WO2001085795A1

WO2001085795A1 - Trivalent antibody constructs having variable regions that are stabilized by disulfide bridges

Info

Publication number: WO2001085795A1
Application number: PCT/EP2001/005161
Authority: WO
Inventors: Stefan Dübel; Andreas Schmiedl
Original assignee: Universität Heidelberg
Priority date: 2000-05-05
Filing date: 2001-05-07
Publication date: 2001-11-15
Also published as: EP1287036A1; AU6028501A; DE10021678A1

Abstract

The invention relates to recombinant antibody constructs, to methods for the production thereof, to pharmaceutical compositions and diagnostic agents, which contain these recombinant antibody constructs, as well as to the use of these recombinant antibody constructs for diagnosing and treating cancerous diseases, autoimmune diseases, allergies, immunological hyperreactions, infections or noxae.

Description

description

TRIVALENT ANTI-BODY CONSTRUCTS WITH DISULFID BRIDGES STABILIZED VARIABLES

REGIONS

The present invention relates to recombinant antibody constructs, processes for their preparation, pharmaceutical compositions and diagnostic agents which contain these recombinant antibody constructs, and the use of these recombinant antibody constructs for the diagnosis and treatment of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxae.

Bispecific antibodies are best prepared recombinantly, since in the conventional quadroma or hybrid hybridoma technique a mixture of 10 different products occurs, from which the only correct one can only be separated with considerable effort (Milstein and Cuello, 1983). However, bispecific antibodies are necessary as agents for new types of therapeutic procedures that could not be carried out without such molecules. They are used in particular to stimulate the immune response against tumor cells (Segal et al., 1999; Hombach et al., 1993; Manzke et al, 1999). T-lymphocytes are specifically introduced to tumor cells and often additionally activated by one of the binding arms of the bispecific agent. Various molecular formats have been developed for the production of therapeutic antibodies in general and bispecific antibodies in particular (Breitling and Dübel, 1997; Carter and Merchant, 1997).

Extensive studies on tumor localization of different antibody formats (scFv, diabody, minibody, complete IgG) with an identical antigen binding site (tumor marker carcinoembryonic antigen, CEA) but with different molecular weights (approx. 30 kDa, 60 kDa, 90 kDa and 150 kDa) showed that that the two smaller formats due to their rapid filtration through the kidney to a much lower concentration ratio Tumor / tissue resulted as constructs whose molecular weights were above the filtration limit of the kidney (Wu et al., 1996; Hu et al., 1996). The best tumor localization was achieved by the so-called minibody (about 90 kDa), which consists of two identical scFv fragments, which dimerize through fused CH3 domains (Hu et al., 1996). This construct is bivalent, but monospecific. The production of bispecific antibodies according to this pattern is difficult because it can lead to homologous pairings and thus to undesirable and difficult-to-separate by-products.

The use of CH1 and CL _(k a _pp a ₎ domains for dimerization (Müller et al., 1998) solved the problem of by-products in the production of bispecific minibodies, but the affinity is limited by the monovalence of the respective antigen binding sites.

The stabilization of Fv fragments by disulfide bridges (dsFv, Brinkmann et al., 1993; Reiter et al., 1995) improved their serum half-life compared to scFvs and led to an increased localization of these constructs in the tumor tissue (Reiter et al., 1994a, b, c; Webber et al., 1995).

Bispecific diabodies, such as those often produced for therapy, are smaller than the pharmacokinetically optimal construct for tumor therapy in that they are composed of only two antigen-binding antibody fragments (Hollinger et al., 1993, 1996, 1999; Arndt et al., 1999 ; Helfrich ef a /., 1998).

The use of dsFv-dsFv 'antibodies, ie constructs from two disulphide bridge-stabilized Fv fragments, which are linked by a short linker peptide (Breitling and Dübel, 1997; Schmiedl et al., 2000) eliminates the stability problems of the diabodies , the construct also has a too low molecular weight for optimal tumor localization. The present invention is therefore based on the object of providing new antibody constructs for diagnostic, therapeutic and research purposes which are intended to eliminate the disadvantages of the prior art listed above.

This object is achieved by the embodiments characterized in the claims. In particular, a recombinant antibody construct with at least three antigen-binding antibody fragments is provided, at least one antigen-binding antibody fragment having a disulfide bridge between the variable domains.

The recombinant antibody construct according to the invention is preferably at least bispecific, the number of specificities depending on the diversity of the antigen-binding antibody fragments. For example, the recombinant antibody construct according to the invention can have three different antigen-binding antibody fragments, which is therefore trispecific. The valence of the recombinant antibody constructs according to the invention generally corresponds to the number of antigen-binding antibody fragments. The recombinant antibody construct according to the invention preferably has a molecular weight of approximately 90 kDa.

The two polypeptide chains of the antigen-binding antibody fragment with at least one disulfide bridge (hereinafter also referred to as “dsFv”) are preferably different from one another.

In a preferred embodiment of the present invention, these polypeptide chains are selected from the variable domains (hereinafter also referred to as “Fv”) of immunoglobulins, such as IgA, IgD, IgE and IgG, and biologically active fragments of these variable domains. biological active fragment "here means that these fragments have or result in an essentially identical or improved antigenic binding property, such as the naturally present Fvs.

According to this embodiment, these two polypeptide chains can be in any combination of the variable parts of the H and L chains from one immunoglobulin ("monoclonal") or two different immunoglobulins of one class, such as IgG, or different classes, such as IgG and IgA , occurrence.

In a further embodiment of the present invention, the recombinant antibody construct can have one or more antigen-binding antibody fragments (hereinafter also referred to as “scFv”) which have a polypeptide chain with two peptide sequences which are different from one another and from the primary structures of the variable domains of immunoglobulins and the biologically active portions thereof are selected. The term "biologically active portion" here means that these portions have or result in substantially identical or improved antigenic binding properties to that of naturally occurring Fvs. These two peptide sequences can occur in any combination of the primary structures of the variable parts of the H and L chains from one immunoglobulin or two different immunoglobulins of one class or different classes.

The recombinant antibody construct according to the invention is linked to one another in accordance with the number n of antigen-binding antibody fragments present via (n-1) peptide linkers. For example, a recombinant antibody construct with three antigen-binding

Antibody fragments two peptide linkers present (see also Figure 2). The recombinant antibody construct according to the invention can also contain, in part or in place of the variable parts of the H and L chains of immunoglobulins, at least one corresponding domain of another protein, in particular a member of the immunoglobulin superfamily, for example variable regions or parts of T cell receptors , MHC proteins (class I and II), cell surface proteins, cytokines or growth factors.

The recombinant antibody construct according to the present invention can further comprise at least one effector domain, which for example consists of interleukin 2, interferon-α, interferon-β, B7.1, B7.2, TNF-α, complement cascade components, toxins such as ricin, PE, DT , and RNAsen and biologically active fragments thereof are selected. The term "effector domain" includes domains that catalyze or inhibit a biochemical reaction.

The recombinant antibody construct according to the invention can further contain at least one radioactive substance which is covalently bound by chemical reaction.

Furthermore, further substances, for example cytokines, antibodies, receptors, complement proteins or low molecular weight compounds, can be coupled to the recombinant antibody construct according to the invention, the destruction of which of the cells in the vicinity of the V-bodies is caused by the body's own or endogenous effectors, for example by other cells such as Immune system cells, e.g. T lymphocytes or macrophages, or by molecules of the immune system, such as complement proteins.

Anchor domains can also be added to the recombinant antibody construct according to the invention, which allow coupling to one of the above-mentioned effectors or effector domains. examples for anchor domains of this type are avidin, streptavidin, or biologically active derivatives or mutations thereof, biotin, streptavidin / avidin binding peptides, bacterial immunoglobulin binding molecules such as protein A, protein G, protein H, protein L, calmodulin, calmodulin binding molecules, fragments of RNAsen , non-natural sequences that bind a fragment of RNAsen, and leucine zippers.

In addition, one or more polyethylene molecules can be covalently coupled to the recombinant antibody construct according to the invention.

The figures show:

1 shows the development of the invention into embodiments in (C) from the prior art in (A) and (B) using the example of immunoglobulins.

2 and 3 show further preferred embodiments of the present invention.

Figure 4 is a graphical representation of the vector used in Example 3 to produce a bispecific antibody construct.

Another object of the present invention is a method for producing the recombinant antibody constructs defined above, wherein the antigen-binding antibody fragments by means of recombinant DNA technology and introduction of a disulfide bridge into at least one antigen-binding antibody fragment via suitable mutagenesis, for example "site-specific mutagenesis" or PCR Mutagenesis of the nucleic acid sequences coding for the two polypeptide chains. The antibody constructs according to the invention are recombinantly produced, for example, in E. coli, insect cell cultures, Pichia patoris, CHO cell culture cells or transgenic plants. For this purpose, vectors are produced by means of DNA cloning according to the prior art, which, after transfection into the corresponding host cells or organisms, enable the subunits to be produced recombinantly. The DNA sequences which code for the subunits of the V-bodies are constructed for this purpose by PCR mutagenesis or other methods according to the prior art such that at least one of the Fv regions involved is mutated in the recombinantly produced protein (i) of two at the contact point between VH and V ^* . opposite amino acid positions (one each in VH and V _L ) to cysteine can be stabilized with an interface disulfide bridge (Brinkmann et al., 1993, Proc. Natl. Acad. Sei., USA 90 (16), 7538-4) and (ii) the recombinant antibody constructs according to the invention (cf. FIGS. 1 (C), 2, 3) are generated. The latter is achieved in that, by introducing compatible restriction sites at the ends of the gene fragments for the various variable domains and subsequent ligation or by PCR assembly, the various variable domains are arranged in the corresponding expression vectors in such a way that they are arranged for those in FIG. 1 (C), 2 or 3 encode polypeptide chains shown. Bi- trivalence resp. Bi- trispecificity and minibody size are achieved by assembling the DNA fragments coding for the variable regions in such a way that the resulting product consists of 3 Fv fragments. The resulting molecular mass corresponds to the advantageous minibody size. The optimal arrangement (sequence) of the different VH and V * _ chain genes in relation to each other in the vector can be optimally selected individually for each construct. Possible by-products of the recombinant production which can arise can be separated off using chromatographic methods known in the art.

Further subjects of the present application are therefore also nucleic acids which are at least partially those for the invention contain recombinant antibody constructs or antigen-binding antibody fragments encoding nucleic acid sequences, as well as suitable methods known in the art for the expression of the recombinant antibody constructs or antigen-binding antibody fragments.

The method according to the invention provides antibody constructs which, for example, enable a bivalent binding to a tumor or effector and thus a much higher off-rate during dissociation. This results in a longer localization in the target tissue and also does without any dimerization domains. As a result, excellent binding is achieved at a suitable molecular weight, preferably about 90 kDa, which cannot be achieved in the same way with any of the constructs previously described in the prior art. The molecular design described here therefore combines for the first time the previously separately found positive properties of previous constructs:

1. stabilization by disulfide bridges,

2. preferably trivalence in addition to at least bispecificity and

3. preferably mini body size.

The antibody constructs according to the invention in particular do not contain constant domains, since these domains are no longer necessary for dimerization, and the constructs according to the invention are sufficiently stabilized through the use of //7terc/.a/t.-disulfide bridges. The antibody constructs according to the invention are therefore also referred to below as "V-bodies", since they may be composed exclusively of variable regions except for short spacer peptides.

In contrast to other methods for producing bispecific antibodies, the method according to the invention enables the construction of the V bodies from human V regions. This enables the entire construct to be made from exclusively human protein fragments - this makes one Avoid immune reaction (e.g. HAMA) against the therapeutic agent, as occurs in constructs that achieve their bivalence or bispecificity by fusing the antigen-binding domains to heterologous fusion partners (such as streptavidin, zipper motifs, protein A fusions, etc.). Variations of the V-bodies consist of 2, 3 or 4 polypeptide chains, different degrees of disulfide stabilization being used (Fig. 1 (C), 2, 3). Trispecific antibodies can be produced without any additional effort compared to bispecific antibodies if the recombinant expression vectors are constructed in such a way that two polypeptide chain constructs are used, for example, each from an scFv and a variable domain of a dsFv fragment (see FIGS. 2 and 3, each above right).

The present invention further relates to a pharmaceutical composition which contains the recombinant antibody construct according to the invention in a pharmaceutically effective amount and optionally a pharmaceutically acceptable carrier and / or diluent. The pharmaceutical composition of the invention can be used, for example, to prevent or treat cancer, i.e. systemic and solid tumors, metastases and metastasis, autoimmune diseases, allergies, immunological overreactions, infections or noxious agents are used.

Another object of the present invention is a diagnostic agent which contains the recombinant antibody construct according to the invention. This diagnostic agent can be used for in vitro and / πu / Vo diagnostics, for example for the detection of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxious substances.

The present invention is illustrated by the following examples.

1. Trispecific antibodies

While maintaining the optimal molecular size for in vivo targeting, trispecific antibodies can be produced without special Trimerization motifs must be used in addition to the antigen-binding regions of the antibodies. This is used to produce therapeutic agents that can bind several tumor markers on one cell, or several different epitopes of a tumor marker. A combination of a tumor marker and a tissue-specific antibody can also be used. This increases the tumor specificity significantly compared to monovalent or multivalent-monospecific binding, and greatly reduces the burden on the patient due to the previously very common localization of the antibodies in non-specific tissues (both by cross-reactions and by expression of the tumor marker on other tissues). Apparent affinity is also improved by the increased avidity. The remaining binding site of the trispecific antibodies serves to strengthen the immune response against the tumor, in particular by binding to CD3 or CD28 of the T lymphocytes. Activation of the natural killer (NK) cells against the tumor by binding to CD16 is also possible. Alternatively, this antibody fragment can also activate complement cascades.

2. Bispecific antibodies with improved tumor accumulation and optimized pharmacokinetics

By producing two different antibody chains in E. coli using compatible expression vectors (e.g. pOPE and pDOPE), V-bodies are assembled in the periplasm of E. coli which contain a binding site for human CD3 and two binding sites for a tumor marker, e.g. MUC1, erbB2 or similar, or differentiation markers, e.g. CD19. The proteins are purified from the periplasm and used intravenously to bring T lymphocytes to the tumor. It is also possible to produce the proteins in eukaryotic cell lines (e.g. CHO or baculovirus) or transgenic plants.

3. Production of a bispecific V-body

To produce a bispecific body, two single-chain Fv fragments (scFv) were used, which are against the largest subunit of RNA polymerase II by Drosophila Melanogaster (Krämer et al., 1980; Kontermann et al., 1995; Schmiedl et al., 2000a) with the aid of a disulfide bridge-stabilized Fv fragment (dsFv) which is attached to the hapten 4-ethoxymethylene-2- phenyl-2-oxazolin-5-one (phOx) binds (Marks et al., 1992; Schmiedl et al., 2000a) by short but flexible peptide - // n / fer linked together covalently.

In order to generate the two monovalent anti-215 scFv fragments of the V body, the C-termini of the variable domains of their heavy chains were combined with the N-termini of the variable domains of their light chains with a flexible // n comprising 18 amino acids / cer peptide, which is composed of the first amino acids of the CH1 domain and the linear α-tubulin epitope EEGEFSEAR (Yol-Tag) of the monoclonal antibody Yol1 / 34 (Kilmartin et al., 1982; Breitling and Little, 1986 ; Schmiedl et al., 2000a). One of the variable domains of the anti-phox dsFv fragment was fused to the C-termini of the light chains. In order to ensure the covalent linkage of the two phOx domains by an interchenar disulfide bridge, the two amino acids Lys ^{H105 of} the heavy and Ala ^{L43 of} the light chain of the anti-phOx Fv fragment were previously replaced by cysteines (Schmiedl et al., 2000a). Additional carboxy-terminal tags at the end of both anti-phOx domains serve to detect and purify the two fusion proteins (Dübel et al., 1992). A short peptide of the human 62 kDa proto-oncogene product c-myc, which contains the linear epitope EEKLISEEDL of the monoclonal antibody Myd-9E10 (Evan et al., 1985), is located near the C-termini of both polypeptide chains before a 6xHis- sweeps the fusion proteins scFv 215 (Yol) -10-V _H (phOx) and scFv 215 (Yol) -10-V _L (phOx).

vector construction

The vector pOPE111-215HphOx / 215LphOx was generated to produce the V-body described above. It codes for the two fusion proteins scFv 215 (Yol) -10-V _H (phOx) and scFv 215 (Yol) - 10-V ^* _ (phOx) described above. The two gene fragments were each separated, with formation of ORFs behind pelB- / eacfer sequences from Erwinia carotovora (Lei et al., 1987) for the secretion of the translation products in the cloned periplasmic space. The vector pOPE111 also has a ColE1 origin of replication and the b-lactamase gene as a selection marker for ampicillin resistance. The expression of the encoded V-body constructs is determined using a synthetic / ac promoter P / A1 / 04 which can be induced by IPTG / 03 and two strong ribosome binding sites (RBS).

expression

A 50 mL overnight culture of E. coli cells transformed with pOPE111-215HphOx / 215LphOx was 1/20 in dYT medium (16 g / L Bacto-Trypton, 10 g / L yeast extract, 5 g / L NaCI) with 100 mM Giucose and 100 μg / mL ampicillin diluted and cultivated at 37 ° C. and 230 rpm to an OD ₆ oo of 0.6 before the promoter was induced by adding 20 mM isopropyl-bD-thiogalactopyranoside. After 3 h of incubation at 22 ° C. and 230 rpm, the bacteria were cooled on ice for 10 min and pelleted by centrifugation at 4 ° C. and 5000 × g.

Preparation of periplasmic extracts

For the preparation of periplasmic extracts, the pelleted bacteria were cooled in 1/10 volume (based on the starting volume of the culture) of shock solution (50 mM Tris / HCl, pH 8.0, 20% (w / v) sucrose, 1 mM EDTA) resuspended, incubated for 20 min with occasional shaking on ice and centrifuged again for 10 min at 6200 xg and 4 ° C. The periplasmic extract in the supernatant was centrifuged for a further 30 min at 30,000 × g and 4 ° C., dialyzed against PBS and analyzed with SDS-PAGE, immunoblot and ELISA. The cell pellet was resuspended in 1/10 volume (based on the initial volume of the culture) 5 mM MgSO ₄ , incubated for 20 min with occasional shaking on ice and also centrifuged for 30 min at 30,000 xg and 4 ° C. The extract thus obtained was also dialyzed against PBS and analyzed with SDS-PAGE, immunoblot and ELISA.

Enrichment of the antibody fragments by IMAC

A column was loaded with 2 ml / liter bacterial culture Ni-NTA-Sepharose and equilibrated in SSP (PBS, pH 7.5, 10 mM imidazole, 1 M NaCl). The dialyzed periplasmic extracts were pooled, adjusted to the same buffer conditions and added to the column. After washing the column with 5 Sepharose volumes SSP, 5 Sepharose volumes SWP (PBS, pH 7.5, 50 mM imidazole, 1 M NaCI) and 5 Sepharose volumes PBS, the proteins were 10 Sepharose volumes elution buffer (PBS, pH 7, 5, 500 mM imidazole, 1 M NaCl) competitively eluted. The collected fractions were analyzed by SDS-PAGE (Laemmli, 1975) and Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b). Fractions containing protein were pooled.

Mono Q chromatography

The antibody fragments obtained via IMAC were dialyzed against 30 mM Tris / HCl, pH 8.0 and at 0.5 mL / min on the column equilibrated in the same buffer (Mono Q HR / 5R; Amersham Pharmacia, Freiburg; Schmiedl et al., 2000b). After washing the column with 5 mL 30 mM Tris / HCl, pH 8.0, the proteins were competitively eluted using a two-phase NaCI gradient. For this purpose, the salt concentration was first increased to 20 mM over 20 mL, then over 1 mL to 1 M NaCl. The column was rinsed with 5 ml of Mono Q elution buffer (30 mM Tris / HCl, pH 8.0, 1 M NaCl) and readjusted to Mono-Q buffer. The collected fractions were analyzed by SDS-PAGE (Laemmli, 1975) and Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b). Fractions containing protein were pooled and their content determined with Bradford's solution.

ELISA (enzyme linked immunosorbent assay)

96-well Maxisorp plates (Nunc, Karlsruhe) were initially above sea level. at 4 ° C with 100 μ / well 0.1 M Na ₂ CO ₃ (pH 9.6), in which 1 mg of an antigen was dissolved, before unspecific binding sites were saturated with MPBS (400 μUwell) for 2 h at RT , The antigens Fp b-galactosidase-215, Fp b-galactosidase-control, BSA-phox and BSA were used to detect functional antibody fragments. Fp b-galactosidase-215 is a fusion protein of the bacterial b-galactosidase with a peptide which contains the mAb 215 epitope. b-Galactosidase fused with a comparable peptide served as a control. The recombinant b-galactosidase fusion proteins were previously obtained by expression in E. coli (Kontermann er al., 1995). Phoxylated BSA was prepared by incubating a 20-fold molar excess of 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (phOx) with BSA in 0.1 M NaCO ₃ (pH 8.5) for two hours. Uncoupled 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one was removed by repeated dialysis against PBS. Unmodified BSA served as a control.

Unspecific binding sites of the Maxisorp plate were saturated by blocking with 400 mL / well MPBS for at least 3 h at RT. The periplasmic extracts were diluted 1: 1 in MPBST and 100 ml / well were incubated for 2 h at RT. The antibody mAb Myc1-9E10 (Calbiochem, Schwalbach; 1/1000 in MPBST) was used in combination with HRP-conjugated goat anti-mouse immunoglobulins (Dianova, Hamburg; 1/2000 in MPBST) to detect bound antibody fragments and with 100 mLl well TMB substrate (10 mL 100 mM sodium acetate, pH 6.0, 25 μL TMB solution (40 mg / mL in DMSO), 8 μL 30% (v / v) H ₂ O ₂ ) was detected. After the color reaction had been stopped by adding 50 μUwell 1 MH ₂ SO, the analysis was carried out using an ELISA reagent at 450 nm.

To demonstrate the bispecificity of the antibody construct, 100 mL / well antibody extract were bound to BSA-phOx coated Maxisorp plates for 2 hours. HRP-conjugated streptavidin (8/1000 in PBST) was treated with 0.1 nmol of the N-terminal biotinylated peptide LPHFIKDDYGPESRGFVENSYLAGLTPSE (ZMBH, Heidelberg), which contains the epitope of the antibody mAb 215, or with the correspondingly biotinylated control peptide KESQHQKQEQRQKKEKQKEQKQKQMKFKRKKPKQEQFKRKKKPKQEKRKKKPKQEKRKKDKKQKQFKRKKKPKQKQKQFKRKKDKQKQKQDKKKFKRKKKDKKQKQDKHKKKDKQKQDKHKKDKKKKDKQKQDKRKKDKKKDKHKKDKKKDKHKKKDKHKKDKKE ZMBH, Heidelberg) incubated at RT for one hour before 100 mU well were applied. The bispecificity was demonstrated after 2 hours of incubation at RT with TMB substrate.

literature

Breitling, F. and Little, M. (1986) C-terminal regions on the surface of tubulin and microtubules. Epitope locations of Yol1 / 34, DM1A and DM1 BJ Mol. Biol. 789, 367-70. Dübel, S., Breitling, F., Klewinghaus, I. and Little, M. (1992) Regulated secretion and purification of recombinant antibodies in E. coli. Cell Biophysics 21, 69-79.

Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, J.M. (1985) Isolation of Monocional Antibodies Specific for Human c-myc Proto-Oncogene Product. Mol. Cell. Bio !. 5, 3610-6.

Kilmartin, J.V., Wright, B. and Milstein, C. (1982) Rat monocional antitubulin antibodies derieved by using a new nonsecreting rat cell line. J. Cell. Biol. 93, 576-82.

Kontermann, R.E., Liu, Z., Schulze, R.A., Sommer, K.A., Queitsch, I., Dübel, S., Kipriyanov, S.M., Breitling, F. and Bautz, E.K.F. (1995) Characteristic of the epitope recognized by a monocional antibody directed against the largest subunit of Drosophila RNA polymerase II. Biol. Chem. Hoppe-Seyler 376, 473-81.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-5.

Lei, S.-P., Lin, H.-C, Wang, S.-S., Callaway, J. and Wilcox, G. (1987) Characterizatiön of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 769, 4379-83.

Marks, J.D., Griffiths, A.D., Malmqvist, M., Clackson, T.P., Bye, J.M. and Winter, G. (1992) By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N.Y.) 10, 779-83.

Schmiedl, A., Breitling, F., Winter, C.H., Queitsch, I. and Dübel, S., (2000a) Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli. J. Immunol. Methods. 242, 101-14.

Schmiedl, A., Breitling, F. and Dübel, S. (2000b) Expression of a bispecific dsFv-dsFv 'antibody fragment in Escherichia coli. Protein Eng. 13, 725-34.

Towbin, H., Staehelin, T. and Gordon, l. (1980) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Be. U.S.A. 76, 4350-4.

Claims

claims

1. Recombinant antibody construct with at least three antigen-binding antibody fragments, wherein at least one antigen-binding antibody fragment has a disulfide bridge between the two antibody domains.

2. Recombinant antibody construct according to claim 1, which is at least bispecific.

3. Recombinant antibody construct according to claim 1 or 2, which has a molecular weight of about 90 kDa.

4. Recombinant antibody construct according to one of claims 1 to 3, wherein the two polypeptide chains of the antigen-binding antibody fragment having a disulfide bridge are different from one another.

5. A recombinant antibody construct according to claim 4, wherein the polypeptide chains are selected from the variable domains of immunoglobulins and biologically active fragments thereof.

6. Recombinant antibody construct according to claim 5, wherein the polypeptide chains are each selected from the variable parts of the H and L chains of one immunoglobulin or two different immunoglobulins of one class or different classes.

7. Recombinant antibody construct according to one of claims 1 to 6, wherein one or two antigen-binding antibody fragments has a polypeptide chain with two peptide sequences which are different from one another and are selected from the primary structures of the variable domains of immunoglobulins and the biologically active sections thereof.

8. Recombinant antibody construct according to claim 7, wherein the peptide sequences are each selected from the primary structures of the variable parts of the H and L chains of one immunoglobulin or two different immunoglobulins of one class or different classes.

9. Recombinant antibody construct according to one of claims 1 to

8, the antigen-binding antibody fragments being connected to one another via at least two peptide linkers.

10. Recombinant antibody construct according to one of claims 1 to

9, which contains instead of the variable parts of the H and L chains of immunoglobulins at least one corresponding domain of another protein, in particular a member of the immunoglobulin superfamily.

11. Recombinant antibody construct according to claim 10, wherein the members are selected from T cell receptors, MHC proteins, cell surface proteins, cytokines and growth factors.

12. Recombinant antibody construct according to one of claims 1 to 11, which further comprises at least one effector domain, which catalyzes or inhibits a biochemical reaction.

13. Recombinant antibody construct according to claim 12, wherein the effector domains are selected from interleukin 2, interferon, interferon-β, B7.1, B7.2, TNF-α, complement cascade components, toxins or RNases.

14. Recombinant antibody construct according to one of claims 1 to

13, which further contains at least one radioactive substance which is covalently bound by chemical reaction.

15. Recombinant antibody construct according to one of claims 1 to

14, which also contains substances that destroy the cells in the Effect around the recombinant antibody constructs by endogenous effectors.

16. A recombinant antibody construct according to claim 15, wherein the endogenous effectors are selected from cells of the immune system or molecules of the immune system.

17. Recombinant antibody construct according to one of claims 1 to 16, which further contains at least one anchor domain, which effects a coupling to an effector or an effector domain, as defined in one of claims 12 to 16.

18. Recombinant antibody construct according to claim 17, wherein the anchor domain from avidin, streptavidin or biologically active derivatives thereof, biotin, streptavidin / avidin binding peptides, bacterial immunoglobulin binding molecules, calmodulin binding molecules, fragments of RNases, non-natural sequences which attach to fragments of RNases bind, or leucine zipper is selected.

19. Recombinant antibody construct according to one of claims 1 to 18, to which further one or more polyethylene glycol molecules are covalently coupled.

20. A method for producing recombinant antibody constructs according to one of claims 1 to 19, wherein the antigen-binding antibody fragments are produced by means of recombinant DNA technology and introduction of a disulfide bridge into at least one antigen-binding antibody fragment via suitable mutagenesis of the nucleic acid sequences coding for the polypeptide chains.

21. A pharmaceutical composition containing the recombinant antibody construct according to one of claims 1 to 19 in a pharmaceutical effective amount and optionally a pharmaceutically acceptable carrier and / or diluent.

22. Diagnostic agent containing the recombinant antibody construct according to one of claims 1 to 19.

23. Use of the recombinant antibody construct according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 21 for the prevention or treatment of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxae.

24. Use of the recombinant antibody construct according to any one of claims 1 to 19 or the diagnostic agent according to claim 22 for the detection of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxae.