MONOMERIC AND DIMERIC ANTIBODY-FRAGMENT FUSION PROTEINS

Abstract: The present invention describes a new class of antigen binding molecules which contain Fv-fragments of an antibody but do not use the constant antibody domains. They can also dimerize with other antibody fragment molecules or with non-antibody fragment molecules to form bi- or multifunctional antibody-fragment fusion proteins and so-called miniantibodies, respectively. The new fusion proteins can be used in the broad field of diagnostic and therapeutical medicine.

Description: Monomeric and Dimeric Antibody-Fragment Fusion Proteins The present invention describes a new class of antigen bin¬ ding molecules which contain Fv-fragments of an antibody but do not use the constant antibody domains. They can also dimerize with other antibody fragment molecules or with non-antibody fragment molecules to form bi- or multifunctio¬ nal antibody-fragment fusion proteins and so-called ininan- tibodies, respectively. The new fusion proteins can be used in the broad field of diagnostic and therapeutical medicine. Background of the invention Since a few years there is a great interest in the biotechno- logical field to modify naturally occuring antibodies in order to obtain more specified and more individual antibody species. Therefore, attempts have been made to produce (modi¬ fied) antibody fragments. All naturally occuring antibodies of all classes have at least two binding sites. This enables them to bind to a surface with a greater functional affinity (also called avidity) than monovalent fragments, such as Fab fragments. Over the last few years, methods have been described (Skerra and Pluckthun, 1988, Science 240, 1038-1040 ; Better et al., 1988, Science 240, 1041-1043) with which functional antibody fragments can be produced in Escherichia coli. These include the Fv fragment (the heterodimer consisting of V H and V L ) and the Fab fragment (con¬ sisting of the complete light chain with the domains V L and C L as well as the first two doi ains of the heavy chains V H and C H ι) . The Fv fragment, however, has a tendency to dissociate into V H and V L and therefore, it is advantageous to link the two domains covalently. One particular way of linking them is by designing a peptide linker between them, either in the orien¬ tation V H -linker-V L or V L -linker-V H (Bird et al.,1988. Science 242, 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879) The resulting fragments are called single-chain Fv fragments. All these fragments are, however, conovalent. We describe in this invention a method to engineer small dimerization domains based on peptides forming amphipathic helices. These peptides will be referred to as "intercalating", but this term is only meant to express the ability of targeted association and not a restriction referring to a particular structure of the dimerization interface. While the methodology described here, is in principle appli¬ cable to either Fab, Fv or scFv fragments, it is the latter for which their use is most advantageous. In this case biva¬ lent fragments can be constructed cf very small size, and still the dissociation into V L and V H as well as the wrong matching of fragment chains, e.g. V L _V L can be prevented. Antibody fragments of small size are of particular advantage in many applications. In diagnostic applications (e. g. ELISA, RIA, etc.), the smaller molecules surface decreases the problems of nonspecific interactions, which are known to frequently involve the constant domains. The same is true in using antibody fragments as ligands in affinity chromatogra¬ phy. In tumor diagnostics or therapy, it is important that a significant proportion of the injected antibody penetrates tissues and localizes to the tumor, and is dependend on the molecular dimensions (Colcher et al., 1990, J. Natl. Cancer Inst. 82, 1191-1197) . Expression yields and secretion efficiency of recombinant proteins are also a function of chain size (Skerra & Pluckthun, 1991, Protein Eng. 4, 971) and smaller proteins are preferred for this reason. There- fore, molecules of a small size are advantageous for several reasons. Previously, decreasing the molecular dimensions of the anti¬ body meant the preparation of proteolytic fragments. The smallest bivalent fragments, (Fab)' 2 fragments, are still about twice the size of the present fragments of this inven¬ tion. Therefore, these new fragments combine three features: (a) small size, (b) bivalence or bifunctionality and (c) ability of functional expression in E. coli. There is great interest in bifunctional antibodies in a large number of areas. Bifunctional antibodies may be defined as having two different specificities for either two different antigens or for two epitopes of the same antigen. There are currently a number of methods how to produce bifun¬ ctional antibodies. However, none of the existing methods allows to produce exclusively bifunctional antibodies in vivo, but rather a mixture of molecular species always occur, requiring complicated and expensive separation procedures. Four principal methods can be distinguished. In the first, chemical crosslinking is used, which may advantageously use heterobifunctional crosslinkers. By this method, whole anti- bodies (Staerz et al., 1985, Nature ' 314, 628; Perez et al, 1985, Nature 316, 354-356), Fab fragments (Carter et al., 1992, Biotechnology 10, 163) and scFv fragments (Cumber et al., 1992, J. Immunol. 149, 120) have been chemically crosslinked after purification. The second previous method involved the fusion of two hybri- domas to give a so-called heterohybridoma or "quadroma". In this method, any light chain can pair with any heavy chain, and the two heavy chains can give homodimers or heterodimers resulting in very complicated product mixtures (Milstein & Cuello, 1983, Nature 305, 537) . The third method is related to the second and consists of transfecting two expression plasmids into a hybridoma cell, encoding the heavy and light chain of the second antibody (Lenz & Weidle, 1990, Gene 87, 213) or a retroviral vector ( De Monte et al., 1990, Acad. Sci. 87, 2941-2945). However, once introduced, the product mixture is identical as in the second procedure. Finally, antibodies have been reduced, mixed and reoxidized (Staerz & Bevan, 1986, Immunology Today 7) . Again, very complicated product mixtures are obtained requiring sophisti¬ cal separation and quality control procedures. Thus a method is still needed allowing the isolation of exclusively heterodimeric antibodies directly without the complicated preparation required from chemical crosslinking. In the present invention, this problem is solved by (i) covalently linking corresponding VH and VL domain in a scFv fragment and (ii) using dimerization domains only allowing the formation of heterodimers, such as certain leucine zip¬ pers and derivatives. Another important consideration in the present invention was the desire to make the MW of the bispecific antibody as small as possible for reasons explained above in detail. This was achieved by using scFv fragments. A number of uses of bispecific antibodies bave been descri¬ bed, and most of them would benefit from this new technology. For example, bispecific antibodies are of great interest in tumor therapy. One arm of the antibody may bind to a tumor marker, the other arm to a T-cell epitope, a toxin, or a radionuclide binding peptide or protein to bring a killing function close to the tumor cell. In diagnostics, one arm may bind to the analyte of interest and the other to a principle which can easily be quantified, e. g. an enzyme. Finally, in cellular applications, it may be advantageous to obtain higher selectivity in binding, if two different epitopes or the same protein complex can be recognized or if two diffe¬ rent proteins can be recognized on the same cell surface.- Thus, it was object of the invention to create new individual and stable antibody fragment fusion proteins with bi- or even mulitfunctional binding sites. It has been found that antibody fragment fusion proteins containing Fv-fragments could be produced by genetic enginee¬ ring methods which show specified and improved properties. Object of the invention is, therefore, a monomeric antibody- fragment fusion protein essentially consisting of a Fv-frag*^ ment of an antibody and a peptide which is capable to dime¬ rize with another peptide by noncovalent interaction. The term "noncovalent interaction" means every existing under normal condititions stable linkage which is not related to a covalent binding, for example linkage by Van der Waal's forces, (steric) interdigitation of amphiphilic peptides, especially peptide helices, or peptides bearing opposite charges of amino acid residues. The correspondingly effective peptides are called above and below interactive or intercala¬ ting peptides. The amphiphilic peptides consist of up to 50 amino acids. Preferrably they consist of 10 to 30 amino acids. In a pre- ferred embodiment of the invention the interactive peptide is a peptide helix bundle (comprising of a helix, a turn and another helix, see above) . In another embodiment the interac¬ tive peptide is a leucine zipper consisting of a peptide having several repeating amino acids, in which every seventh amino acid is a leucine residue. In other cases according to the invention the peptide bear positively or negatively charged residues, e.g. lysine (positively charged) or gluta- mic acid (negatively charged) in a way that this peptide can bind to another peptide (of a second monomeric unit) bearing opposite charges. The Fv-fragment and the intercalating peptide are linked together either directly or by a linker peptide, preferrably by a linker peptide. In a preferred embodiment the linker peptide is a hinge region sequence of an antibody. As defined, the Fv-fragment consists of the V L and V H region of an antibody. The Fv-fragment according to the invention is preferrably a single chain fragment. Single chain fragments can be obtained by standard techniques using standard linker molecules. Furthermore, object of the invention is a di eric fusion protein essentially consisting of two monomeric fusion pro¬ teins, wherein the linkage of the monomeric units bases on noncovalent interaction of identical or different peptides, characterized in that at least one monomeric unit is an antibody-Fv-fragment fusion protein as defined above. If the dimer contains two Fv-fragments, the Fv-fragments may be the same (identical antigen binding sites) or may be different (different antigen binding sites) . In these cases mono- and bispecific (Fv)- miniantibodies can be obtained. According to the invention bispecific mini-antibodies are preferred. The interactive peptides may be the same or may be different; preferrably, they are identical. The intercalating peptides may be associated in parallel or in antiparallel fashion. Object of the invention is, therefore, above all, a dimeric fusion protein consisting of two Fv-fragments with different specificity (antigen binding sites) and identical intercala¬ ting helix peptides, the antibody fragments and the peptides are linked together by a hinge region sequence. Furthermore, object of the invention is a dimer consisting of a monomeric unit containing a Fv-fragment and another monome¬ ric unit wherein the Fv-Fragment was replaced by a non-anti¬ body peptide. The non-antibody peptide may be a toxin, like ricin, a chelator- or metal binding peptide, or an enzyme (e.g. marker enzyme), or a peptide bearing a detectable lable (e.g. a radioisotope) . The non-antibody peptide can also bear a corresponding bin- ding site for said groups, binding sites directed to T-cells or T-cell fragments included. Furthermore, the invention relates to monomers and dimers, as defined above, wherein the interactive peptide(s) is (are) additionally fused at the C-terminus to target proteins/pep¬ tides as mentioned above, the corresponding binding sites included. Thus, the resulting fusion proteins and miniantibo¬ dies, respectively, are multifunctional. The invention relates, furthermore, to a process for prepara¬ tion of a monomeric antibody fusion protein as defined above, characterized in that the genes coding for the Fv-fragment, the interactive peptide and, if desired, the linking peptide are cloned into one expression plasmid, a host cell is trans¬ formed with said expression plasmid and cultivated in a nutrient solution, and the monomeric fusion protein is expressed in the cell or secreted into the medium. Object of the invention is, finally, a process for prepara- • tion of a dimeric fusion protein as defined above, characte¬ rized in that the genes coding for the complete monomeric fusion proteins or parts of it are cloned at least into one expression plasmid, a host cell is transformed with said expression plasmid(s) and cultivated in a nutrient solution, and either the complete dimeric fusion protein is expressed in the cell or into the medium, or the monomeric fusion proteins are separately expressed and the noncovalent lin¬ kage between the two monomeric units is performed in the medium or in vitro, and in the case that only parts of the fusion proteins were cloned, protein engineering steps are additionally performed according to standard techniques. The dimeric Fv-fragments containing fusion proteins according to the invention show a high avidity against corresponding antigens and a satisfying stability. These novel bivalent or bifunctional molecules can be prepared as folded and assem¬ bled molecules in E. coli. These miniantibodies are compa¬ tible with functional expression by secretion. Detailed description of the invention The oligomerization domains were selected for having a fairly small molecular weight and for being compatible with trans- port of the fusion protein through the membrane. They are based on two different types of amphiphilic helices. Amphiphilic helices are known to predominantly, but not exclusively, associate in two different molecular structures: Four helix bundles and coiled coils. The design and formation of helix bundles has been studied previously (Eisenberg et al., 1986, Proteins 1, 16-22; Ho and deGrado, 1987, J. Am. Chem. Soc. 109, 6751-6758; Regan and deGrado, 1988, Science 241, 976-978 ; Hill et al., 1990, Science 294, 543-546 ) . This molecule association is also known from natural proteins (Richardson, 1981, Adv. Prot. Chem. 34, 167 ) . The four helix bundle may be formed from either four separate molecules (each contributing one helix) , two molecules con- taining two helices each (connected as helix-turn-helix) or one molecule containing a helix-turn-helix-turn-helix-turn- helix motif. For dimerization or multimerization, only the first two are suitable. Three variations of this latter theme were tested. In the first, one helix of the sequence given in Eisenberg et al. (1986) ( Proteins 1, 16-22 ) was used. In the second, this sequence was extended by a small hydrophilic peptide ending in a cysteine. Once the helices are associated, the hydrophi- lie peptides are held in sufficiently close contact that they can collide and a disulfide bond can form under oxidizing conditions, as in the periplasm of E. coli. In the third variation, two helices are used in tandem, separated by a short turn encoding peptide. In the second design, peptides are used*which can form so- called coiled-coil structures. Such peptides occur in tran¬ scription factors such as e. g. GCN4 from yeast and have been called leucine zippers (Landschulz et al., 1988, Science 240, 1759-1764 ) . The crystal structure of this has been solved recently (O'Shea et al., 1991, Science 254, 539-544) and showed a parallel arrangement of the helices. A covalent attachment of the helices is possible by a small peptide extension, again containing a cystein. Since the helices are now parallel, the peptide extension can be much shorter, since the distance is much smaller. The various dimerization devices (intercalating helices) were however not fused to the antibody domain directly. It is advantageous to introduce a flexible peptide between the end of the scFv fragment and the beginning of the helix. As an example, the upper hinge region of mouse IgG3 has been used. However, a variety of hinges can be used. It is not required for dimerization per se, but provides a spacing of the two scFv domains similar to the antigen binding sites of a whole antibody. This way, the two binding sites span a greater distance in space and therefore can reach neighboring anti¬ gens on a solid surface. The naturally occuring hinges of antibodies are preferred embodiments of hinges in bivalent miniantibodies. In the case of bifunctional miniantibodies, the hinges may be shorter, since frequently molecules from different surfaces are to be crosslinked as close as possible, and flexibilty of the dimer is not necessary. The choice of the hinge is governed by the desired residue sequence,1 ength (Argos, 1990, J. Mol. Biol. 211, 943-958) , compatibility with folding and stability of the amphiphilic helices (Richardson & Richardson, 1988, Science 240, 1648-1652) , secretion and resistance against proteases . The present invention deals with peptides as dimerization devices, which should be as small as possible. One preferred embodiment is the use of peptides which can form amphipathic helices. Such helices shield the hydrophobic surface by dimerization or even multimerization. Helices of this type are characterized by their having hydrophobic patches on one face of the helix, and containing a sufficient number of helix-forming residues. Rules for such peptides are discussed in Eisenberg et al. 1986, O'Shea et al.,.1991 (Science 254, 539-544) ,1992 (Cell 68, 699-708) . Natural peptides of this type are found as the so-called leucine zippers, characterized by a periodic occurence of leucine (every seventh residue) and other hydrophilic resi¬ dues (e. g. valine) also every seventh residue. As these priniples are now understood (O'Shea et al. 1991, 1992, literature cited) , the sequence can be varied to incorporate residues which make the association of homodimers unfavor¬ able, but favors the association of heterodimers. Such sequence alteration can e. g. involve the incorporation of charge bridges, such that in the homodimers, like charges repel each other and in the heterodimer, opposite charges attract each other (see below) . The present invention can also be extended to bifunctional miniantibodies. In this case, dimerization devices (inter- calation peptides) have to be used which will only allow the formation of heterodimers, but not homodimers. A preferred embodiment of this part of the invention are two different coiled-coil helices, such as in naturally occuring leucine zippers, e. g. from the transcription factor proteins jun and fos (O'Shea et al.,1989. Science 245, 646-648 ). In a further embodiment of the invention, the constant scFv- hinge-helix can be extended at the C-terminus to result in a fusion protein. For example, a fusion to an enzyme may be made to use such bivalent constructs in diagnostics. Such enzymes are e. g. alkaline phosphatase, luciferase or horse radish peroxidase. The advantage of such a antibody-enzyme fusion protein would be that the bivalence of the antibody would lead to an enhanced binding to the surface-bound anti- gen. The advantage over a fusion protein prepared by conven¬ tional technology (i. e. chemical coupling of the antibody to the enzyme of choice) would be a greater batch-to-batch consistency; homogeneity of the product and the much simpler method of preparation, namely from E. coli in a single step. In the same fashion, the miniantibodies may be extended at the C-terminus to incorporate a toxin. Such immunotoxins would be bivalent or even bispecific and thus combine the advantages of such antibody fragments linked above with the advantages in tumor therapy known for immunotoxins. Simi¬ larly, a metal binding peptide or protein could be linked genetically to be used in radioimmunotherapy or in tumor imaging. The same advantages for any genetically encoded hybrid protein hold true as given above for the antibody-en- zyme fusions. In another embodiment of the invention, a construct of the type scFv-hinge-helix may be made to dimerize with another protein fused to a dimerization domain, in complete analogy as described above for the formation of bispecific minianti¬ bodies. In this fashion, the scFv fragment would e. g. be fitted with the helix of the fos protein. Such foreign pro¬ tein, which could be made to form heterodimers with the scFv fragment, include enzymes useful in diagnosis, toxins, metal- binding peptides or proteins useful in radioimmunotherapy or radio-imaging. Using the principles of this invention, the dimerization domains presented here can also serve for purification purpo- ses. A recombinant protein of any kind can be fused to a dimerization domain, e.g. to hinge-fos-zipper. After coex- pression with a scFv-hinge-jun, the heterodimer can be puri¬ fied in one step with an affinity column for the scFv-speci- ficity. In an alternative approach, the 'opposite' zipper, linked to a column support, 'catches' the protein-hinge-zip¬ per when passing through the column as a crude cell extract. ι 5 The elution of the pure fusion protein from the column is possible using the unfolding temperature of the zipper. A subsequent separation from the dimerization domain is achie¬ vable by introduction of a proteolytic site, e.g. for blood clotting factor Xa, into the hinge (Nagai & Thogerson, 1987, Meth. Enyzmol. 152, ,461-481) . A particular advantage of the miniantibodies described in this invention is the ability to assemble functionally in Escherichia coli. In the case of homobivalent constructs, a dimerization principle is used which allows the formation of homodimers. Examples described above include the coiled-coil helix (leucine zipper) of the yeast protein GCN4 or the helices from an antiparallel 4-helix bundle. In this case, the scFv fragment is expressed in the presence of a bacterial signal sequence and carries at the end of the gene of the scFv fragment the codons for a hinge and the dimerization helix or helix-turn-helix. The helices are compatible with secretion to the periplasmic space in E. coli, where protein folding, disulfide formation and assembly occurs. Under these conditions, the homodimeric proteins form by themselves and can directly be isolated in the dimeric form. If heterobivalent constructs are desired, two different scFv fragments or one scFv fragment associating with a different protein need to associate. In the preferred embodiment of this invention, both proteins to be assembled are expressed in the same cell, preferably on the same plasmid, preferably as a dicistronic operon. The design of artificial dicistronic operons is explained e.g. in Skerra et al. (1991, Protein Eng. 4, 971) . Since the assembly must take place in the periplasm, because the scFv fragment can only fold in the oxidizing milieu, both proteins must be transported and both must be fitted with a signal sequence. The dimerization peptides must be chosen such that they promote the associa¬ tion of two different proteins, but prevent the association of the respective homodimers. Examples of such proteins are the leucine zipper peptides of the proteins fos and jun (see above) . When not expressed in the same cell, the different scFv- hinge-zipper constructs have to be mixed together as a crude cell extract or purified protein and treated with raised temperature. In absence of the 'opposite' zipper, e.g. a scFv-hinge-jun-zipper construct is able to form homodimers. After short heating to the melting temperature of around 40~C, the zippers of the unwanted homodimer unfold and form a much more stable heterodimer (O'Shea et al., 1992, Cell 68, 699-708) . Without raising the temperature, formation of heterodimers in vitro is not possible, as tested in experi¬ ments. Short Description of the Figures and the Sequence Listing Fig. 1 scFv-Expression vector pLISC-SE containg the scFv- fragment. Fig. 2 Dicistronic scFv-hinge-zipper expression vector pACKxFyJ. Fig. 3 Functional ELISA; • The concentrations of the affinity purified proteins, measured by OD 280 (vertical axis) , refer to the molar number of binding sites per well (horizontal axis) . The ELISA plates were coated with phosphocholine-BSA, and the purified phosphocholine-specific minianti- body-proteins were bound and detected by an anti- McPC603 antiserum. (a) Comparison of various miniantibodies. (b) Comparison of miniantibody scHLXc with ScFV and whole IgA. Fig. 4 Functional Anti-lysozyme ELISA; PC-affinity purified samples of coexpressed anti-PC- anti-lysozyme bispecific miniantibody. + and - on the horizontal axis means: plus inhibitor (+) and without inhibitor (-) . The attached sequence listing refers to sequence identity numbers (S.I.N. ) : S.I.N. 1: Whole nucleotid- and amino acid sequence of the pLISC-SE vector. S.I.N. 2: Gene cassette of intercalating GCN4-leucine zip¬ per (nucleotid- and amino acid sequence) . S.I.N. 3: Gene cassette encoding intercalating antiparallel helix-turn-helix (nucleotid- and amino acid sequence) . S.I.N. 4: Gene cassette encoding intercalating jun-zipper and IgG3-hinge region. S.I.N. 5: Gene cassette encoding intercalating fos-zipper and IgG3-hinge region. S.I.N. 6: Gene cassette encoding intercalating jun-zipper and designed linker. S.I.N. 7: Gene cassette encoding intercalating fos-zipper and designed linker. Example 1: Construction of vectors for secreted single-chain fragments, containing a restriction site for introducing genes for intercalating peptides. Recombinant DNA-techniques were based on Sambrook et al. (1989, Molecular Cloning: A laboratory manual. Second edi¬ tion. Cold Spring Harbor Laboratory, New York) . Functional expression of the single-chain Fv fragments and the minian¬ tibodies in E. coli M83 was carried out with vectors similar to pASK-lisc (Skerra et al., 1991, Protein Eng. 4, 971) . Site directed mutagenesis was directly performed in these vectors according to Kunkel et al. (1987, Meth. Enzy- mol. 154, 367-382) and Geisselsoder et al. (1987, Biotechni- ques 5, 186-191) using the helper phage M13K07 (Vieira & Messing, 1987, Meth. Enzymol. 153, 3-11) . SDS-PAGE was car¬ ried out as described by Fling and Gregerson (1986, Anal. Biochem. 155, 83-88) . Concentrations of affinity-purified proteins were measured by OD 2 so using calculated extinction coefficients (Gill & von Hippel, 1989, Anal. Biochem. 182, 319-326) .A vector such as pASK40 (Skerra et al., 1991, Pro¬ tein Eng. 4, 971) is used, which contains an origin of repli¬ cation, a regulatable promotor, a bacterial signal sequence followed by a multiple cloning site, a transcription termina¬ tor and an origin for single stranded phages.The gene for the single-chain Fv fragment is designed as follows: The nucleo- tide sequence of a V H domain is directly followed by a linker sequence encoding preferably about 15 residues, preferably of the sequence (Gly 4 Ser) 3 , followed directly by the sequence of" the V L domain. Alternatively, the sequence of the V L domain may be directly followed by the sequence of the linker, followed by the sequence of the V H domain. If the antibody is of known sequence, the complete gene of the scFv fragment may be assembled from synthetic oligonuc- leotides. A detailed experimental procedure for such a gene synthesis of an antibody gene is e.g. given in Plϋckthun et al. (1987, Cold Spring Harbor Symp. Quant. Biol. 52, 105-112) . If the genes of the V H and V L domains are present in other vectors, the gene for the scFv fragment may be assembled from restriction fragments. For example, a restriction fragment encoding most of the V H domain may be excised from another plasmid, and a fragment encoding most of the V L domain may be excised from a plasmid. The remaining pieces of V L and V H and the linker for the scFv fragment can be provided by cassettes of synthetic oligonucleotides, which need to be ligated by standard methodology (Sambrook et al., 1989, literature cited) . The mixture of fragments is ligated into the vector pASK40 or a similar plasmid containing a pair of suitable restriction sites. If the genes of the antibody have not been cloned before, they may be directly obtained from the hybridoma cell pro- ducing the antibody by the polymerase chain reaction (PCR; PCR methodology is described in McPherson et al., 1991, PCR-A Practical Approach Oxford University Press, New York) . Pri¬ mers suitable for amplification of V H and V L domains have been given by Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA 86, 3833-3837; Huse et al., 1989, Science 246, 1275-1281; Larrick et al., 1989, Bio-technology 7, 934-938. The methodology of obtaining mRNA from hybridoma is described in these referen¬ ces as well. The separate V H and V L genes may be cloned into separate vectors, and the scFv gene assembled according to the principles explained above. If the ligated fragments do not result in a correct reading frame of the scFv fragment, a precise fusion with the signal sequence codons resident on the plasmid may be generated by site directed mutagenesis. The design of the oligonucleotides and the execution is possible for anyone skilled in the art. 2 t The scFv expression plasmid so obtained contains the codons for a bacterial signal sequence, directly followed by the first variable domain (V H or V L ) , a linker and the second variable domain (V L or V H ) under the control of a regulatable promotor. At the 3' end of this genes, corresponding to the C-terminus of the scFv protein, a unique restriction site is introduced into the expression plasmid to allow insertion of cassettes coding for the intercalating peptides. The restriction site is introduced by site directed mutagenesis using the method of Kunkel (1987, Meth. Enzymol. 154, 367-382) . An example of the complete sequence of a suitable single- chain Fv expression plasmid pLISC-SE for receiving an inter¬ calation peptide is shown in Fig. 1 and Sequence Identity No. (S.I.N.) 1 (see Squence Listing) . Example 2: Design and construction of a gene cassette enco¬ ding intercalating peptides of a leucine zipper. The gene cassette, fitted with restriction sites to be compa¬ tible with the restriction site at the 3' end of the scFv fragment gene, must encode the sequence of a hinge (con¬ nection the scFv fragment to the intercalating peptide) and the intercalation peptide itself. The hinge region, may however also be obmitted. As an example the sequence of the upper hinge region of mouse IgG3 (Dangl et al., 1988, EMBO J. 7, 1989-1994), followed by the sequence of the leucine zipper sequence of the yeast protein GC 4 (Oas et al., 1990, Biochemistry T29, 2891-2894), 7. 2 is back-translated into frequently used E. coli codons (S.I.N.: 2). Oligonucleotides are synthesized, and ligated into the vector pLISC-SE, previously digested with EcoRI and Hind III. Example 3: Design and construction of a gene cassette enco¬ ding intercalating peptides of a four-helix bundle. Analogous to Example 2, the sequence of the upper hinge region of mouse IgG3, followed by the sequence of the helix- turn-helix of a four helix bundle (Eisenberg et al., 1986, literature cited) is backtranslated into frequently used E. coli codons (S.I.N.: 3) . Oligonucleotides are synthesized, and ligated into the vector pLISC-SE, previously digested with Eco RI and Hind III. Example 4: Design and construction two gene cassettes enco¬ ding intercalating peptides of a leucine zipper and their co-expression. Analogous to Example 2, the sequence of the upper hinge region of mouse IgG3 followed by the sequence of the zipper sequence of the jun protein (O'Shea et al., 1992, literature cited) , is backtranslated into frequently used E. coli codons (S.I.N.: 4) . Oligonucleotides are synthesized, and ligated into the vector pLISC-SE, previously digested with EcoRI and Hind III. In a parallel reaction, the sequence if the upper hinge region of mouse IgG3, followed by the sequence of the zipper sequence of the fos protein (O'Shea et al., 1992, Cell 68, 699-708), is backtranslated into frequently used E. coli codons (S.I.N.: 5) . Oligonucleotides are synthesized, and ligated into the vector pLISC-SE, previously digested with Eco RI and Hind III.The two vectors thus each code for a different antibody scFv fragment, followed by a hinge peptide and a different leucine zipper peptide. To co-express the two scFv fragments, the whole scFv-hinge-zipper gene of the fos-containing product is excised from the vector as a Xba I-Hind III fragment and ligated into the vector, pLISC-SE- scFv-jun, containing already the scFv gene of the other antibody. The newly obtained vector then expresses the scFVi-linker- t - fos-zipper and the scFv 2 -linker 2 -jun-zipper from a single promoter as a dicistronic operon. An improved sequence for the hinge region in the context of fos and jun zippers is given in S.I.N.: 6 and 7. This hinge is shorter and therefore not as susceptible to proteolysis. In cases, where the distance between the two binding sites is of less importance, such shortened hinges may be advanta¬ geous. In this case, the "tail" of the scFv fragment has been shortened and the EcoRI site, which receive the genes for the intercalating peptides has been moved four residues upstream. Example 5: Purification of bivalent miniantibody from E. coli. E. coli M83, harboring a plasmid constructed as in examples II and III, are grown to an O.D. 550 of 0.5 and induced with IPTG at a final concentration of 1 mM. The cells are centri- fuged, resuspended in BBS buffer ( 200 mM Na-borate, 160 mM NaCl, pH 8.0) and the suspension is passed through a French press. In these examples, a phosphorylcholine binding minian- tibody is used. The miniantibody is purified by passage over a phosphorylcholine affinity chromatography as described (Chesebro and Metzger, 1972, Biochemistry 11, 766-771) . Example 6: Purification of a bispecific miniantibody from E. coli E. coli JM83, harboring a plasmid constructed as in examples II and III and containing a dicistronic structural gene for two different scFv (Fig. 2), are grown to an O.D. 550 of 0.5 and induced with IPTG at a final concentration of ImM. The cells are centrifuged, resuspended in BBS buffer (200 M Na-borate, 160 mM NaCl, pH 8.0) and the suspension is passed through a French press. In this example, a bispecific miniantibody is used containing both a specificity for phosphorylcholine as well as benzoyl- ampicillin. The miniantibody is purified by passage over a phosphorylcholine affinity chromatography as described (Che¬ sebro and Metzger, 1972, literature cited) . Example 7: Surface binding of bivalent miniantibodies The ELISA-plates (Nunc, Macrosorp) were coated with 400 ~g/ml phosphocholine-BSA in PBS buffer (20 mM phosphate, pH 7.2, 115 mM NaCl) . The hapten reagent was prepared from nitro- phenyl phosphocholine (Sigma) , which was reduced and diazoti- zed essentially as described (Chesebro & Metzger, 1972, literature cited) , and reacted by azo-coupling to BSA (Sigma) in borate-saline buffer (52.5 mM sodium borate, pH 9, 120 mM NaCl) at 4~C for 48 hours with subsequent dialysis against PBS. After blocking the non-coated plate surface with 5% skim milk (Nestle) in PBS buffer for at least 2 hours, the peri- plasmic extract or the purified protein was incubated in BBS buffer on the plate for 90 min at room temperature. After thorough washing (3 times) , remaining functional antibody fragments were detected according standard procedures (Harlow & Lane, 1988, "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory, 555-592) with rabbit anti-McPC603 serum and anti-rabbit immunoglobulin linked to peroxidase (Sigma) according to Gallati (1979, Clin. Chem. Clin. Biochem. 17, 1-4) . An enormous gain in binding, and thus sensitivity, is obser¬ ved for all miniantibody constructs, compared to the monome- ric scFv fragment. This is consistent with the simultaneous binding of two or even more binding sites to the same sur¬ face. _These avidity of the fusion protein scHLXc was compa¬ rable to the natural antibody McPC603, which could be detected with antigen-coated ELISA, while the monomeric scFv fragment could only be detected with hundred-fold higher concentrations (Fig. 3 a, b) . All binding is nearly totally inhibitible with soluble hapten, except of the monomeric scFv fragment. The thermodynamic affinity of the natural antibody to soluble phosphocholine is about 1.6 $ 10 s M- 1 and thus relatively weak (Metzger et al., 1971, Proceedings of the I st Congress of Imunology. Academic Press, New York, pp. 253-267), and this is apparently not sufficient for a monome¬ ric fragment-hapten complex to survive the repeated washing steps of a functional ELISA (Kemeny & Challaco be, 1988, "ELISA and other solid phase immunoassays", Wiley & Sons, New York) . Example 8: Surface binding of bifunctional miniantibodies Coexpressed bifunctional miniantibodies recognizing phos- phorylcholine with one arm and lysozyme with the other arm were purified by phosphocholine (PC) affinity chromatography and tested for lysozyme specificity. An ELISA-plate was coated with lysozyme, the ELISA was carried out as described in Example VII.Three different preparations show binding to the antigen-surface, which is completely inhibitible with soluble lysozyme (Fig. 4) . SEQUENCE LISTING: SEQ ID NO: 1 ACCCGACACC ATCGAATGGC GCAAAACCTT TCGCGGTATG GCATGATAGC 50 GCCCGGAAGA GAGTCAATTC AGGGTGGTGA ATGTGAAACC AGTAACGTTA 100 TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG TTTCCCGCGT 150 GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG 200 CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG 250 GCGGGCAAAC AGTCGTTGCT GATTGGCGTT GCCACCTCCA GTCTGGCCCT 300 GCACGCGCCG TCGCAAATTG TCGCGGCGAT TAAATCTCGC GCCGATCAAC 350 TGGGTGCCAG CTGTGTGGTG TCGATGGTAG AACGAAGCGG CGTCGAAGCC 400 TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA GTGGGCTGAT 450 CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT 500 GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC sso AACAGTATTA TTTTCTCCCA TGAAGACGGT ACGCGACTGG GCGTGGAGCA 600 TCTGGTCGCA TTGGGTCACC AGCAAATCGC GCTGTTAGCG GGCCCATTAA 650 GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG GCTGGCATAA ATATCTCACT 700 CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT GGAGTGCCAT 750 GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA 800 CZGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC 850 ATTACCGAGT CCGGGCTGCG CGTTGGTGCG GATGTCTCGG TAGTGGGATA 900 CGCAGATACC GAAGACAGCT CATGTTATAT CCCGCCGTTA ACCACCATCA 950 AACAGGATTT TCGCCTGCTG GGGCAAACCA GCGTGGACCG CTTGCTGCAA 1000 CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC CCGTCTCACT 1050 GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC 1100 GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG 1150 GAAAGCGGGC AGTGAGCGCA ACGCAATTAA TGTGAGTTAG CTCACTCATT 1200 AGGCACCCCA GGCTTTACAC TTTATGCTTC CGGCTCGTAT GTTGTGTGGA 1250 ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCTATG ACCATGATTA 1300 CGAATTTCTA GATAACGAGG GCAAAAA ATG AAA AAG ACA GCT ATC 1345 Met Lys Lys Thr Ala lie 1 5 GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT ACC GTA GCG CAG 1387 Ala lie Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Gin 10 15 20 GCC GAA GTT AAA CTG GTA GAG TCT GGT GGT GGT CTG GTA CAG 1429 Ala Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gin 25 30 CCG GGT GGA TCC CTG CGT CTG TCT TGC GCT ACC TCA GGT TTC 1471 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe 35 40 45 ACC TTC TCT GAC TTC TAC ATG GAG TGG GTA CGT CAG CCC CCG 1513 Thr Phe Ser Asp Phe Tyr Met Glu Trp Val Arg Gin Pro Pro 50 55 60 GGT AAA CGT CTC GAG TGG ATC GCA GCT AGC CGT AAC AAA GGT 1555 Gly Lys Arg Leu Glu Trp lie Ala Ala Ser Arg Asn Lys Gly 65 70 75 AAC AAG TAT ACC ACC GAA TAC AGC GCT TCT GTT AAA GGT CGT 1597 Asn Lys Tyr Thr Thr Glu Tyr Ser Ala Ser Val Lys Gly Arg 80 85 90 TTC ATC GTT TCT CGT GAC ACT AGT CAA TCG ATC CTG TAC CTG 1639 Phe lie Val Ser Arg Asp Thr Ser Gin Ser lie Leu Tyr Leu 95 100 CAG ATG AAT GCA TTG CGT GCT GAA GAC ACC GCT ATC TAC TAC 1681 Gin Met Asn Ala Leu Arg Ala Glu Asp Thr Ala lie Tyr Tyr 105 110 115 TGC GCG CGT AAC TAC TAT GGC AGC ACT TGG TAC TTC GAC GTT 1723 Cys Ala Arg Asn Tyr Tyr Gly Ser Thr Trp Tyr Phe Asp Val 120 125 130 TGG GGT GCA GGT ACC ACC GTT ACC GTT TCT TCT GGT GGT GGT 1765 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 135 140 145 GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GAT ATC 1807 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie 150 155 160 GTT ATG ACC CAG TCT CCG AGC TCT CTG TCT GTA TCT GCA GGT 1849 Val Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 165 170 GAA CGT GTT ACC ATG TCT TGC AAA TCT TCT CAG TCT CTG CTG 1891 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu 175 180 185 AAC TCT GGT AAC CAG AAA AAC TTC CTG GCG TGG TAT CAG CAA 1933 Asn Ser Gly Asn Gin Lys Asn Phe Leu Ala Trp Tyr Gin Gin 190 195 200 AAG CCT GGC CAA CCG CCG AAA CTG CTG ATC TAC GGT GCG TCG 1975 Lsy Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser 205 210 215 ACC CGT GAA TCT GGT GTT CCG GAC CGT TTT ACC GGT AGC GGT 2017 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 220 225 230 AGC GGT ACC GAC TTC ACT CTG ACC ATC TCT TCT GTA CAG GCT 2059 Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Val Gin Ala 235 240 GAA GAT CTG GCT GTT TAC TAC TGT CAA AAC GAC CAC TCT TAC 2101 Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp His Ser Tyr 245 250 255 CCG CTG ACC TTT GGC GCC GGC ACC AAA CTG GAA CTG AAG CGC 2143 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 260 • 265 270 GCT AAC GGT GAA TTC-TGATAAGCTTGA CCTGTGAAGT GAAAAATGGC 2190 Ala Asn Gly Glu Phe * 275 GCACATTGTG CGACATTTTT TTTGTCTGCC GTTTACCGCT ACTGCGTCAC 2240 GGATCCCCAC GCGCCCTGTA GCGGCGCATT AAGCGCGGCG GGTGTGGTGG 2290 TTACGCGCAG CGTGACCGCT ACACTTGCCA GCGCCCTAGC GCCCGCTCCT 2340 TTCGCTTTCT TCCCTTCCTT TCTCGCCACG TTCGCCGGCT TTCCCCGTCA 2390 AGCTCTAAAT CGGGGCATCC CTTTAGGGTT CCGATTTAGT GCTTTACGGC 2440 ACCTCGACCC CAAAAAACTT GATTAGGGTG ATGGTTCACG TAGTGGGCCA 2490 TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT CCACGTTCTT 2540 TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG 2590 TCTATTCTTT TGATTTATAA GGGATTTTGC CGATTTCGGC CTATTGGTTA 2640 SEQ ID NO : 2 GGT GAA TTC CCC AAA CCT AGT ACT CCC CCT GGC AGC AGC CGC ATG 45 Gly Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Arg Met 1 f 5 10 15 I lgG3-hinge ' ' AAA CAG CTG GAA GAT AAA GTT GAA GAG CTT CTT TCG AAA AAC TAC so Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr 20 25 30 GCN4-zipper CAC CTC GAA AAT GAA GTT GCG CGC CTC AAA AA CTT GTT GGT GAA 135 His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu 35 40 45 CGC TGATAAGCTT GAC 151 Arg f —-'stop SEQ ID NO: 3 GGT GAA TTC CCC AAA CCT AGC ACC CCC CCT GGC AGC AGT GGT GAA 45 Gly Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Glu I f 5 10 15 ' IgG3-hinge ' ' CTG GAA GAG CTG CTT AAG CAT CTT AAA GAA CTT CTG AAG GGC CCC 90 Leu Glu Glu Leu Leu Lys His Leu Lys Glu Leu Leu Lys Gly Pro 20 25 30 bundle-helix A — ' *-— CGC AAA GGC GAA CTC GAG GAA CTG CTG AAA CAT CTG AAG GAG CTG 135 Arg Lys Gly Glu Leu Glu Glu Leu Leu Lys His Leu Lys Glu Leu 35 40 45 — urn — - *■ ' bundle-helix B — CTT AAA GGT GAA TTC TGATAAGCTT GACCTGTGAA GTGAAAAAAT G 191 Leu Lys Gly Glu Phe f 50 t 1 st o p SEQ ID NO: 4 GGT GAA TTC CCC AAA CCT AGT ACT CCC CCT GGC AGC AGC CGT ATC 45 Gly Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Arg lie 1 5 10 15 I IgG3-hinge ' ' GCT CGT CTC GAG GAA AAA GTT AAA ACC CTG AAA GCT CAG AAC TCC 90 Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gin Asn Ser 20 25 30 -— — — — jun-zipper — GAA CTG GCT TCC ACC GCT AAC ATG CTG CGT GAA CAG GTT GCT CAG 135 Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin 35 40 45 — — jun-zipper — — — — . CTG AAA CAG AAA GTT ATG AAC TAC TGATAAGCTT GACCTGTGAA G 180 Leu Lys Gin Lys Val Met Asn Tyr 50 -J stop SEQ ID NO: 5 GGT GAA TTC CCC AAA CCT AGT ACT CCC CCT GGC AGC AGC CTG ACC 45 Gly Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Leu Thr 1 4 5 10 15 I IgG3-hinge 1 ' GAC ACC CTG CAG GCT GAA ACC GAC CAG CTG GAA GAC AAA AAA TCC 90 Asp Thr Leu Gin Ala Glu Thr Asp Gin Leu Glu Asp Lys Lys Ser 20 25 30 — — — — — _ _ —_ fos-zipper — ——.__—_ — GCT CTG CAG ACC GAA ATC GCT AAC CTG CTG AAA GAA AAA GAA AAA 135 Ala Leu Gin Thr Glu lie Ala Asn Leu Leu Lys Glu Lys Glu Lys 35 40 45 — — — — • fos-zipper — —.— — — — CTG GAA TTT ATC CTG GCT GCT TAC TGATAAGCTT GACCTGTGAA G 180 Leu Glu Phe lie Leu Ala Ala Tyr . 50 ' — — — -— — -.-.-—. 1 s op SEQ ID NO: 6 GGT GAA TTC CCG TCT GGT AAC GAA GCT CGT ATC GCT CGT CTC GAG 45 Gly Glu Phe Pro Ser Gly Asn Glu Ala Arg lie Ala Arg Leu Glu 1 f 5 10 15 I linker • ' GAA AAA GTT AAA ACC CTG AAA GCT CAG AAC TCC GAA CTG GCT TCC 90 Glu Lys Val Lys Thr Leu Lys Ala Gin Asn Ser Glu Leu Ala Ser 20 25 30 — jun-zipper — - — — — — — — — ACC GCT AAC ATG CTG CGT GAA CAG GTT GCT CAG CTG AAA CAG AAA 135 Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin Leu Lys Gin Lys 35 40 45 — jun-zipper — — — GTT ATG AAC TAC TGATAAGCTT GACCTGTGAA GTGAAAAATG GCG 180 Val Met Asn Tyr j SEQ ID NO: 7 GGT GAA TTC GGT CCG TCT GGT AAC GAA CTG ACC GAC ACC CTG CAG 45 Gly Glu Phe Gly Pro Ser Gly Asn Glu Leu Thr Asp Thr Leu Gin 1 f 5 10 15 I—— linker ——J I —— fos-zipper ————— GCT GAA ACC GAC CAG CTG GAA GAC AAA AAA TCC GCT CTG CAG ACC 90 Ala Glu Thr Asp Gin Leu Glu Asp Lys Lys Ser Ala Leu Gin Thr 20 25 30 — — fos-zipper — — — — — — — GAA ATC GCT AAC CTG CTG AAA GAA AAA GAA AAA CTG GAA TTT ATC 135 Glu lie Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe lie 35 40 45 fos-zipper — — — — — ~ — — — ____ CTG GCT GCT TAC TGATAAGCTT GACCTGTGAA GTGAAAAATGGCG 180 Leu Ala Ala Tyr 1 s top

Claims: Patent claims 1. Monomeric antibody-fragment fusion protein essentially consisting of a Fv-fragment of an antibody and a peptide which is capable to dimerize with another peptide by noncovalent interaction. 2. Monomer according to claim 1 characterized in that the Fv-fragment is a single chain fragment. 3. Monomer according to claim 1 or 2 characterized in that the interactive peptide consists of 10 to 50, preferrably 10 to 30 amino acids. 4. Monomer according to one of the claims 1 or 3 characteri¬ zed in that the peptide consists of at least one helix. 5. Monomer according to claim 4 characterized in that the helix peptide consists of a helix, a turn and another helix. 6. Monomer according to claims 4 characterized in that the peptide contains a leucine zipper molecule, having several repeating amino acids, in which every seventh amino acid is a leucine. 7. Monomer according to claims 4 characterized in that the peptide bears charged residues. 8. Monomer according one of the claims 1 to 7 characterized in that a linking peptide is between the Fv-fragment and the peptide. 9. Monomer according to claim 8 characterized in that the linking peptide is a hinge region sequence of an antibody or a fragment thereof. 10. Process for preparation of a monomeric antibody fusion protein as defined in claims 1 to 9, characterized in that the genes coding for the Fv-fragment, the interac- tive peptide and, if desired, the linking peptide are cloned into one expression plasmid, a host cell is trans¬ formed with said expression plasmid and cultivated in a nutrient solution, and the monomeric fusion protein is expressed in the cell or secreted into the medium. 11. Process according to claim 10 characterized in that the host cell is E. coli. 12. Dimeric fusion protein essentially consisting of two monomeric fusion proteins, wherein the linkage of the monomeric units bases on noncovalent interaction of identical or different peptides, characterized in that at least one monomeric unit is an antibody-fragment fusion protein as defined in claims 1 to 9. 13. Dimeric fusion protein according to claim 12 wherein the interactive peptides are the same. 14. Dimer according to claim 12 or 13 characterized in that the second monomeric unit is an antibody-fragment fusion protein as defined in claims 1 to 9 having different specificity. 15. Dimer according to claim 12 or 13 characterized in that the second monomeric unit is a fusion protein as defined in claims 1 to 9, wherein the antibody-fragment (Fv) is replaced by a non-antibody protein or peptide. 16. Dimer according to claim 15 characterized in that the protein or peptide is a toxin, a chelator peptide, a metal binding protein or an enzyme, or has the correspon¬ ding specific binding site. 17. Dimer according to claim 15 characterized in that the protein or peptide has a T-cell-, or a T-cell fragment specific binding site. 18. Dimer according to one of the claims 12 to 17, wherein another protein is fused at the C-terminus of one or both of the intercalating peptides. 19. Dimer according to claim 18, wherein the fused protein is a toxin, a chelator peptide, a metal binding protein or an enzyme, or has the corresponding specific binding site, or has a T-cell (fragment) specific binding site. 3S 20. Process for preparation of a dimeric fusion proteinas defined in claims 12 to 19 characterized in that the genes coding for the complete monomeric fusion proteins or parts of it are cloned at least into one expression plasmid, a host cell is transformed with said expression plasmid(s) and cultivated in a nutrient solution, and either the complete dimeric fusion protein is expressed in the cell or into the medium, or the monomeric fusion proteins are separately expressed and the noncovalent linkage between the two monomeric units is performed in the medium or in vitro, and in the case that only parts of the fusion proteins were cloned protein engineering steps are additionally performed. 21. Process according to claim 20 characterized in that the gene coding for the first monomeric fusion protein is cloned into a first expression plasmid, and the gene coding for the second monomeric fusion protein is cloned into a second expression plasmid. 22. Process according to claim 20 characterized in that the noncovalent linkage between the monomeric units forming the dimeric fusion protein is performed in vitro. 23. Process according to one of the claims 20 to 22 characte¬ rized in that the host cell is E. coli. 24. Construction kit for preparation of seletive dimers of antibody-fragment fusion proteins as defined in claims 12 to 19 containing(a) a monomeric antibody-fragment fusion protein as defined in claims 1 to 8, and (b) a second monomeric fusion protein as defined in (a) , wherein the antibody fragment has the same or another antigen speci- ficity, or wherein the antibody fragment unit is replaced by a non-antibody protein/peptide.

Inventors: PACK PETER, PLUECKTHUN ANDREAS, PACK, PETER, PLUECKTHUN, ANDREAS
Applicants: MERCK PATENT GMBH
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, A61K39, C07K14, C07K16, C07K19, C12N15, C12P21, C12R1, G01N33