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WO1998008875A1 - Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie - Google Patents

Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie Download PDF

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Publication number
WO1998008875A1
WO1998008875A1 PCT/EP1997/004493 EP9704493W WO9808875A1 WO 1998008875 A1 WO1998008875 A1 WO 1998008875A1 EP 9704493 W EP9704493 W EP 9704493W WO 9808875 A1 WO9808875 A1 WO 9808875A1
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Prior art keywords
component
biological
agent
antibody
combination preparations
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PCT/EP1997/004493
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German (de)
English (en)
Inventor
Heribert Bohlen
Original Assignee
Viva Diagnostika Diagnostische Produkte Gmbh
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Publication date
Priority claimed from DE19634730A external-priority patent/DE19634730A1/de
Priority claimed from DE1997103699 external-priority patent/DE19703699A1/de
Application filed by Viva Diagnostika Diagnostische Produkte Gmbh filed Critical Viva Diagnostika Diagnostische Produkte Gmbh
Priority to AU41193/97A priority Critical patent/AU4119397A/en
Publication of WO1998008875A1 publication Critical patent/WO1998008875A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • 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 -
  • 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 -
  • the combination according to the invention is generally used in immunology, both in immunodiagnostics and as a prophylactic and therapeutic agent.
  • Body area for example topically, can be applied.
  • 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.
  • 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”).
  • 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.
  • 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
  • IgM immunoglobulin-like molecules
  • 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).
  • cross-linking cross-linking
  • 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.
  • Antibody fragments of the IgG2a type (Fab and F (ab ') 2> which react specifically with target cells (melanoma) and against CD3 and CD28, respectively.
  • 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 ⁇ 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 ).
  • heteroconjugated bispecific antibodies 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.
  • 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.
  • 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).
  • TNP 2,4,6 -Trinitrophenyl
  • 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).
  • bispecific antibodies are suitable for use in immunodiagnostic assays (ELISA) and in immunohistochemistry.
  • ELISA immunodiagnostic assays
  • An example of this application is described by MILSTEIN, C. & CUELLO, AL, Nature 305, 537-540, 1983.
  • 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.
  • NIP hapten p-nitrophenyl phosphate
  • bispecific 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.
  • 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.
  • T cells are the main mediators in ?
  • ant ⁇ -CD3 ant ⁇ -CD25 bispecific monoclonal antibodies cause cytolysis of CD25-bearing alloreactive T cells and in cytotoxic T lymphocytes (CTL) with undefined specificity.
  • 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.
  • CD3 CD19 bispecific antibody T cells from patients with chronic lymphocytic leukemia, in combination with monospecific, bivalent anti-CD28 antibodies, could be activated.
  • 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.
  • only the combination anti-CD3: anti-CD19 plus anti-CD28 could achieve cytotoxicity against autologous malignant B cells.
  • 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- ⁇ 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
  • 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.
  • an antigen e.g. epitope, target cell, surface receptor
  • the production of a completely new bispecific antibody is necessary.
  • large molecules e.g.
  • 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.
  • the number of conjugates to be synthesized is the same is the number of molecules selected per antibody.
  • 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).
  • the object of the present invention was to provide means and new methods which overcome the disadvantages of the prior art.
  • a combination preparation consisting of at least one with a defined specific, immunologically reactive determinant Ha -
  • Ha2 or is or are connected with several such determinants Ha n , where Ha -
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the combinations according to the invention are equally outstanding in the case of immunization or for vaccination, in particular with tumor proteins.
  • 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 .
  • 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-
  • 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 ant ⁇ -Ha n specificity of an at least bispecific component 2 (with anti-Ha -
  • 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.
  • 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.
  • , 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 .
  • Component 1 (T): X— Ha -]
  • Component 2 anti-Ha -
  • Component 3 Ha2 - Z or Ha n - Z
  • (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 Adscosionsmolekülbindu ⁇ gsstelle, a sugar residue, a drug, a toxin, what, without the involvement of a determinant Ha j, X - Ha
  • X is a reagent equipped or linked with a determinant Ha -], which can be m ⁇ nospecific 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,
  • T target location
  • 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-
  • 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.
  • 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.
  • determinant Ha2 or H a n
  • component 3 determinant 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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-
  • 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 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 -
  • a combination preparation as mentioned above is preferred,
  • 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
  • a very particularly preferred combination preparation is that X is a with a hapten Ha-
  • a special, exemplary embodiment of the combination preparation according to the invention can then exist that Ha-
  • NP nitrophenylde ⁇ vat
  • DNP 2,4-d ⁇ n ⁇ trophenol
  • Z is a steroid hapten, for example digoxigenin ( ⁇ 20:22 -3 / ?, 1 2 / 914,21 - tetrahydroxy
  • 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 -
  • KLH Keyhole Limpet Hemocyanin
  • OKT3 antibody which with DNP as hapten Ha -
  • 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.
  • 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
  • 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
  • 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.
  • blood coagulation disorders pathological blood clots within a mammalian organism
  • 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).
  • ARC antigen Presenting Cells
  • 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
  • Rejection reactions to achieve immunosuppression, to prevent disease exacerbation, to predict or to determine
  • 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.
  • inventions 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.
  • 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.
  • kits-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.
  • component 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.
  • 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
  • determinants e.g. defined haptenized antigens
  • marker or reporter function Ha2 - Z or Ha n - Z, according to Table 1).
  • 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.
  • 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.
  • 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.
  • 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.
  • binding-specific component e.g. an antibody
  • 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.
  • agent encompasses compounds or substances which enter into a preferably non-covalent bond with a partner.
  • components 1, 2 and 3 can be regarded as reagents that react with one another non-covalently.
  • “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.
  • an agent as defined for Z in Table 1.
  • 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.
  • targeting 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.
  • 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 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.
  • 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
  • Antibodies or immunoglobulins are bound non-covalently. Antigens with
  • Immune system activating properties which induce the antibody synthesis, are called "immunogenes”.
  • 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
  • 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 ⁇ -amino groups or with SH groups (for example the "hinge” region) of antibodies, or which activate thiols on Fab fragments.
  • linker molecules are also included that can combine biomolecules with other substances, for example the avidin-biotin system or the streptavidin-biotin system.
  • Effective 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 ⁇ emitters, for example 99m Tc, l 1 1 ln, 1 3 1 J, 1 23 J, 90 Y, 1 86 Re
  • 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
  • marker substances such as dyes, fluorescent substances (e.g. fluorescein isothiocyanate), enzyme (e.g. lucifera
  • 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-s
  • 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 K ⁇ HLER, G. & MILSTEIN C, 1975,
  • 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.
  • 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.
  • antibodies in particular monoclonal antibodies, fragments or derivatives thereof, are preferred.
  • Bispecific antibodies can be produced in principle using three different processes.
  • 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).
  • the second parental hybridoma cells are preferably pretreated beforehand with a lethal amount of a suitable cytocidal reagent (for example 10 mM Iodoacetamide).
  • 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).
  • PEG polyethylene glycol
  • 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 ⁇
  • the hybridomas can be selected in a suitable medium.
  • 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.
  • the procedure is similar to that for the production of hybridomas.
  • one of the hybridoma cells to be hybridized is selected for sensitivity, for example for HAT sensitivity.
  • tetradomes 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.
  • the heavy and light 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 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.
  • 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.
  • antibodies produced by recombinant DNA 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.
  • 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: Fc ⁇ or CD4: CD3) et al., Nature 344, 667-670, 1990).
  • bispecific (fusion) antibody can be genetically engineered according to ASHKENAZI, A. et al., Proc. Natl. Acad. Be. USA 88 (23), 10535-10539, 1 991.
  • the fusion protein concerned an immunoglobulin (immunoadhesion) which inhibits inflammation by interfering with another adhesion molecule.
  • gene transfer via retroviral shuttle vectors for the production of bispecific antibodies is proposed.
  • bispecific antibodies 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).
  • diabodies 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.
  • 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
  • the bacterial product can then fold in such a way that the binding sites V j _ ] 1 and V
  • 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.
  • 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-
  • a target antigen for example a tumor marker
  • an effector cell of the immune system for example, so that the latter is transported to the target tissue (for example a tumor).
  • 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.
  • diabodies can be used advantageously for the purpose of the combination preparations according to the invention, in particular for component 2.
  • 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' ⁇ ) 2 antibodies are constructed via thioether-linked Fab' ⁇ fragments using o-phenylenedimaleimide. Bispecific antibodies can also be prepared by chemical recombination of fragments of monoclonal antibodies (BRENNAN, M.
  • the Fc 'portions of the antibody in question for example IgG
  • pepsin hydrolysis for example 2% w / w pepsin in 0.1 M sodium acetate, pH 4.2, 18 hours, 37 ° C.
  • F from '> 2 parts obtained.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ -methyl- (2-pyridyldithioo) toluene fSMPT], ⁇ / -succ ⁇ nim ⁇ yi 3- (2-py ⁇ dyldithio) prop ⁇ onate [SPDPj, sulfosuccmimidyl 6- [3- (2-pyridyldithio) prop ⁇ onam ⁇ do] hexanoate, sulfosuccinimidyl 4- ( ⁇ -maleimidomethyl) cyclohexane-1-carboxylate 4 - succinimidyl -maleimidomethyl) cyclohexane-1-carboxylate [SMCC], ⁇ /
  • DTSSP 3,3'-dithio ⁇ / s (sulfosuccinimidyl propionate) [DTSSP], bis (sulfosuccinimidyl) suberate,
  • 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.
  • 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).
  • 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.
  • suitable immunoglobulins for example an IgG
  • 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.
  • 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.
  • a bispecific reagent of high stability results via the avidin (streptav ⁇ din) -B ⁇ ot ⁇ n 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.
  • 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.
  • bispecific antibodies produced via hybrid hybridoma fusions for example in the case of lymphomas, are regarded as preferred component 2 (Y).
  • 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).
  • DNP 2,4-dinitrophenol
  • DIG digoxigenin
  • 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.
  • PEG polyfunctional alcohol
  • electrofusion for example according to VIENKEN, J., ZIMMERMANN, U
  • 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,
  • Antibody fragments containing the idiotype of the molecule can also be produced by known methods.
  • 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 ⁇ m) 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 (S ⁇ 4> 2 / 0.1 M Na2HP ⁇ 4 (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.
  • the corresponding combination preparation can be further investigated with regard to its effectiveness in vivo.
  • 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.
  • 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.
  • the combination preparations according to the invention can be used in all known serological-immunological diagnostic methods.
  • 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), ⁇ -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
  • IFMA Immunofluorometric Assay
  • TR-FIA Time Resolved Fluorescence Immunoassay
  • 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.
  • CELIA chemiluminescence immunoassay
  • SPALT solid phase antigen luminescence technique
  • ILMA immunoluminometric assay
  • ILSA immunoluminal labeled second antibody assay
  • immunoassays that can deliver the desired data within a short time can be preferred.
  • those immunoassays come into consideration which are based on a sandwich test, competitive test, colorimetric test (such as, for example, the CEDIA TM 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.
  • 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.
  • 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 detectable agent for example 2,2'-azino-ö / s (3-ethylbenzothiazoline-6-sulfonate (ABTS).
  • ABTS 2,2'-azino-ö / s (3-ethylbenzothiazoline-6-sulfonate
  • 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.
  • a suitable, detectable agent for example 2,2'-azino-o / s (3-ethylbenzothiazoline-6-sulfonate) be measured spectrophotometrically by known methods.
  • 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 -
  • 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.
  • 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.
  • a carrier material for example conventional microtiter plates
  • a suitable buffer Is brought into contact With the appropriate component 2 added beforehand, a suitable component can then be added tl
  • equipped reagent for example an antibody
  • a suitable buffer system an IgG-peroxidase conjugate equipped with a determinant Ha2 and a suitable reagent (for example o ⁇ phenylenediamine)
  • a suitable reagent for example o ⁇ phenylenediamine
  • 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.
  • 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.
  • 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.
  • 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.
  • the carrier material can be made with a hollow body (for example, tubes, tubes, cuvettes) or with at least
  • 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).
  • 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, ⁇ -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-d ⁇ [3-ethylbenzth ⁇ azol ⁇ n 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
  • 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.
  • 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
  • Biotinylation 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).
  • biotmylated antigens or the corresponding antibody complexes can be identified, for example, using avidin-conjugated alkaline phosphatase (ExtrAvidin, Sigma, Deisendorf, Germany).
  • 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.
  • DIG digoxigenin
  • a bispecific reagent which can be used as an immunolinker (component 2) Ha-] (ant ⁇ -DNP): ant ⁇ -Ha2 (anti-DIG) has specificities.
  • 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 .
  • component 3 can be added after the reaction of components 1 and 2, after a time delay .
  • a further meaning in the use of the combination preparations according to the invention is that they are suitable for "imagining".
  • Pathologically modified cells and tissues can be malignant cells or tissues, for example.
  • imaging can be carried out by defining X and Z according to Table 1 with two different determinants Ha-
  • 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.
  • a radioactive isotope all known ⁇ and ⁇ emitters can be used, but preferably those with a short half-life.
  • radioactive agents known to the person skilled in the art are available, for example, 1 25, 1 23
  • s Fluorescent dye is, for example, a fluorescein (for example fluorescein isothiocyanate) and a contrast agent, for example a paramagnetic contrast agent.
  • a combination preparation can easily detect and localize cell and tissue types and parts thereof.
  • the determinants for example haptens
  • 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.
  • 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
  • hapten 20 is a hapten, or n to selected haptens determinants and Ha, which is or which is bound to a carrier (Z).
  • Z a carrier
  • 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
  • binding acid amide preferably to peptides or proteins (Z).
  • 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
  • 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
  • bivalent haptens in particular hydrophilic in nature, preferred over monovalent, hydrophobic haptens.
  • divalent haptens in question would be dinitrophenyl derivatives or derivatives of Nt-dinitrophenylaminocaproic acid, which with 125
  • Such haptens are commercially available
  • mono- and bivalent haptens which can be labeled with, for example, ⁇ 25, 0 ( -
  • 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
  • radioisotopes can be transported directly to the desired location with the combination preparations according to the invention.
  • 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.
  • suitable chelators are ethylenediaminetetraacetic acid or bisthiosemicarbazones of 1, 2- or 1, 3-dicarbonyl compounds.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • a time delay in this case likewise iv
  • Y - anti-Ha2 anti-Ha2 site of component 2
  • 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.
  • the behavior of component 3 in a mammalian organism can be predetermined and the modes of administration (amount,
  • 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.
  • component 1 in the case of an antibody, preferably a humanized antibody if a person is to be examined
  • component 2 systemically or topically, for example IV, im or sc, into the Proximity, to or in the material to be examined.
  • 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.
  • radioisotopes examples include 1 5
  • 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.
  • DNP 2,4-dinitrophenol
  • anti-Ha2 properties for example anti-DNP: anti-DIG, if Ha2 is a digoxigenin [DIG]
  • component 3 for example a " * ⁇ unmarked or 123
  • 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.
  • 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
  • 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 a)
  • 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
  • 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-
  • agents with biological, chemical or physical properties 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
  • 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
  • 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-
  • 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.
  • 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
  • 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 ⁇ , ⁇ or ⁇ 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.
  • a determinant Ha2 for example haptenized, (for example alkaline phosphatase), a cytokine (for example interferon ⁇ , ⁇ or ⁇ or derivatives thereof, an interleukin such as IL-2)
  • a determinant for example
  • the radioimmunoconjugates which can be prepared by known methods are suitable for this purpose.
  • 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 ⁇ -emitters are particularly suitable as radioisotopes, for example 1 5 ,, 1 23 ,, 1 3 1 l, 1 l 1
  • radioisotopes for example 1 5 ,, 1 23 ,, 1 3 1 l, 1 l 1
  • radioisotopes for example 1 5 ,, 1 23 ,, 1 3 1 l, 1 l 1
  • 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).
  • Ha2 means an exerting agent (for example an enzyme, a cytokine, a T-lymphocyte, an adhesion molecule, etc.).
  • an exerting agent for example an enzyme, a cytokine, a T-lymphocyte, an adhesion molecule, etc.
  • 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.
  • the size and type 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.
  • the combination preparations according to the invention regardless of which antitumor agent is associated with it can.
  • multiple, systemic applications, preferably iv injections, of component 3 are advantageous.
  • localizable tumors for example the lymph nodes or the peritoneum, will require local application, possibly several times.
  • the period of administration of component 3 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.
  • binding affinity component 1 for example consisting of a haptenized monoclonal antibody with specific reactivity for a specific tumor antigen
  • 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.
  • 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.
  • 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.
  • prodrug that is to say a drug or active substance precursor
  • 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 .
  • 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.
  • 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.
  • 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.
  • the two-step process is an advantageous and therefore preferred process for using the combination preparations according to the invention.
  • 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.
  • 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).
  • pathogenic pathogens for example anti-nucleoprotein , anti-hemagglutinin from influenza viruses, anti-HIV antigens.
  • 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.
  • TAA tumor-associated antigen
  • 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.
  • 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-din ⁇ trophenyl (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-.
  • DIG digoxigenin
  • 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.
  • 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.
  • a corresponding antibody against a cell surface antigen for example a monospecific anti-CD28 antibody.
  • 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 .
  • 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.
  • 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.
  • 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).
  • undifferentiated tissue such as, for example, nephroblastoma, neuroblastoma, medulloblastoma, retinoblastoma, embryonic rhabdomyosarcoma, human tumor, endocrine formation, and endocrine formation , HCG
  • 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.
  • component 2 can be a bispecific antibody with anti-Ha-
  • component 1 an idiotype endowed with a determinant Ha-, preferably a hapten, for example antigen-presenting cells, for example dendritic cells
  • component 3 one with a determinant Ha2 or more determinants Ha n , preferably Haptens, equipped antibodies, for example an antibody directed against "human ⁇ 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- ⁇ anti-Ha-, - Y - anti- Ha2: Ha2 "Z) for immunization in a mammalian organism via the known routes, for example iv or ip.
  • Combination preparations can be easily applied to these indication areas and transferred analogously.
  • 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.
  • a sample to be examined can be a non-biological liquid medium if it contains agglutinable, markable or detectable antigens and / or antibodies.
  • 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.
  • 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 ⁇ g / ml, or it can be lyophilized, the lyophilisate being prepared by known methods in physiological saline, PBS or can be reconstituted in sterile water.
  • PBS phosphate buffered saline
  • 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 ⁇ g.
  • 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 ⁇ 33 - 436, 1 987, or according to JUNG, G.
  • 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.
  • 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
  • int particles are understood to be particles to which
  • erythrocytes such as Toxocell IHA 0 from Red ⁇ test, SA »Barcelona, or Mast Serodia-Anti HBs FD 41 1 from Mast Diagnostica, Hamburg, or according to BIRD, T. & STEPHENSON, JH, J. clin. Path.
  • cellulose 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 TM 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).
  • PBS phosphate-buffered saline
  • 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 ⁇ range or from approx. 1 ⁇ to 20 ⁇ 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 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), Re ⁇ al Cell Carcinoma Anti
  • antigens which originate from, or are part of, viruses, prokaryotes and eukaryotes which are not assigned to a mammal are also suitable.
  • 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], Brucel
  • 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.
  • 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
  • chemically produced toxins are also included.
  • antigens of plant origin are also included by definition, for example pollen-associated antigens or antigenic substances dissolved in plant juices.
  • non-biological systems or test samples can contain waste water
  • 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.
  • 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.
  • 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.
  • a substance which triggers an immunological reaction for example a protein
  • haptens which according to the invention for Hapte ⁇ leiter the respective components of theticianspraparationen are, 2-nitrophenyl, nitrophenyl, digitoxigenin, digoxigenin, choline chloride, Aminophenylphosphochloride, glycerophosphocholine, nitrophenyl acetate, Trinitrophe ⁇ yl, Trinitrophenylglycin, dimethylsulfoxide, guanidine hydrochloride, 4-hydroxy-nitrophenylacetate 3, 2 , 4-dinitrophenyl, benzyl-EDTA, to name but a few.
  • 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 ⁇ l of, for example, p-nitrophenylphosphate caproic acid o-succinimide ester (in an aprotic solvent, for example dimethylformamide, for example 20 mg / ml).
  • an aprotic solvent for example dimethylformamide, for example 20 mg / ml.
  • PBS phosphate-buffered saline
  • 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.
  • an aprotic solvent for example dimethylformamide
  • pH 8.5 in the presence of 0.1 5 M NaCl
  • 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.
  • cellular structures for example bacteria, viruses, fungi, modified cells
  • CD8 + lymphocytes CD8 + lymphocytes
  • Such surface molecules of T cells. are known.
  • 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 ⁇ - Chain (SANCHEZ-MADRID, F. et al., J. Exp. Med. 1 58. 1785 - 1803, 1983), the latter also known as CD18.
  • KEIZER G., et al., Eur. J. Immunol. 15, 1 142 - 1 147, 1 985
  • SANCHEZ-MADRID F. et al., J. Exp. Med. 1 58. 1785 - 1803, 1983
  • 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.
  • ICAM-1 is known to be localized on the surface of endothelial cells.
  • Y is a bispecific reagent with anti-Ha-
  • 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.
  • 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.
  • 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.
  • the buffers and media known to the person skilled in the art can be used, if appropriate together with a stabilizing agent.
  • a stabilizing agent 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.
  • 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.
  • the techniques known to those skilled in the art of lyophilization and the reconstitution to be carried out before using the corresponding antibodies can be used.
  • the lyophilized form is preferred if these are present as substances accessible for lyophilization.
  • 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.
  • the individual components can be formulated without restrictions according to the known methods as are used and known in pharmaceutical chemistry.
  • 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.
  • PBS physiological saline
  • HSA human serum albumin
  • compositions according to the invention 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 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,
  • 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.
  • the particles in question are to be selected from polymeric materials, for example polyesters, hydrogels, polyamino acids, polylactic acid.
  • microencapsulation of the combination preparations according to the invention it is also possible to achieve such effects by microencapsulation of the combination preparations according to the invention.
  • the known methods for example coacervation
  • 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.
  • the same or equivalent formulations are suitable for in vitro purposes, the physiological safety being of no importance here.
  • 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.
  • 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 entire spectrum known to the person skilled in the art can be considered for administration without restrictions.
  • 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.
  • the application is carried out in the form of a sterile aqueous solution, as described above.
  • amounts in the ng, ⁇ g or mg range can be used, depending on the type of disease to be treated.
  • the doses can be in the ng and ⁇ g range, whereas for in vivo applications doses in the ⁇ g 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.
  • 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.
  • intravenous, intraarterial or intrapleural administration should preferably be used.
  • Intraperitoneal administration is preferred for ovarian tumors or for diseases of the peritoneum.
  • 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.
  • metastatic tumors for example the lungs, the breast or to be treated by the liver.
  • 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.
  • Fig.1 Basic schematic representation of the combination preparations according to the invention.
  • Determinant Ha-] equipped reagent R ( ⁇ ) binds specifically to a target site (T).
  • one anti-Ha 1 part of the bispecific reagent (BiAb ( ⁇ j) 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 ( ⁇ ) BiAb ( ⁇ ) 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
  • 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 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 (dH2 ⁇ ) (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 dH2 ⁇ , 1 mM EDTA, then the dialysis tube was rinsed with dH2 ⁇ and in an aqueous sodium azide solution (0.02% sodium azide (Sigma) in dH2 ⁇ ) at 4 ° C.
  • dH2 ⁇ distilled water
  • 2 mM EDTA Sigma, Deisenhofen, Germany
  • 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)
  • 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.
  • the KLH-DNP conjugate was sterile filtered (filter: 3 ⁇ , Millipore) and briefly at 4 ° C, for longer storage at -20 ° C.
  • 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.
  • 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.
  • 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 described further procedure.
  • 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 ⁇ g antibody per ml PBS (Gibco BRL, Gaithersburg, USA) and 50 ⁇ l per hole at 4 ° C immobilized overnight.
  • the free binding sites on the plastic surface were saturated with 100 ⁇ l 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 ⁇ l per well and the incubation time was 30 minutes at room temperature. It was then washed 3 times with 250 ⁇ l PBS / 1% BSA per hole.
  • mice 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.
  • DNP 2,4-dinitrophenol
  • DIG digoxigenin
  • 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 a2HP ⁇ 4, 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.
  • mice 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.
  • PEG polyethylene glycol
  • RPMI 1 640 medium Gibco BRL
  • 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.
  • selection medium or HAT medium RPMI 1640 with Glutamax KGibco BRL
  • FCS fetal calf serum
  • FCS fetal calf serum
  • HGPRT defect hyperxanthine-guanine phosphoribosyl transferase
  • Hybridomas were checked in the ELISA and then subcloned.
  • Defect mutants of the DIG hybridomas were isolated in analogy to the DNP hybridomas.
  • the anti-DNP and the anti-DIG antibodies were in the same dilutions as in Example 2.3.
  • 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 ⁇ g / ml Streptomyci ⁇ and 0.3 mg / ml glutamine) performed.
  • cell culture medium RPMI 1640 (GIBCO BRL), 5% fetal beetle serum (GIBCO BRL) 100 lU / ml penicillin, 100 ⁇ g / ml Streptomyci ⁇ and 0.3 mg / ml glutamine
  • 100 ⁇ l 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.
  • 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.
  • 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.
  • RPMI 1 640 with Glutamax KGibco BRL 10% fetal calf serum
  • hybrid hybridomas tetradomes
  • BSA bovine serum albumin
  • FCS fetal calf serum
  • 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 ⁇ l per hole) and incubated for 30 minutes at room temperature After washing three times (PBS / 10% FCS, 200 ⁇ l 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
  • 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 (S ⁇ > 2). The bispecific antibodies were further purified on a
  • Phenyl-Superose column (Pharmacia) from which it is continuous
  • Example 5 Production of cytotoxic T cells (effector cells)
  • PBMC Human mononuclear blood cells
  • 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
  • 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
  • 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.
  • 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.
  • a second, hapten (DNP) - coupled antibody is added, which in turn specifically binds to the test antibody.
  • the bispecific anti-DNP: anti-DIG antibody is added and then washed.
  • 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.
  • anti-DNP anti-DIG - (component 2) bispecific antibody
  • 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 ⁇ g antibody / ml PBS and 50 ⁇ l / hole at 4 ° C. overnight.
  • the free binding sites on the plastic surface were saturated with 100 ⁇ l 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 ⁇ l / well and the incubation time was 30 minutes at RT. It was then washed 3 times with 250 ⁇ l PBS / well.
  • the bispecific anti-DNP: anti-DIG antibody (reagent 3 or component 2) was then added at a dilution of 0.1 ⁇ g / ml and incubated for one hour at RT. After washing three times, 100 ⁇ l of DIG-conjugated HRP (reagent 4 or component 3) were added.
  • Substrate buffers 200 ⁇ l / 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
  • Anti-DNP anti-DIG bispecific antibody (as component 2)
  • PBMC peripheral blood mononuclear cells
  • Example 9 Immunization with tumor proteins by focusing tumor antigens on dendritic cells
  • 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)
  • 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
  • Component 2 Bispecific anti-DNP: anti-DIG antibody.
  • Component 2 horseradish peroxidase (HRPO), haptenized with DIG.
  • HRPO horseradish peroxidase

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Abstract

L'invention concerne des préparations combinées comprenant des constituants s'utilisant de manière ciblée en immunologie aussi bien en termes de diagnostic que de thérapie. La combinaison est fondée sur l'utilisation universelle d'un immuno-segment de liaison comme constituant des préparations combinées, qui peut lier deux autres constituants différents présentant des déterminants distincts.
PCT/EP1997/004493 1996-08-28 1997-08-18 Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie WO1998008875A1 (fr)

Priority Applications (1)

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AU41193/97A AU4119397A (en) 1996-08-28 1997-08-18 Novel combination preparations and their use in immunodiagnosis and immunotherapy

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19634730A DE19634730A1 (de) 1996-08-28 1996-08-28 Neue Kombinationspräparationen und ihre Verwendung in der Immundiagnostik und Immuntherapie
DE19634730.0 1996-08-28
DE1997103699 DE19703699A1 (de) 1997-02-03 1997-02-03 Neue Kombinationspräparationen und ihre Verwendung in der Immundiagnostik und Immuntherapie
DE19703699.6 1997-02-03

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WO2001032207A1 (fr) * 1998-10-30 2001-05-10 United States Army Medical Research And Materiel Command Procede permettant d'appliquer une immunotherapie active/passive
US7138103B2 (en) 1998-06-22 2006-11-21 Immunomedics, Inc. Use of bi-specific antibodies for pre-targeting diagnosis and therapy
US7387772B1 (en) 1999-06-22 2008-06-17 Immunimedics, Inc. Chimeric, human and humanized anti-CSAP monoclonal antibodies
US7405320B2 (en) 1998-06-22 2008-07-29 Immunomedics, Inc. Therapeutic and diagnostic conjugates for use with multispecific antibodies
US7429381B2 (en) 1998-06-22 2008-09-30 Immunomedics, Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
WO2010052014A1 (fr) * 2008-11-07 2010-05-14 Micromet Ag Traitement de la leucémie lymphoblastique aiguë
WO2010052013A1 (fr) * 2008-11-07 2010-05-14 Micromet Ag Nouveau traitement de la leucémie lymphoblastique aiguë de l’enfant et de l’adolescent
US7833528B2 (en) 1998-06-22 2010-11-16 Immunomedics, Inc. Use of multispecific, non-covalent complexes for targeted delivery of therapeutics
WO2011003557A1 (fr) * 2009-07-06 2011-01-13 F. Hoffmann-La Roche Ag Complexe d'anticorps bispécifique et de digoxigénine conjugué à un agent thérapeutique ou de diagnostic
DE102013105144A1 (de) * 2013-05-17 2014-11-20 Arno Thaller Pharmazeutisches Kombinationspräparat mit einem anti-idiotypischen Antikörperfragment

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US7833528B2 (en) 1998-06-22 2010-11-16 Immunomedics, Inc. Use of multispecific, non-covalent complexes for targeted delivery of therapeutics
US7641891B2 (en) 1998-06-22 2010-01-05 Immunomedics, Inc. Chimeric, human and humanized anti-CSAp monoclonal antibodies
US7138103B2 (en) 1998-06-22 2006-11-21 Immunomedics, Inc. Use of bi-specific antibodies for pre-targeting diagnosis and therapy
US7914787B2 (en) 1998-06-22 2011-03-29 Immunomedics, Inc. Production and use of novel peptide-based agents with bispecific antibodies
US7405320B2 (en) 1998-06-22 2008-07-29 Immunomedics, Inc. Therapeutic and diagnostic conjugates for use with multispecific antibodies
US7414121B2 (en) 1998-06-22 2008-08-19 Immunomedics, Inc. Chimeric, human and humanized anti-CSAp monoclonal antibodies
US7429381B2 (en) 1998-06-22 2008-09-30 Immunomedics, Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
US7553953B2 (en) 1998-06-22 2009-06-30 Immunomedics, Inc. Chimeric, human and humanized anti-CSAp monoclonal antibodies
US7560110B2 (en) 1998-06-22 2009-07-14 Immunomedics, Inc. Production and use of novel peptide-based agents with bispecific antibodies
US8158573B2 (en) 1998-06-22 2012-04-17 Immunomedics, Inc. Therapeutic and diagnostic conjugates for use with multispecific antibodies
US7666415B2 (en) 1998-06-22 2010-02-23 Immunomedics, Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
US7943343B2 (en) 1998-06-22 2011-05-17 Immunomedics, Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
WO1999066951A3 (fr) * 1998-06-22 2000-06-15 Immunomedics Inc Utilisation d'anticorps bi-specifiques pour diagnostic et therapie de pre-ciblage
US7820164B2 (en) 1998-06-22 2010-10-26 Immunomedics, Inc. Chimeric, human and humanized anti-CSAP monoclonal antibodies
WO2001032207A1 (fr) * 1998-10-30 2001-05-10 United States Army Medical Research And Materiel Command Procede permettant d'appliquer une immunotherapie active/passive
US7387772B1 (en) 1999-06-22 2008-06-17 Immunimedics, Inc. Chimeric, human and humanized anti-CSAP monoclonal antibodies
WO2010052013A1 (fr) * 2008-11-07 2010-05-14 Micromet Ag Nouveau traitement de la leucémie lymphoblastique aiguë de l’enfant et de l’adolescent
WO2010052014A1 (fr) * 2008-11-07 2010-05-14 Micromet Ag Traitement de la leucémie lymphoblastique aiguë
US11597766B2 (en) 2008-11-07 2023-03-07 Amgen Research (Munich) Gmbh Treatment of acute lymphoblastic leukemia
CN102209729A (zh) * 2008-11-07 2011-10-05 米克罗麦特股份公司 小儿急性淋巴细胞白血病的治疗方法
WO2011003780A1 (fr) * 2009-07-06 2011-01-13 F. Hoffmann-La Roche Ag Anticorps bispécifiques de liaison à la digoxygénine
CN102470171A (zh) * 2009-07-06 2012-05-23 弗·哈夫曼-拉罗切有限公司 双特异性抗体和与治疗或诊断剂缀合的地高辛配基的复合物
CN102481367A (zh) * 2009-07-06 2012-05-30 弗·哈夫曼-拉罗切有限公司 结合地高辛配基的双特异性抗体
JP2012532174A (ja) * 2009-07-06 2012-12-13 エフ．ホフマン−ラ ロシュ アーゲー 二重特異性ジゴキシゲニン結合抗体
US8907069B2 (en) 2009-07-06 2014-12-09 Hoffmann-La Roche Inc. Complex of bi-specific antibody and digoxigenin conjugated to a therapeutic or diagnostic agent
EP2823821A1 (fr) * 2009-07-06 2015-01-14 F. Hoffmann-La Roche AG Anticorps se liant spécifiquement à la digoxigénine
CN102481367B (zh) * 2009-07-06 2015-04-29 弗·哈夫曼-拉罗切有限公司 结合地高辛配基的双特异性抗体
US9050375B2 (en) 2009-07-06 2015-06-09 Hoffmann-La Roche, Inc. Bi-specific digoxigenin binding antibodies
US9574016B2 (en) 2009-07-06 2017-02-21 Hoffmann-La Roche Inc. Complex of bi-specific antibody and digoxigenin conjugated to a therapeutic or diagnostic agent
WO2011003557A1 (fr) * 2009-07-06 2011-01-13 F. Hoffmann-La Roche Ag Complexe d'anticorps bispécifique et de digoxigénine conjugué à un agent thérapeutique ou de diagnostic
DE102013105144A1 (de) * 2013-05-17 2014-11-20 Arno Thaller Pharmazeutisches Kombinationspräparat mit einem anti-idiotypischen Antikörperfragment

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