EP1287036A1 - Trivalent antibody constructs having variable regions that are stabilized by disulfide bridges - Google Patents
Trivalent antibody constructs having variable regions that are stabilized by disulfide bridgesInfo
- Publication number
- EP1287036A1 EP1287036A1 EP01933941A EP01933941A EP1287036A1 EP 1287036 A1 EP1287036 A1 EP 1287036A1 EP 01933941 A EP01933941 A EP 01933941A EP 01933941 A EP01933941 A EP 01933941A EP 1287036 A1 EP1287036 A1 EP 1287036A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- recombinant antibody
- construct according
- antibody construct
- binding
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/624—Disulfide-stabilized antibody (dsFv)
Definitions
- 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).
- 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).
- minibody about 90 kDa
- scFv fragments two identical scFv fragments, which dimerize through fused CH3 domains.
- 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.
- CH1 and CL (k a pp a ) domains for dimerization 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.
- 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).
- 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.
- 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.
- 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 are preferably different from one another.
- 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.
- Fv 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.
- 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.
- 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.
- scFv antigen-binding antibody fragments
- 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.
- 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.
- n-1 the number of antigen-binding antibody fragments present via (n-1) peptide linkers.
- Antibody fragments two peptide linkers present 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- ⁇ , interferon- ⁇ , B7.1, B7.2, TNF- ⁇ , complement cascade components, toxins such as ricin, PE, DT , and RNAsen and biologically active fragments thereof are selected.
- 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.
- cytokines for example cytokines, antibodies, receptors, complement proteins or low molecular weight compounds
- cytokines for example cytokines, antibodies, receptors, complement proteins or low molecular weight compounds
- 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.
- 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.
- polyethylene molecules can be covalently coupled to the recombinant antibody construct according to the 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.
- 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.
- 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.
- 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.
- 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:
- 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.
- 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.).
- HAMA e.g. antigen-binding domains
- 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 / ⁇ u / Vo diagnostics, for example for the detection of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxious substances.
- 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.
- NK natural killer
- 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.
- scFv single-chain Fv fragments
- dsFv disulfide bridge-stabilized Fv fragment
- phOx 4-ethoxymethylene-2- phenyl-2-oxazolin-5-one
- 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 ⁇ -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.
- 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).
- 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).
- 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 ⁇ g / 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.
- 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.
- shock solution 50 mM Tris / HCl, pH 8.0, 20% (w / v) sucrose, 1 mM EDTA
- 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.
- 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).
- SSP SSP
- the dialyzed periplasmic extracts were pooled, adjusted to the same buffer conditions and added to the column.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ L TMB solution (40 mg / mL in DMSO), 8 ⁇ L 30% (v / v) H 2 O 2 ) was detected. After the color reaction had been stopped by adding 50 ⁇ Uwell 1 MH 2 SO, the analysis was carried out using an ELISA reagent at 450 nm.
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Abstract
Description
Beschreibung description
TRIVALENTE ANTIKORPERKONSTRUKTE MIT DISULFID-BRÜCKEN STABILISIERTEN VARIABLENTRIVALENT ANTI-BODY CONSTRUCTS WITH DISULFID BRIDGES STABILIZED VARIABLES
REGIONENREGIONS
Die vorliegende Erfindung betrifft rekombinante Antikörperkonstrukte, Verfahren zu deren Herstellung, pharmazeutische Zusammensetzungen und diagnostische Mittel, welche diese rekombinanten Antikörperkonstrukte enthalten, sowie die Verwendung dieser rekombinanten Antikörperkonstrukte zur Diagnose und Behandlung von Krebserkrankungen, Autoimmunerkrankungen, Allergien, immunologischen Überreaktionen, Infektionen oder Noxen.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.
Bispezifische Antikörper sind am besten rekombinant herzusteilen, da bei der herkömmlichen Quadroma- bzw. Hybrid-Hybridoma-Technik ein Gemisch aus 10 verschiedenen Produkten auftritt, aus denen das einzige korrekte nur unter erheblichem Aufwand abgetrennt werden kann (Milstein und Cuello, 1983). Bispezifische Antikörper sind aber als Agenzien für neuartige Therapieverfahren notwendig, die ohne solche Moleküle nicht durchgeführt werden könnten. Anwendung finden sie im besonderen bei der Stimulierung der Immunantwort gegen Tumorzellen (Segal et al., 1999; Hombach et al., 1993; Manzke et al, 1999). Dabei werden T-Lymphozyten spezifisch an Tumorzellen herangeführt und oft noch zusätzlich durch einen der Bindungsarme des bispezifischen Agens aktiviert. Verschiedene molekulare Formate wurden zur Herstellung von therapeutischen Antikörpern im allgemeinen und von bispezifischen Antikörpern im speziellen entwickelt (Breitling und Dübel, 1997; Carter und Merchant, 1997).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).
Bei ausführlichen Untersuchungen zur Tumorlokalisation von verschiedenen Antikörperformaten (scFv, Diabody, Minibody, komplette IgG) mit identischer Antigenbindestelle (Tumormarker carcinoembryonic antigen, CEA) aber mit unterschiedlichem Molekulargewicht (ca. 30 kDa, 60 kDa, 90 kDa und 150 kDa) zeigte sich, daß die beiden kleineren Formate aufgrund ihrer raschen Filtrierung durch die Niere zu einem weit geringeren Konzentrationsverhältnis Tumor/Gewebe führten als Konstrukte, deren Molekulargewichte über der Filtrationsgrenze der Niere lagen (Wu et al., 1996; Hu et al., 1996). Beste Tumorlokalisation erbrachte der sog. Minibody (etwa 90 kDa), der aus zwei identischen scFv-Fragenten besteht, welche durch fusionierte CH3 Domänen dimerisieren (Hu et al., 1996). Dieses Konstrukt ist zwar bivalent, aber monospezifisch. Die Herstellung von bispezifischen Antikörpern nach diesem Muster ist schwierig, da es wiederum zu homologen Paarungen und damit zu unerwünschten und schwer abtrennbaren Nebenprodukten kommen kann.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.
Die Verwendung von CH1 und CL(kappa)-Domänen zur Dimerisierung (Müller et al., 1998) löste zwar das Problem der Nebenprodukte bei der Herstellung bispezifischer Minibodies, jedoch ist die Affinität durch die Monovalenz der jeweiligen Antigenbindestellen eingeschränkt.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.
Die Stabilisierung von Fv-Fragmenten durch Disulfidbrücken (dsFv, Brinkmann et al., 1993; Reiter et al., 1995) verbesserte deren Serum-Halbwertzeit gegenüber scFvs und führte zu einer erhöhten Lokalisierung dieser Konstrukte im Tumorgewebe (Reiter et al., 1994a, b, c; Webber et al., 1995).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).
Bispezifische Diabodies, wie sie vielfach für die Therapie hergestellt wurden, sind kleiner als das pharmakokinetisch optimale Konstrukt für die Tumortherapie, indem sie lediglich aus zwei antigenbindenden Antikörperfragmenten zusammengesetzt sind (Hollinger et al., 1993, 1996, 1999; Arndt et al., 1999; Helfrich ef a/., 1998).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).
Die Verwendung von dsFv-dsFv '-Antikörpern, d.h. Konstrukten aus zwei Disulfidbrücken-stablisierten Fv-Fragmenten, die durch ein kurzes Linker-Peptid verbunden sind (Breitling und Dübel, 1997; Schmiedl et al., 2000) beseitigt zwar die Stabilitätsprobleme der Diabodies, das Konstrukt hat aber auch ein zu geringes Molekulargewicht für eine optimale Tumorlokalisation. Somit liegt der vorliegenden Er indung die Aufgabe zugrunde, neue Antikörperkonstrukte für diagnostische, therapeutische und Forschungszwecke bereitzustellen, welche die vorstehend aufgeführten Nachteile des Standes der Technik beseitigen sollen.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.
Diese Aufgabe wird durch die in den Ansprüchen gekennzeichneten Ausführungsformen gelöst. Insbesondere wird ein rekombinantes Antikörperkonstrukt mit mindestens drei antigenbindenden Antikörperfragmenten bereitgestellt, wobei mindesten ein antigenbindendes Antikörperfragment eine Disulfid-Brücke zwischen den variablen Domänen aufweist.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.
Das erfindungsgemäße rekombinante Antikörperkonstrukt ist vorzugsweise mindestens bispezifisch, wobei die Anzahl der Spezifitäten von der Diversität der antigenbindenden Antikörperfragmente abhängt. Beispielsweise kann das erfindungsgemäße rekominante Antikörperkonstrukt drei unterschiedliche antigenbindende Antikörperfragmente aufweisen, welches somit trispezifisch ist. Die Valenz der erfindungsgemäßen rekominanten Antikörperkonstrukte entspricht im allgemeinen der Anzahl der antigenbindenden Antikörperfragmente. Das erfindungsgemäße rekominante Antikörperkonstrukt weist vorzugsweise ein Molekulargewicht von etwa 90 kDa auf.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.
Die zwei Polypeptid-Ketten des antigenbindenden Antikörperfragments mit mindestens einer Disulfid-Brücke (im Folgenden auch als „dsFv" bezeichnet), sind vorzugsweise unterschiedlich zueinander.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 einer bevorzugten Ausführungsform der vorliegenden Erfindung werden diese Polypeptid-Ketten aus den variablen Domänen (im Folgenden auch als „Fv" bezeichnet) von Immunglobulinen, wie IgA, IgD, IgE und IgG, sowie biologisch aktiven Fragmenten dieser variablen Domänen ausgewählt. Der Begriff „biologisch aktives Fragment" bedeutet hier, daß diese Fragmente eine im wesentlichen identische oder verbesserte antigene Bindungseigenschaft aufweisen bzw. zur Folge haben, wie die natürlich vorhandenen Fvs.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.
Gemäß dieser Ausführungsform können diese zwei Polypeptid-Ketten in jedweder Kombination aus den variablen Teilen der H- und L-Ketten von einem Immunglobulin („monoklonal") oder zwei unterschiedlichen Immunglobulinen einer Klasse, wie IgG,, oder unterschiedlichen Klassen, wie IgG und IgA, vorkommen.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 einer weiteren Ausführungsform der vorliegenden Erfindung kann das rekombinante Antikörperkonstrukt ein oder mehrere antigenbindende Antikörperfragmente (im Folgenden auch als „scFv" bzeichnet) aufweisen, welche eine Polypeptid-Kette mit zwei Peptidsequenzen, die unterschiedlich zueinander sind und aus den Primärstrukturen der variablen Domänen von Immunglobulinen und den biologisch aktiven Abschnitten davon ausgewählt sind, aufweisen. Der Begriff „biologisch aktiver Abschnitt" bedeutet hier, daß diese Abschnitte eine im wesentlichen identische oder verbesserte antigene Bindungseigenschaft aufweisen bzw. zur Folge haben, wie die natürlich vorhandenen Fvs. Diese zwei Peptidsequenzen können in jedweder Kombination aus den Primärstrukturen der variablen Teile der H- und L-Ketten von einem Immunglobulin oder zwei unterschiedlichen Immunglobulienen einer Klasse oder unterschiedlichen Klassen vorkommen.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.
Das erfindungsgemäße, rekombinante Antikörperkonstrukt ist entsprechend der Anzahl n der vorhandenen antigenbindenden Antikörperfragmente über (n-1 ) Peptid-Linker miteinander verbunden. Beispielsweise sind bei einem rekombinanten Antikörperkonstrukt mit drei antigenbindendenThe 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
Antikörperfragmenten zwei Peptid-Linker vorhanden, (vgl. auch Figur 2). Das erfindungsgemäße rekombinante Antikörperkonstrukt kann ferner teilweise oder anstelle der variablen Teile der H- und L- Ketten von Immunglobulinenen mindestens eine entsprechende Domäne eines anderen Protein, insbesondere eines Mitglieds der Immunglobulin-Superfamilie enthalten, beispielsweise variable Regionen bzw. Teile von T-Zell-Rezeptoren, MHC-Proteine (Klasse I und II), Zeiloberflächenproteine, Cytokine oder Wachstumsfaktoren.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.
Das rekombinante Antikörperkonstrukt gemäß der vorliegenden Erfindung kann ferner mindestens eine Effektordomäne, welche beispielsweise aus Interleukin 2, Interferon-α, Interferon-ß, B7.1 , B7.2, TNF-α, Komplementkaskaden- Komponenten, Toxinen wie Ricin, PE, DT, und RNAsen und biologisch aktiven Fragmenten davon ausgewählt sind, enthalten. Der Begriff „Effektordomäne" umfaßt Domänen, welche eine biochemische Reaktion katalysieren oder inhibieren.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.
Das erfindungsgemäße rekombinante Antikörperkonstrukt kann weiter mindestens eine radioaktive Substanz enthalten, welche durch chemische Reaktion kovalent gebunden ist.The recombinant antibody construct according to the invention can further contain at least one radioactive substance which is covalently bound by chemical reaction.
Ferner können an das erfindungsgemäße rekombinante Antikörperkonstrukt weitere Substanzen, beispielsweise Cytokine, Antikörper, Rezeptoren, Komplementproteine oder niedermolekurlare Verbindungen, gekoppelt sein, deren Zerstörung der Zellen in der Umgebung der V-Bodies durch körpereigene bzw. endogene Effektoren bewirken, beispielsweise durch andere Zellen, wie Zellen des Immunsystems, z.B. T-Lymphocyten oder Makrophagen, oder durch Moleküle des Immunsystems, wie Komplementproteine.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.
An das erfindungsgemäße rekombinante Antikörperkonstrukt können auch Ankerdomänen angefügt werden, welche eine Koppelung an einen der obengenannten Effektoren bzw. Effektordomänen erlauben. Beispiele für derartige Ankerdomänen sind Avidin, Streptavidin, oder biologisch aktive Derivate bzw. Mutationen davon, Biotin, Streptavidin/Avidin-Bindepeptide, bakterielle Immunglobulin-Bindemoleküle wie Protein A, Protein G, Protein H, Protein L, Calmodulin, Calmodulin-Bindemoleküle, Fragmente von RNAsen, nicht-natürliche Sequenzen, welche ein Fragment von RNAsen binden, und Leucin-Zipper.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.
Darüber hinaus können an das erfindungsgemäße rekombinante Antikörperkonstrukt ein oder mehrere Polyethylenmoleküle kovalent gekoppelt werden.In addition, one or more polyethylene molecules can be covalently coupled to the recombinant antibody construct according to the invention.
Die Figuren zeigen:The figures show:
Fig.1 zeigt die Entwicklung der Erfindung zu Ausführungsforme in (C) aus dem Stand der Technik in (A) und (B) am Beispiel von Immunglobulinen.1 shows the development of the invention into embodiments in (C) from the prior art in (A) and (B) using the example of immunoglobulins.
Fig. 2 und 3 zeigen weitere bevorzugte Ausführungsformen der vorliegenden Erfindung.2 and 3 show further preferred embodiments of the present invention.
Fig. 4 ist eine graphische Darstellung des in Beispiel 3 verwendeten Vektors zur Herstellung eines bispezifischen Antikörperkonstrukts.Figure 4 is a graphical representation of the vector used in Example 3 to produce a bispecific antibody construct.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung der vorstehend definierten rekombinanten Antikörperkonstrukte, worin die antigenbindenden Antikörperfragmente mittels rekombinanter DNA- Technologie und Einführen einer Disulfid-Brücke in mindestens ein antigenbindendes Antikörperfragment über geeignete Mutagenese, beispielsweise „site-specific mutagenesis" oder PCR-Mutagenese der für die zwei Polypeptid- Ketten kodierenden Nukleinsäure-Sequenzen hergestellt werden. Die rekombinante Herstellung der erfindungsgemäßen Antikörperkonstrukte erfolgt beispielsweise in E. coli, Insektenzellkulturen, Pichia patoris, CHO- Zellkulturzellen oder transgenen Pflanzen. Dazu werden Vektoren mittels DNA- Klonierung nach dem Stand der Technik hergestellt, welche nach Transfektion in die entsprechenden Wirtszellen bzw. -Organismen die rekombinante Proteinproduktion der Untereinheiten ermöglichen. Die DNA-Sequenzen, welche für die Untereinheiten der V-bodies codieren, werden dafür solcherart durch PCR- Mutagenese oder andere Methoden nach dem Stand der Technik konstruiert, daß im rekombinant produzierten Protein (i) mindestens eines der beteiligten Fv- Regionen durch -Mutation von zwei an der Kontaktstelle zwischen VH und V*. gegenüberliegenden Aminosäurepositionen (jeweils eine in VH und VL) zu Cystein mit einer interface-Disulfid-Brücke stabilisiert werden (Brinkmann et al., 1993, Proc. Natl. Acad. Sei., USA 90(16), 7538-4) und (ii) die erfindungsgemäßen rekombinanten Antikörperkonstrukte (vgl. Figuren 1(C), 2, 3) erzeugt werden. Letzteres wird dadurch erreicht, daß über die Einführung von kompatiblen Restriktionsschnittstellen an den Enden der Genfragmente für die verschiedenen variablen Domänen und darauffolgende Ligation oder durch PCR-Assembly die verschiedenen variablen Domänen in den entsprechenden Expressionsvektoren so angeordnet werden, daß sie für die in Fig. 1 (C), 2 oder 3 gezeigten Polypeptidketten codieren. Bi- Trivalenz resp. Bi- Trispezifität und Minibody-Größe werden dadurch erreicht, daß die für die variablen Regionen codierenden DNA- Fragmente so zusammengesetzt werden, daß das resultierende Produkt aus 3 Fv- Fragmenten besteht. Die resultierende Molekularmasse entspricht dadurch der vorteilhaften Minibody-Größe. Die optimale Anordnung (Reihenfolge) der verschiedenen VH- und V*_-Ketten-Gene zueinander im Vektor kann dabei individuell für jedes Konstrukt optimal gewählt werden. Mögliche entstehende Nebenprodukte der rekombinanten Produktion können mit Hilfe im Stand der Technik bekannten chromatographischer Methoden abgetrennt werden.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.
Weitere Gegenstände der vorliegenden Anmeldung sind somit auch Nukleinsäuren, welche mindestens teilweise die für die erfindungsgemäßen rekombinanten Antikörperkonstrukte oder antigenbindenden Antikörperfragmente codierenden Nukleinsäuresequenzen enthalten, sowie geeignete, im Stand der Technik bekannte Verfahren zur Expression der rekombinanten Antikörperkonstrukte oder antigenbindenden Antikörperfragmente.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.
Das erfindungsgemässe Verfahren stellt Antikörperkonstrukte zur Verfügung, welche beispielsweise eine bivalente Bindung an einen Tumor oder Effektor und damit eine weit höhere off-rate bei der Dissoziierung ermöglichen. Dies hat eine längere Lokalisation im Zielgewebe zur Folge, und kommt außerdem ohne jegliche Dimerisierungsdomänen aus. Dadurch wird bei einem geeigneten Molekulargewicht, vorzugsweise etwa 90 kDa, eine ausgezeichnete Bindung erreicht, die mit keinem der bisher im Stand der Technik beschriebenen Konstrukte in gleicher Weise erreicht werden kann. Das hier beschriebene molekulare Design vereint deshalb erstmals die zuvor separat gefundenen positiven Eigenschaften bisheriger Konstrukte: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. Stabilisierung durch Disulfidbrücken,1. stabilization by disulfide bridges,
2. vorzugsweise Trivalenz zusätzlich zur mindestens Bispezifität und2. preferably trivalence in addition to at least bispecificity and
3. vorzugsweise Minibody-Größe.3. preferably mini body size.
Die erfindungsgemäßen Antikörperkonstrukte enthalten insbesondere keine konstanten Domänen, da diese Domänen für die Dimerisierung nicht mehr notwendig sind, und die erfindungsgemäßen Konstrukte durch den Einsatz von //7terc/.a/t.-Disulfidbrücken ausreichend stabilisiert sind. Die erfindungsgemäßen Antikörperkonstrukte werden deshalb im weiteren auch als "V-Bodies" bezeichnet, da sie gegebenenfalls bis auf kurze Spacer-Peptide ausschließlich aus variablen Regionen zusammengesetzt sind.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.
Das erfindungsgemässe Verfahren ermöglicht im Gegensatz zu anderen Methoden zur Herstellung bispezifischer Antikörper, die Konstruktion der V-bodies aus humanen V-Regionen. Dies ermöglicht die Herstellung des gesamten Konstruktes aus ausschließlich humanen Protein-Fragmenten - damit wird eine Immunreaktion (z.B. HAMA) gegen das therapeutische Agens vermieden, wie sie bei Konstrukten auftritt, die ihre Bivalenz oder Bispezifität durch Fusion der antigenbindenden Domänen an heterologe Fusionspartner erreichen (wie Streptavidin, Zipper-Motive, Protein A-Fusionen etc.). Variationen der V-bodies bestehen aus 2, 3 oder 4 Polypeptidketten, wobei unterschiedliche Grade der Disulfidstabilisierung zum Einsatz kommen (Fig. 1 (C), 2, 3). Trispezifische Antikörper sind ohne Mehraufwand gegenüber bispezifischen Antikörpern herzustellen, wenn die rekombinanten Expressionsvektoren so konstruiert werden, daß zwei Polypeptidketten-Konstrukte zum Beispiel aus je einem scFv und einer variablen Domäne eines dsFv-Fragmentes eingesetzt werden (vgl. Fig. 2 und 3, jeweils oben rechts).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).
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft eine pharmazeutische Zusammensetzung, die das erfindungsgemäße rekombinante Antikörperkonstrukt in einer pharmazeutisch wirksamen Menge und gegebenenfalls ein pharmazeutisch verträglichen Träger und/oder Verdünnungsmittel enthält. Die erfindungsgemäße pharmazeutische Zusammensetzung kann beispielsweise zur Verhinderung oder Behandlung von Krebserkrankungen, d.h. systemischen und soliden Tumoren, Metastasen und Metastasenbildung, Autoimmunerkrankungen, Allergien, immunologischen Überreaktionen, Infektionen oder Noxen verwendet werden.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.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein diagnostisches Mittel, welche das erfindungsgemäße rekombinante Antikörperkonstrukt enthält. Dieses diagnostische Mittel kann zur invitro und /πu/Vo-Diagnostik, beispielsweise zum Nachweis von Krebserkrankungen, Autoimmunerkrankungen, Allergien, immunologischen Überreaktionen, Infektionen oder Noxen verwendet werden.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.
Die vorliegende Erfindung wird durch die nachfolgenden Beispiele näher erläutert.The present invention is illustrated by the following examples.
1. Trispezifische Antikörper1. Trispecific antibodies
Unter Beibehaltung der für das in vivo-targeting optimalen Molekülgröße lassen sich trispezifische Antikörper herstellen, ohne daß besondere Trimerisierungsmotive zusätzlich zu den antigenbindenden Regionen der Antikörper eingesetzt werden müssen. Dies wird eingesetzt, um therapeutische Agentien herzustellen, die zum einen mehrere Tumormarker auf einer Zelle binden können, oder mehrere verschiedene Epitope eines Tumormarkers. Ebenso kann eine Kombination aus einem Tumormarker und einem gewebespezifischen Antikörper eingesetzt werden. Dadurch erhöht sich die Tumorspezifität erheblich gegenüber monovalenter oder multivalent-monospezifischer Bindung, und verringert stark die Belastung der Patienten durch die bisher sehr häufig auftretende Lokalisierung der Antikörper in unspezifischen Geweben (sowohl durch Kreuzreaktionen wie durch Expression des Tumormarkers auf anderen Geweben). Zudem wird die apparente Affinität durch die erhöhte Avidität verbessert. Die verbleibende Bindestelle der trispezifischen Antikörper dient zur Verstärkung der Immunantwort gegen den Tumor, insbesondere durch Bindung an CD3 oder CD28 der T-Lymphozyten. Auch eine Aktivierung der Natural Killer (NK)-Zellen gegen den Tumor über Bindung an CD16 ist möglich. Alternativ kann dieses Antikörperfragment auch Komplementkaskaden aktivieren.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. Bispezifische Antikörper mit verbesserter Tumor-Anreicherung und optimierter Pharmakokinetik2. Bispecific antibodies with improved tumor accumulation and optimized pharmacokinetics
Durch Produktion von zwei verschiedenen Antikörperketten in E. coli unter Benutzung von kompatiblem Expressionsvektoren (z.B. pOPE und pDOPE), werden V-Bodies im Periplasma von E. coli zusammengesetzt, welche eine Bindestelle für humanes CD3 enthalten, und zwei Bindestellen für einen Tumormarker, z.B. MUC1 , erbB2 o.a., oder Differenzierungsmarker, wie z.B. CD19. Die Proteine werden aus dem Periplasma gereinigt, und intravenös eingesetzt, um T-Lymphozyten an den Tumor heranzuführen. Eine Produktion der Proteine in eukaryontischen Zellinien (z.B. CHO oder Baculovirus) oder transgenen Pflanzen ist zur Herstellung ebenfalls möglich.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. Herstellung eines bispezifischen V-bodies3. Production of a bispecific V-body
Zur Herstellung eines bispezifischen -bodies wurden zwei single-chain Fv- Fragmenten (scFv), welche gegen die größte Untereinheit der RNA-Polymerase II von Drosophila Melanogaster gerichtet sind (Krämer et al., 1980; Kontermann et al., 1995; Schmiedl et al., 2000a) mit Hilfe eines Disulfidbrücken-stabilisierten Fv- Fragments (dsFv), welches an das Hapten 4-Ethoxymethylen-2-phenyl-2-oxazolin- 5-on (phOx) bindet (Marks et al., 1992; Schmiedl et al., 2000a) durch kurze, aber flexible Peptid-//n/fer kovalent miteinander verknüpft.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.
Um die beiden monovalenten anti-215 scFv-Fragmente des V-bodies zu generieren, wurden die C-Termini der variablen Domänen ihrer schweren Ketten mit den N-Termini der variablen Domänen ihrer leichten Ketten jeweils mit einem 18 Aminosäuren umfassenden, flexiblen //n/cer-Peptid verbunden, welches sich aus den ersten Aminosäuren der CH1 -Domäne und dem linearen α-Tubulin-Epitop EEGEFSEAR (Yol-Tag) des monoklonalen Antikörpers Yol1/34 zusammensetzt (Kilmartin et al., 1982; Breitling and Little, 1986; Schmiedl et al., 2000a). An die C- Termini der leichten Ketten wurde jeweils eine der variablen Domäne des anti- phOx dsFv-Fragments fusioniert. Um die kovalente Verknüpfung der beiden phOx-Domänen durch eine interchenare Disulfidbrücke zu gewährleisten, wurden zuvor die beiden Aminosäuren LysH105 der schweren und AlaL43 der leichten Kette des anti-phOx Fv-Fragments durch Cysteine ersetzt (Schmiedl et al., 2000a). Weitere, carboxyterminale tags am Ende beider anti-phOx Domänen dienen zur Detektion und Aufreinigung der beiden Fusionsproteine (Dübel et al., 1992). Jeweils ein kurzes Peptid des humanen 62 kDa Proto-Oncogenprodukts c-myc, welches das lineare Epitop EEKLISEEDL des monoklonalen Antikörpers Myd- 9E10 beinhaltet (Evan et al., 1985), ist nahe der C-Termini beider Polypeptidketten lokalisiert, bevor ein 6xHis-feg die Fusionsproteine scFv 215(Yol)-10-VH(phOx) und scFv 215(Yol)-10-VL(phOx) abschließt.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).
Vektorkonstruktionvector construction
Zur Herstellung des oben beschriebenen V-bodies wurde der Vektor pOPE111-215HphOx/215LphOx generiert. Er kodiert für die beiden oben bechriebenen Fusionsproteine scFv 215(Yol)-10-VH(phOx) und scFv 215(Yol)- 10-V*_(phOx). Die beiden Genfragmente wurden jeweils separat, unter Ausbildung von ORFs hinter pelB-/eacfer Sequenzen aus Erwinia carotovora (Lei et al., 1987) zur Sekretion der Translationsprodukte in den periplasmatischen Raum kloniert. Der Vektor pOPE111 besitzt ferner ein ColE1 origin of replication und das b-Lactamase-Gen als Selektionsmarker für die Ampicillinresistenz Die Expression der kodierten V-body-Konstrukte wird mit Hilfe eines, durch IPTG induzierbaren, synthetischen /ac-Promotors P/A1/04/03 und zwei starken ribosome binding sites (RBS) reguliert.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).
Expressionexpression
Eine 50 mL Übernachtkultur von E. coli Zellen transformiert mit pOPE111- 215HphOx/215LphOx wurde 1/20 in dYT-Medium (16 g/L Bacto-Trypton, 10 g/L Hefe-Extrakt, 5 g/L NaCI) mit 100 mM Giucose und 100 μg/mL Ampicillin verdünnt und bei 37 °C und 230 rpm bis zu einer OD6oo von 0,6 kultiviert, bevor der Promotor durch Zugabe von 20 mM Isopropyl-b-D- thiogalactopyranosid induziert wurde. Nach 3 h Inkubation bei 22 °C und 230 rpm wurden die Bakterien 10 min auf Eis abgekühlt und durch Zentrifugation bei 4 °C und 5000 x g pelletiert.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.
Präparation periplasmatischer ExtraktePreparation of periplasmic extracts
Zur Präparation periplasmatischer Extrakte wurden die pelletierten Bakterien in 1/10 Volumen (bezogen auf das Ausgangsvolumen der Kultur) gekühlter Schock- Lösung (50 mM Tris/HCI, pH 8,0, 20 % (w/v) Saccharose, 1 mM EDTA) resuspendiert, für 20 min unter gelegentlichem Schütteln auf Eis inkubiert und erneut für 10 min bei 6200 x g und 4 °C zentrifugiert. Der periplasmatische Extrakt im Überstand wurde für weitere 30 min bei 30000 x g und 4 °C zentrifugiert, gegen PBS dialysiert und mit SDS-PAGE, Immunoblot und ELISA analysiert. Das Zellpellet wurde in 1/10 Volumen (bezogen auf das Ausgangsvolumen der Kultur) 5 mM MgSO4 resuspendiert, für 20 min unter gelegentlichem Schütteln auf Eis inkubiert und ebenfalls für 30 min bei 30000 x g und 4 °C zentrifugiert. Der so erhaltene Extrakt wurde ebenfalls gegen PBS dialysiert und mit SDS-PAGE, Immunoblot und ELISA analysiert.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.
Anreicherung der Antikörper-Fragmente durch IMACEnrichment of the antibody fragments by IMAC
Eine Säule wurde mit 2 mL/Liter Bakterienkultur Ni-NTA-Sepharose beladen und in SSP (PBS , pH 7,5, 10 mM Imidazol, 1 M NaCI) äquilibriert. Die dialysierten periplasmatischen Extrakte wurden vereinigt, auf dieselben Pufferbedingungen eingestellt und auf die Säule gegeben. Nach Waschen der Säule mit 5 Sepharose-Volumen SSP, 5 Sepharose-Volumen SWP (PBS, pH 7.5, 50 mM Imidazol, 1 M NaCI) und 5 Sepharose-Volumen PBS wurden die Proteine 10 Sepharose-Volumen Elutionspuffer (PBS, pH 7,5, 500 mM Imidazol, 1 M NaCI) kompetitiv eluiert. Die gesammelten Fraktionen wurden durch SDS-PAGE (Laemmli, 1975) und Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b) analysiert. Protein enthaltende Fraktionen wurden vereinigt.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-ChromatographieMono Q chromatography
Die über IMAC erhaltenen Antikörperfragmente wurden gegen 30 mM Tris/HCI, pH 8,0 dialysiert und mit 0,5 mL/min auf die im selben Puffer äquilibrierte Säule (Mono Q HR/5R; Amersham Pharmacia, Freiburg; Schmiedl et al., 2000b) gegeben. Nach Waschen der Säule mit 5 mL 30 mM Tris/HCI, pH 8,0 wurden die Proteine mit Hile eines zweiphasigen NaCI-Gradienten kompetitiv eluiert. Dazu wurde die Salzkonzentration zunächst über 20 mL auf 250 mM, dann über 1 mL auf 1 M NaCI gesteigert. Die Säule wurde noch mit 5 mL Mono Q-Elutionspuffer (30 mM Tris/HCI, pH 8,0, 1 M NaCI) gespült und wieder auf Mono-Q-Puffer eingestellt. Die gesammelten Fraktionen wurden durch SDS-PAGE (Laemmli, 1975) und Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b) analysiert. Protein enthaltende Fraktionen wurden vereinigt und ihr Gehalt mit Bradford- Lösung bestimmt.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)ELISA (enzyme linked immunosorbent assay)
96-well Maxisorp Platten (Nunc, Karlsruhe) wurden zunächst ü.N. bei 4 °C mit 100 μ /well 0,1 M Na2CO3 (pH 9.6), in denen je 1 mg eines Antigens gelöst wurden, inkubiert, bevor unspezifische Bindungsstellen durch MPBS (400 μUwell) für 2 h bei RT abgesättigt wurden. Zum Nachweis funktioneller Antikörper-Fragmente wurden die Antigene Fp b-Galactosidase-215, Fp b-Galactosidase-control, BSA- phox bzw. BSA verwendet. Fp b-Galactosidase-215 ist ein Fusionsprotein der bakteriellen b-Galactosidase mit einem Peptid, welches das mAb 215 Epitop beinhaltet. b-Galactosidase fusioniert mit einem vergleichbaren Peptid diente als Kontrolle. Die rekombinanten b-Galactosidase-Fusionsproteine wurden zuvor durch Expression in E. coli gewonnen (Kontermann er al., 1995). Phoxyliertes BSA wurde durch Inkubation eines 20fachen molaren Überschusses von 4- Ethoxymethylen-2-phenyl-2-oxazolin-5-on (phOx) mit BSA in 0,1 M NaCO3 (pH 8,5) für zwei Stunden dargestellt. Nicht gekoppeltes 4-Ethoxy-methylen-2- phenyl-2-oxazolin-5-on wurde durch wiederholte Dialyse gegen PBS entfernt. Nicht-modifiziertes BSA diente als Kontrolle.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.
Unspezifische Bindungsstellen der Maxisorp-Platte wurden durch Blocken mit 400 mLlwell MPBS für mindestens 3 h bei RT abgesättigt. Die Periplasma- Extrakte wurden 1 :1 in MPBST verdünnt und je 100 mLlwell für 2 h bei RT inkubiert. Der Antikörper mAb Myc1-9E10 (Calbiochem, Schwalbach; 1/1000 in MPBST) wurde in Kombination mit HRP-konjugierten Ziege-anti-Maus Immunglobulinen (Dianova, Hamburg; 1/2000 in MPBST) zum Nachweis gebundener Antikörperfragmente eingesetzt und mit 100 mLl well TMB-Substrat (10 mL 100 mM Natriumacetat, pH 6,0, 25 μL TMB-Lösung (40 mg/mL in DMSO), 8 μL 30 % (v/v) H2O2) detektiert. Nach Stoppen der Farbreaktion durch Zugabe von 50 μUwell 1 M H2SO erfolgte die Analyse mit Hilfe eines ELISA-Reaαfers bei 450 nm.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.
Zum Nachweis der Bispezifität des Antikörper-Konstrukts wurden 100 mL/well Antikörper-Extrakt für 2 Stunden an BSA-phOx gecoatete Maxisorp-Platten gebunden. HRP-konjugietes Streptavidin (8/1000 in PBST) wurde mit 0,1 nmol des N-terminal biotinylieten Peptids LPHFIKDDYGPESRGFVENSYLAGLTPSE (ZMBH, Heidelberg), welches das Epitop des Antikörpers mAb 215 enthält, bzw. mit dem entsprechend biotinylierten Kontoll-Peptid KESRAKKFQRQHMDEGEEF (ZMBH, Heidelberg) für eine Stunde bei RT inkubiert, bevor je 100 mU well aufgetragen wurden. Der Nachweis der Bispezifität erfolgte nach 2 Stunden Inkubation bei RT mit TMB-Substrat.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.
Literaturliterature
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| DE10021678A DE10021678A1 (en) | 2000-05-05 | 2000-05-05 | Recombinant polyspecific antibody constructs, useful for diagnosis and treatment of cancer, comprises three antibody fragments,where at least one comprises a disulfide bridge |
| DE10021678 | 2000-05-05 | ||
| PCT/EP2001/005161 WO2001085795A1 (en) | 2000-05-05 | 2001-05-07 | Trivalent antibody constructs having variable regions that are stabilized by disulfide bridges |
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| EP2014680A1 (en) * | 2007-07-10 | 2009-01-14 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Recombinant, single-chain, trivalent tri-specific or bi-specific antibody derivatives |
| WO2009040562A1 (en) | 2007-09-26 | 2009-04-02 | Ucb Pharma S.A. | Dual specificity antibody fusions |
| SI2334705T1 (en) * | 2008-09-26 | 2017-05-31 | Ucb Biopharma Sprl | Biological products |
| PT2417156E (en) | 2009-04-07 | 2015-04-29 | Roche Glycart Ag | Trivalent, bispecific antibodies |
| US9676845B2 (en) | 2009-06-16 | 2017-06-13 | Hoffmann-La Roche, Inc. | Bispecific antigen binding proteins |
| RU2015153109A (en) | 2009-09-16 | 2019-01-15 | Дженентек, Инк. | SUPERSPIRAL AND / OR BINDING PROTEIN COMPLEXES AND THEIR APPLICATIONS |
| AR080793A1 (en) | 2010-03-26 | 2012-05-09 | Roche Glycart Ag | BISPECIFIC ANTIBODIES |
| KR101586128B1 (en) | 2010-08-24 | 2016-01-15 | 에프. 호프만-라 로슈 아게 | Bispecific antibodies comprising a disulfide stabilized - fv fragment |
| AR085403A1 (en) | 2011-02-28 | 2013-09-25 | Hoffmann La Roche | MONOVALENT PROTEINS THAT JOIN ANTIGENS |
| CA2825081A1 (en) | 2011-02-28 | 2012-09-07 | Birgit Bossenmaier | Antigen binding proteins |
| CA2922912A1 (en) | 2013-10-11 | 2015-04-16 | F. Hoffmann-La Roche Ag | Multispecific domain exchanged common variable light chain antibodies |
| WO2016087416A1 (en) | 2014-12-03 | 2016-06-09 | F. Hoffmann-La Roche Ag | Multispecific antibodies |
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| DE69232137T2 (en) * | 1991-11-25 | 2002-05-29 | Enzon Inc | MULTIVALENT ANTI-BINDING PROTEINS |
| GB9221657D0 (en) * | 1992-10-15 | 1992-11-25 | Scotgen Ltd | Recombinant bispecific antibodies |
| AUPO591797A0 (en) * | 1997-03-27 | 1997-04-24 | Commonwealth Scientific And Industrial Research Organisation | High avidity polyvalent and polyspecific reagents |
| EP1015480A2 (en) * | 1997-08-18 | 2000-07-05 | Innogenetics N.V. | Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders |
| AU2719099A (en) * | 1998-01-23 | 1999-08-09 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Multipurpose antibody derivatives |
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