WO1998008875A1

WO1998008875A1 - Novel combination preparations and their use in immunodiagnosis and immunotherapy

Info

Publication number: WO1998008875A1
Application number: PCT/EP1997/004493
Authority: WO
Inventors: Heribert Bohlen
Original assignee: Viva Diagnostika Diagnostische Produkte Gmbh
Priority date: 1996-08-28
Filing date: 1997-08-18
Publication date: 1998-03-05
Also published as: AU4119397A

Abstract

The invention concerns combination preparations comprising three components which are used for specific purposes in immunology, diagnosis and therapy. The combination is based on the universal use as a component of the combination preparations of an immuno-linker which can link two other different components provided with different determinants.

Description

CO BINATION PREPARATIONS AND THEIR USE IN IMMUNIAGNOSTICS AND IMMUNOTHERAPY

description

Technical field

The present invention relates to a combination of three preparations or formulations (components) that can be used in immunology, consisting of at least one first component equipped or connected with a defined specific, immunologically reactive determinant Ha-], an at least bispecific second component with binding specificity for Ha - _|

15 (anti-Ha -]) and for at least one further binding specificity for a defined specific, immunologically reactive determinant Ha2 (anti-Ha2) or with further binding specificities for several such determinants Ha _n (anti-Ha _π ), which differ from one another, and a third component which is or is or are equipped with at least one defined, specific, immunologically reactive determinant Ha2 or with a plurality of such determinants Ha _n , Ha-j, Ha2 and Ha _{n being} different from one another.

The combination according to the invention is generally used in immunology, both in immunodiagnostics and as a prophylactic and therapeutic agent.

State of the art

0 The targeted use of substances for diagnostic or immunohistochemical and therapeutic purposes in order to specifically and efficiently reach certain tissues and cells of the animal body, in particular humans, is based on the idea of selecting suitable agents or active ingredients for desired, to transport and bind specific sites outside (in vitro) or inside (in 5 vivo) the body and to exclude other tissues and cells from this "targeting" in vitro or in vivo. In principle, three methods are available to achieve this goal. On the one hand, a substance that exerts a desired effect can be local to a l

Body area, for example topically, can be applied. However, this method has the disadvantages that only parts of the body can be reached which are directly accessible and that the substance in question, due to diffusion processes, does not only act at the desired location and there with rapidly decreasing intensity.

On the other hand, there is the possibility, based on the certain chemical and physiological properties of certain agents, of having these substances accumulate in certain parts of the body (organs, tissues) through their bio-distribution. The disadvantage of these methods is in particular that the use of these substances is narrowly limited by their chemical nature, so that biodistribution and site-specific effects can often not be reconciled in the manner hoped for.

As a third and most modern option, attempts have been made for around two decades to use certain proteins, in particular antibodies, as vehicles for the targeted transport of active substances in order to bring such substances to desired locations in vitro or in vivo for immunodiagnostic or immunohistochemical and immunotherapeutic purposes . Due to the availability of monoclonal antibodies since the mid-1970s (KÖHLER, G. & MILSTEIN, C, Nature 256, 495 - 497, 1 975), means were put in hand, intensive diagnostic, immunohistochemical and therapeutic examinations and tests with appropriately modified perform monoclonal antibodies (Mabs).

Because of the inability of the monoclonal antibodies to be therapeutically or diagnostically efficient per se, efforts have recently been made to increase this efficiency by using different agents or effector molecules, e.g. bacterial or vegetable toxins, radionuclides or cytotoxic substances were conjugated with Mabs or linked covalently. Such immunoconjugates have since been used in diagnosis and therapy (GOLDENBERG, D.M., Immunol. Today 10, 286-288, 1989).

Not least because of various disadvantages of these immunoconjugates, alternative molecules for "targeting" have been produced. Bispecific antibodies have been developed which have two different antigen-specific binding sites: one for tumor-associated antigen, also called "target binding arm", and another for the effector molecule ("effector binding arm"). Such bispecific antibodies have a spectrum of potential uses both in immunodiagnostics, immunohistochemistry, immunohistology, immunocytology and in immunotherapy for the targeted application of cytotoxic, cytocidal or cytostatic substances.

The bispecific antibodies have various advantages over the immunoconjugates: cytotoxic substances can be released at the desired target sites without prior cleavage of the covalent bond and non-specific toxic effects can be reduced. Bispecific m antibodies (for example of the IgG class) are structurally bivalent or multivalent (especially IgM) but functionally monovalent for each antigen binding site.

An overview of the production and use of bispecific antibodies in diagnostics and therapy is given, for example, by SONGSIVILAI, S.

15 & LACHMANN P.J., Clin exp. Immunol. 79, 31 5-321, 1990; RENNER, C. & PFREUNDSCHUH, M., Immunol. Rev. 5, 1 79-209, 1995; HAMILTON, R.G., Clin. Chem. 39 (9), 1988-1997, 1,993; FANGER, M.W. & GUYRE, P.M., Tibtech 6, 375-380, 1,991; Fanger, M. et al., Crit. Rev. Immunol. 12 (3,4), 101-124, 1992; FANGER, W.M. et al. , Immunomethods 4, 72-81, 1994, and BRISSINCK, 0 J. et al., Drugs of the Future 12 (1 1), 1003-1010, 1992.

It is known to use bispecific antibodies with dual specificity in immunoassays, in tumor "targeting", for cross-linking ("cross-linking") cellular antigens and in "targeting" effector cells, for the specific release of active substances to target cells or for triggering effector cells which mediate cellular cytotoxicity after stimulation (for example phagocytic cells, natural killer cells [NK cells] or T-lymphocytes).

Bispecific antibodies are known which have been produced via hybrid hybridomas, which on the one hand have specificity against the CD3 protein (T-cell receptor complex) on the effector cell and on the other hand are specific against the IgK-1 b chain of the immunoglobulin of the rat (GILLILAND, LK et al., Proc. Natl. Acad. Sci. USA 85, 7719-7723, 1 988). This bispecific antibody was able to lyse the target cell in vitro in cooperation with T cells (mouse thymon). BERG, J. et al., Proc. Natl. Acad. Be. USA 88-4723-4727, 1,991, describe a bispecific antibody, one arm of which is from the Fusion protein CD4 γ1 / κ (L) chain and its other arm consisted of a CD3 γ 1 / κ pair. The arm with the fusion protein bound HIV gp1 20, the opposite arm reacted with CD3 (T cell receptor) and activated the cytotoxic T cell. It could be shown that this antibody lysed HIV-infected cells. Further bispecific antibodies with tumor-destroying properties or trigger function of cellular cytotoxicity are known from, for example, BRISSINCK, J. et al., J. Immunol. 147, 4019-4026, 1 991. In the publication by JUNG, G. et al., Eur. J. Immunol. 21, 2431 2435, 1 991, become bispecific

Antibody fragments of the IgG2a type (Fab and F (ab ') 2> which react specifically with target cells (melanoma) and against CD3 and CD28, respectively. These anti-melanoma x anti-CD3 and anti-melanoma x anti-CD28 conjugates destroyed the Target cells in vitro and open a systematic application in vivo for tumor therapy via T-cell stimulation.

A bispecific antibody was produced via chemical coupling ("cross-linking"), which was directed both against an epitope of the β chain of fibrin and against urokinase (scuPA). This bispecific construct was able to increase both the fibrinolytic efficiency and the fibrin specificity of scuPA by significantly increasing fibrin monomers and plasma clumping with this antibody compared to the sole action of scuPA (CHARPIE, JR et al., Biochemistry 29, 6374-6378, 1990 ). In another case it could be shown that a bispecific antibody constructed via "cross-linking", which on the one hand was specific for tPA (tissue plasminogen activator) on the other hand for fibrin, could increase the fibrinolytic activity of tPA (BODA, C. et al. , J. Biol. Chem. 264, 944-948, 1,989).

An antiviral effect of heteroconjugated bispecific antibodies is described by STAERZ, UD et al., Eur. J. Immunol. 17, 571-574, 1 987. The antibodies used were specific against T cell antigen receptor and specific against hemagututinin (HA) or against nucleoprotein of an influenza virus. As a result, the cells infected by the virus could be lysed. In another case, hybrid-specific bispecific antibodies with dual specificity for TCR x HA influenza A (fusion between IgG2b specifically against influenza virus HA and IgG2a specifically against Vß8 of TCR) were successfully used to inhibit influenza virus replication. Cell lysis of cells infected with HIV by bispecific antibody conjugates (antι-HIV-1 gp1 10: antι-CD3 / TCR complex or antι-gp1 1 0: antι-CD1 6Fcγ-R) has been described by ZARLING, JM et al., J Immunol. 140, 2609 261 3, 1 988. Bispecific antibody heteroconjugates with the specificities anti-CD3: anti-HSV-1 gpC or anti-FcγRIII: anti-HSV-1 gpD increased immunity to herpes simplex virus (HSV) infections or destroyed HSV infected cells (PAYA, CV et al., J. Immunol. 142, 666-671, 1,989).

Targeting CD4 + helper / killer cells by an anti-CD3 x anti-glioma bispecific antibody together with IL-2 activated CD4 + helper / killer cells caused an increase in cytotoxicity against glioma cells in vitro, so that an adoptive tumor immunotherapy was hereby proposed. It was also possible to inhibit colon tumor growth with a bispecific antibody of the specificity ant-c-erb-B2: ant-CD3 plus CD4 + positive cells (NISHIMURA, T. et al., J. Immunol. 148, 285-291, 1 992). In vivo, the effect against malignant glioma with an adoptive immunotherapy of this type using a chemically conjugated anti-CD3 monoclonal antibody plus anti-glioma antibody with lymphokine (recombinant IL-2) activated killer cells was demonstrated in 10 patients (NITTA, T. et al ., The Lancet 335, 368-371, 1,990). DONOHUE, JH et al., Cancer Research 50, 6508-6514, 1990, similarly describes that a bispecific reagent (immunoheteroaggregate) with the specificity of anti-CD3 x anti-tumor antigen by in vitro activated peripheral blood lymphocytes (PBLs) Ovarian carcinoma cells lysed in vitro. In a previous work (RAMMENSEE, H.-G. et al., Eur. J. Immunol. 17, 433-436, 1987), antibodies specific for Vß8 ⁺ T cell receptors were identified with 2,4,6 -Trinitrophenyl (TNP) haptenized and linked to receptors of TNP-specific B cells. This construct lysed TNP-specific B hybridomas and blast cells from mice that were transgeπ with regard to μ, a TNP-specific antibody. The effector functions of cytotoxic T-lymphocytes (CTL) could thus be targeted via antigen-specific receptors to antigen-specific B-lymphocytes and the number of B-cells manipulated (reduced) in vivo.

In addition to B and / or T cells, activated macrophages have also been proposed as effector cells in conjunction with bispecific antibodies. Initial clinical trials with such triggered macrophages are said to have confirmed increased tumor lysis (BAUER, T. & DRAKEMAN, DL, Vox Sang 61, 1 56 - 1 57, 1 991). Peripheral mononuclear blood cells (PBMCs) as effector cells are described by MENARD, S. et al., Int. J. Biol. Markers 4 (3), 1 31 - 1 34, 1 989, wherein an anti-CD3 x anti-ovarian cancer antibody was used as the bispecific reagent for the lysis of the target cells (ovarian cancer).

It is also known that bispecific antibodies are suitable for use in immunodiagnostic assays (ELISA) and in immunohistochemistry. An example of this application is described by MILSTEIN, C. & CUELLO, AL, Nature 305, 537-540, 1983. In this case bispecific antibodies could be immunohistochemically treated with an anti-matostatin x anti-peroxidase antibody in certain pituitary regions be detected.

MICHAELSEN, TE, Eur. J. Immunol. 21, 1 1 - 1 6, 1991, produced chimeric anti- (4-hydroxy-3-nitrophenyl) acetyl (NP) antibodies which also reacted with the hapten p-nitrophenyl phosphate (NIP) to provide complement-mediated lytic properties of IgG isotypes. A labeling of two different haptens revealed a distribution of two different antigens on the target cells. According to the publication by JU, S.-T. et al., Mol. Immunol. 26 (3), 283-292, 1989, were secreted by fusion of a myeloma cell line which produced α / λ2 antibodies and a hybridoma line which produced μ / λ-j antibody inter alia immunoglobulins of the serotypes IgM and IgA which were bispecific for both the hapten 2,4-dinitrophenyl (DNP) and the hapten 4-hydroxy-3-nitrophenylacetyl (NP). The basis of the investigation was to determine which molecular classes of the L and H chains after myeloma-hybridoma fusions which haptens (NP and / or DNP) were able to bind with which avidity and to determine the meaning of the Vλ- _| and Vλ2 sequences (L chains) in the development of antibody activity. A dual binding property was found for K, λ-j and λ2 of the anti-NP antibodies.

In addition to the aforementioned uses, specific, dual bonds to CD3 and CD25 (p55 chain of the IL-2 receptor) have been described for bispecific antibodies. Such an anti-CD3: anti-CD25 bispecific antibody was able to selectively bind to activated CD25-expressing T cells and lymphoma, both antigens CD3 and CD25 being recognized and being co-expressed by activated T cells. As is known, T cells are the main mediators in ?

Rejection reactions and in a number of autoimmune diseases derailing or dysregulated T cells thus play an important role in many autoimmune diseases and lymphoproferative diseases (eg lymphomas). By MACLEAN, JA et al. , J. Immunol. 150, 1 61 9 1 628, 1 993, could be shown that antι-CD3: antι-CD25 bispecific monoclonal antibodies cause cytolysis of CD25-bearing alloreactive T cells and in cytotoxic T lymphocytes (CTL) with undefined specificity. Since such "redirected" cytolysis requires close spatial contact of CTL with CD25 ⁺ cells, a therapeutic effect is to be expected for those tissues and cells that are infiltrated by activated CTL, as is the case in organs with cell rejection reactions or autoimmune effects. In the present case, an effective CTL stimulation could be achieved by the anti-CD3: anti-IL-2 receptor (anti-CD25) bispecific antibody in order to stimulate T-cells stimulated by CDA antigen-bearing PHA (phytohemagglutinin) and IL-2 receptor positive tumor cells lyse.

In another case, CD3: CD19 bispecific antibody T cells from patients with chronic lymphocytic leukemia, in combination with monospecific, bivalent anti-CD28 antibodies, could be activated. In particular, CD4 ^"1" T cells were activated and stimulated to proliferate. It was clearly possible to achieve a sharp decrease in the number of CD19 + malignant B cells, whereas both with anti-CD3: anti-CD1 9 bispecific antibodies on their own and with the individual antibodies anti-CD3 plus anti-CD19 plus anti -CD28 no such decrease could be achieved, but also no stimulation of CD2 +, CD4 + and CD25 + T lymphocytes. In this case, only the combination anti-CD3: anti-CD19 plus anti-CD28 could achieve cytotoxicity against autologous malignant B cells. Such T cell activation was obtained even with very low doses of the antibodies used (1 ng / ml). Stimulation of T cells with the combination of bispecific and monospecific, bivalent antibodies mentioned was associated with a sharp increase in IL-2, IL-6 and interferon-γ secretion of the T cells in question. Such a procedure is proposed for the therapy of chronic lymphocytic leukemia (BOHLEN, H. et al., Blood 82, 1 803 - 1 81 2, 1 993). 2

Further uses of bispecific antibodies, also in combination with other anti-CD antibodies, are described in the diverse literature and are known to the person skilled in the art. Inter alia, the corresponding review articles and the others in the publication by BOHLEN, H. et al. , 1 993, loc cit., Literature cited.

Furthermore, successful cytolytic effects of bispecific antibodies are known in renal cell carcinomas (VAN, DJ. Et al., Int. J. Cancer 43, 344, 1989), melanomas (JUNG, G. et al., Proc. Natl. Acad. Sei USA 84, 461 1, 1987), gliomas (NITTA, T. et al., J. Neurosurg. 72, 426, 1 990), lymphomas (GRAVELLE, M. & OCHI, A., J. Immunol. 142, 4079 , 1989), leukaemias (OSHIMI, K. et al., Blood 77, 1044, 1991) and in drug-resistant tumor cells (VAN, DJ et al., Int. J. Cancer 44, 738, 1 989).

However, the bispecific and in some cases also trispecific antibodies described in the prior art and associated with them the determination methods and therapeutic methods and measures carried out with these reagents and agents have a number of serious disadvantages. For every change of at least one of two or more binding specificities of an antigen (e.g. epitope, target cell, surface receptor) to which a bispecific antibody is to bind, the production of a completely new bispecific antibody is necessary. This makes the previously known bispecific systems of "targeting", triggering or marking, complex, inflexible and expensive. When large molecules (e.g. marker, reporter or effector molecules) are coupled directly to a bispecific antibody, the binding properties of the bispecific antibody in question can be changed or deteriorated, which leads to problems with, for example, immunohistochemical or immunocytological labels with dyes and enzymes or with diagnostic Test systems, but can also lead to immunotherapy measures.

Another disadvantage arises with the previous direct coupling of dyes, enzymes or effector molecules to an antibody in that for each newly selected molecule (for example effector molecule) for which the antibody has a specificity, a new coupling must always be carried out. As a result, the number of conjugates to be synthesized is the same is the number of molecules selected per antibody. In such a case, a large number of end products result. For example, for 100 antibodies with different specificity and for example 5 different reporter or effector molecules, 500 different products would have to be produced in order to bring the desired reporter or effector molecules to the desired location (for example for test purposes).

Each new introduction of an antigen into a test system or into a therapy scheme, for example in immunodiagnostics, in ELISAs, immunocytochemistry, optical immunoassays, etc., as well as in immunotherapy for targeting target cells or tumor markers, etc., draws for everyone according to the known prior art new combination a complex and expensive development, characterization and production of a new antibody construct. Presentation of the invention and preferred embodiments

The object of the present invention was to provide means and new methods which overcome the disadvantages of the prior art.

The object was achieved according to claim 1 in that a combination preparation is provided, consisting of at least one with a defined specific, immunologically reactive determinant Ha - | equipped or connected first component, an at least bispecific second component with binding specificity for Ha- | (anti-Ha- |) and for at least one further binding specificity for a defined specific, immunologically reactive determinant Ha2 (anti-Ha2) or with others

Binding specificities for several such determinants Ha _n (anti-Ha _n ), which differ from one another, and a third component, which have at least one specific, immunologically reactive determinant

Ha2 or is or are connected with several such determinants Ha _n , where Ha - | , Ha2 and Ha _n are different from each other.

To simplify matters, the term “defined specific, immunologically reactive determinant” is replaced by the term “determinant” to be understood synonymously or by the terms “shark”, “Ha2” and “Ha _n ”. OK

The first binding-specific component 1 associated with a determinant Ha-] comprises a reagent with specific binding properties which enables it to bind to cells, tissues or specific structures of the animal and human body in vitro and in vivo. In particular, the reagent comprises a protein, preferably an immunoglobulin or antibody or a fragment or derivative thereof or any ligand with the above-mentioned binding properties or specificities for an antigenic binding site, for example a lectin, or adhesion molecules, cytokines or chemokines, which are known, that they recognize receptors and other binding sites of tissues, cells or structures of the body and can attach them there specifically and specifically. An antibody is preferred for this first component.

The second, at least bispecific component 2 comprises an agent or reagent with at least bispecific properties, for example an inert particle or a protein with specificity for at least two determinants, it being possible for the protein to be a ligand, for example an immunoglobulin or antibody or a lectin, or from any linker molecule which has binding specificity for the desired determinants Ha-], Ha2 or Ha _n , where at least one determinant recognition site is complementary for Ha i ₍ another for Ha2 or one or more others for Ha _{n is} preferred a bispecific antibody with anti-Ha-] and anti-Ha2 specificity, which can react with at least two different determinants, one reaction partner being the determinant Ha-] and the other Ha2, with Hai and Ha2 being different.

The third component 3 of the combination according to the invention is an effector molecule or a reporter molecule associated with a determinant Ha2, preferably an antibody, any marker substance, an enzyme, a radioactive substance, a contrast agent, biologically active substances, a cytostatic, cytocidal or cytotoxic effect Agents or any substances and antigens, as well as adhesion molecules, cytokines or chemokines, which are known to recognize specific binding sites on cells or structures of the body of a mammal and to attach them there specifically and in a targeted manner. //

The combination preparations according to the invention find broad, universal use in immunology, in particular in the field of immunodiagnostics, immunohistochemistry or immunohistology, immunocytology and immunotherapy. However, the area of application also includes the prognosis and determination of disease states in which the combination preparations according to the invention have an effect, but also the prophylactic use, for example for preventing the outbreak or the development of a disease state or for preventing an exacerbation of a disease state.

In addition, the combination preparations according to the invention are broad and universally usable in the therapeutic treatment of diseases which are known to be accessible to immunological, therapeutic methods. This applies in particular to infectious diseases and diseases that are based on a tumor. However, the combinations according to the invention are equally outstanding in the case of immunization or for vaccination, in particular with tumor proteins.

Accordingly, the present invention consists of three components or formulations, the core of the combination according to the invention being the use of an at least bispecific agent or reagent which has specificity for at least two mutually different determinants Ha-] and Ha2 or Ha _n .

In this respect, the present invention does not only include the combination of the three preparations mentioned. Another object of the invention is therefore the use of an agent or immunolinker in vivo and in vitro with binding specificity for at least two different determinants for connecting at least two agents or reagents equipped with different determinants both in diagnostics, in therapy and in immunotherapy or for immunization purposes.

The principle on which the invention is based is that those with at least one determinant Ha- | equipped component 1 with a specific binding site, for example on the surface of a cell, for example with an antigen binding site, an epitope, a receptor binding site, with a II

Adhesion molecule binding site or with a sugar residue specifically reacts and binds to them via their determinant Ha -j or determinants Ha _n with the corresponding anti-Ha -] or antι-Ha _n specificity of an at least bispecific component 2 (with anti-Ha - | and antι-Ha2 or antι-Ha _n specificities) reacts and that at least one further component 3 equipped with a determinant Ha2 or several different determinants Ha _n binds to the antι-Ha2 or antι-Ha _n recognition site of component 2.

According to the invention, the individual components are to be understood as a functional unit, according to which both a common targeted use of these three individual components and the spatial juxtaposition of these individual means (as kit-of-parts) are included. However, the use of component 2 per se for immunological purposes is also included in the manufacture of a preparation for connecting at least two different agents or molecules or reagents each equipped with at least two mutually different determinants.

As determinants Ha- | , Ha2 or Ha _n are, according to the invention, haptens, specific, couplable antigenic structures, epitopes, paratopes, idiotopes. Haptens are included as preferred determinants Ha-, Ha2 and Ha _n .

According to Table 1, the general definition for the combination preparations according to the invention is based on the following scheme for the composition of the three components:

B

Table 1

I.

Component 1: (T): X— Ha -]

Component 2: anti-Ha - | - Y - anti-Ha2 or

anti-Ha - _] - Y - anti-Ha _n

Component 3: Ha2 - Z or Ha _n - Z

wherein (T) a target or a "target", in particular a specific, biological binding structure or binding site, for example on the surface of a cell or a membrane or a part thereof, for example an antigen, an allergen, an epitope, a receptor, a Adhäsionsmolekülbinduπgsstelle, a sugar residue, a drug, a toxin, what, without the involvement of a determinant Ha _j, X - Ha _| can bind, whereby (T) is not to be understood as a component of component 1 according to the invention, but only for explanatory purposes and was therefore shown in brackets,

X is a reagent equipped or linked with a determinant Ha -], which can be mσnospecific with regard to its target location (T), for example comprising a protein, an immunoglobulin or an antibody or a derivative or fragment thereof, a ligand, a lectin, a receptor binding molecule, an adhesion molecule, a cytokine, a chemokine, a lymphokine,

Ha-j a defined specific, immunologically reactive determinant, for example comprising a hapten, a specific couplable antigenic structure, an epitope, a paratope, an idiotope, Y an at least for at least two determinants Ha- _| and Ha2 or Ha _n bispecific reagent, for example, comprising an inert particle, a protein, an immunoglobulin or an antibody, a diabody, with the corresponding anti-Ha -], anti-Ha2 or anti-Ha _n specificities, Ha2 one of Ha -] various, defined specific, immunologically reactive determinants, for example comprising a hapten, a specific linkable antigenic structure, an epitope, a paratope, an idiotope, Ha _n any other of Ha- | and Ha2 different defined specific, immunologically reactive determinants, for example comprising a hapten, a specific couplable antigenic structure, an epitope, a paratope, an idiotope, Z a biological, chemical or physical one equipped or connected with at least one determinant Ha2 or Ha _n active or detectable couplable agent, for example comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a marker substance, an enzyme, a radioactive substance, a radioactively labeled substance, a contrast agent, a biologically active substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with effector marker or reporter function, a prodrug, an adhesion molecule, a cytokine, a lymphokine, a chemokine, a ligand.

For example, if X is monospecific in terms of its destination (T), for example in the event that X is an antibody, a receptor binding molecule, an adhesion molecule or a lectin, the reagent X can bind to a specific biological binding structure or to parts thereof, for example comprising a cell surface structure , an antigen, an allergen, an epitope, a receptor, an adhesion molecule binding site or parts of the structures mentioned, or a sugar residue, a drug, a toxin.

Such a specific, biological binding structure or parts thereof can be of natural origin or can be produced synthetically or semi-synthetically via DNA recombination, or, in particular with regard to antigens and epitopes, can originate from or be contained in biological and non-biological liquids.

For a basic understanding, it should be explained that component 2 is to be understood as a universally applicable "immunolinker" or immunological linker module, which has the function of being equipped, for example, with at least one determinant (Ha2 or H _a n) haptenized, component 3, as defined above via a determinant Ha- with a certain determinant different from Ha2 or Ha _n ] equipped or connected, for example haptenized, monospecific component 1, as defined above, to a targeted location (T), as defined above. This means that the function of the actual "targeting" is carried out by component 1, which has specificity for the relevant target location at which an effect is desired. Component 3 is therefore to be understood in the narrower sense as a carrier which enables the desired effect. The variability of this system on which the invention is based is thus made possible by components 2 and 3, in which any determinants Ha2 or Ha _n , which are different, for example different haptens among themselves, are coupled to Z and any at least bispecific reagents Y with corresponding anti-Ha2 or anti-Ha _n specificities can bind non-covalently with each other. It is crucial here that the individual determinants are chosen so that they are different from one another.

A combination preparation is preferred in which X and Z are reagents or agents equipped or linked with haptens, in which Y is an at least bispecific antibody which has anti-Ha - and anti-Ha2 or anti-Ha _n specificities and where Z is a biologically, chemically or physically active or detectable agent equipped or connected with at least one hapten Ha2.

In this respect, a combination preparation is preferred, wherein a binding-specific component 1 connected to a hapten Ha-] comprises a reagent with specific binding properties which enables it to bind to cells, tissues or specific structures of the animal and human body. In particular, the reagent comprises a protein, preferably an immunoglobulin or antibody or a fragment or derivative thereof or any ligand with the above-mentioned binding properties or specificities for an antigenic binding site, for example a lectin, or adhesive molecules, cytokines or chemokines, which are known, that they recognize receptors and other binding sites of tissues, cells or structures of the body and can attach them there specifically and specifically.

The second, at least bispecific component 2 preferably comprises an agent or reagent with at least bispecific properties, in particular an inert particle or a protein with specificity for at least two haptens, it being possible for the protein to be a ligand, for example an immunoglobulin or Antibodies or a lectin, or from any linker molecule which has binding specificity for the desired haptens Ha -], Ha2 or Ha _n , where at least one hapten recognition site is complementary for Ha- _] another for Ha2 or another for Ha _n . Preferred is a bispecific antibody with anti-Ha -j and antt Ha2 specificity, which can react with at least two different haptens, one reaction partner being the hapten Ha-] and the other Ha2, where Ha- | and Ha2 are different. The third component 3 of the combination according to the invention is preferably an effector molecule or a reporter molecule connected to a hapten Ha2, preferably an antibody, any marker substance, an enzyme, a radioactive substance, a contrast medium, biologically active substances cytostatic, cytocidal or cytotoxic agent or any substances and antigens, as well as adhesion molecules, cytokines or chemokines, which are known, for example, to recognize specific binding sites on cells 5 or structures of the body of a mammal and to attach them there specifically and in a targeted manner.

A combination preparation in which X is a protein equipped or linked with a hapten Ha -], in which Y is an at least 0 bispecific antibody which has anti-Ha-] and anti-Ha2 or anti-Ha _n specificities is particularly preferred and in which Z is a biologically, chemically or physically active or detectable agent equipped or connected with at least one hapten Ha2 or Ha _n .

A combination preparation is very preferred, in which X is an antibody linked to a hapten Ha -], which reacts with an antigen as a specific binding structure, in which Y is a bispecific antibody, the anti-Ha - _| and has anti-Ha2 properties and in which Z is an enzyme linked to a hapten Ha2, which is different from Ha-], a radioactive substance, a contrast agent, a fluorescent molecule or a marker substance, if the combination preparation, preferably in vitro , for immunological, immunohistochemical, immunohistological or immunocytological purposes or for immunoassays. A combination preparation as mentioned above is preferred,

■ ■ but with the peculiarity that Z is an effector molecule linked to a hapten, an antibody, a radioactive substance, a biologically active one Substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent, if the combination preparation, preferably in vivo, for immunotherapeutic or prophylactic purposes or in the prognosis and

Determination of disease states should be used.

A very particularly preferred combination preparation is that X is a with a hapten Ha- | is connected monoclonal antibody and Y is a bispecific antibody which antι-Ha - | and antι-Ha2 has properties, where Z is as defined above and is connected to a hapten Ha2.

A special, exemplary embodiment of the combination preparation according to the invention can then exist that Ha- | of X is a nitrophenyldeπvat (NP), in particular 2,4-dιnιtrophenol (DNP), and Ha2 of Z is a steroid hapten, for example digoxigenin (Δ ^20:22 -3 / ?, 1 2 / 914,21 - tetrahydroxynorcholensaurelakton) (DIG ) and Y is a monoclonal antibody with anti-NP: anti-DIG specificity, in particular with anti-DNP: anti-DIG specificity, and with any specificity of X and any function of Z within the scope of the definition described above.

A further exemplary embodiment of the combination preparations according to the invention can be that X is a protein, for example KLH (Keyhole Limpet Hemocyanin) or OKT3 antibody, which with DNP as hapten Ha - | is connected, Y is a bispecific, monoclonal antibody which has anti-DNP: anti-DIG specificities and where Z is an enzyme, a dye or a T cell which are conjugated or linked to DIG.

However, the invention also encompasses the use of an agent provided with at least bispecific specificity for at least two different haptens, according to the definition for Y given in Table or according to claim 2 and the dependent claims dependent thereon, for producing a combination preparation for use in immunology for diagnostic purposes , prophylactic, therapeutic purposes and for immunizations.

A still further aspect of the present invention is the use of the combination preparations for the production of an agent for immunodiagnostic purposes. An additional aspect of the present invention is the use of the combination preparations for the preparation of agents for immunohistochemical, immuncytochemical, immunohistological purposes and for immunoassays.

A further additional aspect is the use of the combination preparations according to the invention for "imaging". i.e. for the detection and localization of cells and / or tissues in vitro and in vivo, which can be both in normal, non-pathological form and may be degenerate or can be specifically changed by other pathological events and processes.

Another aspect of the present invention is the use of the combination preparations for the production of an agent or a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the prognosis and determination in vitro and in vivo of disease states in or outside a mammalian organism.

Another aspect of the present invention is the use of the combination preparations for producing a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the immunotherapy of malignant tumors.

A still further aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the therapy of infectious diseases caused by viruses, bacteria, fungi or parasites.

An additional aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the therapy of inflammatory diseases.

Another additional aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiological Q compatible carriers and additives, for the therapy of

Autoimmune diseases and for immunosuppression.

A still further aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the acute treatment of rejection reactions.

An additional aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the prophylactic treatment of tumor, inflammatory, infectious or autoimmune diseases or

Rejection reactions or to prevent exacerbation of such an existing disease.

Furthermore, the use of the combination preparations according to the invention for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives, for the therapy and prophylaxis of diseases which are associated with blood coagulation disorders (pathological blood clots within a mammalian organism) and which support or increase fibrinolysis.

In addition, a further aspect of the present invention is the use of the combination preparations for the production of a pharmaceutical composition, optionally together with physiologically compatible carriers and additives for the immunization or vaccination of animals and humans, with, for example, tumor proteins by means of modulating antigens, for example those on ARC (antigen Presenting Cells).

However, the invention also includes the use of a component with at least bispecific specificities as defined in Table 1 (anti-Ha-] - Y - anti-Ha2 or anti-Ha-] - Y - anti-Ha2). for the production of an agent which can be used in immunodiagnostics and immunotherapy or a pharmaceutical preparation with the functional property of an immunolinker, in particular in immunohistochemistry, immunocytology, the 2o immunohistology, in immunoassays as well as in prognosis, prophylaxis and

Therapy of malignant tumors, of inflammatory diseases, of

Infectious diseases, from autoimmune diseases, to avoid

Rejection reactions, to achieve immunosuppression, to prevent disease exacerbation, to predict or to determine

Disease state or course of the disease.

In addition, the invention accordingly includes pharmaceutical formulations and their uses, as described above, which consist of components 1, 2 and 3 or of components 1 and 2 or which consist only of component 2.

Further aspects of the present invention include methods for determining whether agents with biological, chemical or physical properties in in vitro or in vivo diagnostics or immunodiagnostics, in immunotherapy (including prophylactic treatments) or in immunizations, in the sense of the uses indicated above, can develop or show suitable biological, chemical or physical effects that are considered as potential candidates, but of which the desired effect is not known a priori.

Yet further aspects of the present invention include methods for screening (searching, testing) a plurality of agents with biological, chemical or physical properties for the purposes of in vitro or in vivo diagnostics or immunodiagnostics, for immunotherapy (including prophylactic treatments) or for Immunizations, in the sense of the above-mentioned possible uses, which can develop or show suitable biological, chemical or physical effects, which are considered as potential candidates, but of which the desired effect is not known a priori.

Further aspects of the present invention relate to the use of an agent consisting of a combination preparation comprising components 1, 2 and 3 or comprising components 1 and 2 or comprising component 2 in one of the abovementioned determination or screening methods. In addition, the invention encompasses methods for producing in-vitro and in-vivo immunodiagnostics, for immunoprophylaxis, for immunotherapy or for immunization, usable agents or pharmaceutical preparations, after which the provision of new diagnostics, immunotherapeutics or immunoprophylactics or Vaccine is made possible.

Another aspect of the present invention relates to a kit-of-parts consisting of components 1, 2 and 3 or of components 1 and 2 or in which component 1 is a component according to the claims or according to the definition for X, Y and Z in Table 1.

Additional aspects of the present invention are set out in the subclaims.

It has been found that the combination preparations according to the invention have significant advantages over the prior art: since component 1 (X-Ha- | according to Table 1), for example a monospecific reagent suitable for targeting (for example a specific antibody) with only one determinant Ha-], for example with a hapten, has to be coupled (haptenized), its binding properties do not change, whereas the direct coupling of macromolecules to antibodies according to the prior art, the binding of a relevant antibody to the target site (for example a cell surface structure or an epitope of a surface receptor of a cell) adversely affected.

Since the binding properties of the central, at least bispecific reagent of component 2 (anti-Ha-] - Y - anti-Ha2 according to Table 1) always remain constant for defined combinations of determinants, for example a hapten combination (coupled to X and Z according to Table 1) , it suffices to use the same binding-specific component 2 for binding mediation between all possible reagents equipped with defined determinants, for example with defined haptenized reagents (for example a protein, an antibody) as target object (X-Ha-], according to Table 1) and all with defined ones To use determinants (e.g. defined haptenized antigens) with effector, marker or reporter function (Ha2 - Z or Ha _n - Z, according to Table 1). Any complex redesign, for example bispecific Antibodies to different, desired new combinations with regard to effector cells, marker or reporter substances, enzymes, biologically active proteins (eg cytokines, hormones) or other substances which are supposed to cause markings or biological, chemical or physical effects at the target location are not required.

In the past, when providing reagents, for example, antibody conjugates, a specific antibody had to be conjugated directly with the corresponding reagent (dye, enzyme, toxin, etc.) according to the prior art, so that a correspondingly large number of individual conjugates had to be prepared if different effector systems were used should. As a result, a corresponding number of predefined end products had to be provided from each specific antibody.

With the combination preparations according to the invention, the required number of end products is drastically reduced and they additionally bring the advantage of a hitherto nonexistent flexibility for the use of immune reagents in in vitro and in vivo diagnostics as well as for the use of immune agents in prophylaxis, therapy and Immunizations. If the determinants Hai and Ha2 or Ha _n are, for example, haptens, a user is enabled to use the at least two specific haptens component 2 (for example an anti-Ha-: anti-Ha2 bispecific antibody) a variety of different combinations on the one hand targeted reagents of component 1 (e.g. a specific antibody) and on the other hand from different components 3 (where Z can be defined according to Table 1), each haptenized with different haptens, light, fast, specific, flexible, stoichiometric (if the relevant Components 1 and 3 each have the haptens Ha -] and Ha2 in a molar ratio of 1: 1) and, last but not least, inexpensive and tailor-made for their needs and problem solutions.

So far, a user of, for example, conjugated antibodies for use in a wide variety of immunological purposes in vitro or in vivo has only had the choice of either producing very specific, individual and finished constructs in a laborious manner or of purchasing them individually at a high price if the same specificity of the binding reagent ( usually an antibody) for example a label should be changed or the coupling with another Effector system was desired. In the inventive

Combination practitioners immediately decide which marking to use, and they can produce the desired product in a modular system in a short time, usually within a few minutes. It is completely irrelevant whether the problem to be solved is in wf / O systems (for example for immunodiagnostic purposes, for assays or in general in cell systems) or in wvo systems (in living mammal organisms, for example in immunotherapy).

On the basis of these advantages, those compositions or substances which are known to have biological, chemical or physical properties or effects can be determined or identified in a simple manner with the combination preparations according to the invention, which are then used in in vitro and in vivo diagnostics immunotherapy or immunoprophylaxis or in immunizations can be used advantageously. Likewise, the combination preparations in question are outstandingly suitable for screening in order to detect from a multiplicity of potential candidates or agents, which are known to have biological, chemical or physical properties or effects, those which are suitable for the purposes according to the invention seem suitable. Such potential candidates are described in more detail below.

The provision of the invention becomes important

Combination preparations but also in processes for the production of agents to be used in in vitro and in vivo immunodiagnostics and also for the production of pharmaceutical preparations which can be used for immunoprophylaxis, immunotherapy and for immunizations. It is of particular importance here that diagnostic or therapeutic agents can be provided as process products via methods of this type, as described further below and in the claims, which, according to the prior art, are not obtained at all or only with great difficulty or only by chance can be. The advantages of the combination preparations according to the invention and their uses, described briefly above, make it plausible to the person skilled in the art that the present invention provides means and methods for diagnosing and to produce therapeutically valuable substances and these according to known

Apply methods for each purpose.

The universality of the invention is based on these findings ^; Combination preparations and therefore include all those applications which are described in the prior art for the reagents previously used in immunology, for example for the conventional antibody constructions, or to which reference is made by way of example in the present description. The advantages of the combination preparations according to the invention can be summarized briefly as follows:

No adverse influence on the binding-specific component 1, e.g. an antibody, by coupling the same with biologically, chemically or physically active or detectable effector systems or

15 macromolecules (Z according to Table 1).

• There is no need for new designs of at least bispecific reagents in the case of different use of couplable agents or effector systems which are intended to have the desired effects at specific destinations. 0 • Drastic lowering of the desired end products for use in immunology in e.g. Diagnosis, therapy, prophylaxis, immunoassays or immunizations.

• High system flexibility, making it quicker and easier to use.

• Inexpensive production of individually desired combinations.

25 • Universal application for the entire field of immunology in vitro and in vivo.

• Universally applicable immunolinker (component 2).

• Easy adaptation to and use for established and proposed in the prior art with conventional immunological means

30 solutions in vitro and in vivo for diagnosis, therapy, prophylaxis, at

Immunoassays or immunizations.

• Easily identify, determine or screen agents that are diagnostic and immunotherapeutic (including prophylaxis and immunization) applicable.

15. Easy and efficient procedures for the production of diagnostics, therapeutics and vaccines. 15 Because of the universal usability and the very broad application possibilities of the combination preparations according to the invention in the immunological area in vitro and in vivo, the selection of the individual components of the components, as shown in Table 1, is completely uncritical and does not have to be specified a priori. The person skilled in the art knows which constituents he considers advantageous and can select and adapt them according to his special needs. In this respect, one cannot speak of preferred, very preferred or very particularly preferred combination preparations, since the degree of preference is closely related to the respective purpose and goal of the specific application which the person skilled in the art would like to carry out to solve a specific immunological task. In the following, the broad usability of the combination preparations according to the invention is to be clarified further, and the person skilled in the art can recognize from this detailed description that he can easily select the components and components thereof that are considered to be necessary in the sense of a modular system to solve his specific problems.

The individual components of the combination preparations according to the invention and their preparation are either known to the person skilled in the art and described exhaustively in the literature, or he can readily produce them on the basis of the disclosure on which the present description is based.

According to the present description, the combination preparations according to the invention can be used for apparently very different areas of application. It should be emphasized at this point, however, that the various described application aspects in vitro and in vivo in immunodiagnostics, immunohistochemistry, immunohistology, immunocytology, immunotherapy including the prognosis and determination of disease states of a mammalian organism, immunization or vaccination and the prophylactic use are connected by a single, unified inventive concept in that the combination preparations according to the invention can be used universally in the entire field relating to immunology and therefore the inventive principle of these uses must be considered as such. This is expressed, for example, by the fact that immunotherapy and a previous diagnosis of a disease state in order to determine the type of disease which can be concretized with other means of in vitro immunodiagnostics (for example by means of an immunoassay or via histological / cytological examinations), regularly goes hand in hand and decide on the selection of the suitable therapeutic or prophylactic use of the combination preparations on which the invention is based. As a result, these can

Aspects of application are combined by linking the combination preparations according to the invention. The same applies if non-biological systems are the basis of immunological tests. However, the uniformity is expressed as such by the universal applicability of the combination preparations according to the invention.

The terms and terminology used in the present specification are intended to be briefly defined as an overview, as follows, although they will be understood by those skilled in the art per se or in the context of the present disclosure unless a more specific explanation or definition is provided elsewhere .

The term "reagent" encompasses compounds or substances which enter into a preferably non-covalent bond with a partner. In particular, with regard to Y according to Table 1, this means an at least bispecific agent which is equipped with at least one agent (X) equipped or connected with at least one specific, immunologically reactive determinant Ha- _] or with at least one defined specific, immunologically reactive determinant Ha2 or Ha _n equipped or connected agent (Z) as a corresponding reaction partner non-covalently binds, for example in the sense of an antigen-antibody reaction. In this respect, components 1, 2 and 3 can be regarded as reagents that react with one another non-covalently.

For the purposes of the present invention, “defined specific, immunologically reactive determinants” are to be understood as meaning specific biological structures recognizable for a reagent (component 2), to which this reagent can bind non-covalently due to its specificities, and with which the corresponding reaction partners , for example a protein, or to which they are bound. Such structures include, for example, surface-specific antigenic structures (antigenic determinants) of cells or components thereof, particulate antigens, epitopes, paratopes, idiotopes or haptens. The term "agent" is understood to mean an active ingredient compound which has a therapeutic or prophylactic (preventive) or inhibitory effect in vivo or in vitro (effector function), or has a marker or reporter function. In particular, an agent as defined for Z in Table 1.

The term "hapten" subsumes low molecular weight substances which do not cause antibody reactions in themselves, but which, after binding to an immunogen, can trigger an immune reaction, with antibodies directed against them being formed.

The term “monospecific reagent” or “binding-specific component” does not include an agent as defined for X in Table 1, which is capable of being targeted to a defined location or to a “target structure” or to “target cells” bind covalently (known as "targeting"), where the defined location can be a target cell or a part thereof, a particulate antigen or an immunogen. In the case of a reagent which is not monospecific in this sense, any means, for example proteins (for example keyhole limpet hempcyanin, KLH), are encompassed to which a certain determinant of the definition given above is bound.

The term “at least bispecific component” or “at least bispecific reagent” encompasses reagents and agents which have binding specificities against various defined, specific, immunologically reactive determinants (Y in Table 1), for example with anti-hapten specificities.

"Binding-specific structures of the animal and human body" or "specific biological binding structures" encompass those structures within and on the surface of cells, tissues and organs which are referred to in the immunological sense as antigens, epitopes or antigenic determinants or receptor binding sites or Binding sites for adhesion molecules include and to which specific antibodies, receptor binding substances, adhesion molecules etc. as well as derivatives and fragments thereof or antibody-like substances (such as lectins, inert particles equipped with specific structures) are able to bind. In this respect, the term "binding-specific structures of the animal and human body" or "specific biological binding structures" as a generic term summarizes defined cell surface structures, antigens, epitopes, receptors, adhesion molecule binding sites or parts of the structures mentioned, and also sugar residues, drugs or toxins 2g

Naturally occurring or chemically synthesized as "antigens"

Understand molecules that are specifically directed against these antigens

Antibodies or immunoglobulins are bound non-covalently. Antigens with

Immune system activating properties, which induce the antibody synthesis, are called "immunogenes".

The term "epitope" encompasses discrete areas on a macromolecule (for

Example a protein) which trigger the antibody synthesis and are synthesized against the specific antibodies or with which antibodies can react specifically, whereby these bind non-covalently to the epitope in question (or the antigenic determinant).

"Lectins" the person skilled in the art understands proteins or ligands with two or more binding sites for very specific sugar residues on glycoproteins and glycolipids on cell surfaces, as a result of which, like antibodies, these can cross-link the corresponding molecules and also on correspondingly equipped or linked with a determinant , for example haptenized, are able to bind binding partners.

"Inert particles" include those particles which do not contain any of the body's own

Are capable of triggering an immune reaction, to which haptens, antigens or epitopes or antigenic determinants can be bound ("coated") and can react specifically with corresponding binding-specific structures and components as reaction partners analogously to antibodies.

“Linker molecules” mean chemically synthesized connecting molecules which can covalently or non-covalently connect two proteins, in particular antibodies, derivatives or fragments thereof, to form hetero aggregates. In particular, this encompasses hetero- and / or homobifunctional compounds, for example those which react with ε-amino groups or with SH groups (for example the "hinge" region) of antibodies, or which activate thiols on Fab fragments. However, other linker molecules are also included that can combine biomolecules with other substances, for example the avidin-biotin system or the streptavidin-biotin system.

"Effector molecules" include those known agents, molecules, compounds or particles of biological or non-biological origin which develop or mediate action, in particular therapeutic action, at the target site, such as, for example, lytic, cytostatic, cytocidal, cytotoxic, fibrinolytic, immunosuppressive, anti-inflammatory , anti-infectious or immunizing effects. In particular mediated through biologically active sg ^•

Substances such as, for example, antigens (for example hormones, enzymes, cytokines or modulating antigens on antigen-presenting cells, for example HLA class II) or effector cells (for example T cells, NK cells, macrophages) or by means of suitable radioactive substances or Isotopes in particular [3- and γ emitters, for example ^99m Tc, ^{l 1 1} ln, ^{1 3 1} J, ^{1 23} J, ⁹⁰ Y, ^{1 86} Re

The term "reporter olekul" encompasses such known agents which are suitable for triggering a detectable signal on organic or biological structures (cells and tissues and components thereof, toxins, antigens, etc.) or parts thereof, or for causing such a signal, e.g. by marker substances such as dyes, fluorescent substances (e.g. fluorescein isothiocyanate), enzyme (e.g. luciferase), contrast agents (e.g. a paramagnetic contrast agent), radioisotopes e.g. of halogens, technetium, lead, thalliums, indiums or mercury, such as e.g. el 25 |, 1 23 ,, 131 ,, 1 1 1 | _n , 99m _Tc , 203 _{Pb #} 201 _{TI |} 198 _Hg , 90 _{Y /} 186 _Re .

In order to provide a more detailed description, for the purposes of the present invention, the term “immunoglobulins” or synonymously “antibodies” means at least bispecific bivalent or polyvalent, monoclonal antibodies, in particular the isotypes IgG, IgM, IgA, IgE and IgD and its subclasses and derivatives thereof, but also those which are fragments thereof and derivatives thereof, including those which contain the idiotypic region of the antibody molecule, in particular those which contain the Fv region, for example the F (ab> 2 and Fab Fragments, but also a priori chimeric antibodies or hybrid antibodies with at least two antigen or epitope binding sites, or bispecific recombinant antibodies (for example "double-single-chain" antibodies, diabodies), anti-idiotypic antibodies and those thereof that have been chemically modified and are to be understood as derivatives of these antibodies and are either via DNA recombination, mi by means of hybridoma technology or antibody engineering or synthetically or semi-synthetically by processes known per se and having the known and described reaction properties with regard to the methods of immunology known and described to the person skilled in the art.

From the diverse literature on the production of monoclonal antibodies and derivatives thereof, reference is only made to the work of KOHLER, G.

& MILSTEIN, C, Nature 256, 495-497, 1975; BIOCCA, S. et al., EMBO J. 9, 101 3o

- 1 08, 1 990; BIRD, R.E. et al. Science 242, 423-426, 1,988; BOSS, M.A. et al. Nucl. Acids Res. 1_2, 3791-3806, 1984; BOULIANNE, G.L. et al. , Nature 31, 2,643-646, 1,984; BUKOVSKY, J. & KENNETT, R.H., Hybridoma 6, 21 9-228, 1 987; DIANO, M. et al. , Anal. Biochem. 166, 223 229, 1 987; HUSTON, J.S. et al. , Proc. Natl. Acad. Be. USA 85, 5879-5883, 1 988; JONES, P.T. et al. , Nature 321, 522-525, 1,986; LANGONE, J.J. & VUKANIS, H.V. (ed.), Methods Enzymol. 121, Academic Press, London, 1 987; MORRISON, S. et al. Proc. Natl. Acad. Be. USA 8J_, 6851-6855, 1984; Ol, V.T. & MORRISON, S.L., BioTechniques 4, 214-221, 1,986; RIECHMANN, L. et al., Nature 332, 323-327, 1,988; TRAMONTANO, A. et al. , Proc. Natl. Acad. Be. USA 83, 6736-6740, 1 986; WOOD, C.R. et al., Nature 314, 446-449, 1,985; BRILES, DE & KEARNEY, JF, Methods in Enzymoiogy 1 16, 1 74 - 189, 1985. Also included are humanized antibodies or CDR-grafted antibodies, for example according to GUSSOW, D. & SEEMANN, G., Methods in Enzymoiogy 203, 99 - 121, 1 991, or according to EP-A 0239 400, WO 92/05274 or WO 92/1 5683, and diabodies, for example according to HOLLIGER, P. et al. Proc. Natl. Acad. Be. USA 90, 6444-6448, 1 993.

Monoclonal antibodies, which can very preferably contain the combination preparations according to the invention, can be obtained by any known technique which is available for the production of antibodies by culturing cell lines. Such known techniques include, for example, those of KÖHLER, G. & MILSTEIN C, 1975, | oc. German, or TAGGART & SAMLOF, Science 219, 1 228 - 1 230, 1 983, described methods with hybridoma cells or those with B Zeil hybridomas (KOZBOR et al. Immunology Today 4, 72 - 79, 1 983) or by means of hybrid hybridomas (TAKAHASHI, M et al., Methods in Enzymoiogy, 203, 31-2327, 1991).

Alternatively, of course, commercially available monoclonal antibodies are also possible, for example those as mentioned in EP-A 0 466 637, pages 4 to 6, including their commercial sources, and the X in component 1 can be used.

With regard to the production of polyclonal antibodies, there are also one

Number of known methods available. For this purpose, for example, different animals can be injected with a suitable or desired antigen, which is of natural origin, via DNA Recombination or synthetic or semi-synthetic, or parts thereof, can be immunized and the desired polyclonal antibodies can be obtained and purified from the sera or blood samples obtained thereafter according to methods known per se. Preferred antigens are those derived from or representing prokaryotes and eukaryotes from viruses. The methods of producing polyclonal antibodies, for example via immunization of an animal, are known to the person skilled in the art and are described, for example, in "Practicai Immunology", Hudson, L. & Hay FC (eds.), Third Edition, Blackwell Scientific Publications, 1989. As an alternative, too intact cells are used. Various adjuvants to increase the desired immune response to the antigen exposure can also be used, depending on the animal selected for the immunization - for example complete Freund's adjuvant, mineral gels such as aluminum hydroxide, surface-active substances such as polyanions, peptides, olemulsions, hemocyanins, dinitrophenol or Lysolecthin.

However, antibodies, in particular monoclonal antibodies, fragments or derivatives thereof, are preferred.

Bispecific antibodies can be produced in principle using three different processes.

One of the earlier methods involves the fusion of two hybridoma cells to tetradomes (MILSTEIN, C, & CUELLO, AC Nature 305, 537-540, 1983). A bispecific antibody can accordingly be produced, for example, by a suitable number of parental hybridoma cells (approx. 10 ^ to 10 ^), which are made sensitive, for example for hypoxanthine, aminopterin and thymidine (HAT), by corresponding selection for a hypoxanthine phosphoryl transferase negative Variant. These are fused to the second parental hybridoma cells in a suitable ratio (for example 1: 1 to 10: 1). For this purpose, the second parental hybridoma cells are preferably pretreated beforehand with a lethal amount of a suitable cytocidal reagent (for example 10 mM Iodoacetamide). The fusion itself can take place in the presence of a polyfunctional alcohol, for example in a solution of polyethylene glycol (PEG, approx. 50% w / v). The excess PEG is removed and the cells are plated as usual in a concentration of approximately 10 ^ to 10 'cells / ml in a suitable, buffered medium 34 ^■

(For example bicarbonate buffered Dulbecco medium, or RPMI 1 640 medium, optionally supplemented with a serum, in particular fetal calf serum (approx. 5% v / v)). After culturing the cells at, for example, 37 ° C., the hybridomas can be selected in a suitable medium.

Basically, the fusion of two hybridoma cells leads to tetradomes (so-called due to the lineage of a total of four original cells), which leads to antibody-producing cells that secrete bispecific molecules that are comparable in structure and size to the naturally occurring immunoglobulins. Technically, the procedure is similar to that for the production of hybridomas. First, one of the hybridoma cells to be hybridized is selected for sensitivity, for example for HAT sensitivity. This is induced by culturing the cells in medium with increasing concentrations of, for example, 8-azaguanine and means that the resulting cell clone has a genetic defect, for example for the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), and therefore does not grow in cell culture media containing hypoxanthine, aminopterin and thymidine (HAT medium). The other hybridoma can either be sensitized to another biological substance or irreversibly inactivated, for example with iodoacetamide. After the fusion of the parental hybridomas, only those hybridomas which contain the genetic information of both parental hybridomas can then grow in the HAT medium. After subcloning, stable tetradome cell lines are then established which secrete the highest possible proportion of bispecific molecules. Such bispecific molecules can be used as component 2.

Another possibility for the production of tetradomes is the possibility of surface marking the cells to be fused with different fluorescent dyes, and sorting double-stained cells after hybridization using flow cytometry (FACSort, "fluorescent activated cell sorting").

The resulting tetradome cell thus contains the genetic material of both original hybridoma cells and thus the information for the formation of the four parental heavy or light antibody chains. After stochastic reassociation, there are a total of ten possible combinations of the heavy and light chains. Of these, however, only three are functionally correct antibody molecules in which the binding sites of the two halves of the molecule Heavy and light chain originate from the same parental hybridoma. These are the two parental (monospecific) antibodies, as well as the newly formed bispecific antibody, the halves of which come from a heavy and light chain each from a parental hybridoma. The remaining seven possible combinations, so-called "mismatches", represent functionally defective immunoglobulins, which, however, arise to a much lesser extent due to the most preferred association of the heavy and light chain of the same parental antibody. The proportion of correctly secreted bispecific antibodies in the tetradome product can vary between 5% and 50% of the total immunoglobulins produced in the different cell lines. Since the later purification of the bispecific antibody fraction usually results in a differentiation via the protein-chemical properties of the constant parts of the heavy chain, care must be taken during the fusion that hybridomas are combined with one another, which preferably secrete immunoglobulins of different isotypes. Thus, for example in the use of directed against human antigens mouse antibody combination of the isotypes _IgG] and IgG 2a and IgG -, and IgG2b ^'m with the subsequent differentiation of parental and bispecific antibodies are particularly suitable. In order to achieve a differentiation within the immunoglobulin fraction, which is heterogeneous despite the monoclonality, further purification methods can preferably be connected following an affinity chromatography. Additional chromatographic purification steps are available here, for example ion exchange and hydrophobic interaction chromatography, the latter also being able to be carried out without prior immunoglobulin enrichment, since in a single chromatography step both differentiation between immunoglobulins and other protein components of the cell culture medium and adequate separation within the antibody fraction. However, other isolation methods, such as electrophoretic separation using isoelectric focusing, which use the different isoelectric points of the parental and bispecific antibodies for differentiation, can also be used successfully. Component 2 can be considered for the above-described methods known from the prior art for the preparation of bispecific antibodies, as well as the other preparation methods which lead to bispecific molecules. However, antibodies produced by recombinant DNA (so-called engineered antibodies) are also included in the scope of the present invention, for example those which are obtained by the described methods according to COLOMA, MJ et al. , J. Immunol. Methods 152, 89-104, 1992; "Methods in Enzymoiogy" 1 78, 1 989 or EP A 0 1 25 023 can be produced.

For example, a bispecific antibody can be obtained by this method in that single-chain proteins from V, _ and V ^ domains of the antibodies (Fv) are linked to the C-terminus of one domain and the N-terminus of the other domain (BIRD, RE & WALKER, BW, Trends in Biotechnol. 9 (4), 132, 1991). The method of TRAUNECKER, A. et al., EM.BO J. 10 (1 2), 3655, 1991 and of BRYN, RA leads to a comparable fusion protein with bispecific properties (for example CD4: Fcγ or CD4: CD3) et al., Nature 344, 667-670, 1990). Another bispecific (fusion) antibody can be genetically engineered according to ASHKENAZI, A. et al., Proc. Natl. Acad. Be. USA 88 (23), 10535-10539, 1 991. In this case, the fusion protein concerned an immunoglobulin (immunoadhesion) which inhibits inflammation by interfering with another adhesion molecule. By DEMONTE, L.B. et al., Proc. Natl. Acad. Be. USA 87 (8), 2941-2945, 1990, gene transfer via retroviral shuttle vectors for the production of bispecific antibodies is proposed. Another method of producing bispecific antibodies is that certain transcription factors (Jun and Fos) are genetically fused with anti-Tac or anti-CD3 Fab2 'regions via a "leucine zipper" ("leucine zipper"). The bispecific antibodies are then formed by mixing the two constructs together (KOSTELNY, S.A. et al., J. Immunol. 148, 1 547-1 553, 1992).

Small antibody fragments with two antigenic binding sites, so-called “diabodies”, which can also be considered for the combination preparations according to the invention, can also be produced by genetic engineering methods. Such fragments, which consist of a variable domain of the heavy chain (V ^), which are connected to a variable domain of the light chain (M) on the same protein chain (-J ^ → - V _[ _) can for example according to HOLLIGER, P . et al. Proc. Natl. Acad. Be. USA 90, 6444-6448, 1993. Similar to single-chain antibodies, diabodies represent genetically engineered proteins using known methods. The binding sequences of the heavy and the Light chains of the (mouse) immunolglobulins can be expressed according to known methods in bacterial systems, the variable part of the heavy chain (V ^ l) of one antibody being linked to the light chain sequence (V [_2) of the other antibody and vice versa (Vn2 / V | _1) can be inserted. The bacterial product can then fold in such a way that the binding sites V _j _ _] 1 and V _| _1, as well as V - 2 and V | _2 come to rest in a functionally correct position with each other and thus a bispecific molecule can arise which has comparable binding affinities like a natural antibody, but at the same time only consists of the variable domains of the original immunoglobulins and therefore only a little can be immunogenic in the human system.

The expression of antibody fragments in bacteria belongs to the prior art, so that such recombinant antibody fragment hybridomas are advantageously considered as the main source of bispecific antibodies for medical (prophylaxis, therapy, immunizations) and diagnostic applications in the sense of the present invention. Antibody fragments have advantages over complete antibodies, for example that of cheaper production in microorganisms or cell cultures (for example bacteria, mammalian cells) and, because of their lower molecular weight, better penetration of, for example, body tissues and tumors.

Since diabodies can be bispecific, they can advantageously be used as component 2 (Y). Accordingly, such a component 2 can have two binding sites (anti-Ha- | and anti-Ha2) on the same molecule: one for binding to a corresponding determinant Ha i on a target antigen (for example a tumor marker), another to a correspondingly different determinant Ha2 on an effector cell of the immune system, for example, so that the latter is transported to the target tissue (for example a tumor). In this way, the combination preparation according to the invention using diabodies can be used, for example, to kill killer T cells-activated tumor cells.

Diabodies can not only activate killer T cells: by varying the effector binding site, a whole range of immune effector functions, such as complement activation or antibody-activated cell lysis, can be activated. The advantage of using diabodies is that they can be cleaned in a single chromatography step. 3b

In this respect, diabodies can be used advantageously for the purpose of the combination preparations according to the invention, in particular for component 2.

However, bispecific antibodies can also be produced by chemical means, various methods being known. For example, according to KARPOVSKY, B. et al. , J. exp. Med. 160, 1 686, 1984, heteroconjugates (“heterocross-linked aggregates”) or it can be prepared by the method according to GLENNIE, MJ et al., J. Immunol. 139, 2367 - 2375, 1987, according to which bispecific F (ab'γ) 2 antibodies are constructed via thioether-linked Fab'γ fragments using o-phenylenedimaleimide. Bispecific antibodies can also be prepared by chemical recombination of fragments of monoclonal antibodies (BRENNAN, M. et al., Science 229, 81-83, 1,985). In such a case, the Fc 'portions of the antibody in question (for example IgG) under mild conditions by limited pepsin hydrolysis (for example 2% w / w pepsin in 0.1 M sodium acetate, pH 4.2, 18 hours, 37 ° C.) removed and F (from '> 2 ^parts obtained. Reduction of F (from'> 2 fragments with mercaptoethylamine (for example 1 mM) in sodium phosphate or sodium arsenite (for example 0.1M, pH 6.8)) and EDTA (for example 1 mM) at 25 ° C. for 18 hours leads to the conversion of F (from '> 2 to Fab'. This conversion is preferably carried out with a dithiol complexing agent. The free thiols are preferred, after being prevented with sodium arsenite that Intermolecular SS bonds are formed, stably derivatized by means of a suitable reagent that activates the thiol groups, for example by reaction with Ellman's reagent (5,5'-dithiobis (2-nitrobenzoic acid)). One of the resulting Fab'-thionitrobenzoates (TNB) -Derivate will be you afterwards By reduction with 2-mercaptoethylamine converted into the Fab'-thiols and mixed with equimolar amounts of Fab-TNB, whereby the desired bispecific antibodies are formed.

With regard to two or more distinct proteins which are chemically covalently linked to one another by a linker molecule and thereby at least have a bispecific character, in particular if Y according to Table 1 is two or more distinct, linked antibodies, all molecules known to the person skilled in the art are within the scope of the present invention or "Crosslinkers" or "Cross-Linking Reagents" with which at least 3? two identical or different proteins or peptides according to known

Methods can be covalently linked. The term “crosslinkers” or “cross-linking reagents” is understood to mean those molecules with the properties mentioned which contain functional groups which bind to amino acid residues in proteins and peptides. Accordingly, in the context of the present invention, all crosslinkers or cross-linking reagents are included which can produce intermolecular covalent bonds between two proteins or parts thereof. Therefore, only "binding molecules" are referred to below when referring to such crosslinkers or crosslinking reagents.

According to the invention, preference is given in particular to those connecting molecules whose reactive groups are the same (homobifunctional connecting molecules) or different (heterobifunctional connecting molecules).

From the group of homobifunctional compound molecules, there are those which preferably react with primary amines (amine-reactive) or with sulfhydryl groups (thiol-reactive), for example imido esters or maleimides, alkyl halides, aryl halides, α-haloacyls, pyridyl disulfides.

In the case of the heterobifunctional compound molecules which have at least two different reactive groups, those are particularly suitable which have an amino-reactive functional group at one end and a sulfhydryl-reactive group at the other end, for example those which, as a first group, are an N-hydroxysuccinimide ester group and as a second functional group has a maleimide group.

However, heterobifunctional compound molecules which react with carboxyl groups, for example with glutamic acid or aspartic acid residues as reactants, are also suitable. Examples include carbodiimides. In addition, however, the known photoreactive compound molecules are also suitable which have a functional photoaffine group which only become reactive through exposure to UV light or other visible radiation (photons), for example aromatic azides (arylazides) or benzophenones which are reacted with amines, sulfhydryls, Carbohydrates and carbonyls react.

As examples of some heterobifunctional compound molecules, which are used for the production of at least bispecific reagents, especially antibodies, The following compounds can be considered, although this list is not exhaustive and the person skilled in the art can see further compounds suitable to him from the prior art: 4-succinimidyloxycarbonyl-α-methyl- (2-pyridyldithioo) toluene fSMPT], Λ / -succιnimιyi 3- (2-pyπdyldithio) propιonate [SPDPj, sulfosuccmimidyl 6- [3- (2-pyridyldithio) propιonamιdo] hexanoate, sulfosuccinimidyl 4- (Λ -maleimidomethyl) cyclohexane-1-carboxylate 4 - succinimidyl -maleimidomethyl) cyclohexane-1-carboxylate [SMCC], Λ / -succinimidyl (4-iodoecetyl) aminobenzoate [SIAB], 5, 5'-dithiobis (2-nitrobenzoic acid) [Ellman's reagent],

As examples of some homobifunctional compound molecules, which for the

Production of at least bispecific reagents, in particular antibodies, may be mentioned, the following compounds may be mentioned, although this list is also not complete and the person skilled in the art will find other suitable ones

Connections of this type from the prior art can easily be used:

Dithio.ÖΛs (succinimidyl propionate) [DSP],

3,3'-dithioό / s (sulfosuccinimidyl propionate) [DTSSP], bis (sulfosuccinimidyl) suberate,

Disuccinimidyl suberate [DSS],

Disuccinimidyl tartrate [DST].

The following compounds may be mentioned as examples of some photoreactive compound molecules which are suitable for the production of at least bispecific reagents, in particular antibodies, this list also being incomplete and the person skilled in the art having further suitable ones

Compounds of this type can readily be found in the prior art: azidobenzoyl hydrazide,

Λ -hydroxysuccinimidyl-4-azidosalicylic acid,

Λ / - [4- (p-azidosalicylamido) butyl] -3 '(2'-pyridyldithio) propionamide [ADPD].

The use of such and the other known compound molecules for the production of bispecific antibodies thus also permit multiples

Marking possibilities of the products obtained, such as

Radio markings with radioactive isotopes, for example, via corresponding Iodation of, for example, l - (p-azidosalycylamido) -4- (iodoacetamido) butane or, for example, fluorescent labels by using suitable connecting molecules, for example sulfosuccinimidyl7-azido-4-methylcoumarin-3-acetate.

The named and the other connecting molecules known to the person skilled in the art can be produced by methods known per se or they are commercially available (e.g. Pierce Europe B.V., Oud-Beijerland, Holland). The named and other homo- and heterobifunctional connecting molecules suitable and to be used for the present invention and their applications and reactions with the reactive groups of the proteins or immunoglobulins in question are also described individually by BRINKLEY, M., Bioconjugate Chem., 3, 2- 13, 1992 and by MATTSON, G. et al. , Mol. Biol. Rep. 17, 167-183, 1993. Further connecting molecules suitable for the purpose of producing bispecific antibodies are described individually by SCHEIDTMANN; K.H. In: Protein structure, a practical approach, Creighton, T.E. (Ed.), IRL Press at Oxford University Press, Chapter 4, 93 - 1 1 5, 1989; TRAUT, R. R. et al. In: Protein function, a practical approach, Creighton, T.E. (Ed.), IRL Press at Oxford University Press, Chapter 5, 101 - 133, 1989; TAE, H., Methods in Enzymoiogy 91, 580-609, 1983; BAUMERT, H.G. & FASOLD, H., Methods in Enzymoiogy 1 72, 584-609, 1 989.

The antibodies covalently linked via the connecting molecules described above can be prepared by methods known from the literature, such as are described, for example, in the publications on the connecting molecules already mentioned, including the further literature cited therein (for example BRINKLEY, M., 1992, loc. Cit .; MATTSON, G. et al., 1993, loc.cit .; Pierce catalog).

For example, an at least bispecific reagent, if Y according to Table 1 is a protein, in particular an immunoglobulin or an antibody, can be produced by covalent binding in that suitable immunoglobulins are obtained and purified by a method known per se. These immunoglobulins, for example an IgG, are then reacted with a suitable connecting molecule, for example a homobifunctional connecting molecule, for example an imidoester, under suitable conditions. The reaction products can then according to known Processes are separated from the other components of the reaction solution and purified, for example by dialysis, centrifugation and / or chromatography.

In addition to the hetero- and homobifunctional and photoreactive compound molecules mentioned, it is also possible to combine two reagents, for example antibodies, for the production of Y of component 2 with avidin or streptavidin and biotin. The high avidity to biotin is used here. In order to obtain a bispecific reagent Y to be used advantageously for component 2 (for example a bispecific antibody), one of the antibodies to be coupled is labeled with biotin and the other antibody with streptavidin according to known methods. After equimolar mixing of the two labeled antibodies, a bispecific reagent of high stability results via the avidin (streptavιdin) -Bιotιn coupling. An advantage of this method is that the easy interchangeability of the respective partners (anti-Ha-] and anti-Ha2 or anti-Ha _n ) enables a wide range of different bispecific reagents Y to be produced. For example, a streptavidin-labeled antibody with specificity for a specific determinant Ha-j can be coupled with a large number of biotin-labeled antibodies that recognize different determinants (Ha _n ).

Bispecific antibodies produced by hybrid hybridoma fusion have certain advantages over those heteroconjugates generated by chemical cross-linking. Antibodies chemically generated via, for example, heterobifunctional linkers are dimeric or multiaggregated molecules which are removed much more quickly via the reticuloidal dothelial system and thus have a much shorter half-life in the bloodstream than those which are produced via hybrid hybridomas. Such large heteroconjugates produced via chemical linkers can also have the disadvantage that they are more difficult to penetrate into the extravascular tissue. Because of their bi- or multivalency, they can more easily induce modulations on the target antigen. Since it is known that the membrane immunoglobulin of lymphoma cells is easily modulated when it is cross-linked by bivalent antibodies, bispecific antibodies produced via hybrid hybridoma fusions, for example in the case of lymphomas, are regarded as preferred component 2 (Y). m

In the event that the combination preparations according to the invention are used in vitro, bispecific antibodies can also be used chemically, for example via hetero- or homobifunctional linkers or via avidin (streptavidin) biotin.

A bispecific antibody to be used according to the present invention as defined in Table (component 2) can, for example, be such that an antibody is bispecific for 2,4-dinitrophenol (DNP) and for digoxigenin (DIG). Such an anti-DNP: anti-DIG antibody can be produced, for example, by immunizing mice with suitable DNP and DIG conjugates by known methods, fusing the spleen cells of the immunized mice with myeloma cells, for example with a polyfunctional alcohol (PEG ) or via electrofusion (for example according to VIENKEN, J., ZIMMERMANN, U. FEBS Letters 182: 278-280, 1985), and the resulting hybridomas are selected according to the methods described and fused to tetradomes, for example via PEG fusion.

The antibodies required for the combination preparations according to the invention and which can be prepared by the known processes can be purified by known methods, for example by precipitation, gel filtration, ion exchange chromatography, immunoabsorption chromatography,

Immunoaffinity chromatography, HPLC (High Performance Liquid

Chromatography) or combinations thereof. Antibody fragments containing the idiotype of the molecule can also be produced by known methods. For example, F (ab '> 2 fragments can be obtained by pepsin digestion of the complete poly- or monoclonal antibody. Fab fragments can be obtained, for example, by reducing the disulfide bridges of the relevant F (ab') 2 fragment and Fab fragments can be obtained, for example by treating the antibody molecules with papain and, if appropriate, a reducing agent, such methods are known to the person skilled in the art and belong to the prior art.

A purification can be carried out, for example, in that the antibodies obtained by the methods described above are stored in a buffer (for example Tris

HCI, 20mM, pH 8.0) are diluted to 3 times the volume, then filtered (Filter pores approx. 0.2 μm) and cleaned using a Q-Sepharose Fast Flow column. The antibody fraction can be eluted, for example, using a NaCl gradient. The collected fractions can then be diluted in, for example, NH. (S04) 2 (2 M) and further purified on a suitable column, for example on a phenyl-Superose column, using a linear gradient of, for example, 0.8 M NH4 as the eluent (Sθ4> 2 / 0.1 M Na2HPθ4 (pH 7.2). The fractions obtained can then be tested using conventional assays.

Any known method can be used to identify and select antibodies, fragments or derivatives thereof which react with at least one epitope of the corresponding antigen. For example, that these can be detected after appropriate labeling, if they have bound to isolated or purified antigen or by immunoprecipitation of the antigen which has been purified, for example by gel filtration, chromatography or polyacrylamide gels, or by the fact that antibodies against a corresponding antigen bind with other antibodies compete the same antigen.

The choice, type, source and method of production of the antibodies for the combination preparations according to the invention are not critical for the implementation of the present invention. They can be selected and produced from any class or subclass and by any known method, depending on which one is preferred by the person skilled in the art. In the event that the combination preparations according to the invention are to be used for therapeutic use and for "targeting" cells or tissues to be treated, the antibodies used for them should be selected with regard to their toxicity. An antibody Z conjugated, for example, with an effector molecule (according to Table 1) together with the other two components 1 and 2, which contain X and Y according to Table 1, can initially be tested for cytotoxicity in vitro if a combination preparation for preferably immunotherapeutic purposes on mammalian organisms , especially on people. In the case of known animal models, for example on mice or rats, the corresponding combination preparation can be further investigated with regard to its effectiveness in vivo. In principle, it is readily possible for a person skilled in the art to use an antibody with the function X according to Table 1, preferably a monoclonal antibody, against all those which occur select antigenic determinants and antigens, make them, and test and study them as outlined above. For example, such antigens are tumor, bacterial, virus, parasite, fungal, mycoptasm, histocompatibility, cell surface, cell receptor antigens or antigens based on toxins, enzymes, allergens or other physiologically relevant substances including plant antigens.

With regard to diagnostic methods, detection methods or assays, the combination preparations according to the invention can be used in all known serological-immunological diagnostic methods.

For example, the combination preparations according to the invention are suitable for immunofluorescence tests, for example direct (IFT) or indirect immunofluorescence tests (IIFT); Enzyme immunoassays (EIA); Agglutination tests, such as, for example, agglutination inhibition test, immunosorbent agglutination assay (ISAGA); Radioimmunoassays (RIA); Immunoradiometric Assay (IRMA), Enzyme Linked Immunosorbent Assay (ELISA), μ-capture assay; Enzyme membrane immunoassay (EMIA); fmmuno Enzymo-Metric Assay (IEMA); Double Antibody Liquid Phase Assay (DALP technique); Fluorescence immunoassay (FIA), for example homogeneous fluorescence immunoassay, fluorescence polarization immunoassay, fluorescence enhancement immunoassay, fluorescence quenching immunoassay, fluorescence excitation transfer immunoassay (FETIA), substrate labeled fluorescent immunoassay (SLIFA), heterogenassay

Immunofluorometric Assay (IFMA), Time Resolved Fluorescence Immunoassay (TR-FIA); Luminescence immunoassay, for example chemiluminescence immunoassay (CELIA), solid phase antigen luminescence technique (SPALT), immunoluminometric assay (ILMA), immunoluminal labeled second antibody assay (ILSA); Immunoblot or Western blotting or dot blot techniques. It should be emphasized at this point that the methods described above by way of example in the literature and known to the person skilled in the art and suitable for the use of the combination preparations according to the invention are not and need not be complete, since such methods are used for immunological diagnostics Are available and work on the basis of immunoglobulins or antibodies, which are known to the person skilled in the art. In addition, such processes and techniques have been described in many different ways in the literature, for example by BAUER, S., Lab. Med. 3, 50, 1 979 (Immunofluorescence techniques); DWENGER, A., J. Clin. Chem. Clin. Biochem. 22, 883-894, 1984 (RIA); ADDISON, GM & HALES, CN, In: Kirkham, K .. Hunts, WM eds., Radioimmunoassay methods, Livingstone, Edinburgh, 595, 1971 (IRMA); OELLERICH, M., J. Clin. Chem. Clin. Biochem. 22, 895-904, 1984 and MIYAI, K., Adv. Clin. Chem. 24, 61 - 1 10, 1 985 and LINKE, R., Mitt. Dt. Ges. Klin. Chem., 1986, 291 (EIA); HEMMILÄ, I. , Clin. Chem. 3_1, 359-370, 1985 and WISSER, H., GIT-Labormedizin 1985, 89 (FIA); WEEKS, I. et al. Clin. Science 70, 403-408, 1986 and GADOW, A. et al., J. Clin. Chem. Clin. Biochem. 12, 337, 347, 1984 and WOOD, WG et al., J. Clin. Chem. Clin. Biochem. 22, 349-356, 1984 (luminescence immunoassay); BURNETTE, WN, Anal. Biochem. 1 12, 195 203, 1 981, (Western blotting), DESMONTS, G. et al. , J. Clin. Microbiol. 14, 486 491, 1 981 (ISAGA).

If an early result of technical data is desired, on the basis of which a diagnosis can be made, immunoassays that can deliver the desired data within a short time can be preferred. For this purpose, for example, those immunoassays come into consideration which are based on a sandwich test, competitive test, colorimetric test (such as, for example, the CEDIA ™ test according to HENDERSON, DR, et al, Clin. Chem. 23 (9): 1 637-1 641, 1986) and are applicable as rapid tests and are known to the person skilled in the art.

In the case of the sandwich assay, component 1 can consist of a protein associated with a determinant Ha-], which can be a monoclonal or polyclonal antibody recognizing, for example, an antigen contained in a test sample, in particular a monoclonal antibody which is linked to an anti-Ha - Binding site (of component 2) can bind and on the other hand can recognize a capture antibody immobilized on a carrier material, for example a microtiter plate. Component 2 can then bind to component 1 via its anti-Ha-] site. Furthermore, an agent equipped with a determinant Ha2, for example an enzyme (for example horseradish peroxidase), which can preferably react with a detectable agent, that is to say a labeling or indicator molecule, for example 2,2'-azino-ö / s (3-ethylbenzothiazoline-6-sulfonate (ABTS). A method disclosed for example in EP-B1 0 394 81 9 can easily be adapted for the combination preparations according to the invention. 1 * 5 Another variant can be based, for example, on antibodies conjugated with biotin, which bind to a support coated with streptavidin. By adding to this system of peroxidase-labeled antibodies connected to a determinant Ha2 and the sample to be examined, the color reaction can be carried out in the presence of a suitable, detectable agent (for example 2,2'-azino-o / s (3-ethylbenzothiazoline-6-sulfonate) be measured spectrophotometrically by known methods.

Accordingly, the combination preparations according to the invention can advantageously also be used for a method based on an immunoassay, in which, for example, a first one with a determinant Ha - _| equipped antibody, which can bind to an antigen to be tested in a sample, is linked to a binding protein of a biological binding constant _kjj s> 0 ^' - 2 M / L, for example biotin or a biotin derivative. A corresponding agent equipped with a determinant Ha2, for example a second antibody, which can bind to an antigen in a sample, can advantageously be labeled with an indicator molecule. A protein with binding protein binding properties can be bound to a carrier material. If the binding protein is biotin, avidin or streptavidin or a functional derivative thereof can be used. All known labeling or indicator molecules, for example horseradish peroxidase, can be used as the indicator molecule with which the second antibody which is not linked to a binding protein but is labeled with Ha2 is labeled. The immune complex formed in this way can be visualized using known methods. In principle, such an immunoassay based on streptavidin-biotin binding is described, for example, by MÜLLER-BARDORFF, M. et al., Circulation 92 (10): 2869-2875, 1995, which is readily advantageous for those according to the invention

Combination preparations can be transferred and used.

In a further variant of an ELISA sandwich assay for the detection of antigens in a sample, a carrier material, for example conventional microtiter plates, can be coated ("coated") with antibodies against this antigen, after which the sample is coated with this coated carrier material in the presence of a suitable buffer Is brought into contact. With the appropriate component 2 added beforehand, a suitable component can then be added tl

Determinants Ha - | equipped reagent, for example an antibody, and correspondingly incubated in the presence of a suitable buffer system. Then an IgG-peroxidase conjugate equipped with a determinant Ha2 and a suitable reagent (for example o ■ phenylenediamine) can be added and the reaction can be stopped using a mineral acid (e.g. H2SO4). The absorption can then be measured using customary methods. For example, by TSUBOUCHI, H. et al. , Hepatology 1_3 (1): 1-5, 1991, the method described can readily be applied to the use of the combination preparations according to the invention.

As carrier material which is suitable for the method according to the invention and which can be coated with corresponding binding proteins according to the present description or on which the

Suitable reagents, proteins or specifically antibodies can be immobilized, all known materials that can be used for an immunoassay are suitable. In particular, any water-insoluble material is known which the person skilled in the art knows that it can bind or adsorb proteins or polypeptides on its surface, preferably those made of plastics, such as, for example, polyvinyls, polystyrenes, acrylates, polypropylenes, polycarbonates, silicones. Such support materials consisting of such substances can be of different shape and size, depending on the individual needs of the person skilled in the art who uses the assay. Accordingly, the carrier material can be made with a hollow body (for example, tubes, tubes, cuvettes) or with at least

25 a depression or trough, which can be rounded or angular, provided molded body, which can preferably be flat (for example plates, disks, plates, strips, slides, microtiter plates).

In order to make the agents of component 3 to be used according to the invention detectable in an immunoassay, these can be labeled with labeling or indicator molecules in a suitable manner and according to known methods. Particularly suitable marking or indicator molecules for the method according to the invention are proteins equipped with determinants Ha2 or Ha _n , for example antibodies, enzymes (for example peroxidases, β-35 galactosidases, alkaline phosphatase), and dyes coupled thereto (in particular fluorescent dyes, for example fluorescein isothiocyanate), paramagnetic atoms, chromogenic substrates (for example o phenylenediamine or 2,2'aziono-dι [3-ethylbenzthιazolιn sulfonate (6)], radioisotopes (for example halogens, technetium, lead, thallium, indium or mercury) metals (For example, gold or gold particles or silver) Processes for carrying out such markings are known to the person skilled in the art and are described in the literature. In the event that gold or gold particles are used as the indicator molecule, the gold with a protein with binding protein-binding properties can be used according to the known immune gold technique , for example coated with biotin-binding proteins (for example avidin or streptavidin). The optical detection takes place in interaction with, for example, correspondingly biotinylated antibodies. Such a signal on which the optical detection is based can be additionally amplified by utilizing the ability of the gold particles to presently kind of a suitable reducing agent (for example hydroquinone) to reduce silver ions. In this case, a subsequent contrast is achieved with this immunegold-silver coloration in that silver is deposited on the gold particles, so that a dark brown to deep black coloration is produced. This advantageously increases the sensitivity of the relevant immunoassay for the detection of antigens in an examination sample

Detection via biotin can be carried out via biotinylation using known methods, for example by binding an antigen to biotin in a known manner using N-hydroxysuccinimide biotin (for example in dimethyl sulfoxide). Such biotmylated antigens or the corresponding antibody complexes can be identified, for example, using avidin-conjugated alkaline phosphatase (ExtrAvidin, Sigma, Deisendorf, Germany).

In an exemplary manner, an application in an immunoassay can take place in that component 1 is an antibody (X in Ha-) which is directed against a certain antigen, for example a tumor antigen, and which is associated with an hapten Ha-], for example 2,4-diminrophenyl (DNP). ] - X), preferably a haptenized monoclonal antibody. Component 3 is, for example, a fluorescent dye (Z in Ha2 - Z) associated with a hapten Ha2 (for example digoxigenin (DIG)), for example fluorescein isothiocyanate. A bispecific reagent which can be used as an immunolinker (component 2) Ha-] (antι-DNP): antι-Ha2 (anti-DIG) has specificities. IS

The immunolinker is preferably a bispecific monocoloma antibody with the corresponding specificities. Components 1 and 2 are either brought into contact with component 3 with the sample to be examined (body fluids, cells, tissue or organ parts) by known methods, or component 3 can be added after the reaction of components 1 and 2, after a time delay . A more detailed description of preferred application modalities is given elsewhere in the present description.

A further meaning in the use of the combination preparations according to the invention is that they are suitable for "imagining". This means the specific detection, detection and localization of healthy, non-pathological and pathologically changed specific cells and tissues and parts thereof in vitro and in vivo, that is to say extracorporeally and intracorporeally. Pathologically modified cells and tissues can be malignant cells or tissues, for example. Such "imaging" can be carried out by defining X and Z according to Table 1 with two different determinants Ha- | and Ha2 (or Ha _n ), for example haptens, are conjugated and Y has corresponding anti-Ha-] and anti-Ha2 (or anti-Ha _n ) specificities. In this case, Z can be a carrier molecule labeled with a radioactive substance, with a fluorescent dye or with a contrast agent, for example a protein provided with a determinant, for example an enzyme. With regard to radioactive isotopes, all known β and γ emitters can be used, but preferably those with a short half-life. If a radioisotope is chosen, the radioactive agents known to the person skilled in the art are available, for example, 1 25, 1 23 | are suitable 1 31 |, 1 1 1, n _r 1 13m | _n 99m _Tc , 203 _Pb , 201 _T |, 198 _Hg , 90γ, 186 _Re , 60 _Co , 67 _Ga , 75 _Se , 1 12m _Ag , 198 _Au , 203 _g , 32 _P , 51 _{Cr |} 59p _e , _A | _s Fluorescent dye is, for example, a fluorescein (for example fluorescein isothiocyanate) and a contrast agent, for example a paramagnetic contrast agent. By means of suitable devices known to the person skilled in the art, for example with a gamma scintillation camera, via magnetic resonance measurement or microscopically, such a combination preparation can easily detect and localize cell and tissue types and parts thereof. In such systems, the determinants, for example haptens, can of course themselves be the object of such a label. In this case too, component 3, for example conjugated to an isotope, can be given with a time delay, combined with the advantage of a lower radioactive load in in vivo imaging.

In order to explain in more detail the use of one of the combination preparations on which the invention is based, an even more detailed description will be given below, although the person skilled in the art can use his experience and the literature known to him for this purpose and a number of methods are therefore available to him which he can easily accept, adapt and use for the purpose according to the invention under his laboratory conditions.

With immunimaging, it must be taken into account to what extent a "targeting" of small radiolabeled ligands can be mediated by bispecific reagents (e.g. antibodies), whether this "targeting" is sufficiently specific and what type of radioactively labeled antigen or hapten (Z

1 or Ha2 or Ha _n in Ha2-Z or Ha _n -Z according to Table 1) should preferably be used. To solve such questions, Z, for example an antibody or another protein or fragment thereof, can be labeled directly with a radioisotope by known methods, or the labeling is carried out by coupling the corresponding isotope to a selected determinant Ha2, for example

20 is a hapten, or _n to selected haptens determinants and Ha, which is or which is bound to a carrier (Z). In the case of a hapten, this can consist of a monovalent hapten or a bivalent hapten, which can be hydrophobic or hydrophilic. Dinitrophenyl, for example, comes in as a monovalent, hydrophilic hapten

2 Question which can be labeled with one of the radioisotopes mentioned above and bound to a protein or peptide, for example ^{1 2} 5 | _oc ( _er 1 1 1, _n . Examples of bivalent hydrophilic haptens are derivatives of diethylenetaminopentaacetic acid, in which isotopes, for example ¹ 5 | _oc j _er 1 1 1 | _n, can be present as metal chelates. Such haptens are able to use their carboxyl groups in the Meaning one

30 binding acid amide preferably to peptides or proteins (Z).

If imaging, in particular immunoimaging of tumors, is to be carried out under clinical conditions, Z can preferably be a radioisotope chelated via a bivalent, hydrophilic hapten, for example ^{1 2} 5, _oc j _er ^{1 1 1} In, which can be obtained, for example, by reaction with the cyclic anhydride

I of diethylenetaminopentaacetic acid can be obtained, for example according to LE DOUSSAL, J.-M. et al., Cancer Res. 50, 3445-3452, 1990. For such one So

The case is bivalent haptens, in particular hydrophilic in nature, preferred over monovalent, hydrophobic haptens. Further examples of divalent haptens in question would be dinitrophenyl derivatives or derivatives of Nt-dinitrophenylaminocaproic acid, which with 125 | can be iodized or marked with ^ ^'• - n. Such haptens are commercially available For further examples of the use and production of mono- and bivalent haptens, which can be labeled with, for example, ^ 25, _{0 (} - | _er 1 1 1 | | _n and which are linked to amino acids and thus to peptides and proteins via acid amide formations may be covalently bound, and which are known to be suitable for tumor imaging but also for therapeutic purposes within the meaning of the present invention, reference is made to the literature, for example to LE DOUSSAL, J.-M. et al., J. Nucl. Med 30, 1358-1366, 1989. In general, based on the results of LE DOUSSAL, 1990, loc. Cit., It can be assumed that bivalent haptens have advantages over monovalent haptens .. The methods of labeling proteins, including antibodies, are known to those skilled in the art with radioisotopes from the diverse literature well known, so that it is unnecessary to refer to special literature Radioactive labeling of proteins should be mentioned purely by way of example in EP A 0 237 150 or EP A 0 36 678.

In this way, radioisotopes can be transported directly to the desired location with the combination preparations according to the invention. With regard to the aforementioned use of a chelator, it is clear to the person skilled in the art that not only the chelators mentioned by way of example come into consideration and not only the radioisotopes mentioned by way of example, but that all known chelators can stand for a large number of detectable markers, in particular radiometals or magnetic resonance-enhancing metal ions. Further examples of suitable chelators are ethylenediaminetetraacetic acid or bisthiosemicarbazones of 1, 2- or 1, 3-dicarbonyl compounds. With regard to a suitable application of the three components Ha-, - X, anti-Ha - | - Y - anti-Ha2 or - Y - anti-Ha _n and Ha2 - Z or Ha _n - Z for tumor imaging, these can be given simultaneously, sequentially or consecutively. A step-by-step procedure can be advantageous, however, by first giving component 1 (X) in order to bind at the target site, together or at different times with the immunolinker component (Y), and only then delaying it by minutes, hours or days (depending on Purpose of investigation and Sl ^■ the location, size and type of the target site or tumor), component 3 (Z). Alternatively, this step-by-step process can also take place in that the time interval of the application lies between components 1 and 2 (X or Y) and component 2 (Y) is given together with component 3 (Z). In this way, a “pre-targeting” mediated by component 1 (or components 1 and 2) can be achieved, as a result of which effective radioactive determinants, for example hapten (s), or radioactive Z are taken up can. In addition, a faster clearance of the determinant or the hapten or the component 3 from the plasma is to be expected, which can lead to an improved tumor / non-tumor contrast. It is also possible to apply all three components 1, 2 and 3 with a time delay.

An application mode in which component 3 is given last, delayed by minutes, hours or days, can be used advantageously. The time intervals of the delayed applications between the components 1 and 2 and component 3 is particularly dependent on the selected radioisotope or its half-life (which is between 1, 2 minutes at 1 1 m _A g and 121 days can be at ^^ Se) and the type of material to be examined. All routes known to the person skilled in the art can be considered as application routes, in particular iV, im, sc, ip, ia, ie

A typical use of the combination preparations according to the invention for imaging consists, for example, in that a monospecific reagent according to component 1, which is prepared by known methods and is linked to a hapten Ha ^, in which X is preferably an antibody, is administered, for example iv, to a mammalian organism, so that it is administered binds specifically to a corresponding binding site (for example an antigen or epitope or a "target structure"), for example a tumor cell. Subsequently or simultaneously, component 2 is given (for example iv), which binds to the determinant (Ha-]) via its anti-Ha ^• ] specificity. Component 3, which is labeled with a suitable radioisotope and linked to a suitable determinant Ha2 or suitable determinants Ha _n , is then preferably administered with a time delay (in this case likewise iv), which contains the anti-Ha2 site of component 2 (Y - anti-Ha2 ) reacts. For the purpose of a predetermination regarding the biodistribution of the radioactive agent (component 3) of the combination preparations according to the invention, appropriately treated test animals (mice, rats, rabbits, etc.) can be used after the last application and their organs and blood are taken. For further examination, the blood can be heparinized, centrifuged and the usual

Radioactivity can be measured in plasma. The organs and tumors removed are usually broken down into correspondingly large sections and their radioactivity measured analogously or the decay of the radionuclide in question is counted using a counter. In this way, the behavior of component 3 in a mammalian organism can be predetermined and the modes of administration (amount,

Dose, time administration) can be easily optimized.

For the actual imaging, the mammal to be examined or the patient can first be injected with component 1 (in the case of an antibody, preferably a humanized antibody if a person is to be examined) (systemically or topically, for example IV, im or sc, into the Proximity, to or in the material to be examined). Subsequently or simultaneously with component 1, component 2 and, preferably with a time delay, the radioactively marked component 3 associated with a suitable determinant Ha2 is applied via the corresponding same route. Examples of suitable radioisotopes are ¹ 5 | _unc j 1 1 1 | _n for example in the range from 1 to 500 μCi (3.7-10 ⁴ to 1.85 -10 ⁷ Bq), in particular around 100 μCi (3.7-10 ⁶ Bq). Following such a two-step system, in a very preferred embodiment a monoclonal antibody with anti-tumor properties and with a hapten Ha-], which can be, for example, a dinitrophenyl derivative, for example 2,4-dinitrophenol (DNP) (component 1), coupled with a bispecific monoclonal antibody with anti-Ha-]: anti-Ha2 properties (for example anti-DNP: anti-DIG, if Ha2 is a digoxigenin [DIG]) is given intravenously into a mammalian organism in the usual way. After a suitable time interval of, for example, 24 to 120 hours as a guide value (depending on the affinity of the selected antibody of component 1, the selected isotope and the type of tumor), component 3, for example a ^" * ^ unmarked or 123 | - _mar | The _{first m} j _t QIQ haptenized carrier molecule, for example a protein, administered intravenously, optionally together with a smaller amount of component 2. In this way, the m in or the ^ 23 | _an d _en previously with component 1 or component 2 "pre-marked" tumor can be transported specifically and in a targeted manner and exert the desired effect. A similar but conventional system is described, for example, by STICKNEY, DR et al., Antibody, Immunoconjugates, and Radiopharmaceuticals 2 (1), 1 - 1 3, 1 989 after the last injection are in corresponding time intervals of, for example, 0.5, 1, 2, 4, 6,

10, 24, 48 or 72 hours of scintigraphic recordings were made using a gamma camera and the scintigrams obtained were evaluated as usual. Of course, this exemplary representation represents only one possibility of using the combination preparations according to the invention and the person skilled in the art is readily able to use other variations and to carry out equivalent applications. Attention is therefore drawn to the diverse literature, which cannot be dealt with exhaustively here and, for the reasons mentioned above, does not have to be discussed either.

It is therefore also self-evident that such "pre-targeting" can also be used in vitro for the other immunodiagnostic methods described, for which the sample to be examined first with component 1, then either simultaneously or delayed with component 2 and after one minute or hourly delay is brought into contact with component 3.

A further advantageous use of the combination preparations according to the invention consists in a method for determining whether an agent with biological, chemical or physical properties comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a marker substance, an enzyme, a radioactive substance, or a radioactively labeled one Substance, a contrast agent, a biologically active substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with an effector-marker or reporter function, a prodrug, an adhesion molecule, a cytokm, a lymphokine, a chemokine, a ligand, a biological, chemical or physical effect suitable in vitro and in vivo diagnostics, in immunotherapy or in immunizations or in vivo or in vivo, which is characterized in that a) a first combination preparation ation consisting of components 1, 2 and 3 according to Table 1, with the definitions given in the present description for X, Y, Z, Ha-, and Ha2 or Ha _n , with a first agent to be determined Z with biological, chemical or produces physical properties, b) an at least second combination preparation consisting of components 1, 2 and 3 according to Table 1, with the definitions for X, Y, Z given in the present description, Ha-, and Ha2 or Ha _n , with a different from a) different agent Z to be determined with biological, chemical or physical properties, c) the combination preparations provided according to a) and b) one after the other or simultaneously in an in vitro or in an in vivo system, which may be the same or different, in contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, d) repeating the step according to c) as desired, the means Z to be determined on component 3 is different for each repetition with regard to its biological, chemical or physical properties, e) measures, detects or records the biological, chemical or physical effects that occurred after step c) or d) and f) those according to e) evaluates the results obtained individually or comparing them.

In addition, the combination preparations according to the invention provide a variant of the method described above, which is also advantageously suitable for determining whether an agent with biological, chemical or physical properties comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a marker substance, an enzyme, a radioactive substance, a radioactively labeled substance, a contrast agent, a biologically active substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with effector-marker or reporter function, an active substance precursor (prodrug), an adhesion molecule , a cytokine, a lymphokine, a chemokine, a ligand, a biological, chemical or physical effect in vitro or in vivo which is suitable in in vitro and in vivo diagnostics, in immunotherapy or in immunizations develops or shows, characterized thereby eichnet that a) a combination preparation consisting of component 1 and component 2 according to Table 1, with the definitions for X, Y, Ha -] and Ha2 or Ha _n given in the present description, b) that according to a) Combination preparation provided in an in vitro or in an in vivo system brings into contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, c) a first component 3 according to Table 1 with which Definitions for Z given in the present description, with a first agent Z to be determined with biological, chemical or physical Produces properties, d) component 3 provided according to c) adds the system according to b), e) then a second or further component 3 according to table 1, with the definitions for Z given in the present description, with a means Z to be determined with biological, chemical or physical properties different from c) and each other, adding the system according to b) simultaneously or in succession, f) optionally repeating steps a) to e) as desired, the agent Z to be determined on component 3 with each repetition with regard to its biological, chemical or physical properties are different, g) measures, detects or records the biological, chemical or physical effects that occurred after step e) or f) and h) evaluates the results obtained according to g) individually or comparably with one another.

In addition, the combination preparations according to the invention advantageously provide a method for screening a plurality of agents with biological, chemical or physical properties selected from effector molecules, a receptor binding molecule, immunoglobulins or antibodies, marker substances, enzymes, radioactive substances, radioactively labeled substances, contrast agents, biologically active substances, cytotoxic, cytocidal, cytostatic or cytolytic agents or any antigens with effector-marker or reporter function, active substance precursors (prodrugs), adhesion molecules, cytokines, lymphokines, chemokines, ligands, which are used in in vitro and in vivo diagnostics, in immunotherapy or in immunizations should develop or show suitable biological, chemical or physical effects in vitro or in vivo, which is characterized in that a) a first combination preparation ion consisting of components 1, 2 and 3 according to Table 1, with the definitions given in the present description for X, Y, Z, Ha-, and Ha2 or Ha _n , with a first agent to be determined Z with biological, chemical or produces physical properties, b) an at least second combination preparation according to Table 1, with the definitions given in the present description for X, Y, Z, Ha- | and Ha2 or Ha _n , with a second agent Z to be determined with biological, chemical or physical properties that is different from a), c) the combination preparations provided according to a) and b) one after the other or simultaneously in an in vitro or in an in vivo System, which can be the same or different, s ^■ brings into contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, d) repeating the step according to c) as desired, the agent Z to be determined on component 3 being repeated each time is different with regard to its biological, chemical or physical properties, e) measures, detects or records the biological, chemical or physical effects that occurred after step c) or d) and f) evaluates the results obtained according to e) individually or comparably with one another.

A variant of the aforementioned method for screening a plurality of agents with biological, chemical or physical properties selected from effector molecules, a receptor binding molecule, immunoglobulins or antibodies, marker substances, enzymes, radioactive substances, radioactively labeled substances, contrast agents, biologically active substances, cytotoxic, cytocidal , cytostatic or cytolytic agents or any antigens with effector marker or reporter function, drug precursors (prodrugs), adhesion molecules, cytokines, lymphokines, chemokines, ligands, which are suitable in in vitro and in vivo diagnostics, in immunotherapy or in immunizations Developing or showing biological, chemical or physical effects in vitro or in vivo is characterized in that a) a combination preparation consisting of component 1 and component 2 according to Table 1, with those in the present B Description given definitions for X, Y, Ha- | and Ha2 or Ha _n , b) brings the combination preparation provided according to a) in an in vitro or in an in vivo system into contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, c) a first component 3 according to Table 1, with the definitions for Z given in the present description, with a first agent Z to be determined with biological, chemical or physical properties, d) the component 3 provided according to c) System according to b), e) followed by a second or further component 3 according to Table 1, with the definitions for Z given in the present description, with an agent Z to be determined with biological, chemical or physical properties different from c) the system according to b) adding simultaneously or successively, f) optionally repeating steps a) to e) as desired, the agent Z to be determined on component 3 being different with regard to its biological, chemical or physical properties with each repetition, g) following step e) or f ) measures, detects or records the biological, chemical or physical effects that have occurred and h) evaluates the results obtained in accordance with g) individually or comparably with one another.

The agents with biological, chemical or physical properties, as mentioned by way of example in the present description, are also known to the person skilled in the art or can be found in the diverse literature. Accordingly, the person skilled in the art is able to recognize the suitable biological, chemical or physical effects of the potentially suitable agents. Such effects or measurement parameters can include, for example: extent of cytolysis, cell death or cytotoxicity, occurrence of specific and non-specific reactions of the immune system, degree, type and expression of radioactive labels or radioactive signals or of binding of radioactive substances at target locations, course of tumor growth, degree stimulation reactions, for example by means of adhesion molecules, degree of activation and accumulation of effector cells, type and degree of binding of effector molecules to a target site, course of autoimmune diseases, rejection reactions, infectious diseases or diseases of the coagulation system, extent and efficiency of a "drug targeting" or a "cell targeting" ", Measurement of fibrinolytic potency of fibrinolytically active agents (for example urokinase, streptokinase, tissue plasminogen activators (tPA), measurement of the inhibition of replication or multiplication of viruses, onset of immunizations, M eating of dye reactions, radioactivities, immune reactions (e.g. turbidity, agglutinations) or bindings to target sites (e.g. receptor bindings) in immunoassays.

Furthermore, the combination preparations according to the invention advantageously enable a method for producing an agent which can be used in in vitro and in vivo immunodiagnostics, which comprises the steps a) providing at least one combination preparation consisting of components 1, 2 and 3 according to Table 1, with those in the present description Definitions given for X, Y, Z, Ha- and Ha2 or Ha _n , or b) providing at least one combination preparation consisting of components 1 and 2 according to Table 1, with the definitions for X, Y, Ha given in the present description -] and Ha2 or Ha _n , c) identifying an agent which is biologically, chemically or physically active with regard to its immunodiagnostic properties by means of the combination preparations provided in a) and b) via the determination and screening methods disclosed above d) formulation of the from c) agent obtained in the form of a combination preparation according to Table 1, with the definitions given in the present description for X, Y, Z, Ha-j and Ha2 or Ha _n with the known auxiliaries and excipients, or d) formulation of the from c) obtained by combining with a monospecific reagent, which is able to bind to biological binding structures, according to known methods and with the be known auxiliaries and excipients, or e) formulating the agent obtained from c) all by itself using known methods with the known excipients and excipients.

It is readily apparent to the person skilled in the art that the use of an agent which consists of a combination preparation, comprising components 1, 2 and 3 according to Table 1, with the definitions given in the present description for, is very particularly advantageous for the processes mentioned X, Y, Z, Ha- | and Ha2 or Ha _n , or consisting of a combination preparation comprising components 1 and 2 according to Table 1, with the definitions given in the present description for X, Y, Ha-, and Ha2 or Ha _n , or consisting of component 2 according to Table 1, with the definitions for Y given in the present description, in one of the stated determination, screening or production processes.

It should be emphasized at this point that, of course, the technical features described in the methods and uses of the means for these methods disclosed above apply to the definitions disclosed in the present description.

It should also be emphasized at this point that such pre-targeting with the combination preparations according to the invention can also be used in immunotherapy or in immunizations in vivo, but also in prophylactic and prognostic application and for the determination of disease states and disease courses as well as for the prevention and prevention of the outbreak or the development or exacerbation of a disease state.

A more detailed description of the use of the combination preparations according to the invention in terms of type and dosage in clinical applications is given below. Since therapeutic / prophylactic / prognostic and contraceptive clinical use is closely related to imaging of tumors and other pathologically / clinically meaningful antigens, the proposed measures apply analogously to the purposes of imaging and vice versa.

With regard to the use of the combination preparations according to the invention in therapy, in particular in immunotherapy, a number of possible uses open up.

As with the diagnostic application, it is of course also relevant for the therapeutic use of the combination preparations according to the invention that X and Z (Table 1) are equipped or connected with two different determinants Ha-] and Ha2 (or Ha _n for Z) and that Y has corresponding anti-Ha and anti-Ha2 (or anti-Ha _n ) specificities. Under these specified conditions, Z can be, for example, a lytically active or a cytocidal or cytotoxic active substance (for example a toxin, an enzyme, another biologically active protein or an effector molecule or a radioisotope), which in particular inhibits the multiplication and proliferation rate of malignant tumor cells , suppressed or reduced. In this case, Z in the combination preparation in question can be an enzyme which is provided with a determinant Ha2, for example haptenized, (for example alkaline phosphatase), a cytokine (for example interferon α, β or γ or derivatives thereof, an interleukin such as IL-2) , with a determinant, for example a hapten, effector cells or trigger molecules (for example cytotoxic T-lymphocytes, monocytes, macrophages, NK cells, granulocytes [polymorphonuclear neutrophils, eosinophils], Fc receptors for IgG, CD2, CD3) a cytotoxic toxin or a radioisotope. With regard to toxin conjugations that come into consideration according to the invention, further and more closely described such cytotoxic substances for immunotherapeutic purposes that are suitable for coupling to the corresponding component of the combination preparations according to the invention are described, for example, in EP-A 0 368 68, in particular on pages 6 to 14, and in the literature listed there.

In addition, a "drug targeting" for the A-chain of RICI V, for Vmca alkaloids, saporin, daunomycin, alkaline phosphatase (conversion of mitomycin phosphate to more effective mitomycin alcohol) is clearly in BRISSINCK, J., Drugs of the Future 17, 1003 - 1010, 1992

If one or more radioisotopes come into consideration for therapeutic purposes, the radioimmunoconjugates which can be prepared by known methods are suitable for this purpose. Here, Z is to be selected according to Table 1 as a radioactively labeled molecule which is provided with at least one determinant, for example a hapten, or the determinant itself can be a carrier of radioisotopes, for example a hapten according to the present description and the known literature. Both ß- and γ-emitters are particularly suitable as radioisotopes, for example 1 5 ,, 1 23 ,, ¹ 3 ¹ l, 1 ^{l 1} | _n , 99mj _c , 90γ, 1 86R _Θ Such therapeutic applications are on the "Fourth International Conference of Monoclonal Antibody Immunoconjugates for Cancer", San Diego, California, 30.3. - April 1, 1989 (GOLDENBERG, DM, Immunology Today 10 (9), 286-288, 1989).

For the clinical use of the combination preparations according to the invention, it can also be advantageous to carry out the application described above in an analogous manner, regardless of whether Ha2 in Z is the carrier of radioactivity or whether the Z associated with Ha2 is radioactively labeled or whether Z is another endowed with a determinant Ha2 means an exerting agent (for example an enzyme, a cytokine, a T-lymphocyte, an adhesion molecule, etc.). The proposed application methods for tumor imaging for the inventive

Combination preparations are therefore used appropriately for clinical use. It may also be advantageous if multiple injections of component 3, whether a radioisotope or another cytocidal, cytotoxic or cytostatic agent, or one Marker substance is connected, to be applied especially when it appears necessary to treat over a longer period of time. In this way, higher concentrations of active substance or radioactivity can be achieved at the site of action.In the event that tumor cells respond to treatment within a short time ('1 day) and can only be killed or are only to be marked or detected, a bolus injection would be carried out one or more times in succession can be given, preferably in the manner described above over two steps, advantageous. Such a single use is certainly also suitable for the aforementioned purpose of imaging, since the organ or tissue to be localized only has to emit a signal for a short time.

In general, the size and type (small, large, homogeneous, heterogeneous, extent of vascularization, stage, type of expressed antigen) are important for the treatment of a tumor by means of the combination preparations according to the invention, regardless of which antitumor agent is associated with it can. For heterogeneous tumors that are not or poorly localized, it is desirable to achieve a constant plasma concentration of active substance. In this case, multiple, systemic applications, preferably iv injections, of component 3 are advantageous. On the other hand, localizable tumors, for example the lymph nodes or the peritoneum, will require local application, possibly several times. With regard to the two-step process already mentioned, the period of administration of component 3 (after application of components 1 and 2) depends on which binding affinity component 1 (for example consisting of a haptenized monoclonal antibody with specific reactivity for a specific tumor antigen) for the antigen in question. With a high binding affinity, the time interval can be from one day to several days. The dose of the active ingredient to be used (Z) advantageously follows stoichiometrically the amount of components 1 and 2 previously applied. It follows that the dose of component 1 is ultimately the factor which determines the effect of the active ingredient used (Z in component 3 ) influenced and conditional - provided that all three components are used in a stoichiometric ratio to each other, which of course is basically assumed here, and which also represents one of the advantages on which the invention is based. In this context, it can generally apply that a high tumor concentration or a high concentration on the target organ or on the target tissue is to be aimed for with regard to component 1, so that the choice of a suitable component 1 as a binding-specific reagent is clearly based on its binding affinity at the target site or target antigen.

Guidelines for such applications of antibody-bound radioisotopes, active substances or "prodrugs" can be found in YUAN, F et al, Cancer Res. 5_1, 31 19

31, 30, 1991. The use of a "prodrug", that is to say a drug or active substance precursor, is also included in so far as such a prodrug can be equipped with a determinant, for example a hapten. An example of converting or activating a relatively non-toxic prodrug at the target site (e.g. a tumor) into a cytotoxic agent is the conversion of etoposide phosphate to etoposide. This can be achieved by targeting or pre-targeting with the combination preparations according to the invention with the proviso that component 3 is an alkaline phosphatase provided with a determinant Ha2 and components 1 and 2 have the corresponding described or corresponding properties . The enzyme binds to Y of component 2 via Ha2, and this in turn binds to component 1 via Ha- of component 1, in this case a tumor. After a suitable time delay of approximately 5 to 24 hours, which among other things Depending on the type of tumor and its location (as already described in detail above), etoposide phosphate is applied in the usual way (i.v., i.m., i.p., i.e., s.c.) in a dose in which this substance is not toxic. The toxicity of etoposide phosphate: etoposide is approx. 1: 100. At the destination, the relatively non-toxic etoposide phosphate is converted enzymatically into the cytotoxic etoposide by the alkaline phosphatase (component 3) and only develops the cytotoxic effect on the tumor. In this way, cytotoxic active ingredients can be administered to a mammal in a non-toxic precursor as a prodrug for therapeutic purposes by using the combination preparations according to the invention while largely avoiding undesirable side effects. In this case too, the two-step process is an advantageous and therefore preferred process for using the combination preparations according to the invention.

Successful activations of prodrugs via conventionally conjugated antibodies are described, for example, by SENTER, PD et al., Proc. Natl. Acad. Be. USA 85, 4842-4846, 1 988, and by SENTER, PD et al., Cancer Res. 49, 5789-5792, 1989. As already described at the beginning, bispecific antibodies can mediate a targeted transport of active substances or active molecules or effector cells. The use of the combination preparations according to the invention naturally includes, and in a manner analogous to that already disclosed in detail for imaging, likewise the targeting of such agents. Just to illustrate this state of affairs, for example, Z according to Table 1 can be a CD3 haptenized with Ha2 and X can be an anti-tumor antibody haptenized with Ha, where Y has the bispecific property anti-Ha -]: anti-Ha2. Similarly, Z in Ha2 - Z can be another trigger molecule (e.g. CD4, CD8, CD1 5, CD1 6, CD1 9, CD25, CD28, CD30, CD32, CD58, CD59, CD64) and combinations thereof (e.g. CD3 / CD30, CD3 / CD 19), any enzyme (for example urokinase, tPA, alkaline phosphatase), any effector cell (CTL, NK cell), any cytokine (interleukins, interferons), an antibody directed against pathogenic pathogens (for example anti-nucleoprotein , anti-hemagglutinin from influenza viruses, anti-HIV antigens). For X in Ha - | - X stands for any diagnostically and therapeutically relevant antigen, regardless of whether it is a tumor-associated antigen (TAA) or an antigen produced by microorganisms, viruses, plants, or by these and other biological systems (e.g. bacterial, plant or fungi originating toxi) or antigens occurring in non-biological systems.

In accordance with the diverse and flexible combination possibilities of the combination preparations according to the invention, which represent one of the further fundamental advantages, any effector molecules or trigger or activator molecules can be combined, in particular for tumor immunotherapy, and simultaneously, sequentially or consecutively or, in particular with regard to component 3, applied at any delay. For example, for the treatment of malignant tumors, such as B-cell lymphomas or chronic lymphocytic leukaemias, melanomas, oval carcinomas, activated autologous T cells can be caused to lyse these malignant cells.

"Dormant" T cells can be activated, for example, by choosing an ant-CD3 antibody for X in component 1, which antibody has a suitable determinant Ha-, for example a hapten, for example 2,4-dinιtrophenyl (DNP) or 2-nitrophenyl (NP) was equipped or haptenized. An anti-CD19 antibody can be selected for Z, which is, for example, haptenized with a hapten Ha2, which is different from 61,

Ha-, for example with digoxigenin (DIG) or any other hapten other than Ha-. According to this constellation, Y is an anti-Ha,: anti-Ha2 (anti-DNP: anti-DIG or anti-NP: anti-DIG) bispecific antibody. Component 1 can be administered systemically together with component 2 or separately, as already described, preference being given to application of component 3 which is delayed by the administration of components 1 and 2. According to BOHLEN, H. et al., Blood 82, 1 803-1 81 2, 1993, it can be advantageous if, in addition to the use of the combination preparations according to the invention, the T cells are co-stimulated, in particular by an additional, in particular systemic administration of a corresponding antibody against a cell surface antigen, for example a monospecific anti-CD28 antibody. With such a measure, it could be shown, for example in chronic lymphocytic leukemia (CCL) in vitro, that there is an upregulation of T cell activation markers (CD25, CD38) and cytotoxicity against autologous chronic lymphocytic leukemia cells and allogeneic B cells (BOHLEN, H. et al., 1,993, loc cit.). The stimulation of the resting T cells with such a combination, in particular together with CD28 antibodies, preferably led to the proliferation of CD4 + T cells, the induction being increased 14-fold compared to CD8. The dose required for a T cell activation can be between 1 ng / ml and 1000 ng / ml of the corresponding antibodies (components 1 and 3 and the additional antibody, here anti-CD28), in particular around 100 ng / ml .

With another combination preparation according to the invention, which in addition to the aforementioned combination (Ha-, - antι-CD3 and Ha2 - anti-CD19 for Ha- _| - X or Ha2 -) additionally a second combination of the shape Ha - | - anti-CD28 for component 1 (Ha-] - X) and Ha2 - anti-CD22 for component 3 (Ha2 - Z), a further advantageous application can be considered, where Y has the corresponding bispecific specificity (BOHLEN, H . et al., Cancer Res. 53, 4310-4314, 1993).

According to our own findings, the combination CD3 x CD28 or CD3 x CD19 for X and Z in Ha-j - X or Ha2 - Z, optionally in a further combination with CD19 or CD28, must have a particularly advantageous effect in the immunotherapy of tumors, in particular of B-cell tumors. A combination preparation according to the invention which is preferred in this way consists, for example, in that X in Ha -] - X an anti-CD3 antibody and Z in Ha2 - Z an anti bs

Is CD28 or an anti-CD 19 antibody, with a corresponding bispecific reagent according to anti-Ha -, - Y - anti-Ha2 being selected for component 2. Both a monovalent and a bivalent hapten are suitable for Ha2. Preferred reagents for X and Y are monoclonal antibodies or bispecific monoclonal antibodies with the respective specific activities.

The described uses of the combination preparations according to the invention both in vitro and in vivo in diagnostics and therapy, for prophylaxis, for the prognosis and for the determination of tumors are of course not only restricted to the tumors mentioned by way of example, but also include all solid and diffuse tumors Mammal including humans. In particular, epithelial tumors of the ectoderm and endoderm, carcinomas, mesenchymal tumors of the mesoderm, sarcomas, embryonic tumors from undifferentiated tissue such as, for example, nephroblastoma, neuroblastoma, medulloblastoma, retinoblastoma, embryonic rhabdomyosarcoma, human tumor, endocrine formation, and endocrine formation , HCG), intercranial, neuroepithelial, spinal tumors, melanomas, gliomas, tumors of the lymphatic system (lymphomas), tumors of the blood tissue (leukaemias). Likewise, the use of the combination preparations according to the invention is not limited to the examples and literature citations selected in the present description, but of course and without exception include all targeting systems known and described to the person skilled in the art, insofar as these are associated with antibody / antigen interactions.

In addition, the combination preparations according to the invention are suitable for immunization, for example with tumor proteins or tumor cells (for example lymphoma cells). For these purposes, component 2 can be a bispecific antibody with anti-Ha- | : anti-Ha2 or anti-Ha - | : anti: Ha _n specificities, component 1 an idiotype endowed with a determinant Ha-, preferably a hapten, for example antigen-presenting cells, for example dendritic cells, and component 3 one with a determinant Ha2 or more determinants Ha _n , preferably Haptens, equipped antibodies, for example an antibody directed against "human γ heavy chain". Ha- and Ha2 can be DNP and DIG, for example. Equimolar amounts of the three components can be mixed in vitro and the resulting complex (X-Ha-μanti-Ha-, - Y - anti- Ha2: Ha2 "Z) for immunization in a mammalian organism via the known routes, for example iv or ip.

The principle of the combination preparations according to the invention, which is described in detail in the present description and which is understandable to the person skilled in the art in its entire range of applications, clearly shows that, in addition to malignant tumors, others which are pathologically changed, conspicuous and typical compared to a healthy state of cells, tissues and organs antigenic properties that can be recognized by known methods are also included. This applies in particular to infectious diseases caused by viruses, bacteria, parasites, mycoplasma, fungi and the other antigens associated with pathological conditions mentioned in the present description. However, the use of the combination preparations according to the invention in autoimmune diseases and diseases of the blood coagulation system and the use for the purpose of preventing rejection reactions and for preventing or triggering immunosuppression are also included. Because the uses of the inventive described in detail

Combination preparations can be easily applied to these indication areas and transferred analogously.

All processes relevant for carrying out the invention, such as radioactive labeling, haptenizing, the synthesis of antibodies, fragments and derivatives thereof, assays, chemical linkages of ligands or syntheses of low molecular weight compounds are known to the person skilled in the art or are described in the literature. The same applies to the applications themselves, which can be transferred in a simple manner to the described methods of the conventional systems using the combination preparations according to the invention.

The combination preparations according to the invention are also advantageously suitable for the purpose of determining all possible antibodies in known antigens and vice versa also for determining antigens in known antibodies, for example in the form of the neutralization or agglutination inhibition test. The use of the combination preparations according to the invention is therefore not limited to the field of diagnosis of pathological conditions in the narrower sense, but generally includes searching, recognizing and Determination of any antigen (antigen screening and antigen diagnostics) for the detection or diagnosis of a physiological state of biological systems, for example a mammal or human (for example proof of pregnancy), an irregular state of a biological system, for example a pathological physical state or a disease of a mammal or human (e.g. tumor diagnosis, infection diagnosis) or a condition caused by taking pharmacologically active substances (e.g. misuse of medication, detection of drug or drug metabolites or of active substance or drug accumulation, half-life determination of pharmacologically active substances, bioavailability and clearance the same, drug or drug addiction) in a mammalian organism or in a human, insofar as these conditions are accompanied by the occurrence of antigens and / or antibodies specific for them and Marked are. In addition, a sample to be examined can be a non-biological liquid medium if it contains agglutinable, markable or detectable antigens and / or antibodies.

On the basis of these disclosures, the person skilled in the art accordingly has new and advantageous methods for producing pharmaceutical preparations which can be used for immunoprophylaxis, immunotherapy and for immunization and which comprise the steps a) providing at least one combination preparation consisting of components 1, 2 and 3 according to Table 1 , with the definitions given in the present description for X, Y, Z, Ha -, and Ha2 or Ha _n , or b) providing at least one combination preparation consisting of components 1 and 2 according to Table 1, with those given in the present description Definitions for X, Y, Ha-] and Ha2 or Ha _n , c) Identifying a biologically, chemically or physically active agent with regard to its immunoprophylactic or immunotherapeutic properties or its immunizing properties by means of the combination preparations provided in a) and b) via the open at the top rten determination and screening method d) formulation of the agent obtained from c) in the form of a combination preparation according to Table 1, with the definitions given in the present description for X, Y, Z, Ha -, and Ha2 or Ha _n , with the known auxiliaries and excipients, or d) formulation of the agent obtained from c) by combining with a monospecific reagent, i8 which can bind to biological binding structures by known methods and with the known auxiliaries and carriers, or e) formulating the agent obtained from c) by itself using known methods with the known auxiliaries and carriers

The monospecific reagent can advantageously be a protein, an immunoglobulin or an antibody or a derivative or fragment thereof, a ligand, a lectin, a receptor binding molecule, an adhesion molecule, a cytokine, a chemokine, a lymphokine. The named biological binding structure is defined as disclosed in the description or in the patent claims.

It is thus also clear to the person skilled in the art that the use of an agent which consists of a combination preparation comprising components 1, 2 and 3 according to Table 1, with which in the Definitions for X, Y, Z, Ha-] and Ha2 or Ha _n given in the present description, or consisting of a combination preparation comprising components 1 and 2 according to Table 1, with the definitions for X, Y, Ha given in the present description - | and Ha2 or Ha _n , or consisting of component 2 according to Table 1, with the definitions for Z given in the present description, in one of the above-mentioned production processes for pharmaceutical preparations for the purpose of immunoprophylaxis, immunotherapy or immunizations.

The process products obtained directly from the processes mentioned for the production of both pharmaceutical preparations for immunoprophylaxis, immunotherapy and for the purposes of immunization and of immunodiagnostics to be used in vitro and in vivo can be formulated by known methods and, if appropriate, with the known auxiliaries and excipients to be mixed together. The person skilled in the art has extensive prior art available for the respective type of use and for the dosage of these products. Thus, according to the production method according to the invention, an agent which is advantageously recognized for a specific therapeutic or diagnostic purpose, for example a cytotoxic or cytolytic agent (for example a Antibodies or an antibody heteroconjugate) or a labeled antigen can be taken up or dissolved in PBS (phosphate buffered saline), for example in a concentration range from 1 to 1000 μg / ml, or it can be lyophilized, the lyophilisate being prepared by known methods in physiological saline, PBS or can be reconstituted in sterile water. In general, it can be assumed that, for example in the event that the agent recognized as being advantageous is a protein (for example an antibody) or is present as a constituent in this agent, it is used in the amount range between 1 and 1000 μg. The agents identified according to the invention or the agents provided via the manufacturing methods according to the invention can furthermore be characterized further by suitable assays with regard to their effects and properties, any in vitro or in vivo immunoassay being considered. Examples of this would be an in vitro cytotoxicity assay (for example the "51 Cr release assay"), for example according to RAMMENSEE; H.-G. et al., Eur. J. Immunol. V Λ33 - 436, 1 987, or according to JUNG, G. et al., Proc. Natl. Acad. Be. USA 83: 4497-4483, 1 986, or an in vivo anti-tumor assay as described by, for example, GARRIDO, MA et al. , Cancer Research 50: 4227-4232, 1990.

According to the invention, kits are also made available for the above-mentioned determination, screening and production methods for the advantageous implementation of these methods, the term “kit” also comprising “kit of parts”, and these kits can contain the following constituents: a ) Components 1, 2 and 3 according to Table 1 with the definitions given in the present description for X, Y, Z, Ha-] and Ha2 or Ha _n , or b) Components 1 and 2 according to Table 1 with the in definitions given for X, Y, Ha-] and Ha2 or Ha _n in the present description, or c) component 2 according to Table 1 with the definition given in the present description for Y, or d) components 1, 2 and 3 or 1 and 2 or 2, as defined above, together with at least one further different component 3, optionally together with auxiliaries and carriers. The different components 3 provided in kit d) can differ in terms of their material composition and / or in terms of their biological, chemical and physical properties. With regard to "inert particles", these are particularly suitable for diagnostic purposes (eg immunoassays) and include all the particles known to the person skilled in the art on which proteins or protein fragments can be adsorbed. If proteins are selected as antibodies, these inert particles can be loaded in such a way that they assume bispecific or multispecific properties and can be regarded as particular forms of a bispecific or multispecific antibody. The use of such particles has the advantage that complex chemical or genetic engineering processes for the construction of antibody heteroaggregates, chimeric antibodies or "cross-linked antibodies" can be avoided. Such inert particles loaded with immunoreagents or antibodies ("coated") adsorbed thereon are known to the person skilled in the art and are described in the prior art

In the more specific sense, "inert particles" are understood to be particles to which

1 Can bind or adsorb antibodies or derivatives or fragments thereof. All natural, synthetic, organic or inorganic particles known to those skilled in the art to which antibodies, derivatives and fragments thereof can be adsorbed can be used for this purpose. For example, blood cells (erythrocytes, such as Toxocell IHA 0 from Redιtest, SA »Barcelona, or Mast Serodia-Anti HBs FD 41 1 from Mast Diagnostica, Hamburg, or according to BIRD, T. & STEPHENSON, JH, J. clin. Path. 26, 623 627, 1973, or Serodia-AFP from Fujizuki Pharmaceutical Co., Ltd., Japan), latex or polystyrene balls (for example from Tokuyama Soda Co., Ltd., Japan, or as described in the EP -A 0435851 or from KOLBER MA, J. 5 Immunol. 143, 1461 - 1466, 1989), microorganisms (bacteria and fungi, for example Salmonella, Candida albicans or Yersinia according to Yersinia Agglutube from Labor Diagnostika GmbH, Heiden, Germany , or Serratia according to SAUL, F. et al. Phys. Lab. 23, 407-41 1, 1977 (Blue ASO test), bentonite (REISBERG, MA et al., J. Immunol. 105, 1 1 51 , 1970), carbohydrates

30 such as cellulose (CAMPBELL, DH et al., Proc. Natl. Acad. Sci. USA 37, 575, 1 951; WELIKY, N. & WEETALL, HH, Immunochemistry_9, 967, 1 972), Sepharose (WILCHEK, M . et al., Biochemistry 10, 2828, 1971), glass beads (WEETALL, HH, J. Biochem. 1 17, 257, 1970), styrenes and derivatives thereof (EP-A 04661 70), liposomes, metal oxide particles, charcoal particles, gelatin (For example Serodia's ™ HIV from Fujirebio Inc., Tokyo, Japan). ? /

Proteins, for example antibodies or fragments thereof, can be attached to the

Inert particles known to the person skilled in the art, for example polystyrene balls, are bound in a manner known to the person skilled in the art in that a suitable antibody known to the person skilled in the art, for example from the American Type Culture Collection. Rockville, Maryland, USA, (ATCC), from Linscott's Directory of Immunological and Biological Reagents, 40 Glen Drive, Mill Valley, California, USA, 94941, or from other known commercial sources of publicly available antibodies (e.g. OKT3 [ATCC, Hybridoma CRL8001 ], OKT4 [ATCC, Hybridom CRL8002] or described in EP A 0468 637) in a suitable solution and under suitable conditions, for example NaHCO 3, at room temperature with polystyrene balls, followed by a physiological solution (for example phosphate-buffered saline, PBS) washed with an inert protein (e.g. cattle serum albumin, BSA). The size of the polystyrene balls is not critical. A diameter of these polystyrene spheres in the μ range or from approx. 1 μ to 20μ can be regarded as a standard approximate value. Suitable polystyrene balls are available, for example, from Polysciences, Warrington, PA, USA. An inert particle coated in this way with, for example, a bispecific antibody or derivative or fragment thereof thus receives the properties of a bispecific particulate antibody and can be used according to the description and the examples according to the invention.

Accordingly, inert particles produced analogously as part of Y in component 2 of the combination preparations according to the invention are also included in the invention.

With regard to the term immunogen or antigen on which the present invention is based, all those substances are encompassed which can induce an immune response in a mammalian organism and thus an immunoglobulin (antibody synthesis). In this respect such an antigen can be of natural origin, via DNA recombination or synthetic or have been made or are part of these, such antigens may occur extracellularly, intracellularly, transcellularly (third space) or interstitially, such as blood, serum and fractions thereof, excrement (such as urine, etc.) in biological fluids of a mammalian organism including humans. Faeces), tear fluid, saliva, amniotic fluid, tissue fluid, ascites, seminal fluid, pleural fluid, cyst fluid, Cerebral fluid, pus, secretions of the gastrointestinal tract, cerebrospinal fluid, eye chamber fluid, edema fluid, pathologically occurring fluids in, for example, hay or peritonitis, secretions, cerebrospinal fluid. Plasma, lymph or liquids, including those obtained from tissue extracts and found in tissue and cell cultures.

Antigens which are associated with a cell occurring in the mammalian organism, a tissue, an organ or a synthesis product thereof or which can be assigned to a disease state, for example immunoglobulins or antibodies in the sense of the definition given above, proteins, are particularly suitable. polypeptides, oligopeptides, hormones (for example, steroid hormones such as estrogens, progestins, androgens, glucocorticoids, mineralocorticoids, cholecalciferols, proteohormones, biogenic amines, oxytocin, vasopressin (ADH), insulin, glucagon, parathyroid hormone, calcitonin, erythropoietin, prostaglandins, gonadotropins such as luteinizing Hormone (LH), chorionic gonadotropin (HCG or ß-HCG), follicle stimulating hormone (FSH), prolactin and other placenta hormones, serotonin, histamine, bradykinin, kallikrein, gastrointestinal hormones, thyroid hormones, catecholamines, enzyme, tumorylcholine) Tumor antigens (such as carcinoembryonic antig en (CEA), Reπal Cell Carcinoma Antigen (RAGE), Melanoma Cell Antigen (MAGE), Tumor Associated Antigen (TAA), alpha-1 -Fetoprotein (AFP), CA 15-3, CA 19-9, CA-50, CA -125, Postata-specific acid phosphatase (PAP), neuron-specific enolase (NSE), Bence-Jones protein, alkaline phosphatase (AP), prostate-specific antigen (PSA), hormones endocrine tumors, LDH, Gamma-GT, neopterin, ferritin.

However, antigens which originate from, or are part of, viruses, prokaryotes and eukaryotes which are not assigned to a mammal are also suitable. In particular microorganisms such as Gram-negative and Gram-positive bacteria (for example the families or orders Enterobacteriaceae [for example the genera Escherichia, Salmonella, Shigella, Yersinia], Corynebacteriaceae, Spirochetales [for example the genera Treponema, Leptospira, Borrelia], Pseudomonadaceae [Mycoplasmat for example the genus Mycoplasma], Mikrococcaceae [for example the genus Staphylococcus], Chlamydiae, Bacillaceae [for example the genera Bacillus, Clostridium], Lactobacillaceae [for example the genus Streptococcus], Neisseriaceae [for example] the Genusillissea for example the Vibrio genus], Brucellaceae [for example the genera Hemophilus, Bordeella, Francisellaj, Actinomycetales [for example the genera Mycobacterium, Actinomyces, Nocardia, Streptomyces], Rickettsiaceae [for example the genus Rickettsia], Chlamydiae [for example the genus Chlam of which (for example Candida, Cryptococcus, Blastomyces, Paracoccidoides, Coccidoides, Aspergillus, Histoplasma), and the toxins or endotoxins and exotoxins synthesized by the microorganisms mentioned, furthermore antigens based on DNA and RNA viruses, for example viral capsid antigens or envelope proteins ( for example hepatitis viruses and related antigens, such as, for example, HBe, HBc, HBs, poxviruses, coxsackie viruses, echoviruses, myxoviruses, rhabdoviruses, togaviruses, reoviruses, for example HIV, tumor viruses, picornaviruses, herpesviruses, paramyxoviruses, rump virus, rubellaviruses, rubellaviruses Coronaviruses), endo- and ectoparasites, especially protozoa (such as C estodes, flagellates, amoebas, Leishmania, Trichomonas, Trypanosoma, Sarcocystidae including Toxoplasma, Eimeriidiae, Plasmodium, Cryptosporiidae) and the toxins and metabolites synthesized therefrom and tumor-specific proteins, including those of cells infected or transformed by tumor viruses, which are in the corresponding host Cause immune response. Also included are antigens produced using the known methods of DNA recombination, for example as described for Toxoplasma gondii in EP-A 0431541, and antigens which can be prepared using the known synthetic and semisynthetic processes. However, allergens are also included since, like the antigens described, they initiate antibody synthesis, in particular IgE, for example mites or house dust. Antigens based on drugs and medicaments and metabolites thereof (for example opiates and opioids, cocaine, diazepines, barbiturates, paracetamol, theophylline) and chemically produced toxins are also included.

But antigens of plant origin are also included by definition, for example pollen-associated antigens or antigenic substances dissolved in plant juices.

However, antigens which occur in non-biological systems and are to be investigated are also suitable. For the purposes of the present invention, “non-biological systems” or test samples can contain waste water,

Be drinking water, standing or flowing water or groundwater or Those which are not bound to the occurrence in biological systems or organisms, such as cell cultures or a mammalian organism and which contain insoluble, particulate immunologically active material in the above sense or which is in a soluble form and, if necessary, subsequently bound to particles in a known manner or can be adsorbed or chemically treated according to known methods and modified or polymerized accordingly.

Accordingly, the invention also includes a priori soluble antigens which can be made insoluble by the known methods, for example by polymerization with ethyl chlorocarbonic acid ester according to AVRAMEAS, S. & TERNYNCK, T., J. Biol. Chem. 242, 1,651-1,659, 1967, or by using glutaraldehyde according to AVRAMEAS, S. & TERNYNCK, T., Immunochemistry 6, 53, 1 969, or from ethylienmaleanhydride according to CENTENO, ER & SEHON, A.H., Immunochemistry 8, 887, 1971. Accordingly, the use of the combination preparations according to the invention can be used not only for a priori particulate antigens or for corpuscular antigens bound to inert particles, but also for a priori soluble antigens.

The term “hapten” is understood by the person skilled in the art to mean low-molecular compounds which do not in themselves produce antibody production and generally have a molecular weight of less than 1000 daitons. If such haptens are bound to a substance which triggers an immunological reaction (for example a protein), antibodies can be obtained which are directed specifically against the hapten or haptens in question. Examples of haptens which according to the invention for Hapteπisierung the respective components of the Kombinationspraparationen are, 2-nitrophenyl, nitrophenyl, digitoxigenin, digoxigenin, choline chloride, Aminophenylphosphochloride, glycerophosphocholine, nitrophenyl acetate, Trinitropheπyl, Trinitrophenylglycin, dimethylsulfoxide, guanidine hydrochloride, 4-hydroxy-nitrophenylacetate 3, 2 , 4-dinitrophenyl, benzyl-EDTA, to name but a few. Such low molecular weight compounds suitable as haptens and their preparation are known to the person skilled in the art and are also generally commercially available, for example from Sigma, Deisenhofen, Germany. X and Z can be haptenized by all known methods which are known to the person skilled in the art or can be found in the literature. A haptenization with, for example, a nitrophenyl derivative (for example p-nitrophenyl phosphate) of X and Z can take place, for example, by the agent in question, for example a protein (for example an antibody or a fragment or a derivative thereof) in NaHCÜ3 (for example 0.1 M) , pH 8.5) in the presence of NaCl (for example 0.1 5 M). A volume of approximately 1 ml thereof is added in 10 to 100 μl of, for example, p-nitrophenylphosphate caproic acid o-succinimide ester (in an aprotic solvent, for example dimethylformamide, for example 20 mg / ml). After about 1 hour at room temperature, about 100 μl of a 1 M ethanolamine solution, pH 8.5, are added and dialyzed against phosphate-buffered saline (PBS), pH 7.3. The binding rate of hapten.'protein can then be determined by means of photospectrometry on the basis of the different adsorptions of hapten and hapten protein at different wavelengths.

According to another method, a haptenization of the relevant protein to be haptenized can be carried out in the corresponding activated ester, for example nitrophenylphosphate caproic acid o-succinimide ester, which is dissolved in an aprotic solvent, for example dimethylformamide, and in 0.1 M NaHCO 3, pH 8.5 (in the presence of 0.1 5 M NaCl) is carried out. After a reaction time of approximately 1 hour at room temperature, the haptenization is complete and the material can be dialyzed against PBS.

It should be emphasized at this point that not only the methods of haptenizing are known to the person skilled in the art, but also that all other methods and processes (markings, couplings, conjugations, syntheses and measurements) necessary for carrying out the present invention belong to the prior art .

Cellular adhesion molecules are to be understood as surface molecules of leukocytes which mediate the adsorption or docking on cellular structures (cells, tissues, for example endothelial cells) in order to co-stimulate them against foreign substances and particles (for example bacteria, viruses, fungi, modified cells) Interaction with other factors (such as CD8 + lymphocytes) to act by lysing the cell in question. Such surface molecules of T cells. are known. For example, the "Lymphocyte Function-associated Antigen-1" (LFA-1) heterodimer located on most lymphocytes or the heterodimer Mac-1 located on macrophages, granulocytes and lymphocytes. Such surface molecules have a crucial function in cell adhesion and belong to the family of certain glycoproteins (KEIZER, G., et al., Eur. J. Immunol. 15, 1 142 - 1 147, 1 985) with a chain and a β- Chain (SANCHEZ-MADRID, F. et al., J. Exp. Med. 1 58. 1785 - 1803, 1983), the latter also known as CD18. Such adhesion molecules are included in the invention (component 1) according to the definition of the scheme shown in Table 1 (component 1) - but also ligands that bind to LFA-1, such as intercellular adhesion molecules ICAM-1 (CD54) or ICAM-2, which as Members of the immunoglobulin superfamily are included, as well as derivatives and fragments thereof. For example, ICAM-1 is known to be localized on the surface of endothelial cells. For example, if in component 1 X in the scheme given above, an antibody directed against such an adhesion molecule, a derivative or a fragment thereof, is linked to a hapten Ha-], Y is a bispecific reagent with anti-Ha- | and anti-Ha2 properties and Z a reporter molecule linked to a hapten Ha2 (for example an enzyme, dye molecule, radioisotope), targeted targeting can thus be carried out to localize infection sites and inflammation sites, since it is known that such ligands (for example ICAM-1) be expressed at such locations.

It is known that CD28 is only expressed by about half of all CD8 + lymphocytes, in contrast, practically all CD4 + lymphocytes express CD28 (LINDSLEY, PS & LEDBETTER, JA, Annu. Rev. Immunol. H, 1 91 - 21 2, 1 993). It was found that the cytotoxicity caused by bispecific monoclonal antibodies is mainly mediated by CD8 + lymphocytes and effector cells and that blocking the LFA-1 / ICAM-1 or CD2 / LFA-3 pathway reduces this cytotoxicity. Therefore, adhesion molecules have a crucial function as co-stimulators in T-lymphocytes activated by bispecific monoclonal antibodies, whereby the signal transmission is enhanced by the CD3 / TCR and CD28 pathways. This results in a further therapeutic use of the combination preparations according to the invention, which consists in that Z in Ha- | - Z is an adhesion molecule, X in Ha -, - X is a T cell activating component and Y is anti Ha - | : has anti-Ha2 specificities. With such a system, disease states which are associated with a deficiency of adhesion molecules, for example ICAM-1 or ICAM-2, can be treated or diagnosed, the modes of application described above being transferred for these purposes.

The combination preparations according to the invention can be in liquid or solid form and can be filled and packaged in bottles, ampoules or other containers equivalent thereto, preferably in glass or plastic containers.

With regard to the presence of components 1, 2 and 3 of the combination preparations according to the invention in liquid form, the buffers and media known to the person skilled in the art can be used, if appropriate together with a stabilizing agent. Correspondingly, for example, tris, phosphate (for example phosphate buffered saline, PBS) or carbonate buffer, sterile water, single or double distilled water or physiological saline are suitable. The known and customary additives, such as, for example, albumins or serum albumin (bovine serum albumin [RSA], human serum albumin [HSA]) or other inert proteins, but also biocides, are suitable as stabilizing agents. For veterinary and human medical use, of course, only the toxicologically safe and physiologically inert additives and stabilizers mentioned and known to the person skilled in the art are suitable. However, the combination preparations according to the invention or the parts thereof can also be in lyophilized form if they exist as a product in solid form. In such cases, the techniques known to those skilled in the art of lyophilization and the reconstitution to be carried out before using the corresponding antibodies (for example with one of the buffers and media mentioned) can be used. For components 1, 2 and 3, the lyophilized form is preferred if these are present as substances accessible for lyophilization. This is particularly the case when components 1, 2 and 3 are a protein or a protein associated with a coloring and / or an effector substance (for example a radioisotope, a reporter molecule, a contrast agent). The reconstitution can preferably be carried out using sterile water for injections or using PBS, for example pH 7.0 - 7.2. In general, however, the individual components can be formulated without restrictions according to the known methods as are used and known in pharmaceutical chemistry. In principle, the individual components can be dissolved or suspended in all known physiologically harmless, preferably sterile, liquids, insofar as they are suitable for parenteral administration, such as, for example, physiological saline (PBS), water for injection purposes.

The carriers and their formulations, for example human serum albumin (HSA), suitable for the combination preparations or parts thereof according to the invention are described, for example in Remington's Pharmaceutical Sciences, 16th ed. (Osol, A., Ed.), 1980.

In addition, pharmaceutical methods can also be used to control the duration of the effect of the combination preparations according to the invention. Preparations for the controlled or controlled release of the combination preparations according to the invention (controlled release of active substance) can be obtained by using polymers to which the combination preparations according to the invention (for example the corresponding proteins or antibodies or parts thereof) can adsorb or complex. The controlled release can then be selected by selecting the available polymers or macromolecules, for example polyester, polyvinyl, polyamino acids, methyl cellulose, carboxymethyl cellulose, pyrrolidone,

Ethylene vinyl acetate. In addition, the combination preparations according to the invention can be incorporated into particles, as a result of which a controlled release or release of active substance can also be achieved. For this purpose, the particles in question are to be selected from polymeric materials, for example polyesters, hydrogels, polyamino acids, polylactic acid. It is also possible to achieve such effects by microencapsulation of the combination preparations according to the invention. In such a case, the known methods (for example coacervation) with, for example, hydroxymethyl cellulose or microencapsulations in gelatin capsule or methyl methacrylate or polymethyl methacrylate or colloidal systems, for example liposomes, microspheres (for example albumin), emulsions are available. Such methods are known to the person skilled in the art and can be found in Remington's Phramaceutical Sciences, 1980. ¥ 9

The ready-to-use formulations are preferably used when the combination preparations according to the invention are to be used for in vivo purposes. Of course, the same or equivalent formulations are suitable for in vitro purposes, the physiological safety being of no importance here.

As an exemplary guideline, it can be assumed that the corresponding lyophilisates of components 1, 2 and 3 can be dissolved in a ratio of 1:10 (ml of aqueous solution / mg of the respective component). For example, a ready-to-use formulation can consist of 10 mg of each component 1, 2 or 3, or equimolar amounts thereof, preferably when X, Y or Z is a protein, dissolved in 1 to 10 ml of PBS, pH 7.2 become; or 5 mg per component, or equimolar amounts thereof, can be dissolved in 0.5 to 5 ml sterile water for injections. The combination preparations according to the invention are either in ready-to-use dilution or concentration or they can be further diluted or concentrated as desired, the person skilled in the art knowing which individual concentration or dilution he would like to use depending on his intended use. For example, X, Y or Z according to Table 1, in particular if these components are proteins, for example antibodies, in a titer range from 1: 1 to 1: 10,000, for example 1:10 to 1: 1000, in particular around the range of 1: 100 available. Dosages of 0.1 to 200 mg, in particular 1 to 50 mg, come into consideration depending on the purpose of use and the application methods known to the person skilled in the art. The above-mentioned buffers and media or sterile aqueous solutions, for example PBS, saline solutions or water, can be used to dilute the antibody concentration.

The manner of administration or application of the combination preparations according to the invention in the form of components 1, 2 and 3 for in vitro or in vivo diagnostics and for medical / clinical purposes (therapeutic or prophylactic or for the purpose of immunization) is also in the Description including the prior art cited therein for illustration and can be found in detail in the known literature, so that the applications and administrations for the purposes mentioned are readily possible for the person skilled in the art. Special dosages and application modalities depend on individual circumstances, as in the present description and described in the literature and are thus also known to those skilled in the art. In particular with regard to the medical / clinical applications in vivo for diagnosis, therapy and immunization, including prophylaxis, the entire spectrum known to the person skilled in the art can be considered for administration without restrictions. For example, the administration of components 1, 2 or 3 of the combination preparations according to the invention according to Table 1 can be intravenous, intraarterial, intraperitoneal, intrapleural, intrathecal, subcutaneous, by perfusion, for example via a catheter, or by injection, for example by means of a syringe, systemically or locally, for example by direct intralesional injection or by injection into the vicinity of the organ or tissue to be treated.

In general, the application is carried out in the form of a sterile aqueous solution, as described above. For the individual components 1, 2 and 3 or for X, Y and Z according to Table 1, amounts in the ng, μg or mg range can be used, depending on the type of disease to be treated. For in vitro applications the doses can be in the ng and μg range, whereas for in vivo applications doses in the μg and mg range can be considered depending on the response rate or effect, the disease and the reactions of the treated Patients on the administrations. Based on these parameters, the person skilled in the art knows when the dosages are to be reduced or when they have to be increased. In general, doses between 0.5 and 200 mg, in particular between 5.0 and 100 mg, can be assumed for the individual components in these cases. The application routes also depend on the type, location and degree of the disease. In the case of, for example, lung and breast tumors or in the case of leukemic tumors or inflammatory diseases or for the prevention of rejection reactions, intravenous, intraarterial or intrapleural administration should preferably be used. Intraperitoneal administration is preferred for ovarian tumors or for diseases of the peritoneum. In the case of tumors and inflammatory diseases of the brain, intrathecal and intravenous administration can be considered in particular. Subcutaneous doses will be used, for example, for skin lesions, Hodgkin's disease or lymphomas. Perfusions by means of a catheter can preferably be considered if metastatic tumors, for example the lungs, the breast or to be treated by the liver. In the case of diseases of limbs or organs to be treated locally or in isolation, isolated perfusion can be carried out according to known methods

Methods are more promising than, for example, systemic intravenous administration.

Components 1, 2 and 3 of the combination preparations according to the invention can be present as spatially separate components in the form of a packaging unit, in particular as a kit or kit-of-parts, or components 1 and 2 can be present spatially separated from component 3 in a packaging unit.

Legends for the figures

Fig.1: Basic schematic representation of the combination preparations according to the invention. a) Conventional system with bispecific reagents (BiAb), which specifically have a target site (T) with a binding site BS-] and an effector molecule (EM) with a binding site BS2 via the anti-BS- | and connects anti-BS2 binding sites, b) Combination preparation according to the invention, in which case X is an example of an antibody. A monospecific, with one

Determinant Ha-] equipped reagent R (χ) binds specifically to a target site (T). With the determinant Ha- | one anti-Ha 1 part of the bispecific reagent (BiAb (γj), the other anti-Ha2 part reacts with an effective or detectable agent, for example an effector molecule EM (z), which carries a determinant Ha2, where Ha- | ≠

Ha2 is and Ha-], Ha2, T, R (χ) BiAb (γ) and EM () are defined according to Ha-], Ha2, T, X, Y and Z as in Table 1.

Fig. 2: ELISA representation according to Example 7. The graphic shows the absorption spectrum at 405 nm after 30 minutes of incubation

Room temperature.

I = The plate coated with IgG4 was blocked as described, incubated with haptenized goat anti-human antibodies and then incubated with component 2 (anti-DNP: anti-DIG bispecific antibodies). In the next step, haptenized enzyme was added and, after washing, incubated with substrate. 81 ^■ II = like I, but without anti-DNP: anti-DIG bispecific antibody.

111 = like I, but without goat anti-human antibody.

Fig. 3: Flow cytometric analysis. The B cells were incubated with haptenized CD 19 antibodies. After washing, the lines were incubated with the anti-DNP: anti-DIG bispecific antibody and, after washing twice, stained with haptenized FITC.

A: All components added, (anti-CD19- [hapten], bispecific

Antibody, [Hapten) -FITC]). B: no haptenized CD 19 antibody, (bispecific antibody,

[Hapten] -FITC).

C: no bispecific antibody, (anti-CD19- [hapten], [hapten] -FITC).

D: Irrelevant first antibody; (anti-CD3- [hapten], bispecific

Antibody, [Hapten] -FITC)

The following examples are intended to describe the invention in more detail without restricting it.

EXAMPLE 1 Production of the Keyhole Limpet Hemocyanin (KLH-DNP) protein conjugated with 2,4-dinitrophenol as component 1

1 .1. Keyhole Limpet Hemocyanin (KLH) Dialysis

A dialysis tube with a length of 1 5 cm, a size of 3-20 / 32 "and a diameter of 1 5.9 mm (company Faust, Cologne, Germany) was placed in 1 liter of distilled water (dH2θ) (Fresenius, Germany) for 1 5 minutes. with 2 mM EDTA (Sigma, Deisenhofen, Germany), this solution was poured off and the dialysis tube slowly stirred for a further 15 minutes in 1 liter of dH2θ, 1 mM EDTA, then the dialysis tube was rinsed with dH2θ and in an aqueous sodium azide solution (0.02% sodium azide (Sigma) in dH2θ) at 4 ° C. KLH was concentrated in a concentration of 10 mg / ml in a dialysis volume of 15 ml 3 times against 0.1 M sodium hydrogen carbonate, pH 8.0 (Merck, Darmstadt, Germany ) dialyzed for 1 hour at room temperature for 1 hour at room temperature and then 1 time overnight at room temperature. 1 .2. Haptenization of KLH with 2,4-dinitrophenol (DNP)

The coupling of DNP to KLH (Boehringer Mannheim, Germany) was carried out according to known methods by esterification with an activated succinimide ester. It became 3-iodo-4-hydroxy-5-nitrophenyl-caprionic acid

Succinimide ester (Nip-cap-OSuc, MW 420) (Cambridge Research Biochemicals, U.K.) at a concentration of 1 mg / ml dissolved in N, N-dimethylformamide (DMF) (Merck). The dialyzed KLH and NIP-cap-OSuc were combined in a concentration ratio of 10: 1 (corresponding to a molecular ratio of 40: 1), with the result that each KLH molecule is coupled with at least one NIP-cap-OSuc molecule. The coupling reaction took place within two hours at room temperature, the reaction solution being agitated with a shaker (DPC® Micromix 5, Gwynedd, Wales, UK). The reaction solution was then dialyzed 3 times against 1 liter of phosphate buffered saline, pH 7.0 (PBS) for 2 hours each at room temperature in order to remove residues of DMF and uncoupled nip-cap-OSuc. The coupling was then verified with an ELISA. For further storage, the KLH-DNP conjugate was sterile filtered (filter: 3μ, Millipore) and briefly at 4 ° C, for longer storage at -20 ° C.

Example 2 Haptenization of the monospecific antibody OKT3 (anti-CD3) with 2,4-dinitrophenol (anti-CD3 - DNP)

2.1. Dialysis of the monoclonal, monospecific antibody OKT3

Dialysis of the OKT3 antibody (anti CD3, ATCC CRL 8001, Proc.Nat.Acad.Sci., USA 77: 4914-4917, 1980, U.S. Patent 4,361, 549) was carried out according to Example 1.1.

2.2. Haptenization of OKT3 with 2,4-dinitrophenol (DNP)

The haptenization of OKT3 with DNP was also carried out here by esterification with an activated succinimide ester, whereby according to Example 1 .2. was proceeded. For this, the according to Example 2.1. dialyzed antibody solution (1 mg / ml) with NIP-cap-OSuc in a concentration ratio of 10: 1 8t, (corresponding to a molecular ratio of 40: 1) combined and as in

Example 1 .2. described further procedure.

2.3. Detection of OKT3 (anti-CD3) -DNP monoclonal antibodies in the ELISA

A polyclonal goat anti-mouse IgG (Southern Biotechnology Association, Ine (SBA)., Birmingham, USA) with H and L chains was used as the uncoupled capture antibody. This antibody was on a 96-well plate (Immuno Maxisorp F96, Nunc GmbH, Wiesbaden, Germany) by binding to the plastic surface with a concentration of 5 μg antibody per ml PBS (Gibco BRL, Gaithersburg, USA) and 50 μl per hole at 4 ° C immobilized overnight. The free binding sites on the plastic surface were saturated with 100 μl PBS with 10% cattle serum albumin (BSA, Sigma, Deisendorf, Germany) per hole at an incubation time of 30 minutes and room temperature to prevent non-specific binding of further reagents. The OKT3-DNP antibody was not diluted at 1, 0.5, 0.2, 0.1, 0.05 and 0.01 mg / ml in PBS / 1% BSA and the corresponding positive controls (same isotype, DNP-coupled) and negative controls (different isotype) DNP coupled) pipetted onto the plate. The volume was 50 μl per well and the incubation time was 30 minutes at room temperature. It was then washed 3 times with 250 μl PBS / 1% BSA per hole. Subsequently, the supernatant of the anti-DNP antibody (IgG 2a / λ) ^{in the} dilution of 1: 10 hinzupippetiert and incubated for 30 minutes. After washing 3 times in PBS / 1% BSA, a peroxidase-coupled antibody (goat anti-mouse lambda (Southern Biotechnology Association, Inc.Birmingham, USA) was diluted 1: 2500 in PBS with 1% BSA and 50 μl substrate buffer ( 10 ml 55 mM citric acid, pH 4.0, 100 μl 2,2'-azino-bis [3-ethylbenzthiazoline] -6-sulfonic acid (ABTS) (stock solution: 15 mg / ml), 3.3 μl H2O2 (33 %) per hole and incubated for at least 15 minutes at room temperature The enzymatic color reaction (ABTS) was measured in an ELISA reader at 405 nm (Titertek Multiscan® Plus MK II, Flow Laboratories, USA) the reaction rate and had not exceeded an OD value of 1.0, so that a linear measuring range was ensured. Example 3: Preparation of a bispecific anti-2,4-dinitrophenyl: anti-digoxigenin antibody as component 2

3.1. Immunization of mice with 2,4-dinitrophenol (DNP) and digoxigenin (DIG)

For the immunization of Balb / c mice, the two haptens 2,4-dinitrophenol (DNP) and digoxigenin (DIG) (Sigma, Deisenhofen, Germany) were used using glutaraldehyde (Merck, Darmstadt, Germany) to form KLH conjugates with keyhole limpet hemocyanin (KLH) (Boehringer Mannheim, Germany) cross-linked. For this purpose, 1.0 mg DNP and 1.0 mg DIG with 5.0 mg KLH in a molar ratio of 1: 3 were dissolved in O.1 M a2HPθ4, pH 8.3, and together with 0.05% glutaraldehyde incubated on a shaker (DPC® Micromix 5, Gwynedd, Wales, UK) for 30 minutes at 21 ° C (room temperature). A volume of 2.0 ml of this solution was placed in a dialysis tube (1 5 cm long, size 3-20 / 32 ", diameter 1 5.9 mm, from Faust, Cologne, Germany) and at 4 ° C for 1 hour dialyzed against 0.5 liters of 0.1 M NH4CI, the reaction being stopped, the corresponding DNP or DIG conjugates were dialyzed against 2.0 liters of PBS at 4 ° C. for 3 hours, after which 50 μg of conjugate were combined with complete Freund's adjuvant F-5881 (Sigma) injected intraperitoneally in a total volume of 100 μl per mouse (Balb / c) These immunizations were repeated twice at weekly intervals, the last immunization being carried out in complete Freund's adjuvant has been.

3.2. Polyethylene glycol fusion and isolation of anti-DNP and anti-DIG antibody producing hybridomas

Three days after the last immunization according to example 3.1. myeloma cells (Sp2 / 0, ATCC CRL 1 581) and spleen cells of the mice immunized with both DNP and DIG in a ratio of 1: 2 by polyethylene glycol (PEG) fusion (PEG 4000, Boehringer Mannheim, Germany) in RPMI 1 640 medium (Gibco BRL) fused according to known methods as follows: 50 x 10 ⁶ Sp2 / 0 myeloma cells and 10 x 10 ⁷ spleen cells, the latter having been mechanically homogenized beforehand, were combined. The mixture was then washed twice in RPMI 1640 medium to remove protein complexes (400 × g, 5 minutes, then 200 × g, 5 minutes, each at 21 ° C.). On the & Loose cell pellet obtained was pipetted in with gentle shaking at 37 ° C. warm PEG (PEG 4000, Boehringer Mannheim, Germany) (1.0 ml). The fusion batch was then slowly made up to 50 ml with RPMI 1 640 and centrifuged at 200 × g at room temperature for 5 minutes. This washing step was repeated once more before the fusion mixture was decomplemented in 36 ml of selection medium or HAT medium (RPMI 1640 with Glutamax KGibco BRL), 10% fetal calf serum (FCS) for 30 minutes at 56 ° C. (Gibco BRL), 0 , 1% ciprofloxacin (Bayer AG, Germany), 5x10 ^'3 M hypoxanthine, 2x10 ⁵ M aminopterin, 8x10 " ⁴ M thymidine) was added.

3.3. Selection of hybridomas by HAT sensitivity and iodoacetamide

Defect mutants with HGPRT defect (hypoxanthine-guanine phosphoribosyl transferase) of the respective DNP and DIG hybridomas were used to select the hybridomas formed and to prevent the growth of unfused parental cells. In the presence of 8-azaguanine, only the HGPRT defect mutants can survive.

2 × 10 ⁸ cells of DNP hybridomas were cultivated in 650 ml culture bottles with HAT medium in the presence of 8-azaguanine (Sigma, Deisenhofen, Germany) with a final concentration of 1.32 × 10 ⁴ M for 3 days at 37 ° C. . After that, more than 99% of the cells had died. This 8-azaguanine cultivation was repeated 2 times. Dead cells were determined compared to the surviving cells by diluting the respective homogeneous cell suspension with 2 to 10 parts of trypan blue (0.05 mg / ml; ICN Biochemical GmbH, Meckenheim, Germany), depending on the cell density. After 1 minute, 10 μl of the stained cells were pipetted into a Neubauer counting chamber (Faust, Cologne, Germany) and 16 fields were counted within a square. The chamber factor was 10 ^ and a square had a volume of 0.1 ul. A control in HAT medium was carried out to exclude 8-azaguanine resistant cells by growing in this medium.

The immunoglobulin production of the survivors after the 8-azaguanine cultivation

Hybridomas were checked in the ELISA and then subcloned.

Defect mutants of the DIG hybridomas were isolated in analogy to the DNP hybridomas. For the ELISA according to Example 2.3. proceeded with the difference that a goat anti-mouse anti-κ and anti-λ in a concentration as capture antibody of 5 μg / ml was present that no supernatants of the anti-DNP antibody or the DIG antibody were added and incubated and that the goat anti-mouse IgG horseradish peroxidase conjugate was diluted 1: 2000. The anti-DNP and the anti-DIG antibodies were in the same dilutions as in Example 2.3. indicated (1, 0.5, 0.2, 0.1, 0.05 and 0.01 mg / ml in PBS / 1% BSA) and pipetted onto the plates in equal volumes (50 μl) and the color reaction measured analogously with ABTS. The subcloning or isolation was carried out according to COLLER, HS & COLLER, BS, method in Enzymol. 1 21: 412-417, 1983, after which the hybridoma cells were diluted in medium such that, according to a Poisson distribution, there were statistically 0.3 cells in each well of a culture plate. According to the trypan blue staining described above, the cell number of the respective hybridomas was determined and dilution series (10.3, 1 and 0.3 cells per 0.1 ml and per well) in cell culture medium (RPMI 1640 (GIBCO BRL), 5% fetal beetle serum (GIBCO BRL) 100 lU / ml penicillin, 100 μg / ml Streptomyciπ and 0.3 mg / ml glutamine) performed. For each dilution, 100 μl of the cell suspension dilutions were plated onto the 96-well round-bottom culture plates (Costar, Cambridge, USA) and cultured at 37 ° C., 7% CÜ2 for one week. For the second selection, 5 x 10 ⁷ cells of the DIG hybridomas without HGPRT defect (not HAT-sensitive cells) were incubated for 1 5 minutes at room temperature in 5 ml iodoacetamide, 1 5 μmol, (Sigma) and washed 3 times in RPMI 1640 .

The respective DNP and DIG hybridoma cells (2 × 10 ⁷ cells each) were mixed, washed twice in 50 ml of RPMI 1 640 (1400 rpm or 300 × g, 5 minutes, room temperature), according to Example 3.2. merged and processed analogously.

3.4. Manufacture of tetradomes via polyethylene glycol (PEG) fusion

For the fusion of the two hybridoma cell lines from Example 3.3. (DNP and DIG hybridomas) were taken up 2x10 ⁷ cells in 50 ml RPMI 1 640 medium and washed 3 times (400 xg, 5 minutes, room temperature). The cell pellets were then resuspended with gentle shaking in PEG 4000 (1 ml) (Boehringer Mannheim, Germany) at 37 ° C., made up to 50 ml with RPMI 1640 medium and centrifuged at 200 × g at room temperature for 5 minutes. The fusion mixture was then dissolved in 8.0 ml selection medium (RPMI 1 640 with Glutamax KGibco BRL), 10% fetal calf serum (FCS), decomplemented for 30 minutes at 56 ° C (Gibco BRL), 0.1% ciprofloxacin (Bayer AG, Germany), 5x10 ^-3 M hypoxanthine, 2x10 ^-5 M aminopterin, 8x10 " ⁴ M thymidine, 100 IU IL-6 / ml (Sigma)) and placed in 24 perforated plates (Costar, Cambridge, USA) with 5 x 10 ^ cells / ml and 1, 0 ml / hole under sterile conditions in an incubator (Tecnomara, Heraeus, Osterode, Germany) in a moisture-saturated atmosphere pipetted with a CO2 content of 7% and at a constant temperature of 37 ° C. The growth of hybrid hybridomas (tetradomes) was checked in the inverted microscope and the bispecific immunoglobulin production was detected by ELISA in the supernatant. The procedure for this ELISA was as follows: bovine serum albumin (BSA) was haptenized with DNP according to the protocol of Example 1.1 and 1.2 The BSA-DNP conjugates obtained (10 μg / ml) were bound to 96 perforated plates (PBS, 4 ° C., overnight) Plates with 10% fetal calf serum (FCS) / PBS (100 ul per well) for 30 minutes, the supernatant of the tetradome cultures (50 ul per well) was added and incubated for 30 minutes at room temperature. After 3 washes with PBS / 10% FCS (200 μI / well), DIG-conjugated peroxidase, which was prepared analogously to Examples 1.1. And 1 .2., Was diluted 1: 2000 from a stock solution (1 mg / ml) pipetted in per hole (100 μl per hole) and incubated for 30 minutes at room temperature After washing three times (PBS / 10% FCS, 200 μl per hole), ABTS was added in accordance with Example 2.3 and then analyzed as in Example 2.3 .

Tetradome cultures in which the desired bispecific anti-DNP: anti-DIG antibodies could be detected were then subcloned up to 20 times. The antibodies produced were washed 3 times with Tris buffer (20 mM, pH

8.0) diluted, sterile filtered (0.2 μm, Millipore) and over a Q-Sepharose Fast-

Flow column (Pharmacia) cleaned up. The antibody fraction was eluted by a step gradient against NaCl (0.01-0.5 M NaCl). Those fractions in which antibodies could be detected were further diluted with 2 M NH4 <S0> 2 to a final concentration of 0.85 M (NH4 (Sθ> 2). The bispecific antibodies were further purified on a

Phenyl-Superose column (Pharmacia) from which it is continuous

Gradients of 0.85 M NH (SO ₄ ) ₂ /0.1 M Na HPO ₄ , pH 7.2, until 0.001 M NH4 (SO4) 2 / 0.1 M Na2HP04, pH 7.2, were eluted. Example 4: Preparation of digoxigenin-conjugated horseradish peroxidase (HRP-DIG) as component 3

In analogy to Examples 1 .1. and 1 .2. , horseradish peroxidase (Sigma) was dialyzed and haptenized with DIG using the same protein concentrations, solutions, reagents and conditions.

Example 5 .: Production of cytotoxic T cells (effector cells)

Human mononuclear blood cells (PBMC) were isolated as usual using Ficoll density centrifugation. PBMC were resuspended in a concentration of 5 x 10 ^ cells / ml in DMEM, 4.5 g glucose / l (Gibco, Germany), containing 5 x 10 ^'5 mol / l 2-mercaptoethanol (Sigma, Germany), 10 mmol / l Sodium pyruvate (Gibco, Germany), 100 U / ml penicillin and 100 μg streptomycin. The cells were incubated on a plate precoated with OKT3 (CD3) (ATCC CRL8001), 10 μg / ml, at 37 ° C. and 5% CO2. After 5 days, the cells were harvested and positively enriched (CD8, Milteny) via "magnetic activated cell sorting" (MACS, Mini-Macs, Milteny, Germany).

Example 6: Cytotoxic Assay

According to the method by BOHLEN, H. et al., J. Immun. Math. 1 73 (1: 55-62, 1 994, Europium-labeled EBV-immortalized B-

Cells. The EBV immortalized B cells were obtained by the method of STUART, A.D. et al., Oncogene H (9): 1 71 1 -1 719, 1995. This

Cells were placed as target tents in holes on a 96-hole V-bottom plate

(50 ul / well, 1 x 10 ⁵ cells / ml). Effector cells (1.0 × 10 ⁵ cells / ml each) according to Example 5 and antibody solutions (anti-DIG: anti-DNP from Example 3, anti-CD3-DNP from Example 2 and anti-CD1 9-DIG, haptenized in analogy to Example 1 .1. And 1 .2.

(anti-CD19 was obtained from Becton Dickinson, Heidelberg, Germany)) was then added to a final volume of 200 μl. Maximum and spontaneous release were determined by adding either 1 50 μl of complete medium or by Triton X-100 (0.5%). The plates were for 5

Centrifuged at 300 xg for minutes. After the incubation period, the Marker release by adding 20 μl of supernatant to 200 μl

"enhancement" solution (Pharmacia, Freiburg) measured.

The fluorescence was then evaluated in a DELFIA fluorometer (Wallac, Turku, Finland) using a 613 nm filter for the determination of europium. Background fluorescence was obtained from three extra holes, which

Contained 5000 target cells in 200 μl complete medium. The cytotoxicity was calculated according to

{(Sample count - spontaneous release count)

/ Maximum release count - spontaneous release count)} x 100 =% specific lysis.

To avoid artifacts that could result from accidental Europium contamination, all reagents were checked before the assay.

Example 7: Use of the anti-DNP: anti-DIG bispecific antibodies in an enzyme-linked immunosorbent test (ELISA)

The principle of the ELISA is the specific antigen determination with antibodies. In this example, the test is used to detect immunoglobulin G4 (human). The structure of this test system is based on the sandwich principle. So-called trap antibodies are coupled to a plastic surface, which recognize the antibody to be tested. The supernatant to be tested is then added. In a further step, a second, hapten (DNP) - coupled antibody is added, which in turn specifically binds to the test antibody. In the next step, the bispecific anti-DNP: anti-DIG antibody is added and then washed. In addition, hapten (DIG) labeled enzyme is added and visualized or detected through substrate after incubation and washing. The detection antibody can also be labeled with the enzyme before the ELISA by equimolar mixing of hapten-labeled detection antibodies with anti-DNP: anti-DIG bispecific antibodies and hapten (DIG) -labeled enzyme.

The following materials were used for the assay:

• 96 perforated plates (Immuno Maxisorp F96, Nunc GmbH, Germany) • Phosphate-buffered saline (PBS; Gibco BRL)

• PBS with 1% cattle serum albumin (BSA; Sigma)

• Substrate buffer of the composition: Sl

10 ml 55 mM citric acid (Merck, Darmstadt), pH 4.0 100 ul ABTS (2,2'-azino-bis [3-ethylbenzthiazoline] -6-sulfonic acid) (Boehringer Mannheim); Stock solution: 1 5 mg / ml 3.3 μl H2O2 (33%) (Merck, Darmstadt) • ELISA reader: Titertek Multiscan® Plus MK II (405 nm) (Flow Laboratories)

The following reagents were used:

Reagent No. reagent conjugated to

1 Human lgG unconjugated

2 goats anti human total-DNP DNP and HRP * (1: 2000) * *

(Component 1)

3 anti-DNP: anti-DIG - (component 2) bispecific antibody

4 HRP * DIG (1: 5000) * * (component 3)

* HRP = horseradish peroxidase, * * values in brackets indicate the dilutions used

The unconjugated antibody Human IgG (Reagent 1) (LD Laboratory Diagnostics, Heiden, Germany) was immobilized on the 96-well plate by binding to the plastic with a concentration of 5 μg antibody / ml PBS and 50 μl / hole at 4 ° C. overnight. The free binding sites on the plastic surface were saturated with 100 μl PBS with 1% BSA / hole at an incubation time of 30 minutes and room temperature (RT) in order to prevent non-specific binding of further reagents. The solution to be tested (reagent 2) was pipetted onto the plate in various dilution series (1, 0.5, 0.2, 0.1, 0.05 and 0.01 mg / ml in PBS / 1% BSA) and the corresponding positive and negative controls. The volume was 50 μl / well and the incubation time was 30 minutes at RT. It was then washed 3 times with 250 μl PBS / well. The bispecific anti-DNP: anti-DIG antibody (reagent 3 or component 2) was then added at a dilution of 0.1 μg / ml and incubated for one hour at RT. After washing three times, 100 μl of DIG-conjugated HRP (reagent 4 or component 3) were added. Substrate buffers (200 μl / well) were added after 1 hour of incubation and three washes and incubated for at least 5 minutes at RT. The incubation period was chosen so that the OD value of approx. 1.0 was not exceeded. The 9 * • Enzymatic color reaction (ABTS) was measured in the ELISA reader at 405 nm. The result, which demonstrates the functionality of the combination preparations according to the invention using this example, is shown in FIG. 2.

Example 8: Surface staining of PBMC with fluorescein isothiocyanate

The following materials were used for this experiment: • Antibodies or antibody fragments Fa (b) against the surface structure CD19 (anti-CD19) (Becton-Dickinson) haptenized with DNP (as component 1)

Anti-DNP: anti-DIG bispecific antibody (as component 2),

• BSA haptenized with DIG and conjugated with fluorescein isothiocyanate (as component 3)

PBMC (1 x 10 ⁶ per test) were isolated by known methods via Fico® and incubated with the DNP haptenized anti-CD19 antibody (5 μg / ml) (15 minutes, RT). After washing the cells (5.0 ml PBS), the cells were incubated with the anti-DNP: anti-DIG bispecific antibody (1.0 μg / ml) at RT for 15 minutes and further with the DIG-haptenized BSA (5.0 μg / ml), which was conjugated with fluorescein isothiocyanate according to known methods, incubated for 1 5 minutes at RT. The fluorescence activity was determined after washing with 5.0 ml of PBS in a flow cytometer (on 5 × 10 ⁴ cells). FIG. 3 shows the result obtained and at the same time it demonstrates that the combination preparations according to the invention are also very suitable for detectable surface markings.

9 *

Example 9: Immunization with tumor proteins by focusing tumor antigens on dendritic cells

The following reagents were used for this experiment:

. Idiotype (38 CB) (described in BERGMAN, Y. & HAIMOVICH J., Europ. J. Immunol. 7: 413-41 7, 1977 and ESHAR, Z. et al., J. Immunol. 122: 2430-2434, 1979) haptenized with DNP (as component 1), • anti-DNP: anti-DIG bispecific antibody (as component 2). anti-class II antibody (ATCC, HB42) haptenized with DIG (as component 3)

In vitro equimolar amounts of components 1, 2 and 3 described above were incubated with one another for 1 hour at RT in PBS, the antibody concentrations being 50 μg / ml. Subsequently, 10 (n = 10) C3H / He mice (Charles River Wiga, Munich, Germany) each with components 1, 2 and 3 (test) described above, and each with control 1 (components 1 plus component 2 ) Control 2 (component 2 plus component 3), control 3 (component 1 only) control 4 (component 2 only) and control 5 (component 3 only) immunized intraperitoneally, with 50 μg per mouse of the respective component (in 100 μl PBS) were used. The immunization was then carried out by analysis of survival and tumor size. As a result, it was found that after immunization with components 1, 2 and 3, both anti-idiotypic antibodies and 40% long-term surviving mice could be noted. Table 2 below shows the results.

Table 2: Tumor immunity in mice (N = 10) induced by tumor cells (38 CB) with anti-class II antibodies using the combination preparation described above

Days after the i.p. Injection of the respective components

10 1 5 20 25 30 35

Survivors (n = 10)

Test 10 10 10 10 6 4

Control 1 10 10 8 2 0 0

Control 2 10 10 7 3 0 0

Control 3 10 10 8 4 0 0

Control 4 10 10 8 4 0 0

Control 5 10 10 7 2 0 0

Based on this example, the combination preparations according to the invention are eminently suitable for immunization with tumor proteins.

Example 10: Formulation of a combination preparation

Component 1 Anti-human-cytomegalovirus immediate early antigen (IEA) monoclonal antibody, mouse IgG-] (IEA E13, Argene

Biosoft Perc Technologique, Delta Sud BP24, Varilhes,

France, order number: 1 1 -003), haptenized with DNP.

1 vial with 0.5 ml conjugate, concentration 250 μg / ml in PBS pH 7.2 with 1% bovine serum albumin (BSA), 50%

Glycerin and 0.01% thimerosal. Component 2: Bispecific anti-DNP: anti-DIG antibody.

1 vial with 0.5 ml of the bispecific antibody,

Concentration 350 μg / ml in PBS, pH 7.2, with 1% BSA, 50%

Glycerin and 0.01% thimerosal.

Component 2: horseradish peroxidase (HRPO), haptenized with DIG.

1 vial with 0.5 ml conjugate, concentration 140 μg / ml in PBS, pH 7.2, with 1% BSA, 50% glycerin and 0.01%

Thimerosal. 35 In this case it was assumed that 50% of component 1 is conjugated with one molecule of DNP and 50% with two molecules of DNP. Component 2 therefore contains approximately 30% more antibodies than component 1. When concentrating component 3, it was taken into account that 50% of the peroxidase is coupled to one molecule of DIG, 50% to two molecules. So that each binding site of component 2 can bind a molecule of peroxidase, the number of available peroxidase molecules of component 3 was increased by more than 30% compared to the number of bispecific antibodies (component 2). By choosing the concentrations, the user can dilute each component equally. For immunodiagnostic, in particular immunocytochemical, applications, the antibodies are diluted 1:10 in PBS, pH 7.2. A different dilution can be selected depending on the type of sample and the intended use.

Claims

36 Patent claims

1 . Combination preparations consisting of at least one with a defined specific, immunologically reactive determinant Ha- | equipped first component, an at least bispecific second

Component with binding specificity for Ha-] (anti-Ha - \) and for at least one further binding specificity for a specific, immunologically reactive determinant Ha2 (anti-Ha2> different from Ha-] or with further binding specificities for several such determinants Ha _n ( anti-Ha _n ) which are different from one another and a third component which is or are endowed with at least one defined specific, immunologically reactive determinant Ha2 or with several such determinants Ha _n , Ha-], Ha2 and Ha _{n being} different from one another .

2. Combination preparations according to claim 1, characterized in that they are composed of

Component 1: <T): X - Ha- _|

Component 2: anti-Ha - | - Y - anti-Ha2 or

anti-Ha 1 - Y - anti-Ha _n

Component 3: Ha2 - or Ha _n - Z in which

X one with a determinant Ha- | equipped or connected

Reagent is, which can be monospecific with regard to its target location (T), comprising a protein, an immunoglobulin or an antibody or a derivative or fragment thereof, a ligand, a lectin, a receptor binding molecule, an adhesion molecule, a cytokine, a chemokine Lymphokine,

Ha - | a defined specific, immunologically reactive determinant, comprising a hapten, a specific couplable antigenic structure, an epitope, a paratope, an idiotope, Y a bispecific reagent comprising at least two determinants Ha-j and Ha2 or Ha _n, comprising an inert particle, on Protein, an immunoglobulin or an antibody, a diabody, with the corresponding anti-Ha-], anti-Ha2 or anti-Ha _n specificities, Ha2 one of Ha- | various, defined specific, immunologically reactive determinants, comprising a hapten, a specific linkable antigenic structure, an epitope, a paratope, an idiotope,

Ha _n any other of Ha- | and Ha2 various defined specific, immunologically reactive determinants, comprising a hapten, a specific couplable antigenic structure, an epitope, a paratope, an idiotope, Z a biologically, chemically or physically active or associated or linked with at least one determinant Ha2 or Ha _n detectable couplable agent, comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a marker substance, an enzyme, a radioactive substance, a radioactively labeled substance, a contrast agent, a biologically active substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with an effector-marker or reporter function, a prodrug, an adhesion molecule, a cytokine, a lymphokine, a chemokine, a ligand.

3. Combination preparations according to claim 2, characterized in that X can bind to a specific biological binding structure or parts thereof.

4. Combination preparations according to claim 3, characterized in that the specific biological binding structure is a cell surface structure, an antigen, an allergen, an epitope, a receptor, an adhesion molecule binding site or parts thereof or a

Sugar residue, a toxin, includes a drug.

5. Combination preparations according to claims 3 and 4, characterized in that the specific biological binding structure is associated with a prokaryote or a eukaryote.

6. Combination preparations according to claim 5, characterized in that the specific biological binding structure is associated with a cell, a tissue or an organ of a mammal or comes from a biological fluid.

7. Combination preparations according to claim 4, characterized in that the antigen is a cell surface antigen.

8. Combination preparations according to claim 7, characterized in that the cell surface antigen is a histocompatibility antigen

Is cell receptor antigen, a lymphopcyte surface antigen, a surface antigen of epithelial cells or a sugar residue.

9. Combination preparations according to claim 4, characterized in that the antigen or epitope is of viral origin.

10. Combination preparations according to claims 4 and 5, characterized in that the specific biological binding structure or parts thereof are derived from bacteria, endo- and ectoparasites, fungi or from a plant.

1 1. Combination preparations according to claims 3 and 10, characterized in that the specific biological binding structure is of natural origin or has been produced by DNA recombination, synthetically or semi-synthetically or from a non-biological

Liquid stems

1 2. Combination preparations according to claims 3 to 10, characterized in that the specific biological binding structure is associated with a toxin which is produced by a virus, a cell or an organism according to claims 9 and 10.

13. Combination preparations according to claims 3 to 8, characterized in that the specific biological binding structure is one with a pathological condition or one with a non-pathological one 59 ^•

Condition of a cell, a tissue or an organ of a mammalian organism-associated antigen or epitope.

14. Combination preparations according to one of claims 3 to 1 3, characterized in that the specific biological binding structure in

Tissue extracts are contained and occur in tissue and cell cultures.

15. Combination preparations according to one of claims 2 to 14, characterized in that the specific biological binding structure is a tumor antigen.

16. Combination preparations according to one of claims 2 to 15, characterized in that the specific biological binding structure is an allergen.

17. Combination preparations according to claims 3 and 4, characterized in that the specific biological binding structure is associated with a drug, a medicament or a metabolite thereof or a chemically produced toxin.

18. Combination preparations according to claim 4, characterized in that the antigen is an a priori soluble antigen.

19. Combination preparations according to claims 1 to 18, characterized in that component X is a monoclonal antibody, a derivative or a fragment thereof.

20. Combination preparations according to claim 1, characterized in that X is an adhesion molecule is selected from the group LFA, Mac,

ICAM.

21. Combination preparations according to claims 1 to 20, characterized in that the haptens bound to X and Z are univalent or bivalent haptens. 22. Combination preparations according to claims 1 to 21, characterized in that the hapten low molecular weight compounds with a

Molecular weight of less than 1000 daltons.

23. Combination preparations according to claim 22, characterized in that the haptens are selected from the group 2-nitrophenyl, nitrophenyl phosphate, digitoxigenin, digoxigenin, choline chloride, aminophenylphosphochloride, glycerophosphocholine, nitrophenylacetate, trinitrophenyl, trinitrophenylglycine, 4-dimethylsulfychloride, dimethylsulfychloride, nitrophenyl acetate, 2,4-dinitrophenyl,

Benzyl EDTA.

24. Combination preparations according to claims 1 to 23, characterized in that Y is an inert particle which is of synthetic, organic or inorganic origin and to which an antibody, a derivative or fragments thereof are bound.

25. Combination preparations according to claim 24, characterized in that the inert particle consists of polystyrene.

26. Combination preparations according to claims 1 to 25, characterized in that Y is an at least bispecific monoclonal antibody with the property of specifically reacting with at least two different defined specific, immunologically reactive determinants which are bound to different reagents, molecules or substances .

27. Combination preparations according to claim 26, characterized in that the at least bispecific antibody was obtained via fusion of hybridoma cells, via DNA recombination or is a heteroconjugate composed of connecting molecules, consisting of at least two distinct antibodies or fragments thereof.

28. combination preparations according to claim 27, characterized in that the heteroconjugate via -S - S bonds or via 10t homobifunctional or heterobifunctional compound molecules is composed.

29. Combination preparations according to claims 1 to 28, characterized in that the radioactive agent Z is a ß or γ emitter.

30. Combination preparations according to claims 29, characterized in that the ß- or γ-emitter is selected from ^ 25 | 123 | _; 131 |, 1 1 1 | n, 1 13m | _n , 99m _TCr 203 _Pb , 201-π. 198 _H g, 90γ, 186 _{Rβ r} 60 _Cθf 67 _G a, 75 _{Sβ (} 1 12m _A g, 198 _{AU /} 203 _Hg , 32 _P> 51 _Cr , 59 _Fe .

31 Combination preparations according to one of claims 1 to 30, characterized in that Z as an effector molecule or as a cytotoxic, cytocidal, cytostatic or cytolytic agent, a biologically active substance is selected from the group of hormones, enzymes, cytokines,

T cells, NK cells, macrophages, adhesion molecules, prodrugs, trigger molecules.

32. Combination preparations according to claim 31, characterized in that the biologically active substance is a cytotoxic, cytocidal, cytostatic or cytolytic agent and additionally consists of an antibody directed against another, different lymphocyte surface antigen.

33. Use of a combination preparation according to claims 1 to 32 for therapeutic and prophylactic treatment, for immunodiagnostic and prognostic purposes, for determining a disease state in a mammal in vitro and in vivo and for the immunization of a mammal.

34. Use of a combination preparation according to claim 33 for imaging.

35. Use of a combination preparation according to claims 33 and 34 for malignant tumors, infection and inflammation diseases,

Autoimmune diseases, for immunosuppression, to prevent one ICO. ^■ Rejection reaction, to prevent exacerbation of a disease state, in diseases of the blood coagulation system or in diseases that are associated with a deficiency of adhesion molecules.

36. Agent, characterized in that it consists of the components according to claims 1 to 32.

37. Composition consisting of component 2 according to one of claims 1 to 32.

38. Immunolinker, consisting of an at least bispecific reagent Y as defined in claims 2, 26 to 28 with binding specificities for at least two different defined specific, immunologically reactive determinants according to claims 2, 21 to 23.

39. Use of an immunolinker according to claim 37 in vivo and in vitro for connecting at least two reagents equipped with at least two different defined specific, immunologically reactive determinants according to claims 21 to 23 and biologically, chemically or physically active or detectable coupling agents.

40. Use of an immunolinker according to claim 38, characterized in that the immunolinker is an at least bispecific

Is antibody.

41. Method for determining whether an agent with biological, chemical or physical properties comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a

Marker substance, an enzyme, a radioactive substance, a radioactively labeled substance, a contrast agent, a biologically active substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with an effector-marker or reporter function, a prodrug, an adhesion molecule, a cytokine

Lymphokine, a chemokine, a ligand, one used in vitro and in vivo Diagnostics, in immunotherapy or in immunizations suitable biological, chemical or physical effects in vitro or in vivo unfolds or shows, characterized in that a) a first combination preparation consisting of components 1, 2 and 3 according to claims 1 to 32 with a first to be determined

Produces agent Z with biological, chemical or physical properties, b) produces an at least second combination preparation consisting of components 1, 2 and 3 according to claims 1 to 32 with a second agent Z to be determined with biological, chemical or physical properties that is different from a), c) the combination preparations provided according to a) and b) in succession or simultaneously in an in vitro or in an in vivo system, which may be the same or different, in contact with a specific biological binding structure to be examined or a sample to be examined within or outside of an animal or human body, d) repeating the step according to c) as desired, the agent Z to be determined on component 3 being different in each repetition with regard to its biological, chemical or physical properties, e) following step c) or d ) occurred biological, c measures, detects or records hemic or physical effects and f) evaluates the results obtained according to e) individually or comparing them.

42. Method for determining whether an agent with biological, chemical or physical properties comprising an effector molecule, a receptor binding molecule, an immunoglobulin or antibody, a marker substance, an enzyme, a radioactive substance, a radioactively labeled substance, a contrast medium, a biologically active Substance, a cytotoxic, cytocidal, cytostatic or cytolytic agent or any antigen with effector-marker or reporter function, a prodrug, an adhesion molecule, a cytokine, a lymphokine, a chemokine, a ligand, one of which in vitro and in vivo

Diagnostics, suitable for immunotherapy or immunizations Develops or shows biological, chemical or physical effects in vitro or in vivo, characterized in that a) a combination preparation according to one of claims 1 to 28 consisting of component 1 and component 2 is produced, b) the combination preparation provided according to a) in one in vitro or in an in vivo system brings into contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, c) a first component 3 according to one of claims 1, 2, 29 to 32 with a first agent Z to be determined, with biological, chemical or physical properties, d) adding component 3 provided according to c) to the system according to b), e) subsequently adding a second or further component 3 according to one of claims 1, 2 29 to 32 with an agent Z to be determined with c) and different from each other n adding biological, chemical or physical properties to the system according to b) simultaneously or in succession, f) optionally repeating steps a) to e) as desired, the agent Z to be determined on component 3 with each repetition with regard to its biological, chemical or physical properties is different, g) measures, detects or records the biological, chemical or physical effects that occurred after step e) or f) and h) evaluates the results obtained according to g) individually or comparably with one another.

43. A method of screening a plurality of agents with biological, chemical or physical properties selected from

Effector molecules, a receptor binding molecule, immunoglobulins or antibodies, marker substances, enzymes, radioactive substances, radioactively labeled substances, contrast agents, biologically active substances, cytotoxic, cytocidal, cytostatic or cytolytic agents or any antigens with effector marker or

Reporter function, drug precursors (prodrugs), adhesion molecules, Cytokines, lymphokines, chemokines, ligands which are intended to develop or show biological, chemical or physical effects which are suitable in vitro and in vivo diagnostics, in immunotherapy or in immunizations, in vitro or in vivo, characterized in that a) a first combination preparation consisting of components 1, 2 and 3 according to claims 1 to 32 with a first to be determined

Manufactures agent Z with biological, chemical or physical properties, b) an at least second combination preparation consisting of

Component 1, 2 and 3 according to claims 1 to 32 with a different from a) second agent Z to be determined with biological, chemical or physical properties, c) the combination preparations provided according to a) and b) successively or simultaneously in an in vitro or in an in vivo

System, which can be the same or different, in contact with a specific biological binding structure to be examined or a sample to be examined inside or outside an animal or human body, d) repeating the step according to c) as desired, the one to be determined

Agent Z on component 3 is different with each repetition with regard to its biological, chemical or physical properties, e) measures, detects or records the biological, chemical or physical effects that occurred after step c) or d) and f) the results obtained according to e) evaluated individually or comparing with each other.

44. A method of screening a plurality of agents with biological, chemical or physical properties selected from

Reporter function, active substance before stages (prodrugs), adhesion molecules, Praise cytokines, lymphokines, chemokines, ligands which are intended to develop or show biological, chemical or physical effects suitable in vitro and in vivo diagnostics, in immunotherapy or in immunizations in vitro or in vivo, characterized in that a) produces a combination preparation according to one of claims 1 to 28 consisting of component 1 and component 2, b) brings the combination preparation provided according to a) into contact with an in vitro or in an in vivo system with a specific biological binding structure to be examined or one sample to be examined inside or outside an animal or human body, c) producing a first component 3 according to one of the claims, 2, 29 to 32 with a first agent Z to be determined with biological, chemical or physical properties, d) which according to c) Provides provided component 3 to the system according to b), e) connect to it nd adding a second or further component 3 according to one of claims 1, 2 29 to 32 with an agent Z to be determined, having biological, chemical or physical properties different from c) and among one another, to the system according to b) simultaneously or successively, f) optionally Steps a) to e) are repeated as desired, the agent Z to be determined on component 3 with each repetition with regard to its biological, chemical or physical

Properties are different, g) measures, detects or records the biological, chemical or physical effects that occurred after step e) or f) and h) evaluates the results obtained according to g) individually or comparably with one another.

45. Method of making an in vitro and in vivo

Immunodiagnostic agent, characterized by the steps a) providing at least one combination preparation consisting of components 1, 2 and 3 according to one of claims 1 to 32, or lό) b) providing at least one combination preparation consisting of components 1 and 2 according to one of claims 1 to 28, c) identifying an agent which is biologically, chemically or physically active with regard to its immunodiagnostic properties by means of the combination preparations provided in a) and b) via the methods according to claims 41 to 44, d) formulation of the agent obtained from c) in the form of a combination preparation according to one of claims 1 to 32 with the known auxiliaries and carriers, or d) formulation of the agent obtained from c) by combining with a monospecific agent which is able to bind to biological binding structures, by known methods and with the known auxiliaries and carriers, or e) formulating the agent obtained from c) by itself using known methods with the known auxiliaries and carriers.

46. Use of an agent consisting of a combination preparation comprising components 1, 2 and 3 according to one of claims 1 to 32 or consisting of a combination preparation comprising components 1 and 2 according to one of claims 1 to 28 or consisting of component 2 according to one of the Claims 1, 2, 26 to 28 and 38 in a method according to any one of claims 41 to 45.

47. A method for producing a pharmaceutical preparation which can be used for immunoprophylaxis, immunotherapy and for immunization, characterized by the steps a) providing at least one combination preparation consisting of components 1, 2 and 3 according to one of claims 1 to 32, or b) providing at least a combination preparation consisting of components 1 and 2 according to one of claims 1 to 28, c) identify one with regard to its immunoprophylactic or immunotherapeutic properties or its immunization properties biologically, chemically or physically active agents by means of the combination preparations provided in a) and b) via the Process according to claims 41 to 44, d) formulation of the agent obtained from c) in the form of a combination preparation according to one of claims 1 to 32 with the known auxiliaries and carriers, or d) formulation of the agent obtained from c) by combining with a monospecific reagent which binds to biological binding structures bind by known methods, optionally with the known auxiliaries and carriers, or e) formulating the agent obtained from c) by itself using known methods, optionally with the known auxiliaries and carriers.

48. The method according to claims 46 and 47, characterized in that the monospecific reagent is a protein, an immunoglobulin or an antibody or a derivative or fragment thereof, a ligand, a lectin, a receptor binding molecule, an adhesion molecule, a cytokine, a chemokine, is a lymphokine, and that the biological binding structure is defined according to claims 4 to 18

49. Use of an agent consisting of a combination preparation comprising components 1, 2 and 3 according to one of claims 1 to 32 or consisting of a combination preparation comprising

Components 1 and 2 according to one of claims 1 to 28 or consisting of component 2 according to one of claims 1, 2, 26 to 28 and 38 in a method according to one of claims 47 and 48.

50. Kit for carrying out the process according to one of claims 41 to 48 containing a) components 1, 2 and 3 according to claims 1 to 32 or b) components 1 and 2 according to claims 1 to 28 or c) component 2 according to claims 1, 26 to 28, 38 or d) components 1, 2 and 3 or 1 and 2 or 2 together with at least one further different component 3, optionally together with auxiliaries and carriers.