p. 67, ed. ELREINHERZ et al., Springer-Verlag, New York, Berlin, Heidelberg, Tokyo 1986). Another cause of the discrimination problems with the known CD 2 antibodies may be due to insufficient affinity of the monoclonal antibodies.

4 Object of the invention

The object of the invention is to provide a process for the production of monoclonal antibodies which allows, in an easily controllable manner, the cost-effective production of larger amounts of specific antibodies for the CD 2 antigen (glycoprotein, 50 kDa) of the human T lymphocytes.

The monoclonal antibodies are said to have a high affinity and to react with an epitope of the CD 2 antigen expressed on both quiescent and activated T lymphocytes and thereby to be used for qualitative and quantitative flow cytometric analysis, for immunohistological detection of T Cells in cryostat tissue sections and for the elimination of normal and neoplastic T cells from Lymphozytenpraparationen.

The monoclonal antibodies should be stable and storable. They should also be suitable for direct coupling with fluorescent dyes.

By providing such monoclonal antibodies, the medical diagnosis of human T-lymphocytes is to 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 hybridomas are first generated, which are easy and inexpensive to handle and their V cultivation is technically and economically advantageous manner mog'ich. Using these hybridomas, monoclonal antibodies reactive with the 50 kDa human T cell glycoprotein are to be generated in subsequent process steps.

The object is achieved by first isolating lymphocytes from the peripheral blood of healthy donors by the known methods of density gradient centrifugation. By passage over nylon wool or E-rosette formation, T lymphocytes are obtained therefrom. A second variant of T-lymphocyte recovery is human thymus

To prepare cell suspension and the connective tissue fractions by density gradient. Tri fugation separate.

Mi ^ - in the previously described or in another similar manner obtained human T-lymphocytes, mice are immunized. Preferably, 6-8 week old female BALB / c mice are used. 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 with mouse B myeloma cells. The appropriate mouse myeloma cells P3-X63 Ag 8.653 (Keearney et al., J. Immunol. 123, (1979), p.1548) are maintained in RPMI-16-10% fetal calf serum (FCS).

In a culture medium which contains 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 contain antibodies of the desired specificity and of others secrete the properties of the target staging.

The selection of the hybridomas takes place in such a way that the cells are divided into a plurality of separate 200 μΐ cultures immediately after the fusion. The supernatants of the cultures containing the growing hybrid cells are tested for the presence of antibodies which react only with cell immune antigens of human T lymphocytes. / Hierz "all culture supernatants which react with human blood lymphocytes are tested with B lymphocytes obtained by elimination of the T cells by E-rosette technique and / or nylon wool separation, and only those cultures whose antibodies do not bind with B Lymphocytes may well react with the vast majority of the blood lymphocytes and any separated T lymphocytes.The culture supernatants can be tested with the appropriate cells both by indirect radio-linkage technique and by indirect immunofluorescence.

In any case, cultures selected in this way must be checked with the indirect immunofluorescence technique. This precludes the possibility of continuing the production of hybridomas whose antibodies contain B lymphocytes, monocytes, granulocytes,

Platelets and erythrocytes react.

According to the invention, those clones whose antibodies are not double-labeled after binding to lymphocytes and developed with FITC-labeled anti-mouse immunoglobulin and subsequent addition of TRITC-labeled anti-pan B cell antibody are now being searched for in accordance with the invention Cells give. As pan-B-cell antibodies can be used: BL-B 22 or BL-B 20.

In a further Selaktiosschritt is checked by double-labeling with a TRITC-labeled anti-pan-T-Zel1 antibody that all marked by this Pan-T-cell antibody lymphocytes are also marked by the hybridoma supernatant. Pan-T-cell antibodies are particularly suitable: TRITC-BL-TP 3 and TRITC-BL-T / Rec.

In a subsequent selection step, the hybridomas are selected which display a population of double labeled cells in a similar double labeling technique with a TRITC-labeled CD16 antibody.

Among the hybridomas selected in this way, only those whose monoclonal antibodies react with the vast majority of the thymocytes are referred to.

In a further selection step, only hybridomas are then pulled on whose monoclonal antibodies from the surfaces of T lymphocytes and thymocytes isolate an antigen which * has an apparent molecular weight of about 50 kDa.

To determine molecular weight, the surface proteins of native human T-lymphocytes or thymocytes are radioiodinated <125-iodine by the lactoperoxidase method or otherwise labeled. The Triton Χ-1Θ0 - lysates of such labeled T cells are incubated with the hybridoma to be examined and then these mouse antibodies precipitated by the addition of anti-mouse immunoglobulin serum.

The precipitate ©, washed free of unspecific components, is dissolved in sodium dodecyl sulfate buffer and then subjected to NDS electrophoresis on polyacrylamide gels (5-10 7,) . "Reference proteins are the apparent molecular weight of the radioiodinated, specific by the corresponding monoclonal antibody separated T cell antigens

certainly. There are selected those hybridomas whose monoclonal antibodies react with cell surface antigens of human T-cells or thymocytes, which have an apparent molecular weight of 50 kDa in the reduced state.

In a subsequent selection step, all of the hybridomas whose antibodies stain human T lymphocytes sufficiently strongly by indirect labeling are further cultured therefrom, so that Jia monoclonal antibodies are used for both qualitative and quantitative analysis in flow cytometric methods such as. analysis with a fluorescence activated cell sorter (FACS 440).

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

The hybridomas obtained by such selection are recloned and highly productive subclones are stably secreting the desired monoclonal antibodies. The resulting hybridomas are termed H-BL-TP 2.

The preparation of the monoclonal antibody BL-TP 2 is based on a method which is directed to the in vitro or in vivo preparation of the hybridoma H-BL-TP 2. The hybrid in H-BL-TP 2 is cultivated at the Life Sciences Section of the Karl Marx University in Leipzig, or is cryopreserved and accessible to interested parties. The monoclonal antibody BL-TP 2 has the registration number ΖΙΜ-Φ156 in the Central Institute of Molecular Biology AdW of the GDR.

The monoclonal antibody BL-TP 2 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.

The reaction pattern of monoclonal antibody with permanently growing cell lines 1 st in Table 2 listed.

The monoclonal antibody BL-TP 2 can be coupled to FITC, TRITC, phytocythrin 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 monoclonal antibody BL-TP 2 belongs to the IgGl subclass.

1st embodiment

Selection of the Hvbrldoms H-BL-TP 2

First, lymphocytes are isolated from the peripheral blood of healthy donors by the known method of density gradient centrifugation. By passage over nylon wool or E - Rosettenbi tion from T lymphocytes are obtained.

These human T lymphocytes are immunized with 6-8 week old female BALB / c mice. 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 with <1-B myeloma cells by treatment with 50% polyethylene glycol. The appropriate mouse myeloma cells P3-X63 Ag 8.653 (Keearney et al., J. Immunol. 123, (1979), p.1548) are described in RPMI-1640 as 10 * /. fetal calf serum (FCS).

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 takes place in such a way that the cells are divided immediately after the fusion in 576 separate 200 μΐ cultures. The supernatants of the cultures containing the mature hybrid cells are tested for the presence of antibodies that react only with cell surface antigens of human T lymphocytes. For this purpose, culture supernatants which react with 60 to 90% of the human blood lymphocytes are tested with B lymphocytes obtained by elimination of the T cells by E-rosette technique and / or nylon wool separation. Only those cultures will continue to be used whose antibodies do not react with B lymphocytcin but with the vast majority of blood lymphocytes and all nylon wool-separated T lymphocytes. The testing of the culture supernatants with the corresponding cells is carried out by means of indirect immunofluorescence.

By the indirect immunofluorescence technique is excluded that hybridomas are moved on, whose antibodies react with B lymphocytes, monocytes, granulocytes, platelets and erythrocytes.

Among the types of these types of viruses are now

those clones are sought 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 anti-pan B cell antibody. As Pan-B-2ell - antibodies can be used: BL-B 22 or BL-B 2Θ.

In a further selectivity step, double-labeling with a TRITC-labeled anti-Pan-T-Zel1 antibody reveals that all lymphocytes marked by this Pan-T cell antibody are also labeled by the hybridoma supernatant. Ale Pan-T cell antibodies are particularly useful: TRITC-BL-TP 3 and TRITC-BL-T / Rec.

In a subsequent selection step, the hybridomas are selected, which in a similar double. ¹ labeling technique with a TRITC-labeled CD 16 - antibody display a population of double-labeled cells.

Among the hybridomas selected in this way, only those whose monoclonal antibodies react with the overwhelming majority of the thymocytes are referred to.

In a further selection step, only hybridomas are then pulled on whose monoclonal antibodies isolate from the surfaces of T lymphocytes and thymocytes an antigen which has an apparent molecular weight of about 50 kDa.

To determine the molecular weight, the surface proteins of native human T-lymphocytes or thymocytes are radioiodinated by the lactoperoxidase method (125-iodine). The Triton X-100 lysates of such labeled T cells are incubated with the hybridoma supernatants to be examined, and subsequently these mouse antibodies are precipitated by addition of anti-mouse immunoglobulin serum.

The precipitate washed free of unspecific components are dissolved in sodium dodecyl sulfate buffer and then subjected to NDS electrophoresis on polyacrylamide gels (5-10%). The apparent molecular weight of the radioiodinated T-cell antigens specifically separated by the corresponding monoclonal antibody is determined on the basis of reference proteins. Those hybridomas are selected, whose monoclonal antibodies react with cell surface antigens of human T-cells or thymocytes, which in the reduced state an apparent

1Θ molecular weight of 50 kDa.

In a subsequent selection step, all of the hybridomas whose antibodies stain human T lymphocytes sufficiently strongly by indirect labeling are further cultured therefrom, so that the monoclonal antibodies are used both for qualitative and quantitative analysis in flow cytometry methods, e.g. can be used with a fluorescence-activated cell sorter (FACS 440).

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 tissue and that they have no side reactions with other tissues.

The hybridomas obtained by such selection are recloned and highly productive subclones are stably secreting the desired monoclonal antibodies. The resulting hybridomas are termed H-BL-TP 2.

2nd embodiment

Recovery of Monoclonal Antibody BL-TP 2

2 × 10 'cells of the hybridoma stored on liquid nitrogen are thawed and, after washing twice, taken up in 4 ml RPMI 1640 cell culture medium containing 10/4 fetal calf serum. Of these, four separate 1 ml cultures are grown in polystyrene tissue culture plates.

After proliferation of the cells, successively 5 ml, 20 ml and 50 ml cultures are placed in tissue culture flasks.

The cell-free culture samples contain the monoclonal antibody BL-TP 2. The monoclonal antibody can be isolated or enriched by a conventional method.

41-272

Table 1

Reaction pattern of the monoclonal antibodies BL-TP 2 with human blood cells and cells from lymphoid organs

Cell type labeled cells C%]

PBL 78 +/- 8

T lymphocytes 98 +/- 2

B lymphocytes <3

LGL (NK) 42 +/- 9

Monocytes 0 Granulocytes 0

Erythrocytes 0

Lymphoblasts (PHA-stim.)> 90

i_ymphobl <8L-TRec-stim.)> 90

Thymocytes 9G +/- 3

Tonsillar lymphocytes 47 +/-

Table 2

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

Zeil line reaction deB mAK BL-TP

with the cells

T-lines:

CEM

MOLT-3

MOLT-4

JURKAT

HSB-2

SKW-3

B-lines:

REH RAJI IM-09 GM-607 ^ HMy-2

Myeloid line: C U-937

Erythroid line: K-562

Detection by indirect immunofluorescence +++; 100 X strongly marked ++, 100% weakly marked +; <100> 25 X. + / -; <25% -; unmarked

Claims (1)

Method for the production of monoclonal antibodies against a 50 kDa anti-human T lymphocyte 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 whose monoclonal antibodies react specifically with a glycoprotein of about 50 kDa are specifically selected in such a way that that T lymphocyte-specific clones are selected first, those hybridomas whose monoclonal antibodies are detected by three consecutive selection steps, which are performed 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, Pan- Based on T-cell and CD16 antibodies that guarantee Pan-T-Zel1 specificity, in a fifth selection step it is ensured that the antibodies react with the whole of the thymocytes, in a further selection step, hybridomas are selected whose monoclonal antibodies react with a radiolabelled surface antigen solubilized by detergent treatment of the T-cells, having a molecular weight of about 50 kDa, in a seventh selection step only those mono-V konal antibodies are taken into account which have an affinity which is sufficiently high for stable labeling in the case of indirect immunofluorescence and flow cytometric analysis, in an eighth step, the antibodies confirm their ability to label the T-cells in cryostat tissue sections and do not undergo side reactions with other non-lymphoid tissues, that the thus determined hybridoma H-BL-TP 2 (registration number ZIM-0156) is cultured in vitro and in vivo and the secreted monoclonal antibody BL-TP 2 is isolated, enriched and optionally coupled with markers or to solid phases.