WO2000018806A1 - Bispecific and trispecific antibodies which specifically react with inducible surface antigens as operational target structures - Google Patents

Abstract

According to the invention, an intact bispecific or trispecific antibody is provided which comprises at least the following properties: a) binding to a T cell; b) binding to at least one antigen on a target cell; c) binding by the Fc portion thereof (in bispecific antibodies) or by a third specificity (in trispecific antibodies).The antigen can be induced and is not found on the target cell in a non-induced state (normal state) or it exists in a low number that is insufficient to destroy the target cell.

Description

Bispecific and trispecific antibodies that react specifically with inducible surface antigens as operational target structures

The present invention relates to new bispecific and trispecific antibodies, methods for producing these antibodies and their use in immunotherapy.

Immunotherapy using antibodies, especially bispecific or trispecific antibodies, has become increasingly important in recent years. An essential problem when using such antibodies in immunotherapy is the specific recognition of the target cell by the antibodies, ie a differentiation as precisely as possible from the cells which are not to be attacked by the antibodies, ie the cells in the normal, physiological state. The more specific the detection of target structures on the target cells the antibody, the gentler a therapy with such antibodies is for a patient and the fewer the side effects.

Solving this problem becomes all the more important the more the efficiency of immunotherapy increases with the possibility of severe side effects.

DE 196 49 223 and DE 197 10495 describe intact bispecific and trispecific antibodies which are used for immunotherapy. These bispecific and tri-specific antibodies are capable of binding positive cells (accessory cells) to a T cell, at least one antigen on a target cell and through their Fc part or through a third specificity to Fc receptor.

For example, while monoclonal antibodies, when used in immunotherapy, only activate certain, clearly defined sub-areas of the immune system on a target cell and therefore the side effects that occur in a patient are also limited and therefore relatively easy to control, intact bispecific and trispecific antibodies produce much stronger side effects if the specificity of the binding arm that recognizes an antigen on a target cell is low. The reason is that intact bispecific and trispecific antibodies activate not only T cells, but also accessory cells, for example monocytes, macrophages and dendritic cells. These side effects could be reduced by allowing a highly specific recognition of the target cell by the antibody.

It is therefore an important object of the present invention to provide intact bispecific and trispecific antibodies which recognize a specific antigen on a target cell. According to the invention, this object is achieved by an intact bispecific or trispecific antibody which has at least the following properties:

a) binding to a T cell; b) binding to at least one antigen on a target cell; c) Binding to Fc receptor positive cells by their Fc part (in the case of bispecific antibodies) or by a third specificity (in the case of trispecific antibodies)

and which thereby induces an immune response and / or destroys the target cells, the antibody being selected such that it binds to a surface antigen as target antigen on the target cell which is inducible and does not occur in the uninduced state (normal state) on the target cell or in such a small number that the number is not sufficient to destroy the target cell.

According to the invention, it was possible to show that, after induction, a relatively small number of the target antigens on the target cell is sufficient to bind the intact bispecific and trispecific antibodies and to initiate destruction of the target cell and / or an immune response. According to the invention, “small number” means a number of target antigens on the target cell of more than 100 target antigens per target cell, preferably more than 300 and furthermore preferably more than 1000 target antigens per cell. The inducible target antigens can also be present in an amount of up to 500,000 per target cell, with amounts of up to 400,000, up to 300,000, up to 200,000 or up to 100,000 being inducible per target cell. Another preferred amount range in which the target antigens can be is from 50,000 to 100,000 and from 5,000 to 50,000.

Examples of inducible surface antigens on target cells (target antigens) which are suitable for immunotherapy by intact bispe- specific and trispecific antibodies can be used are heat shock proteins and "MHC class I-related" MIC molecules. Heat shock proteins (Hsp) are synthesized by the cell in response to cell stress. According to the invention, "cell stress" includes, for example, degeneration of the cell, the action of radiation, chemical substances and of increased temperature on the cell, and infection of the cell by microorganisms. Four families of heat shock proteins are currently known, which are differentiated on the basis of their different molecular weights. These families are referred to as Hsp25, Hsp60, Hsp70 and Hsp90, the number representing the approximate molecular weight of the stress proteins in kilo-daltons. The heat shock proteins are characterized by highly conserved amino acid sequences, the degree of conservation being over 35% amino acid identity, preferably 35-55%, further preferably 55-75% and most preferably between 75% to 85% amino acid identity .

Reference is made to the following overview articles: Annu. Rev. Net. 27, 437-496 (1993); Biochimie 76, 737-747 (1994); Cell. Mol. Life Be. 53: 80-129, 168-211 (1997); Experientia 48, 621-656 (1992); Kabakov et al. Gabai, Heat Shock Proteins and Cytoprotection, Berlin: Springer 1997.

Heat shock proteins are normally not expressed on healthy tissue. However, there are studies which show that heat shock proteins can be expressed, for example, on tumor cell lines and on tumor material from patients (Melcher et al., Nature Medicine, 4: 581, 1998). However, the expression of the heat shock proteins on the tumor cell lines is relatively weak and is therefore unsuitable for previously known immunotherapeutic approaches with monoclonal antibodies and incomplete (F (ab) 2) bispecific antibodies.

Examples of heat shock proteins are Hsp60 and Hsp70 / 72. The same applies to MIC molecules. These molecules can also be induced by cell stress, as defined in more detail above. MIC molecules are MHC I-related molecules that are under the control of heat shock promoter elements (Groh et al., PNAS 93: 12445, 1996). Examples of MIC molecules are MIC A and MIC B. It was also possible to show for MIC molecules that they are not expressed on normal tissue or only in such a small amount that they act as a target structure for detection by an antibody in order to destroy the target cell are not suitable, while they are expressed on, for example, epithelial tumors in such an amount that they can be used as target antigens in immunotherapy due to the bispecific and trispecific antibodies provided according to the invention.

The intact bispecific or trispecific antibodies provided according to the invention are selected such that they are directed against at least one target antigen on a target cell which is inducible and does not or does not occur essentially on the target cell in the non-induced state. For example, this number is approximately 100 target antigens / cell. However, this does not mean that such target antigens cannot occur on other cells, ie non-target cells. For example, heat shock proteins occur on rapidly regenerating tissue, for example the mucous membrane cells of the gastrointestinal tract, even in a physiological state. However, these heat shock proteins are not included in the present invention, since they are not inducible, but already occur on normal tissue. When using the antibodies according to the invention which are directed against heat shock proteins, certain mucosal cells of the gastrointestinal tract can also be destroyed, since, as explained above, these constitutively express heat shock proteins on their cell surface. The possible destruction of these cells is associated with certain disadvantages for a patient, which, however, compared to the achievable tumor destruction are to be added.

The rapidly regenerating tissue is thus not the primary target of the antibodies according to the invention, but can be "hit" by the antibodies if the antibody according to the invention not only recognizes the inducible target antigens on the target cell, but these antigens e.g. also occur on rapidly regenerating tissue. However, since this tissue divides quickly, the original state of this tissue can be restored relatively quickly after stopping the antibody therapy so that it can again perform its physiological task. The invention is therefore not aimed at the bispecific and trispecific antibodies according to the invention antigens on e.g. recognize rapidly regenerating tissue, which may be constitutively expressed there, but the invention only includes those antibodies which are directed against target antigens which are inducible on the target cell and, after induction, for example also constitutively, are expressible.

According to the invention, “inducible” is understood to mean target antigens which here are tumor-specific operations and which do not occur in the physiological state on the cell or only occur in such a number that an immune response against them cannot be induced or a destruction of the target cells cannot or due to this small number takes place to an insignificant, therapeutically usable extent.

Inducible target antigens on a target cell are not only to be understood as those on a tumor cell, but also antigens that are induced when the target cell is infected, for example, by a microorganism. According to the invention, “microorganism” is understood to mean any organism which influences the target cell in such a way that a target antigen induced by the microorganism is expressed on its surface to the extent described above. Microorganisms are invented According to the invention, for example, bacteria, viruses, protozoa and fungi are understood. Bacteria include, for example, gram-positive and and gram-negative bacteria, mycoplasmas and rickettsia. The protozoa in particular include plasmodes. The viruses include, for example, retroviruses, adenoviruses, herpes viruses, hepatitis viruses, togaviruses, poxviruses, etc. It is crucial that in cells which have been infected by one or more of these microorganisms, antigens are expressed on the cell surface which are caused by the microorganisms. Infection can be induced. These are antigens that are produced by the target cells in response to the microorganism infection and appear on the cell surface (host antigens), but not target antigens that are produced by the microorganisms themselves. The infection of a target cell by a microorganism also leads to a cell stress situation for the cell, which in response induces the expression of certain proteins, for example heat shock proteins and MIC proteins.

The intact bispecific and trispecific antibodies provided according to the invention not only bring one type of immune cell, but T cells and accessory cells to the target cell, and for this reason they are particularly suitable for identifying inducible surface antigens as operational target structures. It could be shown that only a few target antigens in an amount of 100 to 5000 per cell on the target cell are sufficient to destroy them. The in vitro experiments carried out for this purpose with stem cell preparations (PBSZ) are described in Example 1 below. Thus, the class of intact bispecific and trispecific antibodies according to the invention is capable of destroying tumor cells or target cells even with a very low expression of the target antigens or of initiating an immune response after their recognition. The antibodies according to the invention, since they are directed against inducible antigens, can help to solve the problem of missing target cell-specific target antigens which are necessary for immunotherapy on tumor cells and on cells infected by microorganisms. Since such inducible antigens do not only occur on tumor cells, but are generally produced in stressful situations, diseases can also be tackled immunotherapeutically, which have been triggered by infection of, for example, viruses, unicellular organisms, bacteria or fungi. These are the MIC molecules and heat shock proteins already described in more detail above.

The intact bispecific or trispecific antibodies described above are used therapeutically to induce an immune response and / or to destroy the target cell. The antibodies are administered in the form of a pharmaceutical preparation, which may contain conventional carriers and / or excipients, in order to enable stability and a favorable form of administration combined with high tolerability and effectiveness. By applying the antibodies, prophylaxis and treatment of, for example, tumor diseases can be achieved, so that immunity to the pathogen or to the tumor, preferably long-term immunity, is achieved.

The antibodies provided according to the invention can be used in a relatively small amount, i.e. they are already effective in an amount of 5 μg to 10 mg per patient in order to bring about the immunity according to the invention, for example tumor immunity, or the destruction of the target cell which can be achieved according to the invention. The application amount is preferably in the range from 10 μg to 100 μg per patient, but it can also, if necessary, be above that, ie in the range from 100 μg to 5 mg per patient.

In the case of a tumor patient, since induction of tumor immunity takes a certain amount of time, at the start of therapy a minimal residual disease (MRD) situation with few remaining tumor cells. MRD is the period after the reduction of larger tumor quantities using suitable methods, such as the removal of the primary tumor by surgical measures, in which residual tumor cells still exist, which may not be detectable, but lead to recurrence after a certain time. However, therapy can begin before the tumor is removed to ensure that enough tumor cells are bound by the bispecific or trispecific antibodies and presented to the immune system.

With the small amounts of intact bispecific or trispecific antibodies administered that can be used according to the invention, the risk of serious side effects is significantly reduced. Another advantage is that a humoral immune response is induced with complement-fixing antibodies (the IgGl and IgG3 isotypes in humans, which are able to destroy tumor cells).

Binding of the antibody to the Fc receptor positive cell activates the cell, thereby initiating or increasing the expression of cytokines and / or of co-stimulatory antigens.

The antibodies according to the invention transmit at least one second activation signal, which is required for a physiological activation of the T cell, through the co-stimulatory antigens on the accessory cell to the T cell, this activation being reflected in the upregulation of activation markers, the destruction the target cell and / or in a proliferation of the T cell. After administration of the antibody to the patient, tumor immunity, preferably long-term tumor immunity, is hereby initiated.

The immune response achievable according to the invention is thereby characterizes that in an organism the body's immune system is activated in such a way that there is permanent destruction and / or control of the tumor or the infectious disease. It is also advantageous when using the antibodies described here to reduce the side effects which commonly used antibodies, for example monoclonal antibodies, have. The target structure for the antibodies according to the invention are inducible surface antigens according to the definition of the present invention, that is to say those which do not occur on the target cell or in a negligible amount, so that direct destruction of the target cell or an immune response, in particular a long-lasting immune response or immunity is not induced or at least is not induced to a therapeutically reasonable extent.

According to the invention, any type of tumor that can be treated under the definition given above can be treated. Epithelial tumors, adenocarcinomas, colon carcinomas, breast carcinomas, ovarian carcinomas, lung carcinomas and neck, nose and / or ear tumors can be treated in particular. Non-epithelial tumors such as leukemia and lymphoma can also be treated. Virus-induced tumors, for example liver tumors or cervical carcinomas, can also be treated.

By using the antibodies provided according to the invention, target cells infected by microorganisms and the diseases induced thereby can also be treated. This includes, for example, infections from CMV and HIV.

According to the invention, “target cell” is understood to mean all cells that are degenerate in the form of a tumor cell or that have been infected by a microorganism and that, in response to the cell degeneration or the microorganism infection, express an antigen which is normal in the target cell, ie without degeneration or without infection, is not produced or to such a small extent that an immunotherapeutic pie is not inducible or an immunotherapeutic destruction of the target cell is not possible.

According to the invention, it is not possible to use any antibodies, but these must be intact, i.e. they must have a functional Fc part, and they are preferably heterologous in nature, i.e. the antibodies are then composed of heavy immunoglobulin chains of different subclasses (combinations, also fragments or individually exchanged amino acids) and / or origin (species).

In a preferred embodiment of the present invention, bispecific and / or trispecific antibodies are used which are able to activate the Fc receptor-positive cell. This initiates or increases the expression of cytokines and / or co-stimulatory antigens.

In the case of the trispecific antibodies, the binding to the Fc receptor positive cells is preferably carried out, for example, via the Fc receptor from Fc receptor positive cells or also via other antigens on Fc receptor positive cells (antigen-presenting cells), such as e.g. the mannose receptor.

Some of the heterologous bispecific and / or trispecific antibodies which can be used according to the invention are known per se, but some are also described for the first time in the above application.

The invention is described below in particular with reference to bispecific antibodies. However, the bispecific antibodies represent only one embodiment of the invention. It is also possible to use trispecific antibodies with the properties specified above. Furthermore, the invention is particularly illustrated using the example of induction of tumor immunity. However, this is also only one possible embodiment of the invention, which is very general for immunotherapeutic German purposes can be used. For example, it is also possible to use the antibodies provided according to the invention to achieve immunity to microorganisms which, among other things, induce the target antigens described here on the infected target cells.

With regard to feature (a), the 1st signal is transmitted, for example, via the T cell receptor complex of the T cell and can therefore, viewed in isolation, lead to an unphysiological activation of the T cell. As a result, the cell is anergized and can no longer react appropriately to stimuli mediated by T cells. By means of the bispecific or trispecific antibodies according to the invention, at least a second activation signal is also simultaneously transmitted to the T cell by the costimulatory antigens on the Fc receptor positive cell, which leads to a physiological activation of the T cell and subsequently to destruction the target cell and / or a proliferation of the T cell. As a further criterion for the activation of the T cell, the upregulation of surface antigens such as CD2, CD25 and / or CD28 and / or the secretion of cytokines such as e.g. IL-2 or INF-γ can be used.

With the bsAk used according to the invention, T cells are activated and redirected against the target cells. Unspecific activations of T cells without redirection generally had little success in immunotherapy.

MHC 1 is upregulated on the target cell and the intracellular processing machinery (proteasome complex) is activated due to the release of cytokines (such as INF-γ and TNF-α) in the immediate vicinity of the target cell. The cytokines are released due to bispecific antibody-mediated activation of T cells and accessory cells (see Fig. 1 and 3). This means that the intact bsAk not only destroys or phagocytizes target cells, but also indirectly their immunity, for example against the tumor, is increased.

The activation of the Fc receptor positive cell by the bsAk depends on the subclass or the subclass combination of the bsAk. As could be demonstrated in in-vitro experiments, bsAk of the subclass combination mouse-IgG2a / - rat-IgG2b are able to bind positive cells to Fc receptor and to activate them at the same time, which leads to upregulation or new formation (expression) of costimulatory antigens, such as CD40, CD80 or CD86, on the cell surface of these cells. In contrast, bsAk of the mouse-IgGl / IgG2b subclass combination are able to bind positive cells to Fc receptors (1), but apparently they cannot activate them to a comparable extent (2).

While the intact bsAk binds the T cell with a binding arm (e.g. to CD3 or CD2) and activates it at the same time, costimulatory signals can be transmitted from the Fc receptor positive cell bound to the Fc part of the bsAk to the T cell . That Only the combination of activation of the T cell via a binding arm of the bsAk and the simultaneous transmission of costimulatory signals from the Fc receptor positive cell to the T cell leads to an efficient T cell activation. Target cell-specific T cells that have been insufficiently activated on the target cell and are anergic can also be reactivated after an ex vivo long-term incubation treatment.

Another important aspect in the induction of target cell immunity is the possible phagocytosis, processing and presentation of target cell components by the accessory cells brought up and activated by the bsAk (monocytes / macrophages, dendritic cells and NK "natural killer" cells). This classic mechanism for the presentation of antigens enables both target cell-specific CD4 and CD8 posi tive cells are generated. Target cell-specific CD4 cells also play an important role in inducing a humoral immune response in connection with TB cell cooperation.

Bispecific and trispecific antibodies can bind with one binding arm to the T cell receptor complex of the T cell, with the second binding arm to target cell-associated antigens on the target cell. They activate T cells that destroy the target cells through the release of cytokines or apoptosis-mediating mechanisms. In addition, there is apparently the possibility that T-cells recognize tumor-specific antigens via their receptor as part of the activation with bispecific antibodies, thereby inducing permanent immunization. The intact Fc part of the bispecific or trispecific antibody, which binds to accessory cells such as e.g. Monocytes / macrophages and dendritic cells are mediated and these cause themselves to become cytotoxic and / or at the same time pass on important costimulatory signals to the T cell (Fig. 1). In this way, a T cell response may obviously can also be induced against previously unknown, target-specific peptides.

Redirection of possibly anergized, target-specific T cells to target cells by means of bispecific and / or trispecific antibodies with simultaneous costimulation of such T cells by accessory cells which bind to the Fc part of the bispecific or trispecific antibody, the anergy of cytotoxic T could Cells (CTLs) are canceled. In other words, a T cell tolerance existing in the patient against the target cell can be broken by means of intact heterologous bispecific and / or trispecific antibodies and thus permanent immunity, for example tumor immunity, can be induced. The antibodies used according to the invention are preferably capable of reactivating target-specific T cells which are in anergy. Furthermore, they are able to induce tumor-reactive complement-binding antibodies and thus to induce a humoral immune response.

The binding is preferably via CD3, CD2, CD4, CD5, CD6, CD8, CD28 and / or CD44 to the T cell. The Fc receptor positive cells have at least one Fcγ receptor I, II or III.

Antibodies which can be used according to the invention are capable of binding to monocytes, macrophages, dendritic cells, "natural killer" cells (NK cells) and / or activated neutrophils as Fcγ receptor 1 positive cells.

The antibodies which can be used according to the invention have the effect that the expression of CD40, CD80, CD86, ICAM-1 and / or LFA-3 as co-stimulatory antigens and / or the secretion of cytokines by the Fc receptor positive cell is initiated or increased. The cytokines are preferably IL-1, IL-2, IL-4, IL-6, IL-8, IL-12 and / or TNF-α.

Binding to the T cell is preferably via the T cell receptor complex of the T cell.

The bispecific antibodies which can be used according to the invention are preferred:

an anti-CD3 X anti-operationally inducible target cell-associated antigen and / or anti-CD4 X anti-operationally inducible target cell-associated antigen and / or anti-CD5 X anti-operationally inducible target cell-associated antigen - and / or anti-CD6 X anti-operationally inducible target cell-associated antigen and / or anti-CD8 X anti-operationally inducible target cell-associated antigen and / or anti-CD2X anti-operationally inducible target cell-associated antigen and / or anti-CD28X anti-operationally inducible target cell-associated antigen and / or anti-CD44X anti-operationally inducible target cell-associated antigen antibody.

The trispecific antibodies which can be used according to the invention are preferred:

an anti-CD3 X anti-operationally inducible target cell-associated antigen and / or anti-CD4 X anti-operationally inducible target cell-associated antigen and / or anti-CD5 X anti-operationally inducible target cell-associated antigen - and / or anti-CD6 X anti-operationally inducible target cell-associated antigen and / or anti-CD8 X anti-operationally inducible target cell-associated antigen and / or anti-CD2 X anti-operationally-in- inducible target cell-associated antigen and / or anti-CD28 X anti-operationally inducible target cell-associated antigen and / or anti-CD44 X anti-operationally-inducible target cell-associated antigen antibody.

Specificities of the antibodies according to the invention which can preferably be used and which can be combined with the isotype combinations mentioned below are, for example:

Anti-CD3 X Anti-Hsp70 Anti-CD2 X Anti-Hsp70 Anti-CD28 X Anti-Hsp70 Anti-CD5 X Anti-Hsp70

Anti-CD3 X Anti-MICA Anti-CD2 X Anti-MICA Anti-CD28 X Anti-MICA Anti-CD5 X Anti-MICA Anti-CD3 X Anti-MICB Anti-CD2 X Anti-MICB Anti-CD28 X Anti-MICB Anti-CD5 X Anti-MICB

The trispecific antibodies which can be used according to the invention have at least one T cell binding arm, one target cell binding arm and one binding arm binding to Fc receptor. This last-mentioned binding arm can be an anti-Fc receptor binding arm or a mannose receptor binding arm.

The bispecific antibody is preferably a heterologous intact rat / mouse bispecific antibody.

With the bispecific and trispecific antibodies which can be used according to the invention, T cells are activated and redirected against the target cells. Preferred heterologous intact bispecific antibodies are selected from one or more of the following isotype combinations:

Rat IgG2b / mouse IgG2a, rat IgG2b / mouse IgG2b, rat IgG2b / mouse IgG3,

Rat IgG2b / human IgGl, rat IgG2b / human IgG2,

Rat IgG2b / human IgG3 [oriental allotype G3m (st) = binding to protein A], rat IgG2b / human IgG4,

Rat IgG2b / rat IgG2c,

Mouse IgG2a / Human IgG3 [Caucasian allotypes G3m (b + g) = no binding to protein A, hereinafter marked with *] Mouse IgG2a / Mouse- [VH-CHl, VL-CL] -Human IgGl- [Hinge] - Human IgG3 * - [CH2-CH3]

Mouse IgG2a / rat- [VH-CHl, VL-CL] -human IgGl- [hinge] -human- IgG3 * - [CH2-CH3]

Mouse IgG2a / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Hu- IgG3 * - [CH2-CH3]

Mouse- [VH-CHl, VL-CL] -human IgGl / rat- [VH-CHl, VL-CL] - human-IgGl- [Hinge] -human IgG3 * - [CH2-CH3]

Mouse- [VH-CHl, VL-CL] -human IgG4 / rat- [VH-CHl, VL-CL] -hu-IgG4- [Hinge] -human-IgG4 [N-terminal region of CH2] -human IgG3 * [C-terminal region of CH2:> AS position 251] -Human- IgG3 * [CH3]

Rat IgG2b / Mouse- [VH-CHl, VL-CL] Human IgGl- [Hinge-CH2-CH3]

Rat IgG2b / off [VH-CHl, VL-CL] human IgG2- [Hinge-CH2-CH3]

Rat IgG2b / mouse [VH-CHl, VL-CL] human IgG3 [hinge CH2-CH3, oriental allotype]

Rat IgG2b / Mouse- [VH-CHl, VL-CL] Human IgG4- [Hinge-CH2-CH3]

Human IgGl / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] - Human IgG3 * - [CH2-CH3]

Human IgGl / Rat- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human IgG4 [N-terminal region of CH2] -Human-IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3]

Huan-IgGl / mouse- [VH-CHl, VL-CL] -human-IgGl- [hinge] -human-IgG4 [N-terminal region of CH2] -human-IgG3 * [C-terminal region of CH2:> AS position 251] human IgG3 * [CH3] Human IgGl / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG2 [N-terminal region of CH2] Human IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3]

Human IgGl / mouse [VH-CHl, VL-CL] human IgGl [hinge] human IgG2 [N-terminal region of CH2] human IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3]

Human IgGl / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG3 * - [CH2-CH3]

Human IgGl / Mouse- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3]

Human IgG2 / Human- [VH-CHl, VL-CL] -Human IgG2- [Hinge] -Human- IgG3 * - [CH2-CH3]

Human IgG4 / Human- [VH-CHl, VL-CL] -Human IgG4- [Hinge] -Human- IgG3 * - [CH2-CH3]

Human IgG4 / Human- [VH-CHl, VL-CL] -Human-IgG4- [Hinge] -Human-IgG4 [N-terminal region of CH2] -Human-IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3]

Mouse IgG2b / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG3 * - [CH2-CH3]

Mouse IgG2b / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3]

Mouse IgG2b / Mouse- [VH-CHl, VL-CL] -Human-IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3]

Mouse- [VH-CHl, VL-CL] -human IgG4 / rat- [VH-CHl, VL-CL] - human-IgG4- [Hinge] -human-IgG4- [CH2] -human-IgG3 * - [ CH3] Human IgGl / Rat [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG4 [CH2] Human IgG3 * [CH3]

Human IgGl / mouse [VH-CHl, VL-CL] -Human-IgGl [Hinge] -Human-IgG4- [CH2] -Human-IgG3 * [CH3]

Human IgG4 / Human [VH-CHl, VL-CL] -Human-IgG4- [Hinge] -Human-IgG4- [CH2] -HumanIgG3 * - [CH3].

The antibodies mentioned above are mentioned as examples. These and other antibodies which can be used according to the invention can be produced by the person skilled in the art using special techniques. DE 195 31 346 is mentioned as an example of this.

In particular, Greenwood et. al. describe the possibility of exchanging individual immunoglobulin domains (eg CH2) using suitable cloning techniques. This provides the technology to produce new antibody combinations, for example: human (VH-CHl, VL-CL) -human IgG4- (hinge) -human IgG4 (N-terminal region of CH2) -human IgG3 ^* (C-terminal Region of CH2:> amino acid position 251) -human IgG3 ^* (CH3).

The combination with an antibody: human IgG4 to produce the bispecific antibody: human IgG4 / human- (VH-CHl, VL-CL) -human IgG4- (hinge) -human IgG4 (N-terminal region of CH2) -human IgG3 ^* (C-terminal region of CH2:> amino acid position 251) - human IgG3 ^* (CH3) is by simple cell fusion, as in Lindhofer et al. (J. Immunol. 155: 219, 1995).

The antibodies which can be used according to the invention are preferably monoclonal, chimeric, recombinant, synthetic, semisynthetic or chemically modified intact antibodies with, for example, Fv, Fab, scFv or F (ab) ₂ fragments. Antibodies or derivatives or fragments from humans are preferably used or those which are modified in such a way that they are suitable for use in humans (so-called "humanized antibodies") (see, for example, Shalaby et al., J. Exp. Med. 175 ( 1992), 217; Mocikat et al., Transplantation 57 (1994), 405).

The person skilled in the art is familiar with the production of the various antibody types and fragments mentioned above. The production of onoclonal antibodies, which originate preferably in mammals, e.g. Human, rat, mouse, rabbit or goat can be done using conventional methods such as those described in e.g. in Köhler and Milstein (Nature 256 (1975), 495), in Harlow and Lane (Antibodies, A Laboratory Manual (1988), Cold Spring Harbor) or in Galfre (Meth. Enzymol. 73 (1981), 3).

It is also possible to produce the described antibodies by means of recombinant DNA technology using techniques familiar to the person skilled in the art (see Kurucz et al., J. Immunol. 154 (1995), 4576; Hollinger et al., Proc. Natl. Acad. Sc. USA 90 (1993), 6444).

The production of antibodies with two different specificities, the so-called bispecific antibodies, is possible on the one hand by using recombinant DNA technology, but also by the so-called hybrid-hybridoma fusion technique (see, for example, Milstein et al., Nature 305 (1983), 537 ). Hybridoma cell lines that produce antibodies with one of the desired specificities are fused and recombinant cell lines are identified and isolated that produce antibodies with both specificities.

That of the invention. underlying problem can be solved by both bispecific and trispecific antibodies, provided that they have the properties and characterized in claim 1 Have effects. The production of antibodies with two and three specificities is described in more detail below. The provision of such bispecific and trispecific antibodies is part of the prior art, and full reference is made here to the literature describing such production techniques.

Antibodies with three specificities, so-called trispecific antibodies, by means of which the problem on which the invention is based can also be produced, can be produced, for example, in such a way that a third antigen binding site with a further specificity, e.g., to one of the heavy Ig chains of a bispecific antibody. in the form of a "single chain variable fragment" (scFv). The scFv can, for example, use a

-SS (G ₄ S) _n DI linker must be bound to one of the heavy chains (S = serine, G = glycine, D = aspartate, I = isoleucine).

Analogously, trispecific F (ab) ₂ constructs can be prepared by replacing the CH2-CH3 regions of the heavy chain of a specificity of a bispecific antibody with an scFv with a third specificity, while the CH2-CH3 regions of the heavy chain of the other specificity, for example by inserting a stop codon (at the end of the "hinge" region) into the coding gene, for example by means of homologous recombination (see Fig. 2).

It is also possible to produce trispecific scFv constructs. Here, three VH-VL regions, which represent three different specificities, are arranged in a row (Fig.3).

According to the invention, intact bispecific antibodies are used, for example. Intact bispecific antibodies are made up of two antibody half-molecules (one H and one L immunoglobulin chain each) each represent specificity, composed, and, like normal antibodies, also have an Fc part with the known effector functions. They are preferably manufactured using Quadrom technology. This manufacturing process is described by way of example in DE-A-44 19 399. Reference is made in full to this document, also with regard to a definition of the bispecific antibodies, for complete disclosure. Of course, other production methods can also be used as long as they lead to the intact bispecific antibodies of the above definition that are necessary according to the invention.

The invention has been described above and is described below in particular with reference to bispecific antibodies. Instead of the bispecific antibodies, of course, trispecific antibodies can also be used as long as they meet the conditions.

The invention has been and will be described in more detail with reference to the accompanying figures. The pictures show:

Fig. 1: Representation of the antibodies of the invention and their

Working principle; Fig. 2: Trispecific F (ab) ₂ antibodies Fig. 3: Trispecific scFv antibody

In a further preferred embodiment of the invention, the antibodies according to the invention are used in order to achieve prophylaxis and therapy of tumor diseases and diseases caused by microorganisms by means of an ex vivo immunization method, in particular in order to achieve immunity to a tumor or to infection by microorganisms induce. This ex vivo immunization method using the antibodies provided according to the invention comprises the following steps: a) isolation of autologous target cells; b) treating the target cells to prevent their survival after reinfusion; c) 30 minutes to 4 hours, preferably 30 minutes to 2 hours, before the inactivated target cells are reinfused, the intact bispecific and / or trispecific antibodies according to the invention are infused into the patient; d) reinfusion of the treated target cells in the patient.

According to the invention, target cells are understood to mean both tumor cells and cells which have been infected by microorganisms, for example by bacteria, viruses or fungi. The preferred embodiment of the invention is illustrated below with the treatment of autologous tumor cells. However, the invention is not restricted to this, but can also be applied to target cells which have been infected by microorganisms and on which host-specific antigens have been induced. For the induction of antigens and their importance for the present invention, reference is made to the above statements.

In the embodiment of the invention now described, heterologous intact bispecific and / or trispecific antibodies are used. In order to prevent the tumor cells from surviving after reinfusion, the tumor cells were treated in a manner known per se, for example by radiation or heat treatment. After irradiation, the tumor cells are brought into contact with the intact heterologous bispecific and / or trispecific antibodies. According to the invention, not any antibodies can be used, but these must be intact, ie they must have a functional Fc part, and they must be heterologous in nature, ie the antibodies are then from heavy immunoglobulin chains of different subclasses (combinations, also fragments) and / or origin (Species) composed.

This method and the use of the antibodies described here ensure that tumor immunity is built up after the antibody has been infused into the patient from whom the tumor cells were previously removed. The treated tumor cells are preferably reinfused into a patient after the treatment of the primary tumor, preferably in patients in a minimal residual disease (MRD) situation. The method provided according to the invention can be used particularly successfully in patients with little remaining tumor cells, but in whom the risk of recurrence may be high.

According to the invention, a distinction is made between two process variants:

1. short-term incubation, and

2. Long-term incubation

In the short-term incubation, the autologous tumor cells are first inactivated after they have been obtained. 30 minutes to 4 hours, preferably 30 minutes to 2 hours, before the treated target cells are reinfused, the intact bispecific and / or trispecific antibodies according to the invention are infused into the patient. The treated tumor cells are then prepared for reinfusion and infused.

In the case of long-term incubation, the autologous tumor cells are also initially inactivated by, for example, radiation or an increase in temperature. Blood cells from the patient, preferably mononuclear cells from peripheral blood (PBMC = peripheral blood mononucleated cells) and the bispecific and / or trispecific antibodies according to the invention are then added to this, and this mixture is then added over a longer period of time, for example 1 to 14 days long, preferably 3 to 10 days and further preferably 6 to 10 days. As a result, a large number of immune cells are "primed" against the tumor ex vivo. Then these are reinfused into the patient. The long-term incubation also internalizes the antibodies and breaks them down.

Initial in vitro results show that tumor cells pretreated in this way can destroy tumor cells without further addition of bispecific and / or trispecific antibodies.

Both in the short-term and in the long-term incubation, T cells are redirected to the tumor cells by means of the intact bispecific and / or trispecific antibodies immobilized on the tumor cells; at the same time, Fc receptor positive cells bind to the Fc part of the bispecific and / or trispecific antibody after reinfusion. The Fc receptor positive cells are activated by binding to the Fc parts of intact bispecific antibodies immobilized (on the T cell or tumor cell).

In order to improve the success of the immunization, the tumor cells treated with the antibodies either by the short-term incubation process or by the long-term incubation process can not only be applied to the patient once, but optionally also administered several times.

MHC 1 is upregulated on the tumor cell and the intracellular processing machinery (proteasome complex) is activated due to the release of cytokines (such as INF-γ and TNF-) in the immediate vicinity of the tumor cell. The cytokines are released due to bispecific antibody-mediated activation of T cells and accessory cells (see Fig. 1 and 3). This means that the intact bsAk not only destroys or phagocytizes tumor cells, it also who also indirectly increases their tumor immunity.

The activation of the Fc receptor positive cell by the bsAk depends on the subclass or the subclass combination of the bsAk. As could be demonstrated in in-vitro experiments, bsAk of the subclass combination mouse-IgG2a / - rat-IgG2b are able to bind positive cells to Fc receptor and to activate them at the same time, which leads to upregulation or new formation (expression) of costimulatory antigens, such as CD40, CD80 or CD86, on the cell surface of these cells. In contrast, bsAk of the mouse-IgGl / IgG2b subclass combination are able to bind positive cells to Fc receptors (1), but apparently they cannot activate them to a comparable extent (2).

While the intact bsAk binds the T cell with a binding arm (eg to CD3 or CD2) and simultaneously activates it, costimulatory signals can be transmitted from the Fc receptor positive cell bound to the Fc part of the bsAk to the T cell . That Only the combination of activation of the T cell via a binding arm of the bsAk and the simultaneous transmission of costimulatory signals from the Fc receptor positive cell to the T cell leads to an efficient T cell activation. Tumor-specific T cells that have been insufficiently activated on the tumor cell and are anergic can also be reactivated after the ex vivo pretreatment according to the invention.

Another important aspect in the induction of tumor immunity is the possible phagocytosis, processing and presentation of tumor components by the accessory cells brought up and activated by the bsAk (monocytes / macrophages, dendritic cells and NK "natural killer" cells). This classic mechanism for the presentation of antigens can be used to generate both tumor-specific CD4 and CD8 positive cells. Tumor-specific CD4 cells play also play an important role in the induction of a humoral immune response in connection with TB cell cooperation.

There are first in vivo data from mouse experiments that indicate such permanent tumor immunity after treatment with a syngeneic tumor and intact bsAk. In these experiments, a total of 14 out of 14 animals that could be successfully treated with bsAk after a first tumor injection survived another tumor injection 144 days after the first tumor injection - without a new dose of bsAk.

Other advantages of ex vivo immunization using bispecific and / or trispecific antibodies are (i) the minimization of possible side effects, (ii) the controlled binding of the tumor binding arm to the tumor cell outside the body, (iii) the lowest possible consumption of bispecific and trispecific antibodies, (iv) and the reproducibility or control of the immunization success by administration of a defined number of tumor cells. In principle, two approaches are possible, which are explained in detail below. An important aspect of long-term incubation is that the bispecific or trispecific antibody used is consumed and broken down within the projected incubation period. This would eliminate the need for tedious drug registration for such immunization.

In the long-term incubation method, the mononuclear cells from the peripheral blood are mixed with the inactivated, autologous tumor cells and incubated together with the antibodies for a period of 1-14 days, more preferably 3 to 10 days, further preferably 6 to 10 days. This incubation is preferably carried out at 37 ° C. under sterile conditions and under GMP conditions (Good Manufacturing Production = GMP) in an incubator. The above-mentioned incubation conditions are only to be understood as examples. Depending on the tumor cells and the antibodies used, other time periods, temperature conditions etc., generally other incubation conditions, can also be selected. The person skilled in the art can determine these conditions by simple experiments.

In the ex vivo immunization, the tumor cells are preferably used in an amount of 10 ⁷ to 10 ⁹ cells, further preferably in an amount of approx. 10 ⁸ cells. The mononuclear cells from the peripheral blood are added in an amount of approximately 10 ⁸ to 10 ¹⁰ cells. Of course, it is also possible for the person skilled in the art to choose other incubation conditions which can be determined by laboratory tests (for example changes in the number of cells and duration of the incubation).

The autologous tumor cells used are irradiated, for example, to prevent further survival of the tumor cells after reinfusion. For example, γ-rays are used, which are used, for example, in a dose strength of 20 to 200 Gy.

The antibodies used according to the invention are preferably capable of reactivating tumor-specific T cells which are in anergy. Furthermore, they are able to induce tumor-reactive complement-binding antibodies and thus to induce a humoral immune response.

IMMUNIZATION PROTOCOLS

Ex vivo immunization (short-term incubation)

1. Preparation of a single cell suspension (10 ⁷ -10 ⁹ cells) from autologous tumor material (or allogeneic tumor cells of the same tumor type) with subsequent γ-radiation (20-100 Gy) or heat treatment (initially approx. 42 ^β C for 30 min; then approx. 60 ° C for 1 hour);

Infusion of the bispecific and / or trispecific antibodies (5-100 μg) 2 to 4 hours before reinfusion of the cell mixture into the patient; Reinfusion of the cell mixture (i.v.)

Ex vivo immunization (long-term incubation)

1. Preparation of a single cell suspension (10 ⁷ -10 ⁹ cells) from autologous tumor material (or allogeneic tumor cells of the same tumor type) with subsequent γ-radiation (50-100 Gy) or heat treatment;

2. Add bispecific and / or trispecific antibodies (5-50 μg), and add PBMC (10 ⁸ -10 ¹⁰ ), [alternatively: 1 x 10 ⁹ cells from T cell apheresis]

3. after 5 to 7 days control of T cell reactivity by transfer of aliquots to e.g. allogeneic breast cancer cells (MCF-7, MX-1)

4. Reinfusion (IV) of the cultured PBMC on days 4 to 14 in the patient (with T cell apheresis cryopreservation)

Abbreviations: PBMC, peripheral blood mononucleated cells = mononuclear cells from peripheral blood; IV, intravenously.

A similar approach, which relied on the addition of cytokines and was carried out with conventional bsAk (no activation of accessory cells by bsAk of the subclass combination rat IgG2B X rat IgGl), demonstrated the principle effectiveness of such an ex vivo immunization in the animal model (5).

In contrast to this, the advantage of the method disclosed here lies in the "self-sufficiency" with those for the high-regulation tion of, for example, MHC 1 on the tumor cell required cytokines (such as INF-γ or TNF-α) by the simultaneous activation of T cells and accessory cells (monocytes / macrophages, Fig.) on the tumor cell. This is achieved by the special subclass combination of the intact bsAk used here mentioned. In short-term incubation, these processes take place in the patient. Other advantages of short-term incubation are (i) avoiding the otherwise necessary cultivation of the cell suspension with serum-containing medium, (ii) this also eliminates the cost-intensive cultivation under GMP guidelines.

The advantage of long-term incubation is that the bsAk consumes itself in vitro after some time (and therefore this method cannot be established as a medication but as a "medical device").

In a further preferred embodiment of the invention, the antibodies according to the invention are used in order to reduce the number of contaminating target cells in stem cell transplants ex vivo. This method is characterized in that the intact bispecific and trispecific antibodies provided according to the invention are brought into contact with stem cell transplants which may contain contaminating target cells for a sufficiently long period of time in order to at least reduce the number of contaminating target cells in the stem cell transplant. For example, stem cell transplants from patients with breast carcinoma or ovarian carcinoma or from patients with leukemia, lymphoma, testicular carcinoma or other chemotherapy sensitive carcinomas are treated here. According to the invention, however, stem cell transplants from patients who are infected by microorganisms, for example by viruses, can also be treated. Examples of stem cell transplants infected with viruses are those infected with CMV or HIV. The bispecific and trispecific antibodies which can be used according to the invention have already been described above. The stem cell transplant is incubated with the bispecific antibodies for a period of 4 to 72 hours, preferably for a period of 24 to 48 hours, for example a temperature in the range from 20 to 25 ° C., preferably room temperature, is selected For example, stem cell transplants have a density of 30,000 to 75,000 cells per μl.

After ex-vivo treatment, the stem cell transplants are reinfused into the patient, possibly after further purification. The full content of the reader is referred to DE 196 49 223.8, the disclosure content of which is fully integrated in the above application.

In this embodiment of the invention with the method according to the invention, contaminating tumor cells in stem cell preparations (leukapheresis products) are eliminated in vitro by means of bispecific or trispecific antibodies. These antibodies have the features specified in claim 1, in particular these antibodies are selected so that they bind to a surface antigen as target antigen on the target cell which is inducible and does not occur in the non-induced state (normal state) or in such a small number that the number is not sufficient for destruction of the target cell or induction of an immune response by the antibody.

Examples of the useful antibodies provided by the present invention have been given. These have the specificities already shown above and can preferably be linked to the isotype combinations shown.

The bispecific antibodies and the stem cells are brought into contact with the contaminating target cells under conditions which both bind the bispecific or trispecific antibodies to the target cells and the T cells as well as maintaining the viability of the stem cells. Adherence to these parameters is necessary for the maintenance and vitality of both stem cells and lymphocytes. For example, the stem cell transplant (leukapheresis product) is incubated for approximately 4-72 hours, preferably 24-48 hours, with bispecific antibodies at room temperature and a cell density of 30,000-75,000 cells / μl, preferably 30,000-50,000 cells / μl, with gentle swirling. With a total cell count of approx. 10 x 10 ⁹ cells / stem cell transplant, a bsAk amount of 5 - 50, 50 - 100, 100 - 500 μg is sufficient for tumor cell destruction. Another important point of the method according to the invention is the use of so-called intact bispecific or trispecific antibodies. These are not only able (due to the specificities used here) to deliver T cells to the tumor cells, but they are also suitable due to the effector functions of the Fc part, by means of complement mediated lysis or by binding of Fc receptor positive cells , such as macrophages, monocytes or activated neutrophil granulocytes, to destroy tumor cells. Intact BSA can thus activate several tumor cell-destroying mechanisms at the same time.

The example below shows that the antibodies provided according to the invention are capable of recognizing and binding inducible surface antigens to target cells and initiating an immune response or destroying the target cell, even if the target structures are present in an extremely small amount on the target cell , for example in an amount of 100 to 10,000 per cell. The target structures can also be present in greater numbers, for example up to 500,000 (further preferred areas are shown above). However, the antibodies according to the invention surprisingly also work when the target structures are present on the target cell in a number of approximately 100, surprisingly, Even with this small number of target structures, only the smallest amounts of the antibody are necessary to carry out an effective therapeutic treatment, ie amounts of approximately 5 μg can be sufficient for the treatment.

In order to improve the success of the immunization, the combination of antibody and tumor cells is preferably administered several times to the patient, the number of infusions depending on the tumor to be treated and on the patient and possibly other factors.

In a further preferred embodiment, the immunogenicity of the tumor cells is increased by subjecting them to a heat pretreatment before administration. A preferred heat pretreatment is carried out over a period of, for example, 1-6 hours, preferably approximately 3 hours, over a temperature range of 41-42 ° C., preferably at a temperature of approximately 41.8 ° C. Both the treatment duration and the treatment temperature depend on the type of tumor to be treated. The temperature to be used and the heat pretreatment period can be determined by the person skilled in the art by experiments.

The invention is illustrated below using an exemplary embodiment. However, the invention is not limited to this specific example, but can be modified within the scope of the present description and the appended claims.

EXAMPLE 1

After the outstanding effectiveness of intact bsAk could be proven not only in vitro but also in vivo in preclinical animal models (Lindhofer et al., Blood, 88: 4651, 1996), the cleaning of stem cell preparations from contaminated developing tumor cells as a further possible application (Lindhofer et al., Exp. Hematol. 25: 879, 1997).

Based on these experiments, subsequent trials with complete stem cell preparations (approx. 2 x 10OlO cells), in particular the effectiveness in the unsaturated region of the target antigens, could be demonstrated. That it was administered in titration experiments so little intact bsAk that in a PBSZ (peripheral blood stem cell) preparation with approx. 6.5 x 10e9 T cells, with a dose of 5 μg antibody / preparation, only 3000 CD3 molecules of approx. 30,000 CD3 molecules / T cell bound to the bsAk (see calculation). Nevertheless, the intact bsAk were able to destroy the target cells (here tumor cell: HCT-8) even under these conditions.

The experiments were structured in such a way that aliquots were taken from PBSZ preparations treated in this way and contaminated with a defined amount of tumor cells. It could be shown that the tumor cells are destroyed even at this low concentration of intact bsAk, in which only a part of the target antigens are occupied.

The evaluation of the experiment described above led to the following results:

Patient WaGr total cell count PBSZ: 2.5 x 10 ¹⁰ + 5 μg bsAk

Aliquot 5xl0 ⁶ PBSZ + bsAk tumor cells

Plate no bsAk tumor cells / mononuclear antibody anti-cells (PBSZ) / hole

CD3Xepcam

24er 6/6 * 0/6 '2xl0 ⁴ / 2xl0 ⁶

96er 12/12 0/12 5000/5 x 10 ⁵

Tumor tumor cell percentage: 1% reduction: none> 51og ° Σ = l, 2xl0 ⁵ tumor numbers / 6 holes

Number of holes with tumor growth, of 6 or 12 plated holes, after 14 days of cultivation.

Titration experiments with the HCT-8 tumor cell line have shown that 1-2 tumor cells / hole are already sufficient to display a clearly visually evaluable tumor growth in 95% of all plated holes after 14 days of cultivation.

Calculation:

An intact bsAk has a molecular weight of 150 KDa. Ie 1 mole is 150 kg and by definition corresponds to 6xl0 ²³ molecules. So 5 μg corresponds to approx. 2xl0 ¹³ molecules.

After ⁹ T cells were determined in a stem cell preparation 6.5xl0 and one T cell carries approx. 30,000 CD3 molecules, comes to a total of 19.5xl0 ¹³ CD3 molecules in the stem cell preparation.

Since each bsAk has an anti-CD3 binding arm, it can be concluded that theoretically, if you compare the two amounts of molecules calculated above, in this specific example no more than approx. 3000 CD3 molecules can be occupied by the bsAk.

L I T E R A T U R

1. Haagen et al., Interaction of human moncyte Fcγ receptors with rat IgG2b, J. Immunolog., 1995, 154: 1852-1860

2nd guest G.C., Haagen I.-A., van Houten A.A., Klein S., Duits A.J., de Weger R.A., Vroom T.M., Clark M.R., J. Phillips, van Dijk A.J.G., de Lau W.B.M., Bast B.J.E.G. CD8 T-cell activation after intravenous administration of CD3 X CD19 bispecific antibody in patients with non-Hodgkin lymphoma. Cancer Immunol. Immunother. 40: 390, 1995

3. Tenny, C, Jacobs, S., Stoller, R., Earle, M., and Kirkwood, J. Adoptive cellualr immunotherapy with high-dose chemotherapy and autologous bone marrow rescue (ABMR) for recurrent breast cancer (meeting abstract). Proc.Annu.Meet.Am.Soc.Clin.Oncol; 11: A88, 1992 ISSN: 0736-7589. CO: PMAODO - 7589 CO, 1993.

4. Early Breast Cancer Trialists' Collaborative Group, Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy - 133 randomized trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Part II Lancet 339: 71-85, 1992

5. Guo et al., Effective tumor vaccines generated by in vitro modification ot tumor cells with cytokines and bispecific monoclonal antibodies. Nature Medicine 3: 451, 1997

6. Lindhofer et al., Preferential species-restricted heavy-light chain pairing in rat-mouse quadromas: Implications for a Single step purification of bispecific antibodies, J. Immunology 1995, 155: 219

Claims

PATENT CLAIMS Intact bispecific or trispecific antibody that has at least the following properties: a) binding to a T cell; b) binding to at least one antigen on a target cell; c) Binding to Fc receptor positive cells by their Fc part (in the case of bispecific antibodies) or by a third specificity (in the case of trispecific antibodies) and which thereby induces an immune response and / or destroys the target cells, the antibody being selected such that it binds to a surface antigen as target antigen on the target cell which is inducible and does not occur in the uninduced state (normal state) on the target cell or in such a small number that the number is not sufficient for the antibody to destroy the target cell. 2. Antibody according to claim 1, characterized in that heat-shock proteins or MHC class I related MIC molecules are used as inducible antigens. 3. Antibody according to claim 1 or 2, characterized in that as heat shock proteins HSP25, HSP60 or HSP70 (HSP72) or HSP90 proteins and as MIC molecules MIC-A and MIC-B molecules be used. 4. Antibody according to one or more of the preceding claims, characterized in that it is directed against target antigens which, after induction, are present in the target cell in a number of at least 100 and at most 500,000 per target cell. 5. Antibody according to claim 4, characterized in that the antibody is further selected so that it is capable of activating Fc receptor positive cells, whereby the expression of cytokines and / or co-stimulatory antigens is initiated or increased. 6. Antibody according to one or more of the preceding claims, that the antibodies are selected so that they are capable of binding Fc receptor positive cells which have an Fcγ receptor I, II or III. 7. Antibody according to one or more of the preceding claims, characterized in that the antibodies for binding to monocytes, macrophages, dendritic cells, "natural killer" cells (NK cells) and / or activated neutrophils as Fcγ receptor I + III positive cells are capable. 8. Antibody according to one or more of the preceding claims, characterized in that the antibodies are capable of inducing target cell-reactive, in particular tumorreaktivεn complement-binding antibodies and thus of inducing a humoral immune response. 9. Antibody according to one or more of the preceding claims, characterized in that the antibodies are selected so that they bind to the T cells via CD2, CD3, CD4, CD5, CD6, CD8, CD28 and / or CD44. 10. Antibody according to one or more of the preceding claims, characterized in that the antibodies are selected so that after their binding to the Fc receptor positive cells the expression of CD40, CD80, CD86, ICAM-1 and / or LFA-3 initiated or increased as costimulatory antigens and / or the secretion of cytokines by the Fc receptor positive cell becomes . 11. Antibody according to one or more of the preceding claims, characterized in that the antibodies are selected so that the secretion of IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, INF -γ as cytokines and / or TNF-α is increased. 12. Antibody according to one or more of the preceding claims, characterized in that the bispecific antibody is selected such that it contains an anti-CD3 X anti-operationally inducible target cell-associated antigen and / or anti-CD4 X anti-operationally inducible target cell -associated antigen and / or anti-CD5 X anti-operationally inducible target cell-associated antigen and / or anti-CD6 X anti-operationally inducible target cell-associated antigen and / or anti-CD8 X anti-operationally inducible target cell associated antigen and / or anti-CD2X anti-operationally inducible target cell-associated antigen and / or anti-CD28X anti-operationally inducible target cell-associated antigen and / or anti-CD44X anti-operationally inducible target cell-associated antigen -Antibody is. 13. Antibody according to one or more of the preceding claims, characterized in that the bispecific antibody is selected from one or more of the following isotype combinations: Rat IgG2b / mouse IgG2a, Rat IgG2b / mouse IgG2b, Rat IgG2b / mouse IgG3, Rat IgG2b / human IgGl, rat IgG2b / human IgG2, Rat IgG2b / human IgG3 [oriental allotype G3m (st) = binding to protein A], rat IgG2b / human IgG4, Rat IgG2b / rat IgG2c, Mouse IgG2a / Human IgG3 [Caucasian allotypes G3m (b + g) = no binding to protein A, hereinafter marked with *] Mouse IgG2a / Mouse- [VH-CHl, VL-CL] -Human IgGl- [Hinge] - Human IgG3 * - [CH2-CH3] Mouse IgG2a / rat- [VH-CHl, VL-CL] -human IgGl- [hinge] -human- IgG3 * - [CH2-CH3] Mouse IgG2a / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3] Mouse- [VH-CHl, VL-CL] -human IgGl / rat- [VH-CHl, VL-CL] - human-IgGl- [Hinge] -human IgG3 * - [CH2-CH3] Mouse- [VH-CHl, VL-CL] -human IgG4 / rat- [VH-CHl, VL-CL] -human- IgG4- [hinge] -human-IgG4 [N-terminal region of CH2] -human- IgG3 * [C-terminal region of CH2:> AS position 251] -Human- IgG3 * [CH3] Rat IgG2b / Mouse- [VH-CHl, VL-CL] Human IgGl- [Hinge-CH2-CH3] Rat IgG2b / Mouse- [VH-CHl, VL-CL] Human IgG2- [Hinge-CH2-CH3] Rat IgG2b / mouse [VH-CHl, VL-CL] human IgG3 [hinge CH2-CH3, oriental allotype] Rat IgG2b / Mouse- [VH-CHl, VL-CL] Human IgG4- [Hinge-CH2-CH3] Human IgGl / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] - Human IgG3 * - [CH2-CH3] Human IgGl / Rat- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human IgG4 [N-terminal region of CH2] -Human-IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3] Human IgGl / mouse [VH-CHl, VL-CL] human IgGl [hinge] human IgG4 [N-terminal region of CH2] human IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3] Human IgGl / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG2 [N-terminal region of CH2] Human IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3] Human IgGl / mouse [VH-CHl, VL-CL] human IgGl [hinge] human IgG2 [N-terminal region of CH2] human IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3] Human IgGl / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG3 * - [CH2-CH3] Human IgGl / Mouse- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3] Human IgG2 / Human- [VH-CHl, VL-CL] -Human IgG2- [Hinge] -Human- IgG3 * - [CH2-CH3] Human IgG4 / Human- [VH-CHl, VL-CL] -Human IgG4- [Hinge] -Human- IgG3 * - [CH2-CH3] Human IgG4 / Human- [VH-CHl, VL-CL] -Human-IgG4- [Hinge] -Human-IgG4 [N-terminal region of CH2] -Human-IgG3 * [C-terminal region of CH2:> AS -Position 251] -human IgG3 * [CH3] Mouse IgG2b / Rat- [VH-CHl, VL-CL] Human IgGl [Hinge] Human IgG3 * - [CH2-CH3] Mouse IgG2b / Human- [VH-CHl, VL-CL] -Human IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3] Mouse IgG2b / Mouse- [VH-CHl, VL-CL] -Human-IgGl- [Hinge] -Human- IgG3 * - [CH2-CH3] Mouse- [VH-CHl, VL-CL] -human IgG4 / rat- [VH-CHl, VL-CL] - human-IgG4- [Hinge] -human-IgG4- [CH2] -human-IgG3 * - [ CH3] HumanIgGl / Rat [VH-CHl, VL-CL] -Human-IgGl [Hinge] -Human- IgG4- [CH2] -HumanIgG3 * [CH3] HumanIgGl / Mouse [VH-CHl, VL-CL] -Human-IgGl [Hinge] -Human-IgG4- [CH2] -Human-IgG3 * [CH3] Human-IgG4 / Human [VH-CHl, VL-CL] -Human-IgG4- [Hinge] -Human- IgG4- [CH2] -HumanIgG3 * - [CH3] 14. Antibody according to one or more of the preceding claims, characterized in that the bispecific antibody from a heterologous Rat / mouse bispecific antibody is selected. 15. Antibody according to one or more of the preceding claims, characterized in that the trispecific antibody has a T cell binding arm, a target cell binding arm and a third specificity for binding to Fc receptor positive cells. 16. Antibody according to one or more of the preceding claims, characterized in that the trispecific antibody is selected such that it contains an anti-CD3 X anti-operationally inducible target cell-associated antigen and / or anti-CD4 X anti-operationally inducible target cell-associated antigen and / or anti-CD5 X anti-operationally inducible target cell-associated antigen and / or anti-CD6 X anti-operationally inducible target cell-associated antigen and / or anti-CD8 X anti-operationally inducible target cell-associated antigen and / or anti-CD2 X anti -operationally inducible target cell-associated antigen and / or anti-CD28 X anti-operationally inducible target cell-associated antigen and / or anti-CD44 X anti-operationally inducible target cell-associated antigen antibody, preferably with an additional anti- Fc receptor is poor in binding. 17. Antibody according to one or more of the preceding claims, characterized in that the target cell is a tumor cell or a cell infected by microorganisms. 18. Pharmaceutical composition, characterized in that it contains one or more of the antibodies according to one or more of the preceding claims, optionally together with one or more conventional pharmaceutical carriers and / or excipients, in a therapeutically effective amount. 19. Use of an antibody according to one or more of the preceding claims for inducing an immune response and / or for destroying the target cell. 20. Use of an antibody according to one or more of the preceding claims for the prophylaxis and therapy of tumor diseases and diseases which have been induced by infection with microorganisms. 21. Use of an antibody according to one or more of the preceding claims for inducing target cell immunity, preferably long-term target cell immunity. 22. Use of an antibody according to one or more of the preceding claims for reducing the number of contaminating target cells in stem cell transplants ex vivo, wherein the antibody is brought into contact with stem cells obtained from transplants, which may contain contaminating target cells, for a sufficiently long period of time to at least reduce the number of contaminating target cells in the stem cell transplant. 23. Use according to one or more of the preceding claims, wherein a tumor cell or a cell infected by microorganisms is used as the target cell.