EP0980390A2

EP0980390A2 - Human-cd28 specific monoclonal antibodies for antigen non-specific activation of t-lymphocytes

Info

Publication number: EP0980390A2
Application number: EP98934848A
Authority: EP
Inventors: Thomas HÜNIG
Original assignee: TeGenero AG
Current assignee: Theramab LLC
Priority date: 1997-05-28
Filing date: 1998-05-28
Publication date: 2000-02-23
Also published as: AU8432498A; JP2002502244A; US20110009602A1; WO1998054225A3; US6987171B1; US8334102B2; DE19722888A1; WO1998054225A2; US20060039909A1

Abstract

The invention relates to human-compatible monoclonal antibodies, which are specific against human-CD28 and which activate non-specifically human-T-lymphocytes of several to all sub-groups without occupying an antigen receptor of said human-T-lymphocytes.

Description

Human CD28 specific monoclonal antibodies for antigen-unspecific activation of T lymphocytes

Description:

The invention relates to monoclonal antibodies which are specific for human CD28 and which activate T-lymphocytes without occupying an antigen receptor of the T-lymphocytes and thus antigen-nonspecifically, hybridoma cells for producing such antibodies, a method for producing such antibodies and uses of such antibodies. - Monoclonal antibodies are antibodies that are produced by hybrid cell lines (so-called hybridomas) that are formed by fusing an antibody-producing B cell of animal or human origin with a suitable myeloma tumor cell. CD28 is a cell surface molecule of known amino acid sequence expressed on T lymphocytes of human and animal origin, which is part of the international "Human

Leukocyte Typing Workshops "was given the abbreviation CD28. Activation of T-lymphocytes means the increase in metabolic activity, increase in cell volume, synthesis of immunologically important molecules and entry into the cell division (proliferation) of T-lymphocytes due to an external stimulus. For example these processes are triggered by occupation of the CD28 molecule on T cells by special CD28-specific monoclonal antibodies The activation of T lymphocytes with the described side effects is part of the physiological immune reaction, but can get out of control there in pathological situations (lymphoproliferative diseases ), or inadequate (immunodeficiency). The following technological background is important for understanding the invention. The activation of resting T cells for proliferation and functional differentiation first requires the occupation of two surface structures, so-called receptors: 1. the antigen receptor, which has a different specificity from cell to cell and for the detection of antigens, e.g. B. viral fission products is necessary; as well as the CD28 molecule expressed equally on all resting T cells, which naturally binds to ligands on the surface of other cells of the immune system. One speaks of the "costimulation" of the antigen-specific immune reaction by CD28. In cell culture, these processes can be simulated by filling the antigen receptor and the CD28 molecule with suitable monoclonal antibodies. In the classic system of costimulation, neither the occupation of the antigen receptor nor that of the CD28 molecule alone leads to T cell proliferation, but the occupation of both receptors is effective. This observation was made on human, mouse and rat T cells.

Monoclonal antibodies of the type mentioned are known. A "direct", ie activation of resting T-lymphocytes by CD28-specific monoclonal antibodies independent of the occupation of the antigen receptor, was observed in the following systems: in the literature Brinkmann et al., J. Immunology, 1996, 156: 4100-4106 showed that a very small proportion (5%) of human T-lymphocytes, which carry the surface marker CD45 RO, which is typical for resting T-lymphocytes, due to the "classic" CD28-specific monoclonal antibody 9.3 when the growth factor interleukin-2 (IL- 2) activated without occupying the antigen receptor. In the work of Siefken et al., Cellular Immunology, 1997, 176: 59-65, it was shown that a CD28-specific monoclonal antibody produced in a conventional manner, ie by immunizing mice with human T cells in cell culture, can activate a subset of human T cells for proliferation without occupation of the antigen receptor if CD28 is occupied by this monoclonal antibody and the cell-bound monoclonal antibody molecules can also be crosslinked by additional antibodies. In both cases, the antibodies described are initially generally not suitable for use in human medicine, since they are mouse antibodies. Furthermore, both of the antibodies described have in common that only a very small proportion of the T cells can be activated "directly".

In the work of Tacke et al., Eur. J. Immunol. , 1997, 27: 239-247, two types of CD28-specific monoclonal antibodies with different functional properties were described: "classic antibodies" which only stimulate the activation of resting T cells when the antigen receptor is occupied; and "direct", which can activate T lymphocytes of all classes in vitro and in the test animal for proliferation without the occupation of the antigen receptor. Both monoclonal antibodies known in this respect result from an immunization with cells on which rat CD28 is expressed and are obtainable by different selections directed towards their respective described properties. It is also shown in this reference that CD28-specific monoclonal antibodies which have the direct activating effect bind to T-lymphocytes much more slowly than classic CD28-specific monoclonal antibodies; however, binding to a mouse fibroblast cell line (L-929), on the surface of which the CD28 molecule is artificially expressed by transfection, is for classic and "directly" stimulating CD28-specific monoclonal antibodies at the same rate. It is concluded from this that the "directly" stimulating CD28-specific monoclonal antibodies known in this respect recognize an active form of the CD28 molecule, the presence of which on suppressed T cells is suppressed by a hitherto unknown mechanism, which, however, occurs when the molecule is expressed in non- T tumor cell lines is accessible. The monoclonal antibodies known in this respect are, on the one hand, specific against rat CD28 and, on the other hand, mouse antibodies. For both reasons, therefore, they are not suitable for therapeutic purposes in humans.

Compared to the state of the art according to the first two references mentioned, the invention is based on the technical problem of providing "direct" human CD28-specific monoclonal antibodies which, on the one hand, are also compatible with humans and, on the other hand, to a large extent on human T cells activate assets.

To solve this technical problem, the invention teaches human-compatible monoclonal antibodies which are specific for human CD28 and which activate human T-lymphocytes from several or all subgroups without occupying an antigen receptor of the human T-lymphocytes and thus antigen-nonspecifically, preferably with human constant components. - Constant components of an antibody are areas that are not important for antigen recognition, in contrast to the variable areas that define the antigen specificity of an antibody. However, constant components differ in the case of antibodies of different types and consequently also animals and humans. The constant ranges of an antibody must correspond to those of antibodies of an organism that is to be treated with the antibodies in order to be compatible. Monoclonal antibodies according to the invention are therefore on the one hand compatible with human beings, either per se or through humanization, and on the other hand can be used to treat various diseases which are based on insufficient T-lymphocyte activity, since the antibodies against human CD28 are specific and because the activation of the T-lymphocytes is comprehensive.

The invention naturally includes a wide variety of derivatives of monoclonal antibodies, provided that the claimed features are met. Derivatives of monoclonal antibodies are to be understood as meaning modifications of the monoclonal antibody which have been produced by customary biochemical or genetic engineering manipulations. This is the case, for example, with the humanization of a mouse monoclonal antibody by partially replacing structural (constant) components of the mouse antibody with those of a human.

In particular, monoclonal antibodies according to the invention are obtainable by: A) producing hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies by immunization with non-T tumor cell lines on which human CD28 is expressed, B) optionally humanization the monoclonal animal antibodies obtainable from hybridoma cells according to stage A by biochemical or genetic engineering exchange of constant components of the animal antibodies with analog constant components of a human antibody or replacement of the components of corresponding genes of the hybridoma cells, C) secretion of the antibodies in hybridoma cell cultures and

Isolation of the antibodies therefrom or production of the antibodies by injection of the hybridoma cells into animals, for example mice, and isolation of the antibodies from the body fluid of the animals. - The essence of the invention is to the closest references Brinkmann et al., J. Immunology ,. 1996, 156: 4100-4106, and Siefken et al., Cellular Immunology, 1997, 176: 59-65, therefore in the finding that "direct" activation of practically all T-lymphocytes can be achieved if the monoclonal antibodies are released by Immunization with non-T tumor cells on which human CD28 is expressed is obtained instead of immunization with T cell lines. This is how monoclonal antibodies can be obtained which are not only specific against human CD28, but which also cause a "direct" activation to a considerable extent. In particular, monoclonal antibodies according to the invention have specificity for determinants of the human CD28 molecule which are difficult to access on the naturally expressed CD28 molecule and whose occupation by the novel monoclonal antibodies leads to the activation of the T cells. A determinant is to be understood as the region of a molecule which is defined by the binding specificity of one or more antibodies.

The basic procedure for the production of hybridoma cells, for the humanization and for the production of the monoclonal antibodies from (humanized) hybridoma cells is well known to the person skilled in the art and need not be explained in more detail here. Basically, all known, freely available cell lines which are customary, in particular, for the production of the hybridoma cells can be used. In addition to the procedure described below, the recombinant expression which is well known to the person skilled in the art can in principle be used to produce the monoclonal antibodies.

In particular, it is preferred if the hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies are obtainable by a) creation a plasmid by inserting human CD28 cDNA into the pHβAPr-1-neo vector after excision of the Sall-Hindlll fragment and production of protoplasts from Escherichia coli (MC1061) which carry the plasmid, b) fusion of the protoplasts with mouse A20J and / or L929 tumor cells using polyethylene glycol, c) culturing the transfected cells obtained in stage b, d) screening the transfected mouse A20J and / or L929 cells for the expression of human CD28 and selection of human CD28 expressing mouse A20J and / or L929 Cells, e) immunization of BALB / c mice with the mouse CD20 expressing mouse A20J and / or L929 cells (for example by injections 6 x ip and then 1 x iV), f) removal of spleen cells from the mice so immunized and fusion of the spleen cells with ("nonproducer" -, ie no antibody producing) cells of the cell line X63-Ag 8.653 using polyethylene glycol, g) selecting the hybridoma cells thus obtained with the proviso that Antibodies are present in the supernatant of selected hybridoma cells which bind to mouse A20J and / or L929 cells expressing human CD28 and h) cultivation / subcloning of the selected hybridoma cells obtained in step g. Instead of steps a) to d), of course, other expression systems familiar to the person skilled in the art can also be used. Human CD28 cDNA is freely available from Dr. A. Aruffo and Dr. B. Seed, who published the sequence and also the following literature: Aruffo, A., and Seed, B, 1987, "Molecular cloning of a CD28 cDNA by a high efficiency COS cell expression system", Proc. Natl. Acad. Be. USA, 84: 8573. The preparation of the human CD28 cDNA can therefore be found in detail in this reference. In addition, any person skilled in the art can easily and quickly produce a human CD28 cDNA clone using the sequence stored in the gene bank and the polymerase chain reaction. The pHßAPr-1-neo Vector is freely available from the authors of the Gunning reference, P, et al., 1987, "A human β-actin expression vector system directs high-level accumulation of antisense transcripts", Proc. Natl. Acad. Be. USA, 84: 4831. "neo" stands for neomycin resistance. Step c) is therefore carried out in the presence of neomycin. The cell lines and / or microorganisms mentioned above are freely available and can be purchased from the American Type Culture Collection (ATCC). Regarding Escherichia coli (MC1061), in addition to the literature Meissner, PS, et al., 1987, "Bacteriophage gamma cloning system for the construction of directional cDNA libraries", Proc. Natl. Acad. Be. USA, 84: 4171.

The invention accordingly also relates to hybridoma cells according to claim 4 and a method for producing antibodies according to the invention according to claims 5 and 6.

However, the use of monoclonal antibodies according to the invention for the production of medicaments, in particular for the treatment of diseases with pathologically reduced CD4 T cell numbers, such as AIDS or in the case of stem cell transplantation after chemotherapy of leukemic diseases, for potentiation and / or qualitative influencing of immune reactions in vaccinations and / or influencing the quality of the T cell reaction, in particular influencing the production of different effector molecules, for example cytokines and chemokines and their receptors, for example in autoimmune diseases and AIDS. The pharmaceutical preparation of the pharmaceuticals for the various administration forms is well known to the person skilled in the art and need not be explained in more detail here. The quality of the T cell reaction is, in particular, the production of certain Understanding cytokine patterns that can be pro- or anti-inflammatory, for example, or can selectively lead to the production of certain immunoglobulin classes in B lymphocytes (classic examples of different qualities of the T cell reaction are the functional TH1 and TH2 phenotypes, as described in the following examples ). The invention also encompasses methods of curing the diseases mentioned above and below using monoclonal antibodies according to the invention.

The invention is explained in more detail below on the basis of exemplary embodiments. In particular, the production of monoclonal antibodies according to the invention is described. These exemplary embodiments also clearly show screening methods with which the inventive monoclonal antibodies or underlying hybridoma cells can be selected. The following examples also show therapeutic applications according to the invention.

The experiments shown and the examples of the effects of "direct" CD28-specific monoclonal antibodies were carried out in the animal model of the rat, the monoclonal antibody JJ319 as an example of a "classic" CD28-specific antibody and as an example of a " direct "activating the monoclonal antibody JJ316 is used. Both antibodies are freely available and commercially available from Pharmingen, San Diego, USA. JJ319 and JJ316 antibodies are otherwise available according to the literature reference M. Tacke et al., Immunology, 1995, 154: 5121-5127, to which express reference is hereby made, also with regard to details of the production of hybridoma cells and monoclonal antibodies. example 1

In this example, the preparation according to the invention, i.e. human CD28-specific monoclonal antibody explained in more detail. These are also referred to as CMY-2 below. Human CD28 from a cDNA library was recombinantly expressed in A20J and / or L929 cell lines. For this purpose, a plasmid was first created by inserting human CD28 cDNA into the pHβAPr-1-neo vector after excision of the Sall-HindIII fragment. Protoplasts carrying the plasmid were produced from Escherichia coli (MC1061). The protoplasts were then fused with mouse A20J and / or L929 tumor cells using polyethylene glycol. The transfected cells thus obtained were cultivated in the usual way. The transfected mouse A20J and / or L929 cells were then screened for the expression of human CD28 and selection of mouse A20J and / or L929 cells expressing human CD28.

Successful expression was demonstrated using a conventional, commercially available fluorescence-labeled antibody with specificity for human CD28 (9.3-phykoerythrin). As a negative control, non-transfected A20J or L929 cells were stained with the same antibody. The transfectants (A20J-CD28 and L929-CD28) showed a higher fluorescence intensity. Since not all cells were CD28 positive, CD28 positive cells were subcloned and used for immunization. As can be seen in FIG. 1 from the upward shift of the point clouds in the two diagrams on the right, these cells reacted with the commercially available antibody, ie they expressed Human CD28 on their surface. The A20J human CD28 cell line was used to immunize BALB / c mice. Cell fusion and screening were carried out as follows: i) Immunization of BALB / c mice with the mouse CD20 expressing mouse A20J cells (injections 6 x ip and then 1 x iV). ii) removal of

Spleen cells of the mice immunized in this way and fusion of the spleen cells with cells of the cell line X63-Ag 8.653 using polyethylene glycol. iii) Selection of the hybridoma cells thus obtained with the proviso that the supernatant of selected hybridoma cells contains antibodies which bind to mouse A20J and / or L929 cells expressing human CD28.

The staining of a mixture of CD28 transfected and untransfected mouse L929 tumor cells served as read-out. FIG. 2 shows that the monoclonal antibody CMY-2 isolated in this way distinguishes transfected and untransfected cells by different fluorescence intensities. Differential screening for antibodies against human CD28 was carried out as follows. 50 μl of supernatant each from cultured cell hybridomas were removed and incubated for 15 min with a mixture of L929 cells and L929-CD28 transfectants. After washing, the cells were stained with DaMIg-PE. Part A shows the negative control. The cells were incubated with DaMIg-PE only. Part B shows staining with a supernatant that was slightly positive but showed no difference in either cell. Part C shows the cells stained with a supernatant from CMY-2.

In non-illustrated experiments, human peripheral blood cells were stained with the newly isolated CMY-2 and the "classic" CD28-specific antibody 9.3. An identical expression pattern was found on the subpopulations of human blood cells. In summary, the experiments show that CMY-2 is a human CD28-specific antibody.

CMY-2 was then tested for classical costimulatory and for "direct" stimulating activity with human T-lymphocytes enriched from peripheral blood on approximately 80%. The T cell proliferation was measured by incorporating ³ H thyme between the 2nd and 3rd day of culture. The following results were achieved:

Costimulation:

Unst uluted cells 276 cpm

CD3-specific antibody 3111 cp CD3-specific antibody + CMY-2 51676 cpm

Direct stimulation:

Solid-phase anti-mouse Ig 379 cpm

Solid-phase anti-mouse Ig + control mAb 258 cpm Solid-phase anti-mouse Ig plus CMY-2 19115 cpm

Explanation: Antι-CD3 provides T cell receptor stimulation (CD3 is part of the TCR complex). CMY-2 was used in the form of an unpurified culture supernatant (50% final volume). Experience has shown that the effective mAb concentration to be expected is suboptimal for direct activation, but sufficient for costimulation. The experiment shows that CMY-2 has direct activating properties.

Hybridoma cells according to the invention which produce CMY-2 have been deposited with the DSMZ, German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig, under the number DSM ACC2353 (20.05.1998). Example 2

In this example, the immunomodulating effect of "direct" CD28-specific monomlonal antibodies is explained in more detail. The aim of the experiments is the detection of the leading cytokines of the TH2 cells, namely IL-4 and IL-10, as a result of the direct activation in vitro and in vivo. IL-4 is crucial for the cooperation of TH2 cells with

B-lymphocytes, for the differentiation of further CD4 T cells to TH2 cells and thus the polarization of the immune system away from the inflammatory and towards the humoral immune response, and IL-10 is the central factor for the suppression of inflammatory (THl) reactions. Only the more important results of the in vivo treatment of rats are shown. Even clearer effects were found in vitro.

FIG. 3 shows the expressed cytokine profile of lymph nodes and spleen cells of young LEW rats three days after ip injection of the directly activating mAb JJ316, the costimulator JJ319, the TCR-specific mAb R73 or the vehicle PBS. The illustration is a so-called RNAse protection test in which radioactively labeled antisense mRNA samples are protected from degradation of added RNAse by hybridization with the RNA extracted from the tissue. These give defined bands on the gel and make it possible to see at a glance the expressed cytokine profile at the mRNA level of a tissue. The two smallest fragments, L32 and GADPH, are "household genes", the uniform expression of which is used to control the same amount of RNA used in the individual batches. The test was carried out using a kit commercially available from Pharmingen.

JJ316, but not JJ319 or R73, massively induce IL-10 and, to a lesser extent, IL-4 mRNA. The effects are particularly evident in the spleen, but are also visible in the lymph nodes.

In Fig. 4, the cytokine IL-4 is detected at the protein and at the single cell level by flow cytophotometric analysis. For this purpose, the cells are first stained with mAb against the surface molecule CD4, then fixed and permeabilized, so that in a subsequent cytoplasmic staining with an IL-4-specific mAb, which is labeled with a second fluorochrome, the IL-4 protein is detected can be. The evaluation is carried out in the flow cytophotometer, each point represents a cell. The quadrants introduced represent the boundaries between the background and the positive reaction. However, the methodology is also described in detail in the Pharmingen catalog.

As shown in Figure 4, injection of the mAb JJ316, but not that of the classic costimulator JJ319, induces the production of IL-4 in a substantial proportion of the isolated CD4 T cells.

A biological effect of the increased IL-4 production is shown in FIG. 5: The level of detectable antibodies of the class IGE increases significantly as a result of the treatment with mAb JJ316 and demonstrates the in vivo effectiveness of the "direct" CD28-specific monoclonal antibodies induced IL-4 secretion. FIGS. 6 and 7 show so-called EMSAs (Electrophoretic Mobility Shift Assays) for the detection of the induction of transcription factors which promote the development of anti-inflammatory TH2 cells. The technique is as follows. T cells are stimulated in vitro for different lengths of time, then the proteins from the cell nuclei are brought into solution and incubated with a radioactively labeled short gene probe, the sequence of which they should recognize as transcription factors. After incubation, the mixture is separated on a polyacrylamide gel. The unbound labeled gene probe runs out of the gel at the bottom (not shown here). Gangs like the ones shown here are e.g. T. non-specific (available everywhere) or selectively induced (strong, weak signals depending on the stimulus).

The following becomes clear from the consideration of FIGS. 6 and 7. Both with a GATA3-specific and with a c-Maf-ResponseElement-specific sample, increased induction during costimulation (TCR + classical CD28) and direct stimulation ("direct" CD28) is shown in comparison to stimulation only via the T cell receptor (TCR ). In summary, it can be said that stimulation of the CD28 molecule promotes the expression of these transcription factors in the cell nucleus, and that this is also possible without TCR stimulation by direct CD28 stimulation. For the importance of these factors for the differentiation of TH2 cells, reference is made to Current Opinion in Immunology 1997, 9: 776-781.

The connections to the immunoregulating or immunomodulating effect of "direct" CD28-specific monoclonal antibodies explained in detail above with regard to the formation of THl and / or TH2 cells consequently make them particularly suitable for the production of medicaments for the treatment of such immune reactions Diseases. These are basically all allergic-inflammatory and autoimmune-inflammatory clinical pictures. The former includes, for example, inflammatory bowel diseases and contact dermatitis, which are grouped under the name "Inflammatory Bowel Disease" (IBD). The latter include type I diabetes and multiple sclerosis. It is also to be expected that strong stimulation of the human CD28 molecule by monoclonal antibodies according to the invention will be able to cure HIV I-infected T cells. Because this allows the viruses to be cellular

Core receptors used chemokine receptors are switched off and the production of chemokines that bind to such receptors and thus block them for HIV I viruses are induced.

Example 3

8 shows the proliferative response of unseparated rat lymph node cells to the "directly" stimulating ^υ CD28-specific monoclonal antibody (JJ316) and the absence of such a response when using a "classic" CD28-specific monoclonal antibody (JJ319). The cells were placed in 0.2 ml medium for two days

(RPMI 1640, available from GIBCO / BRL, containing 5% FCS

[fetal calf serum]) in the presence or absence of the specified

Additives at a density of 1 million cells per ml in gassing

Cultivated incubator. The Zellteilungsaktivitat was by the incorporation of radioactively labeled thymidine (1 uCi / assay f _o r

16 hours, lCi = 37GBq, determination with β detector). 0

In contrast to published results (Siefken et al., Cellular Immunology, 1997, 176: 59-65), this result shows that it is not necessary for T cell activation by directly activating CD28-specific monoclonal antibodies, to artificially cross-link these with one another using a second antibody. Rather, the presence of non-T cells from lymphoid organs, namely B lymphocytes and so-called accessory cells, is sufficient to enable direct activation by soluble added CD28-specific monoclonal antibodies. This is probably done by binding the monoclonal antibodies to so-called Fc receptors of these non-T cells. This result is an important prerequisite for the therapeutic use of "directly" stimulating CD28-specific monoclonal antibodies, in which an artificial crosslinking with anti-immunoglobulin antibodies is not practicable in the whole organism.

Example 4.

"Directly" activating CD28-specific monoclonal antibodies lead to an increase in the number of CD4 T cells in the intact organism. FIG. 9 shows this for lymph nodes of the rat which had received 1 mg on day 0 of the "directly" stimulating CD28-specific monoclonal antibody (JJ316) or of the "classic" CD28-specific monoclonal antibody (JJ319). With directly activating monoclonal antibodies according to the invention with specificity for human CD28 and their ability to stimulate the proliferation of T-lymphocytes, quite analogous effects are achieved. This can then be used in particular in situations in which the proportion of CD4 T cells is pathologically reduced and the normal level is to be approximated again. Such situations exist particularly in the clinical picture of AIDS and after chemotherapy and bone marrow transplantation. In the present example, the CD4 T cell number is only temporarily increased; this is because healthy animals have been treated with normal CD4 T cell counts. The due to the Proliferation stimulation of "excess" cells is broken down by homeostatic mechanisms.

Example 5.

As can be expected from the above conclusions drawn in FIGS. 5-7, directly activating CD28-specific monoclonal antibodies can be used therapeutically, inter alia for preventing an inflammatory autoimmune reaction. FIG. 10 shows an experiment for this, namely for the so-called adjuvant arthritis in the rat, a model system for certain forms of rheumatoid arthritis in humans. "Paw volume increase" indicates the increase in the volume of the paws. "Days" stands for days. "Healthy" data points indicate values for healthy animals. A monoclonal antibody of the same immunoglobulin class with specificity for an irrelevant human cell surface molecule was used for the isotype control. AA stands for adjuvant arthritis. PBS stands for "phosphate buffered saline". W3 / 25 stands for a monoclonal antibody with specificity for the CD4 molecule of the rat. The adjuvant arthritis is mediated by so-called THl cells. THl cells arise from resting CD4 T cells in the course of activation under the influence of certain soluble factors of the immune system, so-called cytokines. The antagonists of the TH1 cells are the anti-inflammatory TH2 cells, the production of which is controlled by other cytokines. In the experiment shown in FIGS. 10 and 11, the development of the adjuvant arthritis, read from the joint swelling (FIG. 10) and the arthritic index (FIG. 11) after immunization with mycobacteria in adjuvant, was determined by the "directly" activating CD28- specific monoclonal antibody JJ316 almost completely suppressed. The "classic" CD28-specific monoclonal antibodies (JJ319) had the opposite effect, ie it worsened the clinical picture. From this it can be seen, also for use in humans, that the immune reaction can be influenced by the application of conventional or according to the invention "directly" stimulating CD28-specific monoclonal antibodies, here in the sense of an "immunodeviation" to THl or TH2. In other words, monoclonal antibodies according to the invention, but also "classic" monoclonal antibodies which are specific for human CD28 (and / or can be obtained by immunization with T cells) can effect an immunomodulation. Such a purpose of "classic" monoclonal antibodies is also not known.

Finally, the invention therefore also relates to the use of monoclonal antibodies specific for human CD28 (obtainable according to the above basic procedures, when immunized with T cell lines expressing human CD28 or non-T cell lines) for the production of medicaments for modulating immune reactions, and namely immunosuppression (for example with human CD28 analogs to JJ319) or immune enhancement (for example with human CD28 analogs to JJ316 such as CMY-2).

Claims

Claims:

1. Human-compatible monoclonal antibodies which are specific for human CD28 and which activate human T-lymphocytes from several to all subgroups without occupying an antigen receptor of the human T-lymphocytes and thus antigen-nonspecifically.

2. Monoclonal antibodies according to claim 1, which are obtainable by

A) Production of hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies by immunization with non-T tumor cell lines on which human CD28 is expressed,

B) if necessary, humanization of the monoclonal animal antibodies obtainable from hybridoma cells according to stage A by biochemical or genetic engineering exchange of constant components of the animal antibodies with analogue constant components of a human antibody or replacement of the components of corresponding genes of the hybridoma cells, C) secretion of the monoclonal antibodies in

Hybridoma cell cultures and isolation of the monoclonal antibodies therefrom or production of the monoclonal antibodies by injection of the hybridoma cells into animals, for example mice, and isolation of the monoclonal antibodies from the

Body fluid of the animals.

3. Monoclonal antibodies according to claim 1 or 2, wherein the hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies are obtainable by a) creating a plasmid by inserting human CD28 cDNA into the pHβAPr-1-neo vector after excision the Sall-Hindlll fragment and production of protoplasts from Escherichia coli (MC1061) which carry the plasmid, b) fusion of the protoplasts with mouse A20J and / or L929 tumor cells using polyethylene glycol, c) cultivation of the transfected cells obtained in step b, d) Screen the transfected mouse A20J and / or

L929 cells for the expression of human CD28 and selection of human CD28 expressing mouse A20J and / or L929 cells, e) immunization of BALB / c mice with the human CD28 expressing mouse A20J and / or L929

Cells, f) removal of spleen cells from the mice immunized in this way and fusion of the spleen cells with cells of the cell line X63-Ag 8.653 by means of polyethylene glycol, g) selection of the hybridoma cells thus obtained with the proviso that antibodies which are present in human cells are contained in the supernatant of selected hybridoma cells. Mouse A20J and / or L929 cells expressing CD28 bind and h) cultivation / subcloning of the selected hybridoma cells obtained in step g. 4) Hybridoma cells for the production of monoclonal antibodies according to one of claims 1 to 3, which are obtainable by the following process steps: a) Creation of a plasmid by inserting human CD28 cDNA into the pHßAPr-1-neo vector after

Excision of the Sall-HindIII fragment and production of protoplasts from Escherichia coli (MC1061) which carry the plasmid, b) Fusion of the protoplasts with mouse A20J and / or L929 tumor cells by means of

Polyethylene glycol, c) culturing the transfected cells obtained in step b, d) screening the transfected mouse A20J and / or L929 cells for the expression of human CD28 and

Selection of human CD28 expressing mouse A20J and / or L929 cells, e) immunization of BALB / c mice with the human CD28 expressing mouse A20J and / or L929 cells, f) removal of spleen cells from the mice so immunized and fusion of the spleen cells with Cells of the cell line X63-Ag 8.653 by means of polyethylene glycol and g) selection of the hybridoma cells thus obtained with the proviso that the supernatant of selected hybridoma cells contains antibodies which bind to mouse A20J and / or L929 expressing human CD28.

5) Method for producing monoclonal antibodies according to one of claims 1 to 3 with the following process stages: A) Production of hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies by immunization with non-T tumor cell lines on which human CD28 is expressed,

B) if necessary, humanization of the monoclonal animal antibodies obtainable from hybridoma cells according to stage A by biochemical or genetic engineering exchange of constant components of the animal antibodies against analog constant components of a human antibody or replacement of the components of corresponding genes of the hybridoma cells,

C) secretion of the monoclonal antibodies in hybridoma cell cultures and isolation of the monoclonal antibodies therefrom or production of the monoclonal antibodies by injection of the hybridoma cells into animals, for example mice, and isolation of the monoclonal antibodies from the body fluid of the animals.

6) Method according to claim 5, wherein the hybridoma cells capable of producing monoclonal human CD28-specific animal antibodies are produced in the following process steps: a) Creation of a plasmid by inserting human CD28 cDNA into the pHβAPr-1-neo vector after excision the Sall-Hindlll fragment and production of protoplasts from Escherichia coli (MC1061) which carry the plasmid, b) fusion of the protoplasts with mouse A20J and / or L929 tumor cells using polyethylene glycol, c) cultivation of the transfected cells obtained in stage b, d) screening of the transfected mouse A20J and / or L929 cells for the expression of human CD28 and selection of human CD28 expressing mouse A20J and / or L929 cells, e) immunization of BALB / c mice with the mouse CD20 expressing mouse A20J and / or L929 cells, f) removal of spleen cells from the mice immunized in this way and fusion of the spleen cells with cells of the cell line X63-Ag 8.653 using polyethylene glycol and g) selection of the hybridoma cells thus obtained with the Provided that the supernatant of selected hybridoma cells contains antibodies which bind to mouse A20J and / or L929 cells expressing human CD28.

7) Use of monoclonal antibodies according to one of claims 1 to 3 for the manufacture of a medicament for the therapeutic treatment of the human body.

8) Use according to claim 7 for the manufacture of a medicament for the treatment of diseases with pathologically reduced CD4 T cell numbers, in particular AIDS or after stem cell transplantation after chemotherapy of leukemic diseases. 9) Use according to claim 7 for the manufacture of a medicament for potentiation and / or qualitative influencing of immune reactions in vaccinations.

10) Use according to claim 7 for the manufacture of a medicament for influencing the quality of the T cell reaction, in particular for influencing the production of different effector molecules, for example cytokines and chemokines and their receptors, for example for autoimmune diseases and AIDS.

11) Use of monoclonal antibodies according to one of claims 1 to 3 for the treatment of diseases of the human body.

12) Method for the therapeutic treatment of the human body, wherein monoclonal antibodies according to one of claims 1 to 3 are used.