Single chain, multiple antigen-binding molecule, its preparation and use

The present invention relates to a single-chain, multi-antigen-binding molecule, with different variable domains of a heavy or a light chain of an immunoglobulin, which are linked in the form of a VH-VL construct, which in turn are linked to one another via a peptide, and their production and use as a drug or diagnostic.

Bispecific antibodies which recognize two different antigens, for example a tumor cell surface antigen and an effector molecule, are widely used in experimental immunotherapy (Fanger et al., Crit. Rev. Immunol. 12, 101-124 (1992); van de Winkel et al ., Immunol. Today 18: 562-564 (1997)). The effector functions that are recruited by bispecific antibodies include those that occur naturally in the organism, such as cytotoxic and phagocytic cells, complement components, cytokines and thrombolytic and fibrinolytic enzymes as well as non-body effector molecules such as toxins, prodrug converting enzymes and radionuclides. For example, the injection of bispecific antibodies against the Hodgkin's tumor-associated antigen CD30 and the T cell antigens CD3 and CD28 in xenografted tumors for the recruitment and stimulation of cytotoxic T cells and for the induction of a tumoricidal activity (Renner et al., Science 264, 833, 1994). In another approach, a bispecific antibody against CD30 and alkaline phosphatase was used to recruit the enzyme to the tumor site and thus convert the non-toxic precursor of an active ingredient into a toxic active ingredient (Sahin et al., Cancer Res. 50, 6944-6948 , 1990).

An alternative to the injection of the purified bispecific antibody is the expression and secretion of these bispecific antibodies by cells transfected in vitro or in vivo. The advantage of this strategy is that the production of the bispecific antibody from the transduced cell takes place in vivo and thus no complex production and purification of the bispecific antibody is necessary before an injection. In addition, by choosing suitable expression systems, the expression of the bispecific antibody can be controlled locally, in organs or in a tumor or also systemically.

Bispecific antibodies can be produced, for example, by chemical crosslinking (Nisonoff et al., Nature 194, 355, 1962). In the chemical crosslinking of monoclonal or polyclonal antibody molecules of animal origin, a not insignificant proportion can be inactivated. In addition, both hetero- and homodimers are formed. Homodimers have to be separated from the desired heterodimers by complex processes. Hybridoma cells that produce bispecific antibodies, so-called hybrid hybridomas, can only be produced with relatively great effort since two different hybridomas have to be fused to one another here (Milstein et al., Nature 305, 537, 1983). The proportion of functional heterodimers is relatively low, theoretically only 10%, since the heavy and light chains of the two antibody molecules can freely associate with each other. Murine monoclonal antibodies are mainly used as starting material, which in turn are immunogenic for humans.

Different bivalent or bispecific antibody molecules can also be produced recombinantly and expressed in bacteria or eukaryotic cells (WO 93/06217). All recombinant antibody molecules have in common that both murine and human starting molecules can be used for their production. Different methods have been developed in order to be able to produce bispecific recombinant antibody molecules as efficiently as possible. Different groups of molecules can be produced using these methods.

In one group of molecules, the variable parts of the antibodies are fused with constant immunoglobulin domains (Fc, CH3, CL) to achieve dimerization (Hu et al., Cancer Res. 56, 3055-3061 (1994); Hayden et al. , Ther. Immunol. 1, 3-15 (1994)). However, there is no selection for heterodimeric molecules, so that predominantly bivalent homodimers are formed in this way. The expression of these molecules in functional form is also restricted to eukaryotic cells.

In another group of molecules, the variable parts of the antibodies are fused with peptides or protein domains from other proteins to produce bivalent or bispecific molecules (Plückthun and Pack, Immunotechnol. 3, 83-105 (1997)). Here too, homo- and heterodimers are usually formed due to the random association. In addition, some of these molecules contain considerable proportion of foreign sequences, so that a clear immunogenicity can be expected.

In a third group of molecules, minor modifications of recombinant Fv fragments, usually single-chain Fv fragments (scFv), are used to produce bivalent or bispecific molecules (Holliger and Winter, Curr. Opin. Biotechnol. 4, 446- 449 (1993)). These include the dimerization by additional cysteines at the C-terminus of the scFv chains (MacCartney et al., Protein Engin. 8, 301-314 (1994)). However, both homo- and heterodimers are formed here and non-functional aggregates are formed when expressed in bacterial cells. Tandem scFv molecules of the structure scFv (A) -linker-scFv (B) (Mallender and Voss, J. Biol. Chem. 269, 199-206, 1994) can be expressed both in bacteria and in eukaryotic cells. Here, however, non-functional associations of the four variable domains can sometimes also occur.

Recombinant antibody technology has led to the development of new small, bivalent or bispecific antibody fragments in recent years. Such molecules are, for example, the "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). These are variable VH and VL domains of the immunoglobulins, which are linked by a very short linker. This linker is too short to cause the VH and VL domains of a chain to assemble, as is the case with single-chain Fv fragments. As a result, the VH and VL domains of two chains are combined to form a dimer, so that molecules with two binding sites are formed (Perisic et al., Structure 2, 1217-1226, 1994). Bispecific "diabodies" result from the expression of two chains of the structure VH (A) -VL (B) and VH (B) -VL (A) in one cell. Here VL means the variable (V) domain of the light (L) chain and VH the variable domain (V) of the heavy (H) chain of an immunoglobulin, these variable domains binding the antigen (A) or (B). The assembly of the VH parts with the VL parts results in heterodimeric fragments with functionally active binding sites. Bacterially expressed bispecific "diabodies" have already been successfully used for the recruitment of various effector molecules, immunoglobulins, C1q or enzymes or effector cells, such as cytotoxic T lymphocytes (Kontermann et al., Nature Biotechnol. 15, 629 (1997); Holliger et al., Protein Engin. 9, 299 (1996); Nature Biotechnol. 15, 632 (1997), Zhu et al., BioTechnol. 14, 192 (1996); FitzGerald et al., Protein Engin. 10, 1221 ( 1997); Krebs et al., J. Biol. Chem. 273, 2858 (1998)).

<img file="EP0952218A2_D0001.tif" />However, diabodies "have the following disadvantages: Since the two VH (A) -VL (B) and VH (B) -VL (A) chains are not physically connected to each other, homo- and heterodimers can be formed to the same extent, which necessitates a very complex cleaning process for the heterodimers. Furthermore, the dimers dissociate, as has already been shown for scFv fragments (Glockshuber et al., Biochem. 29, 1362-1367 (1990)). To solve these problems, disulfide-stabilized "diabodies" (FitzGerald et al., Protein Engin. 10, 1221-1225, 1997) or "knob-into-hole diabodies" (Zhu et al., Protein Sci. 6, 781 -788, 1997). However, their production is associated with considerable effort. In addition, a signal sequence and a ribosome binding site are required for the genetic engineering expression of a bispecific "diabody", which is very complex. In addition, non-equimolar amounts of the variable domains can be expressed, which increases the proportion of non-functional homodimers.

The object of the present invention was therefore to find multi-antigen-binding molecules which have the disadvantages of the so-called <img file="EP0952218A2_D0001.tif" />Diabodies "do not have and which can be produced in a predominantly homogeneous form in a simple manner.

The present invention therefore relates to a single-chain, multi-antigen-binding molecule comprising the following components:<ul id="ul0001" list-style="none" compact="compact"><li>a) a variable domain of an immunoglobulin heavy chain (VH) with a first specificity (A) and functional parts thereof,</li><li>b) a variable domain of an immunoglobulin light chain (VL) with a second specificity (B) and functional parts thereof,</li><li>c) a variable domain of an immunoglobulin heavy chain (VH) with specificity (B) and functional parts thereof, and</li><li>d) a variable domain of an immunoglobulin light chain (VL) with specificity (A) and functional parts thereof,</li></ul> wherein the domains VH and VL in the form of a VH-VL construct or VL-VH construct and the two VH-VL constructs are connected via a peptide (P).

In a further embodiment, the molecule according to the invention contains more than two of the VH-VL constructs mentioned. In this way, molecules can be obtained which contain several specificities (A), (B), (C) etc.

In a preferred embodiment, the individual VH-VL constructs are bound with the same specificity via their variable domain (for example VH (B) -peptide-VL (B)). A particularly preferred structure of a molecule according to the invention is shown in FIG. 1.

In a further preferred embodiment, the individual specificities are essentially identical. The term<img file="EP0952218A2_D0001.tif" />According to the present invention, "essentially identical" means that the molecular structure of the domains can be quite different, but their specificity, ie the specific antigen-binding function, is retained. It follows from this that the molecule according to the invention does not just represent a complete variable domain of a heavy or can contain the light chain of an immunoglobulin, but also its functional parts, ie the parts that have retained their specific antigen-binding properties. For the purposes of the present invention, this also includes, for example, artificial constructs that consist of the individual CDR parts (<img file="EP0952218A2_D0001.tif" />complementary determining regions ") of variable domains of an immunoglobulin (Jones, PT et al. Nature, 321, 522, 1986; Riechmann, L. et al. Nature 332, 323, 1988; Verhoeyen, M. et al. Science, 239, 1534, 1988).

In a preferred embodiment, the domains VH and VL are connected via a peptide linker (L) in the form of a VH-L-VL construct or VL-L-VH construct. The linker should generally be as short as possible, preferably about 1 to 20 amino acids, in particular about 1 to 5 amino acids. A linker with the sequence GGGGS is particularly preferred.

In contrast to the linker (L), the connecting peptide (P) can be of any length. However, a peptide (P) with approximately 12-40 amino acids, in particular with approximately 12-20 amino acids, especially with approximately 14 amino acids, is preferred. Particularly preferred is a peptide (P) which contains the amino acid sequence GGGGSGGRASGGGS or GGGGSGGRASGGGGS and in particular a peptide (P) which consists of the amino acid sequence mentioned.

In a further preferred embodiment, the molecule according to the invention contains an effector (E) as a further component. This effector can be bound to the molecule according to the invention directly or, if appropriate, via a link (B). It is particularly preferred if the link (B) contains a protease cleavage sequence, preferably a PSA (prostate-specific antigen), cathepsin, plasminogen and / or plasminogen activator cleavage sequence.

The protease cleavage sequence is particularly preferred because it enables the effector to be separated from the molecule according to the invention with the aid of a protease. The separation is particularly advantageous if the activity of the effector is inhibited by direct or indirect binding of the effector to the molecule according to the invention.

Since proteases are particularly present in areas of inflammation or in tumors, the effectors, which are preferably inactivated due to their binding to the molecule according to the invention via a protease cleavage sequence, are released locally to a high degree. By selecting suitable target structures for the molecule according to the invention, for example with a specificity for antigens on tumor cells, for antigens on tumor-associated endothelial cells or for antigens on inflammatory cells, such as lymphocytes or macrophages, an additional enrichment of the molecule according to the invention with the effector in the range of, for example, one can Inflammation or a tumor can be reached.

According to the present invention, several effectors can also be bound directly or indirectly to the molecule according to the invention.

Proteases or the associated cleavage sequences are listed in detail in DE19704301.1, for example.

An example of a preferred molecule according to the invention with an effector which is bound to the molecule via a link is shown schematically in FIG. 2.

The selection of the individual components of the molecule according to the invention generally depends on the field of application.

If the molecule according to the invention is to be used as a diagnostic agent, for example in a further embodiment the first specificity (A) is directed against a molecule to be analyzed and the second specificity (B) directly or indirectly against an analyte. For example, the analyte can be a radioactive molecule, a fluorescent molecule or an enzyme which, through its enzymatic activity, converts a precursor of an analyte into an active analyte.

In a further preferred embodiment, the first specificity (A) is directed against a molecule to be analyzed, the second specificity (B) is directed against another molecule to be analyzed and the effector (E) is an analyte, for example a radioactive molecule, a fluorescent molecule and / or an enzyme, as already explained in more detail above.

However, the molecule according to the invention can also be used as a ligand for the target cell-specific binding of a viral or non-viral vector. In a special embodiment, the molecule according to the invention can be used as a so-called multifunctional ligand. For this purpose, it is advantageous if the peptide (P) and / or the effector (E) contains a fusiogenic peptide. The multifunctional ligand is used for the target cell-specific transfer of nucleotide sequences and is generally a protein which contains a target cell-specific ligand, a gene construct-specific ligand and a fusiogenic peptide. With the help of such multifunctional ligands, gene constructs, ie Nucleic acid constructs are specifically bound to a target cell and the fusiogenic peptide enables the penetration of the nucleic acid construct through the cell membrane into the cell nucleus and thus also the release from the endosome.

When using the molecule according to the invention as a ligand for a vector or as a multifunctional ligand, it is advantageous if the first specificity (A) is directed against a target cell and the second specificity (B) against a vector. The vector is generally a nucleic acid, a cationic peptide or protein, a cationic lipid, a cationic polymer or a cationic porphyrin. In a particular embodiment of this invention, the vector is a viral vector, derived for example from adenoviruses (AdV), adeno-associated viruses (AAV), vaccinia virus, RSV, HSV, influenza virus, or lentivirus.

Examples of target cell-specific ligands, for membrane structures on the target cell, for target cell-specific ligands and for gene construct-specific ligands which are derived from immunoglobulins, ie which contain VH and VL domains, and also of peptides with a fusiogenic property are described in detail in DE19649645. 4 described.

The molecule according to the invention is also suitable for prophylaxis and / or as a therapeutic agent.

For this purpose, for example, the first specificity (A) against a cell membrane, such as against lymphocytes, macrophages, monocytes, granulocytes, hematopoietic cells, endothelial cells, smooth muscle cells, striated muscle cells, epithelial cells, liver cells, kidney cells, glial cells, cells of the supporting tissue, tumor cells or leukemia cells , or against proteins of the extracellular matrix, the complement system, the coagulation system, the kinine system, the blood plasma, the supporting tissue, or against cytokines or chemokines, or against endogenous or foreign toxins, or against medicaments, for example digitalis, and / or against infectious agents, such as bacterial, viral and / or parasitic infectious agents.

The second specificity (B) can be directed, for example, against a cell membrane of, for example, lymphocytes, macrophages, monocytes or granulocytes, with the result that their crosslinking with a target structure there can lead to cytotoxic, immunomodulating or inflammatory processes.

It can also be directed against cytokines, chemokines or growth factors, with the result that their crosslinking with a target structure can trigger immunomodulating or proliferative processes there. It can also be directed against proteins of the complement system which initiate, amplify or inhibit its activation. Depending on the choice of the protein, the crosslinking of such a protein with a target structure can be inflammatory and cytolytic or trigger anti-inflammatory and cytoprotective reactions. It can also be directed against proteins of the coagulation system which initiate, amplify or inhibit its activation. The crosslinking of such a protein with the target structure can induce or prevent thrombosis, depending on the choice of the protein. Furthermore, it can be used against fibrinolytic proteins, which lead to the dissolution of fibrin clots on the target structure, against enzymes, which on the target structure convert the ineffective precursor of an active ingredient into an active, e.g. B. can transfer cytotoxic agent, against peptide hormones or steroid hormones, against the constant part of an immunoglobulin, against a mediator, such as histamine, serotonin, leukotriene, prostacyclin or kinin, against infectious agents, such as bacterial, viral and / or parasitic infectious agents or against tumor cells.

Cytokines as target structures or monocytes, macrophages and / or lymphocytes as target structures can be cross-linked with infection or tumor antigens by the molecule according to the invention and thereby trigger an increased immune reaction against the antigen in vivo. It is advantageous here to add the respective antigen from the infectious agent or from the tumor and, if appropriate, also the cytokine to the molecule according to the invention and to administer or inject the crosslinked complex locally.

The second specificity (B) can also be directed against endogenous or foreign toxins, so that either neutralization of the toxin and phagocytosis or a toxic reaction to the target structure can be brought about. It can also be directed against drugs, such as digitalis, so that complexation and elimination of the drug can be effected.

In a further preferred embodiment, the molecule according to the invention with the effector allows two identical or different target structures to be linked with one or more effectors.

Suitable as effectors are, for example, a transmembrane domain, a glycophospholipid anchor, the part of a receptor that binds the ligand; the ligand for a receptor or the partial sequence of the ligand binding the receptor; a peptide hormone; a cytokine; a growth factor; a growth factor inhibitor; a chemokine; an interferon; a mediator; a circulatory peptide; an enzyme which converts an inactive precursor of an active substance into an active active substance; a protein that activates or inhibits coagulation; a protein that activates or inhibits fibrinolysis; a protein that activates or inhibits the complement system; one or more constant domains of an immunoglobulin; a cytotoxic peptide; another, single-chain, single or multiple, in particular double antigen-binding molecule; a tumor antigen or the antigen of an infectious agent, such as a bacterial antigen or a viral antigen; a peptide containing cysteine ​​for the production of dimers of the molecule according to the invention; and / or a di- or multimerizing peptide (Plückthun and Pack, Immunotechnol. 3, 83-105 (1997)).

Another object of the present invention is a nucleic acid coding for a molecule according to the invention. The nucleic acid is generally a DNA or RNA, preferably a double-stranded DNA.

For the desired secretion of the expression product according to the invention, the nucleic acid according to the invention contains at the 5 'end a nucleotide sequence coding for a signal or transmembrane sequence (see, for example, DE19639103.2 or DE19651443.6). An example of suitable signal or transmembrane sequences is the signal sequence for the immunoglobulin (DNA position ≤ 63 to ≥ 107), the signal sequence for the CEA (DNA position ≤ 33 to ≥ 134) or the signal sequence of the Human Respiratory Syncytial Virus Glycoproteins ( cDNA of amino acid sequences ≤ 38 to ≥ 50 or 48 to 65).

In a further embodiment, the nucleic acid according to the invention contains a promoter and / or activator at the 5 'end. The activator is preferably cell-specific, cell cycle-specific, metabolically specific and / or can be activated or suppressed by an active ingredient. Such activator sequences, including their combinations, are described in EP97101507.8, DE19617851.7, DE19639103.2, DE19651443.6, DE19704301.1, EP97102547.3 and DE19710643.9. Particularly preferred nucleic acid constructs according to the invention are shown schematically in FIGS. 3 and 4.

In a preferred embodiment, the nucleic acid according to the invention contains the sequence GCCACC or GCCGCC at the 5 'end of the start codon, which sequence can increase the translation.

Another embodiment of the present invention relates to a vector containing the nucleic acid according to the invention. The vector can be a viral or non-viral vector, preferably a non-viral vector, which is particularly selected from a cationic lipid, a cationic polymer, a cationic peptide or a cationic porphyrin.

To produce the molecules according to the invention, the nucleic acid described is cloned into an expression vector, for example a suitable plasmid, into a suitable cell, for example into a bacterial, yeast, insect or mammalian cell, the cell transformed or transfected in this way is cultivated and the expression product, if appropriate isolated. The methods are generally known to the person skilled in the art and are described, for example, by Sambrook J. et al. Molecular Cloning, A Laboratory Handbook 2nd ed., Cold Spring Harbor Laboratory Press, 1989.

In a particular embodiment of the present invention, these cells express a molecule according to the invention with an effector, this effector preferably being a transmembrane domain.

For example, the DNA sequence coding for the transmembrane sequence of the human macrophage colony-stimulating factor (DNA position ≤ 1485 to ≥ 1554) or the DNA sequence coding for the signal and transmembrane region of the human respiratory syncytial virus (RSV) glycoprotein G (Amino acids 1 to 63 or their partial sequence amino acids 38 to 63) or the DNA sequence coding for the signal and transmembrane region of the influenza virus neuraminidase (amino acids 7 to 35 or the partial sequence amino acids 7 to 27) are inserted between the promoter sequence and the DNA sequence of the molecule according to the invention or at the 3 'end of the gene.

To anchor the active substance in the cell membrane of the cells expressing the molecule according to the invention, however, a nucleotide sequence coding for a glycophospholipid anchor can also be inserted into the nucleic acid construct.

A glycophospholipid anchor is generally inserted at the 3 'end of the nucleotide sequence coding for the molecule according to the invention and can also be used to insert a signal sequence.

Glycophospholipid anchors have been described, for example, for the CEA, for the N-CAM and for other membrane proteins, such as, for example, Thy-1.

Through this transmembrane region, the respective cell expresses the molecule according to the invention on the cell membrane and thus receives a "receptor" which is specific for those target or effector structures which are recognized by the antigen-binding parts of the molecule according to the invention.

Another preferred receptor is the transmembrane or signal transducing domain of a receptor, for example the T cell receptor or the M-CSF receptor. As a result, the cell expressing the molecule according to the invention on the cell membrane can be activated by binding the target structures to specific functions. Such specific functions can be, for example, cytotoxic reactions of T lymphocytes, phagocytosis reactions of macrophages and granulocytes or exocytosis reactions of granulocytes, monocytes and macrophages.

Another object of the present invention is therefore a cell containing a nucleic acid according to the invention or a vector according to the invention, in particular a bacterial, insect, yeast or mammalian cell. In addition to the generally known cells for the expression of nucleic acids, such as, for example, CHO or BHK cells, also a lymphocyte, a macrophage, a glial cell, an epithelial cell, a liver cell, a kidney cell, a bone marrow cell, an endothelial cell, a smooth or striated muscle cell or a fibroblast are suitable.

The last-mentioned cells are particularly suitable for gene therapy use, since these cells, which contain the nucleic acid construct according to the invention, can be injected locally or parenterally, for example intravenously, intraarterially, into a body cavity, into an organ or subcutaneously, in order to prevent them or to serve therapy for a disease.

For the gene therapy treatment of diseases, however, the nucleic acid constructs according to the invention can also be administered directly to the patient locally, into a body cavity, into an organ, into the blood vessel system, subcutaneously or intramuscularly.

Another object of the present invention is therefore also a medicament containing a molecule according to the invention, a nucleic acid according to the invention, a vector according to the invention or a cell according to the invention, and a diagnostic agent containing a molecule according to the invention, which is also directed against an analyte, as already described in more detail above.

The molecule according to the invention, the nucleic acid according to the invention, the vector according to the invention or the cell according to the invention are thus suitable for therapy, prophylaxis or diagnosis of, for example, tumor diseases, autoimmune diseases, inflammatory diseases, diseases of the blood, in particular of the blood coagulation and / or blood circulation system, diseases of the nervous system and / or infectious diseases.

The choice of the individual components generally depends on the use of the objects according to the invention. The individual components and their uses are described in more detail below in general and by way of example:

For the production of an object according to the invention, as already exemplified in FIGS. 3 and 4, the nucleic acid construct according to the invention at its 5 'end with the 3' end of a nucleic acid coding for a signal sequence and this in turn at its 5 'end with the 3 'end of a promoter sequence linked.

The promoter sequences to be selected include, for example, promoters and activator sequences which can be activated without restriction, such as the promoter of RNA polymerase III, the promoter of RNA polymerase II, the CMV promoter and / or enhancer, the SV40 promoter; or viral promoter and activator sequences, such as HBV, HCV, HSV, HPV, EBV, HTLV, HIV.

When using the HIV promoter, the entire LTR sequence including the TAR sequence [position ≤ -453 to--80, Rosen et al., Cell <u>41</u>, 813 (1985)] as a virus-specific promoter.

Other promoter sequences to be selected are, for example, metabolically activatable promoter and enhancer sequences, such as, for example, the hypoxia-inducible enhancer, cell cycle-specific activators, such as the promoter of the cdc25C gene, the cdc25B gene, the cyclin A gene, the cdc2 gene, the B-myb gene , the DHFR gene or the E2F-1 gene or binding sequences for cell cycle-specific occurring or activated transcription factors. These binding sequences include, for example, binding sequences for c-myc proteins. These binding sequences include monomers or multimers of the nucleotide sequence designated as Myc E-Box [5'-GGAAGCAGACCACGTGGTCTGCTTCC-3 '].

Further promoter sequences to be selected are, for example, promoters which can be activated by tetracycline, such as the tetracycline operator in combination with a corresponding repressor, or chimeric promoters. A chimeric promoter represents the combination of an upstream cell-specific, metabolically or virus-specific activator sequence with a downstream promoter module which contains the nucleotide sequence CDE-CHR or E2FBS-CHR, to which suppressive proteins bind, which hereby activates the upstream activator sequence in G₀- and G₁Inhibit phase of the cell cycle (WO96 / 06943; Lucibello et al., EMBO J. <u>14</u>, 132 (1995)).

Other promoter sequences to be selected are, for example, cell-specifically activatable promoters, such as preferably promoters or activator sequences from promoters or enhancers of those genes which code for proteins which are preferably formed in selected cells.

For example, in the context of the invention, promoters for the following proteins are preferably to be used in the following cells:

Promoter and activator sequences that are activated in endothelial cells are gene regulatory sequences from genes that code for the following proteins, for example: Brain-specific, endothelial glucose-1 transporter, endoglin, VEGF receptor-1 (flt-1), VEGF receptor-2 (flk-1, KDR), til-1 or til-2, B61 receptor (Eck Receptor), B61, endothelin, in particular endothelin B or endothelin-1, endothelin receptors, in particular the endothelin B receptor, mannose-6-phosphate receptors, from Willebrand factor, IL-1α, IL-1β, IL- 1-receptor, vascular cell adhesion molecule (VCAM-1) or synthetic activator sequences, that consist of oligomerized binding sites for transcription factors that are preferentially or selectively active in endothelial cells. An example of this is the transcription factor GATA-2, whose binding site in the endothelin-1 gene is 5'-TTATCT-3 '.

Promoters or activator sequences which are activated in cells in the vicinity of activated endothelial cells are gene-regulatory sequences from genes which code for the following proteins, for example: VEGF, the gene-regulatory sequences for the VEGF gene being the 5 'flanking region, the 3' flanking region, the c-Src gene or the v-Src gene, or steroid hormone receptors and their promoter elements, in particular the mouse mammary tumor virus promoter.

Promoters or activator sequences that are activated in muscle cells, especially smooth muscle cells, are gene regulatory sequences from genes that code for the following proteins, for example: Tropomyosin, α-actin, α-myosin, receptor for PDGF, receptor for FGF, MRF-4, phosphofructokinase A, phosphoglycerate mutase, troponin C, myogens, receptors for endothelin A, desmin, VEGF (see above), "artificial" promoters, or Promoters of muscle-specific transcription factors, such as factors of the Helix-Loop-Helix (HLH) family (MyoD, Myf-5, Myogen, MRF4) or the zinc finger protein GATA-4.

The HLH proteins and GATA-4 show muscle-specific transcription not only with promoters of muscle-specific genes, but also in a heterologous context, including with artificial promoters. Such artificial promoters are, for example, multiple copies of the (DNA) binding site for muscle-specific HLH proteins such as the E-Box (Myo D) (e.g. 4x AGCAGGTGTTGGGAGGC) or multiple copies of the DNA binding site for GATA-4 of the α-myosin heavy chain gene ( e.g. 5'-GGCCGATGGGCAGATAGAGGGGGCCGATGGGCAGATAGAGG3 ')

Promoters and activator sequences which are activated in glial cells are, for example, gene regulatory sequences from genes which code, for example, for the following proteins: the Schwann cell-specific protein periaxine, glutamine synthetase, the glial cell-specific protein (Glial fibrillary acid protein = GFAP), the glial cell protein S100b, IL-6, CNTF, 5-HT receptors, TNFα, IL-10, insulin-like growth factor receptor I and II or VEGF (see above)

Promoters and activator sequences which are activated in hematopoietic cells are, for example, promoter sequences for genes of a cytokine or its receptor which are expressed in hematopoietic cells or in neighboring cells such as the stroma.

These include promoter sequences from genes which code, for example, for the following cytokines and their receptors: stem cell factor receptor, stem cell factor, IL-1α, IL-1 receptor, IL-3, IL-3 receptor (α-subunit) , IL-3 receptor (β-subunit), IL-6, IL-6 receptor, GM-CSF, GM-CSF receptor (α chain), interferon regulatory factor 1 (IRF-1), the promoter of IRF-1 is activated by IL-6 as well as by IFNγ or IFNβ, erythropoietin or erythropoietin receptor.

Promoters and activator sequences which are activated in lymphocytes and / or macrophages are, for example, the promoter and activator sequences of the genes coding for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragment of antibodies, for example IL-1 receptor, IL-1α, IL-1β, IL-2, IL-2 receptor, IL-3, IL-3 receptor (α-subunit), IL-3 receptor (β-subunit), IL-4, IL-4 receptor, IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1 (IRF-1), the promoter of IRF-1 being activated by IL-6 as well as by IFNγ or IFNβ, IFNγ Responsive Promoter, IL-7, IL-8, IL-10, IL-11, IFNγ, GM-CSF, GM-CSF receptor (α chain), IL-13, LIF, macrophage colony Stimulating Factor (M-CSF) receptor, type I and II macrophage scavenger receptors, MAC-1 (leukocyte functional antigen), LFA-1α (leukocyte functional antigen) or p150.95 (leukocyte functional antigen).

Promoter and activator sequences which are activated in synovial cells are, for example, the promoter sequences of genes coding for matrix metalloproteinases (MMP), such as MMP-1 (interstitial collagenase), MMP-3 (stromelysin / transin) or tissue inhibitors of metalloproteinases ( TIMP), such as TIMP-1, TIMP-2, TIMP-3.

Promoters and activator sequences which are activated in linseed cells are, for example, promoters of genes which code for the following proteins: HSP-70, bcl-1 / cyclin D-1, bcl-2, IL-6, IL-10, TNFα, TNFβ , HOX-11, BCR-Abl, E2A-PBX-1, PML-RAR (Promyelocytic Leukemia - Retinoic Acid Receptor) or c-myc, whereby c-myc proteins activate and activate multimers of the nucleotide sequence called Myc E-Box ( 5'-GGAAGCAGACCAGCTGGTCTGCTTCC-3 ').

Promoters or activator sequences that are activated in tumor cells are, for example, gene-regulatory nucleotide sequences with which transcription factors, formed or actively interact in tumor cells, interact.

For the purposes of this invention, the preferred promoters or activator sequences include gene regulatory sequences or elements from genes which encode proteins formed especially in cancer cells or sarcoma cells. The promoter of the N-CAM protein is preferably used for small cell bronchial carcinomas, the promoter of the "hepatitis growth factor" receptor or the L-plastin is used for ovarian carcinomas and the promoter of L-plastin or the polymorphic epithelial mucin (PEM) is used for pancreatic carcinomas .

A major advantage of the present invention is that the linking of the individual components favors a heterodimeric association, so that predominantly single-chain, multi-antigen-binding molecules are formed. Furthermore, the dissociation of the dimers is reduced, as has already been shown for scFv fragments (see, for example, Glockshuber et al., Biochem. 29, 1362-1367, 1990). The molecules according to the invention can thus be produced in a less complex and more homogeneous form than the so-called <img file="EP0952218A2_D0001.tif" />Diabodies ", even if they are disulfide stabilized or in the form of <img file="EP0952218A2_D0001.tif" />knob-into-hole diabodies ".

Furthermore, only one signal sequence and one ribosome binding site (RBS) is necessary for the production of the molecule according to the invention. In contrast, the inventive<img file="EP0952218A2_D0001.tif" />Diabodies "requires a signal sequence and an RBS for each chain. Another advantage of the present invention is that in the molecules according to the invention, equimolar amounts of the variable domains are expressed, while in the expression of the two chains of <img file="EP0952218A2_D0001.tif" />Diabodies "non-equimolar amounts can arise and thereby the proportion of non-functional homodimers is increased.

Another advantage of the molecule according to the invention is that it can be expressed simply and in functional form in bacteria as well as in yeasts, baculoviruses and eukaryotic cells. In addition to the secretion of the molecules according to the invention, there is also the possibility of expressing them intracellularly or in the membrane (see, for example, Biocca &amp; Cattaneo, Trends Cell Biol. 5, 248-252 (1995)).

Another advantage of the molecules according to the invention is that they can be widely used both as a diagnostic and as an active ingredient for the prophylaxis and / or therapy of a disease, such as bispecific antibodies.

The genes of effectors and promoter sequences must be selected with a view to the desired application and taking into account the target cell to be transduced. For example, the following combinations of promoter sequences and genes for effectors should be selected for the following diseases:

Therapy of tumors

For the therapy of tumors, the target cells used are, for example, proliferating endothelial cells, or stromal cells and muscle cells adjacent to the endothelial cell, or tumor cells or leukemia cells, as promoters, for example, endothelial cell-specific and cell cycle-specific, or cell-unspecific or muscle cell-specific and cell cycle-specific, or tumor cell-specific, or tumor cell-specific, leukemia-specific (solid) tumor cells- specific (solid) Promoters, and as effectors, for example, the following genes:

Genes of inhibitors of cell proliferation include the retinoblastoma protein (pRb = p110) or the related p107 and p130 proteins. The retinoblastoma protein (pRb / p110) and the related p107 and p130 proteins are inactivated by phosphorylation. Those genes of cell cycle inhibitors which have mutations for the inactivation sites of the expressed proteins are preferred to be used, without their function being impaired thereby. Examples of these mutations have been described for p110. The DNA sequence for the p107 protein or the p130 protein is mutated in an analogous manner. The p53 protein gene is also suitable. The protein p53 is inactivated in the cell either by binding to special proteins, such as MDM2, or by oligomerizing the p53 via the dephosphorylated C-terminal serine. A DNA sequence is therefore preferably used for a p53 protein which is shortened by the serine 392 at the C-terminal. Other suitable genes are the gene from p21 (WAF-1), from p16 protein, from other cdk inhibitors, from GADD45 protein or from bak protein.

Genes from coagulation-inducing factors and angiogenesis inhibitors are, for example, from plasminogen activator inhibitor-1 (PAI-1), PAI-2, PAI-3, angiostatin, interferons (IFNα, IFNβ or IFNγ), platelet factor 4, IL-12, TIMP-1 , TIMP-2, TIMP-3, leukemia inhibitory factor (LIF) or tissue factor (TF) and its coagulation-active fragments.

Genes of cytostatic and cytotoxic proteins are, for example, from perforin, granzyme, IL-2, IL-4, IL-12, interferons, such as, for example, IFN-α, IFNβ or IFNγ, TNF, such as TNFα or TNFβ, Oncostatin M, sphingomyelinase or magainin and magainin derivatives.

Genes from inflammation inducers are, for example, IL-1, IL-2, RANTES (MCP-2) monocyte chemotactic and activating factor (MCAF), IL-8, macrophage inflammatory protein-1 (MIP-1α, -β), neutrophil activating protein-2 (NAP-2), IL-3, IL-5, human leukemia inhibitory factor (LIF), IL-7, IL-11, IL-13, GM-CSF, G-CSF, M-CSF , Cobra venom factor (CVF) or partial sequences from the CVF which functionally correspond to the human complement factor C3b, ie which can bind to the complement factor B and, after cleavage by the factor D, represent a C3 convertase, human complement factor C3 or its partial sequence C3b, of cleavage products of the human complement factor C3, which functionally and structurally resemble the CVF, or of bacterial proteins which activate complement or trigger inflammation, such as porins from Salmonella typhimurium, "clumping" factors from Staphylococcus aureus, Modulins especially from gram-negative bacteria, "Major outer membrane protein" from Legionella or from Haemophilus influenza type B or from glue lines or M molecules from streptococcal group G.

Genes of enzymes for the activation of precursors of cytostatics are, for example, of enzymes which cleave inactive substances (prodrugs) into active cytostatics (drugs). Such substances and the associated prodrugs and drugs have already been described by Deonarain et al. (Br. J. Cancer<u>70</u>, 786 (1994)), Mullen (Pharmac. Ther <u>63</u>, 199 (1994)) and Harris et al. (Gene Ther<u>1</u>, 170 (1994)) has been clearly described. For example, the DNA sequence can use one of the following enzymes: herpes simplex virus thymidine kinase, varicella zoster virus thymidine kinase, bacterial nitroreductase, bacterial β-glucuronidase, vegetable β-glucuronidase from Secale cereale, human β-glucuronidase (human carboxypeptide) Example CB-A of the mast cell, CB-B of the pancreas or bacterial carboxypeptidase, bacterial β-lactamase, bacterial cytosine deaminase, human catalase or Peroxidase, phosphatase, in particular human alkaline phosphatase, human acidic prostate phosphatase or type 5 acidic phosphatase, oxidase, in particular human lysyloxidase or hinmane acidic D-aminooxidase peroxidase, in particular human glutathione peroxidase, hinmane eosinophilic peroxidase or human β-thyroid actinase, Galactosidase. A β-galactosidase is particularly preferred for converting the prodrug daunomycin-β-D-galactopyranoside into the cytostatic agent daunomycin.

Therapy of autoimmune diseases and inflammation

For the therapy of autoimmune diseases and inflammation, the target cells are, for example, proliferating endothelial cells or macrophages and / or lymphocytes or synovial cells, and the promoters are, for example, endothelial cell-specific and cell cycle-specific, or macrophage and / or lymphocyte-specific and / or cell cycle-specific, or synovial cell-specific and / or cell-specific and / or cell-specific and / or cell-specific the following genes as effectors:

Genes for the treatment of allergies are, for example, IFNβ, IFNγ, IL-10, antibodies or antibody fragments against IL-4, soluble IL-4 receptors, IL-12 or TGFβ.

Genes for preventing rejection of transplanted organs are, for example, IL-10, TGFβ, soluble IL-1 receptors, soluble IL-2 receptors, IL-1 receptor antagonists or soluble IL-6 receptors.

Genes for the therapy of antibody-mediated autoimmune diseases are, for example, TGFβ, IFNα, IFNβ, IFNγ, IL-12, soluble IL-4 receptors, soluble IL-6 receptors, immunosuppressive antibodies or their V_H and V_L- containing fragments.

Genes for the therapy of cell-mediated autoimmune diseases are, for example, IL-6, IL-9, IL-10, IL-13, TNFα or TNFβ, IL-13, an immunosuppressive antibody or its V_H- and V_L- containing fragments.

Genes of inhibitors of cell proliferation, cytostatic or cytotoxic proteins and enzymes for the activation of precursors of cytostatics have already been described in more detail above.

Genes for the therapy of arthritis are, for example, structural genes, the expressed protein of which directly or indirectly inhibits inflammation, for example in the joint, and / or promotes the reconstitution of extracellular matrix (cartilage, connective tissue) in the joint.

These include, for example, IL-1 receptor antagonist (IL-1-RA), since IL-1-RA inhibits the binding of IL-1α, β; soluble IL-1 receptor; since soluble IL-1 receptor binds and inactivates IL-1; IL-6, since IL-6 increases the secretion of TIMP and sin peroxides and decreases the secretion of IL-1 and TNFα by synovial cells and chondrocytes; soluble TNF receptor because soluble TNF receptor binds and inactivates TNF; IL-4, since IL-4 inhibits the formation and secretion of IL-1, TNFα and MMP; IL-10, since IL-10 inhibits the formation and secretion of IL-1, TNF∝ and MMP and increases the secretion of TIMP; Insulin-like growth factor (IGF-1) because IGF-1 stimulates the synthesis of extracellular matrix; TGFβ, especially TGFβ1 and TGFβ2, since TGFβ stimulates the synthesis of extracellular matrix; Superoxide dismutase or TIMP, in particular TIMP-1, TIMP-2 or TIMP-3.

Therapy of the hematopoietic system

For the therapy of the deficient formation of cells of the blood, the target cells are, for example, proliferating, immature cells of the hematopoietic system or stromal cells adjacent to the hematopoietic cells, as promoters, for example, specific and / or cell cycle-specific or cell-unspecific and cell cycle-specific promoters, and as effectors, for example The following genes: Genes for the treatment of anemia are, for example, from erythropoietin.

Genes for the therapy of leukopenia are, for example, from G-CSF, GM-CSF or M-CSF.

Genes for the therapy of thrombocytopenia are, for example, from IL-3, leukemia inhibitory factor (LIF), IL-11 or thrombopoietin.

Therapy of the nervous system

For the therapy of damage to the nervous system, target cells, for example, are glial cells or proliferating endothelial cells, promoters, for example, glia cell-specific and cell cycle-specific or endothelial cell-specific and cell cycle-specific, or non-specific and cell cycle-specific promoters, and as effectors, for example, the following genes:

Genes for neuronal growth factors include FGF, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) or ciliary neurotrophic factor (CNTF) .

Genes for enzymes are, for example, genes of tyrosine hydroxylase or dopadecarboxylase.

Genes for cytokines and their inhibitors that inhibit or neutralize the neurotoxic effect of TNFα are, for example, from TGFβ; of soluble TNF receptors, since TNF receptors neutralize TNFα; of IL-10 because IL-10 inhibits the formation of IFNγ, TNFα, IL-2 and IL-4; of soluble IL-1 receptors such as IL-1 receptor I or IL-1 receptor II, since soluble IL-1 receptors neutralize the activity of IL-1; from the IL-1 receptor antagonist or from soluble IL-6 receptors.

Therapy of the blood coagulation and blood circulation system

For the treatment of disorders of the blood coagulation and blood circulation system, target cells, for example, are endothelial cells, proliferating endothelial cells, somatic cells in the vicinity of endothelial cells and smooth muscle cells or macrophages, promoters, for example, cell-unspecific and cell cycle-specific, or endothelial cells, smooth muscle cells or macrophages-specific and cell cycle-specific , and as effectors, for example, the following genes:

Genes for the inhibition of coagulation or for the promotion of fibrinolysis are for example from the tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), from hybrids of tPA and uPA, from protein C, hirudin, from serine proteinase inhibitors ( Serpine), such as C-1S inhibitor, α1 -antitrypsin or antithrombin III, or from the tissue factor pathway inhibitor (TFPI).

Genes for promoting coagulation are, for example, F VIII, F IX, von Willebrand factor, F XIII, PAI-1, PAI-2 or tissue factor and fragments thereof.

Genes for angiogenesis factors are, for example, from VEGF or FGF.

Genes for lowering blood pressure are, for example, "nitric oxide synthase" from kallikrein or endothelial cells.

Genes for the inhibition of the proliferation of smooth muscle cells after injuries to the endothelial layer are, for example, from an antiproliferative, cytostatic or cytotoxic protein or from an enzyme for breaking down precursors of cytostatics into cytostatics, as already mentioned above, or from a fusion protein of one of these active ingredients a ligand, for example an antibody or antibody fragments, which is specific for muscle cells.

Genes for other blood plasma proteins are, for example, from albumin, C1 inactivator, serum cholinesterase, transferrin or 1-antitrypsin.

Prophylaxis and / or therapy of infectious diseases

For vaccinations, for example, muscle cells or macrophages and / or lymphocytes serve as target cells, promoters, for example, non-specific and cell cycle-specific or target cell-specific and cell cycle-specific promoters, and effectors, for example, genes for the prophylaxis of infectious diseases

The active substance selected is generally the DNA of a protein formed by the infectious agent, which leads to neutralization and / or killing of the pathogen by triggering an immune reaction, ie by antibody binding and / or by cytotoxic T-lymphocytes. Such neutralization antigens are already used as vaccine antigens (see eg Ellis, Adv. Exp. Med. Biol.<u>327</u>, 263 (1992).

According to the invention, a nucleic acid coding for neutralization antigens of the following pathogens is preferred: Influenza A virus, HIV, rabies virus, HSV (herpes simplex virus), RSV (respiratory syncytial virus), parainfluenza virus, rotavirus, VZV (varicella zoster virus), CMV (cytomegalo virus), measles virus, HPV (Human papilloma virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HAV (hepatitis A virus), vibrio cholera antigen, Borrelia burgdorferi, Helicobacter pylori or malaria antigen.

Such active substances within the meaning of the present invention also include the DNA of an anti-idiotype antibody or its antigen-binding fragments whose antigen-binding structures (the "complementary determining regions") represent copies of the protein or carbohydrate structure of the neutralizing antigen of the infectious agent (see above).

Such anti-idiotype antibodies and their cleavage products can particularly replace carbohydrate antigens in bacterial infectious agents and are described, for example, by Hawkins et al. (J. Immunother.<u>14</u>, 273 (1993)) and Westerink and Apicella (Springer Seminars in Immunopathol. <u>15</u>, 227 (1993)) is clearly described.

Other effectors are, for example, genes from "tumor vaccines". These include antigens on tumor cells, such as from Sedlacek et al., Contrib. to Oncol.<u>32</u>, Karger Verlag, Munich (1988) and Contrib. to Oncol<u>43</u>, Karger Verlag, Munich (1992) clearly presented.

Further examples are the genes of the following antigens or of the following anti-idiotype antibodies: Sialyl Lewis; Peptides on tumors that are recognized by T cells; proteins expressed by oncogenes; Blood group antigens and their precursors; Antigens on the polymorphic epithelial mucin; or antigens on heat shock proteins.

For the treatment of chronic infectious diseases, the target cells are, for example, liver cells, lymphocytes and / or macrophages, epithelial cells or endothelial cells, promoters, for example virus-specific or cell-specific and cell cycle-specific promoters, and the following genes as effectors:

Genes coding for a protein which has cytostatic, apoptotic or cytotoxic effects, or coding for an enzyme which cleaves a precursor of an antiviral or cytotoxic substance into the active substance.

Genes coding for antiviral proteins, such as antivirally active cytokines and growth factors such as, for example, IFNα, IFNβ, IFN-γ, TNFβ, TNFα, IL-1 or TGFβ, or antibodies of a specificity which inactivates the respective virus or its V_H and V_L containing fragments or its V linked via a linker_H and V_L Fragments as already described. Antibodies against virus antigens are, for example: anti HBV, anti HCV, anti HSV, anti HPV, anti HIV, anti EBV, anti HTLV, anti Coxsackie virus or anti Hantaan virus.

Another antiviral protein is a Rev binding protein. These proteins bind to the Rev-RNA and inhibit Rev-dependent post-transcriptional levels of retrovirus gene expression. Examples of Rev-binding proteins are: RBP9-27, RBP1-8U, RBP1-8D or pseudogenes of RBP1-8.

Genes coding for antibacterial proteins, such as antibodies, which neutralize bacterial toxins or opsonize bacteria. For example, these include antibodies against meningococcal C or B, E. coli, Borrelia, Pseudomonas, Helicobacter pylori or Staphylococcus aureus.

The following figures and examples are intended to explain the invention in more detail without restricting it thereto.

Description of the figures

<dl id="dl0001" compact="compact"><dt>Fig. 1</dt><dd>describes schematically the structure of a single-chain, double-antigen binding molecule</dd><dt>Fig. 2</dt><dd>describes schematically a single-chain, double-antigen-binding molecule with effector.</dd><dt>3 and 5</dt><dd>describe schematically nucleic acid constructs coding for a single-chain, double-antigen-binding molecule</dd><dt>Fig. 4</dt><dd>describes schematically a nucleic acid construct coding for a single-chain, double-antigen-binding molecule with effector.</dd><dt>Fig. 6</dt><dd>shows the recruitment of the β-galactosidase on plastic-bound CEA by the single-chain, double-antigen-binding protein (scDb-CEAGal) according to the invention in comparison to diabodies against CEA and E. coli β-galactosidase (CEAGal). A microtiter plate was coated with BSA as a negative control.</dd><dt>Fig. 7</dt><dd>shows the recruitment of the β-galactosidase by scDb-CEAGal, which was secreted by mammalian cells, using different amounts of scDb-CEAGal and β-galactosidase in CEA-coated microtiter plates. O-Nitrophenyl-β-D-galactopyranoside was used as the substrate. A microtiter plate was coated with BSA as a negative control.</dd><dt>Fig. 8 A</dt><dd>shows the death of LoVo cells in the presence of the single-chain, double-antigen-binding protein (scDb), β-galactosidase (β-gal), daunomycin-β-D-galactopyranoside (prodrug) and / or daunomycin (drug) according to the invention.</dd><dt>Figure 8B</dt><dd>shows control experiments with a CEA negative cell line (A549).</dd></dl>

Examples:

example 1

Production and bacterial expression of a single-chain, double-antigen-binding protein

The production of a single-chain, double-antigen-binding protein is illustrated using the example of a protein which recognizes the antigens "Carcinoembryonic Antigen" (CEA) and E. coli β-galactosidase:

The following DNA sequences were joined in the direction 5 'to 3' as follows:<ul id="ul0002" list-style="dash" compact="compact"><li>LacZ promoter</li><li>bacterial, ribosomal binding structure (AAGGAG)</li><li>bacterial signal sequence pelB (Power et al., Gene 113, 95-99 (1992))</li><li>VH-anti-CEA (Kontermann et al., Immunotechnol. 3, 137 (1997))</li><li>Left GGGS (Kontermann et al., (1997))</li><li>VL anti-β-galactosidase (Kontermann et al., (1997))</li><li>connecting peptide GGGGSGGRASGGGGS</li><li>VH anti-β-galactosidase (Kontermann et al., (1997))</li><li>Left GGGGS</li><li>VL-anti-CEA (Kontermann et al., (1997))</li><li>Myc epitope for antibody 9E10 EQKLISEEDLN (Munro &amp; Pelham, Cell 46, 291-300 (1986))</li><li>Polyhistidine HHHHHH for cleaning using IMAC (Kontermann et al., (1997))</li></ul>

The linkage of the construct was made possible by means of suitable restriction sites which were carried out by PCR amplification at the termini of the different DNA sequences (Kontermann et al., Immunotechnol. 3, 137 (1997)).

The linkage was carried out with the aid of enzymes and DNA ligases known to those skilled in the art and specific for the restriction sites. The enzymes are commercially available. The construct was cloned into the bacterial expression vector pAB1 (Kontermann et al., (1997)).

For cloning, the oligonucleotide primers LMB2 and LMB3 (Kontermann, RE (1997), supra) and<ul id="ul0003" list-style="none" compact="compact"><li>V<img file="EP0952218A2_D0002.tif" />-NotFor: 5'-TTG TTC TGC GGC CGC CCG TTT CAG CTG CAG CTT GGT GCC AGC ACC-3 ',</li><li>scDb-AscBack: 5'-TGC ATG CTA GGG CGC GCC TCG GGC GGA GGT GGC TCA CAG GTG CAG CTG GTG CAA TCT GG-3 ',</li><li>scDb-AscFor<img file="EP0952218A2_D0002.tif" />: GCT CGG TAA GGC GCG CCC AGC GCT GCC ACC GCC TCC ACC TAG GAC GGT CAG CTT GGT CCC-3 'and</li><li>pelB-Metminus: 5'-TTA CTC GCG GCC CAG CCG GCC ACG GCC CAG GT-3 'used.</li></ul>

For this purpose, the fragments VHB-VLA (fragment 1) and VHA-VLB (fragment 2) were amplified from the diabody CEAGal (Kontermann, RE (1997)) with the aid of the primers LMB2 and LMB3 and gel-purified. Fragment 1 was then primed with V<img file="EP0952218A2_D0002.tif" />-NotFor and scDb-AscBack amplified to introduce an AscI site and 7 amino acids of the linker. Fragment 2 was amplified with the primers LMB3 and scDb-AscForλ to insert an AscI site and 8 amino acids of the linker. The fragments were hydrolyzed with AscI and NotI (fragment 1) and SfiI and AscI (fragment 2) and cloned into the bacterial expression vector pAB1 (Kontermann, RE (1997)). The resulting insert encodes a single-chain polypeptide in which VHA-VLB is linked to VHB-VLA via a 15 amino acid long linker with the sequence GGGGSGGRASGGGGS.

The plasmid was then introduced into TG1 bacteria and these were cultivated using the methods familiar to the person skilled in the art (Kontermann et al., (1997)).

The bacteria were disrupted and the single-chain, double-antigen-binding protein was purified from the periplasmic preparation by immobilized metal affinity chromatography (IMAC) (Hochuli et al., Bio / Techn. 6, 1321-1325 (1988)). Details of this method are described in Kontermann et al., (1997).

The purified protein has a molecular weight of 60 kDa, as shown by SDS-polyacrylamide gel electrophoresis and gel filtration. About 200-300 µg of this molecule could be purified per liter of bacterial culture. The bacterially expressed, single-chain, double-antigen-binding protein reacted in the ELISA with CEA and β-galactosidase and was able to recruit the enzyme to plastic-bound CEA, as was shown by the conversion of o-nitrophenyl-β-D-galactopyranoside (Fig. 6). Furthermore, this single-chain, double-antigen-binding protein was able to live and also fix CEA-expressing LoVo cells by recruiting β-galactosidase and converting the substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal ) stain. LoVo cells with 10 µg / ml of the corresponding antigen-binding protein with 10 µg / ml β-galactosidase and X-Gal (0.8 mg / ml in PBS, 3mM potassium iron (III) -cyanide 3mM potassium iron (II) - cyanide) incubated. No staining was observed with various control cells (A549, HEK293) or with a diabody against chicken egg lysozyme and β-galactosidase (HELGal; Kontermann et al., (1997)).

Example 2

Eukaryotic expression of a single chain, double antigen binding protein

For expression in eukaryotic cells, the coding region of the single-chain, double-antigen-binding protein was cloned into a eukaryotic expression vector (pSecTagA, Invitrogen), the bacterial signal sequence being replaced by the Ig-κ signal sequence which is already contained in the vector.

For this purpose, the single-chain construct was amplified with the primers LMB2 and pelB-Metminus, whereby methionine of the pelB leader (position 21) was replaced by threonine. It was then hydrolyzed with SfiI and EcoRI and cloned into the vector pSecTagA. The mature single chain antigen binding protein contains 7 additional amino acids (AAQPATA) at the N-terminus of the VHA domain.

The plasmid was transiently transfected with lipofectamine (Gibco) in eukaryotic HEK 293 cells. Stable cells were selected in the presence of Zeocin. With these cells, a staining of the endoplasmic reticulum ER and the Golgi apparatus could be demonstrated in immunofluorescence experiments. Furthermore, the secretion of the single-chain, double-antigen-binding protein could be demonstrated by immunoprecipitation of a 60 kDa protein from the supernatant 35 S-Met labeled cells and by purification by means of immobilized metal affinity chromatography (IMAC). The purified protein is functionally active in the binding of β-galactosidase and CEA-coated microtiter plates or the recruitment of β-galactosidase to plastic-bound CEA (FIG. 7).

Example 3

In vitro enzyme recruitment by cocultivation with single-chain, double-antigen-binding protein-secreting cells

Recruitment was examined in vitro by coculturing the single-chain, double-antigen-binding protein-producing HEK 293 cells and CEA-positive LoVo cells. For this purpose, the cells producing the protein according to the invention were first cultivated with the LoVo cells in Transwell cell culture dishes (Costar) in which the two cell lines are separated by a membrane. After two days, β-galactosidase (10 µg / ml) was added and the recruitment was verified by adding the substrate X-Gal. A specific staining was found when coculturing with the cells producing the single-chain, double-antigen-binding protein, while control experiments with untransfected HEK 293 cells showed no staining.

Experiments in which the LoVo cells were replaced by A549 cells (CEA-negative) and experiments in which the cells producing the single-chain, double-antigen-binding protein were incubated with enzyme and substrate were also negative. The latter makes it clear that the cells producing the single-chain, double-antigen-binding protein are themselves not able to bind the enzyme.

In a further experiment, the HEK 293 cells secreting the single-chain, double-antigen-binding protein and the LoVo cells were cocultivated directly with an inoculation ratio of 1: 4 to 1:24. In order to distinguish the HEK 293 cells from the LoVo cells, the former were stained with CM-DiI (Molecular Probes) (red fluorescence) before adding the LoVo cells. After two days, β-galactosidase was added and the binding to cells by adding X-Gal (10 µg / ml) or detected by indirect immunofluorescence with an anti-β-galactosidase antibody (Biotrend). These experiments also showed a specific recruitment of the enzyme to the LoVo cells, while the HEK 293 cells were not stained. Control experiments with non-transfected HEK 293 cells were negative. These experiments prove that the single-chain, double-antigen-binding protein specifically and selectively recognizes tumor cells.

The conversion of non-toxic daunomycin-β-D-galactopyranoside to the cytotoxic daunomycin was investigated in further experiments. By incubating LoVo cells with purified, single-chain, double-binding protein (10 µg / ml) for one hour and then incubating at 37 ° for one hour with β-galactosidase (1 µg / ml) and daunomycin-β-D-galactopyranoside (2 µM) a specific nuclear localization of the resulting daunomycin could be demonstrated by means of autofluorescence of the substance, comparable to the coloring by direct incubation with daunomycin. No nuclear staining was observed in the absence of single-chain, double-antigen-binding protein or β-galactosidase and with control cells (HEK 293; single-chain, double-antigen-binding protein-producing HEK 293). These experiments demonstrate that the single chain, double antigen binding protein is able to recruit an enzyme to a tumor cell and that this enzyme can be used to convert a non-toxic precursor to a toxic substance.

In further experiments, this effect can be used to specifically kill tumor cells.

For this purpose, the LoVo cells were incubated in 96-well plates with the single-chain, double-binding protein (10 μg / ml) and then with daunomycin-β-D-galactopyranoside (5 μM) for one hour at 37 ° C. The cell death was analyzed after 2 days by means of a WST test (Boehringer Mannheim) (FIG. 8). The cell death during the conversion of the prodring into the drug was almost as good as in the presence of the bring (FIG. 8A). In the absence of a component or with CEA negative A549 cells, essentially no effect was found (Fig. 8B).

Example 4

Production of constructs for the intracellular expression of single-chain, double-antigen-binding proteins

For the intracellular expression of the single-chain, double-antigen-binding protein, the DNA of the gene for the single-chain, double-antigen-binding protein is amplified with different primers:<ul id="ul0004" list-style="dash"><li>transmembrane single chain, double antigen binding protein (TM-scDAP). A primer is used for this, which inserts the transmembrane domain of PDGFR at the 3 'end of the gene<ul id="ul0005" list-style="dash" compact="compact"><li>(LPFKVVVISAIIALVVLTIISLIILIMLWQKKPRYES)</li></ul></li><li>Endoplasmic reticulum localized single chain (<u>s</u>ingle <u>c</u>grove), double (<u>d</u>ouble) -<u>a</u>binding <u>P</u>red (ER-scDAP). A primer is used for this, which inserts an ER retention signal at the 3 'end of the gene (SEKDEL).</li><li>Cytoplasmic localized single chain, double antigen binding protein (cyto-scDAP). A primer is used for this, which replaces the signal sequence at the 5 'end of the gene with a methionine and a Kozak sequence for optimal translation initiation.</li><li>core localized single chain, double antigen binding protein (nuc-scDb). For this purpose, primers are used which replace the signal sequence at the 5 'end of the gene with a methionine and a Kozak sequence for optimal translation initiation and insert a nuclear localization sequence at the 3' end of the gene (P<u>KKKRK</u>VGGGT; the core localization sequence is underlined).</li></ul>

These fragments are cloned in suitable eukaryotic expression vectors (pSecTagA or pcDNA3; Invitrogen). The resulting constructs (TM-scDAP, ER-scDAP, cyto-scDAP, nuc-scDAP, and sec-scDAP (= secreted protein; see Example 2) are then transiently transfected into eukaryotic cells (3T3) and the localization of the expressed protein carried out Immunofluorescence was investigated using an anti-Myc epitope antibody. For the constructs sec-scDAP, ER-scDAP and TM-scDAP, a staining, as is typical for the secretory pathway, can be demonstrated, while cyto-scDAP shows a diffuse localization in the cytoplasm and nuc-scDAP shows a nuclear localization.