HK1186769B - Detection of a posttranslationally modified polypeptide by a bi-valent binding agent - Google Patents

Description

Detection of post-translationally modified polypeptides by bivalent binding agents

Background

The present invention relates to a bivalent binding agent consisting of a first monovalent binder that binds to a polypeptide epitope of a target polypeptide, a second monovalent binder that binds to a post-translational polypeptide modification on the target polypeptide, and a linker. Further disclosed are methods for detecting a post-translationally modified target polypeptide with the aid of such divalent binding agents, methods for preparing such divalent binding agents, and the use of such divalent agents in histological staining procedures.

The primary structure of a polypeptide (i.e., its sequence) is determined by its encoding nucleic acid. However, knowing the primary structure of a polypeptide is only part of a matter. Many polypeptides (estimated in the range of 50 to 90%) undergo secondary modifications. Polypeptides having the same primary structure may exhibit quite different biological functions depending on e.g. the type of secondary modification, the percentage of modified polypeptides and/or e.g. the exact position/location of the secondary modification.

Secondary protein modifications fine-tune the cellular function of each protein. There is a continuing and intense effort worldwide to understand the relationship between post-translational modifications and functional changes ("posttranslational biologics"), which is not analogous to the human genome project. In combination with separation techniques and mass spectrometry, proteomics makes it possible to dissect and characterize portions of post-translational modifications, and provide systematic analysis.

Approximately a decade ago, proteins were viewed as linear polymers of amino acids, and it became apparent first that such polypeptide chains could be decorated with simple amino acid modifications. However, a very complex modification in proteins has recently been discovered in a number of processes. Multiple chemical modifications have been observed in a single protein, and these modifications, alone or in various combinations, occur in a time and signal dependent manner. Post-translational modifications of a protein determine its tertiary and quaternary structure, and regulate its activity and function. Advances in "post-translational proteomics" have led to much pioneering understanding of the interrelationship of secondary modifications and biological functions, for example, with respect to the regulation of biochemical pathways and disease states involving these proteins.

However, the detection and quantification of secondary modified polypeptides requires sophisticated tools and techniques.

Various types of separation and optionally fragmentation techniques are often combined with mass spectrometry to identify post-translationally modified polypeptides.

Immunological detection of post-translationally modified polypeptides has consistently proven to be quite difficult. Various types of problems may be encountered. Obtaining sufficient purity and quantity of the desired immunogen can be difficult. Antibodies obtained according to standard immunization and screening methods may not have the desired specificity and/or affinity. Especially when highly reproducible, consistent quality antibodies (e.g., monoclonal antibodies) are required, obtaining such antibodies can prove to be very laborious. Such antibodies will have to strongly bind to epitopes consisting of secondary modifications and the polypeptide part carrying it. However, many binding agents generated by conventional procedures exhibit cross-reactivity with other polypeptides having the same post-translational modification, do not exhibit the desired affinity for the recognized epitope and/or exhibit cross-reactivity with unmodified polypeptides.

Many larger polypeptides even contain several sites where one type of post-translational modification occurs. There may be, for example, several threonine residues that are glycosylated in a statistical manner. Assessing the glycosylation state of such polypeptides may require several different antibodies specific for each position potentially carrying the post-translational modification.

From the above non-exhaustive discussion of various possible problems and the prior art protocols and binding agents, it becomes apparent that there is an urgent need to provide binding agents that can reproducibly generate binding polypeptides with high affinity post-translationally modified in virtually unlimited numbers and in unsuitable quality.

Surprisingly, it has been found that a post-translationally modified target polypeptide can be detected by a bivalent binding agent consisting of two monovalent binding agents linked to each other via a linker, wherein the first monovalent binding agent binds to a polypeptide epitope of the target polypeptide and the second monovalent binding agent binds to a post-translational polypeptide modification, wherein each monovalent binding agent has a range of 5x10^-3Second to 10^-4K/sec_DissociationAnd wherein the bivalent binding agent has 3x10^-5K/sec or less_Dissociation。

Summary of The Invention

Post-translational polypeptide modifications are essential for regulating and/or modulating the properties and/or activity of the polypeptide. An advantageous method for detecting certain types of secondary modifications on a target polypeptide would rely on specific binding agents.

The present invention relates to a bivalent binding agent binding to a post-translationally modified target polypeptide, consisting of two monovalent binding agents connected to each other via a linker, wherein the first monovalent binding agent binds to a polypeptide epitope of said target polypeptide, wherein the second monovalent binding agent binds to a post-translational polypeptide modification, wherein each monovalent binding agent has a range of 5x10^-3Second to 10^-4K/sec_DissociationAnd wherein the bivalent binding agent has 3x10^-5K/sec or less_Dissociation。

Also disclosed is a method for obtaining a bivalent binding agent that specifically binds to a post-translationally modified target polypeptide, the method comprising the steps of: selected to be between 5x10^-3Second to 10^-4K between/sec_DissociationA first monovalent binder that binds to an untranslated modified epitope of the target polypeptide, selected at 5x10^-3Second to 10^-4Per second ofK_DissociationBinding a second monovalent binder modified by a post-translational polypeptide, coupling the two monovalent binders through a linker, and selecting the binding site as having a size of 3x10^-5K/sec or less_DissociationBivalent binding agent of value.

Uses of the novel bivalent binding agents, particularly in immunohistochemical procedures, are also described and claimed.

Detailed Description

The present invention relates to a bivalent binding agent binding to a post-translationally modified target polypeptide, which binding agent consists of two monovalent binding agents connected to each other via a linker, wherein a) a first monovalent binding agent binds to a polypeptide epitope of said target polypeptide, b) a second monovalent binding agent binds to a post-translational polypeptide modification, c) each monovalent binding agent has a range of 5x10^-3Second to 10^-4K/sec_DissociationAnd d) wherein the bivalent binding agent has 3x10^-5K/sec or less_Dissociation。

A bivalent binding agent according to the present invention is a binding agent comprising exactly two monovalent binding agents of different specificity.

In one embodiment, by Biacore^TMThe SPR technique characterizes the kinetic rate properties of each monovalent binder and of the divalent binder, as detailed in the examples.

As the skilled artisan will appreciate, the bivalent binding agent described in the present invention may be isolated and purified as desired. In one embodiment, the invention relates to an isolated bivalent binding agent as disclosed herein. An "isolated" bivalent binding agent is a bivalent binding agent that has been identified and separated and/or recovered from, for example, a mixture of reagents used to synthesize such bivalent binding agent. An unwanted component of such a reaction mixture is, for example, a monovalent binder that is not used up in the desired divalent binder. In one embodiment, the bivalent binding agent is purified to greater than 80%. In some embodiments, the divalent binding agent is purified to greater than 90%, 95%, 98%, or 99% by weight, respectively. In case both monovalent binders are polypeptides, the purity is easily determined by SDS-PAGE under reducing or non-reducing conditions, e.g. in protein detection using e.g. coomassie blue or silver staining agents. In case purity is assessed at the nucleic acid level, size exclusion chromatography is applied to separate the bivalent binding agent from the side products and OD at 260nm is monitored to assess its purity.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an antibody" refers to one antibody or more than one antibody.

As used herein, the term "oligonucleotide" or "nucleic acid sequence" generally refers to a short, usually single-stranded, polynucleotide comprising at least 8 nucleotides and at most about 1000 nucleotides. In a preferred embodiment, the oligonucleotide will have a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In a preferred embodiment, the oligonucleotide will have a length of no more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. The description given below for polynucleotides applies equally and fully to oligonucleotides.

The term oligonucleotide is to be understood broadly and includes DNA and RNA as well as analogs and modifications thereof.

For example, an oligonucleotide may contain substituted nucleotides carrying substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), deoxyuridine (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines such as 5-methyl dC, aminoallyl (aminoallyl) dU or dC, 5- (aminoethyl-3-acryloylimino) (acylamino) -dU, 5-propynyl-dU or-dC, 5-halogenated-dU or-dC; n-substituted pyrimidines such as N4-ethyl-dC; n-substituted purines such as N6-ethyl-dA, N2-ethyl-dG; 8-substituted purines such as 8- [ 6-amino) -hex-1-yl ] -8-amino-dG or-dA, 8-halogenated dA or dG, 8-hydrocarbyl dG or dA; and 2 substituted dA such as 2 amino dA.

The oligonucleotide may contain a nucleotide or nucleoside analogue. That is, a naturally occurring nucleobase can be exchanged by using a nucleobase analog such as 5-nitroindole (Nitroindol) d nucleoside; 3-nitropyrrole d-nucleoside, deoxyinosine (dI), deoxyxanthosine (dX); 7 deaza-dG, -dA, -dI or-dX; 7-deaza-8-aza-dG, -dA, -dI or-dX; 8-aza-dA, -dG, -dI, or-dX; d-m-type mycin (Formycin); false dU; a pseudo-differential dC; 4 thio dT; 6 thio dG; 2-thio dT; iso-dG; 5-methyl-iso-dC; n8-linked 8-aza-7-deaza-dA; 5, 6-dihydro-5-aza-dC; and vinylidene-dA or pyrrolo-dC. As will be apparent to the skilled artisan, the nucleobases in the complementary strand must be selected in such a way that duplex formation is specific. If, for example, 5-methyl-iso-dC is used in one strand (e.g., (a)), then iso-dG must be used in the complementary strand (e.g., (a')).

The oligonucleotide backbone may be modified to contain substituted sugar residues, sugar analogs, modifications to the internucleoside phosphate moiety, and/or be PNA.

The oligonucleotide may, for example, contain a nucleotide having a substituted deoxyribose sugar, such as 2 ' -methoxy, 2 ' -fluoro, 2 ' -methylseleno, 2 ' -allyloxy, 4 ' -methyl dN (where N is a nucleobase, e.g., a, G, C, T or U).

Sugar analogs are, for example, xylose; 2 ', 4' bridged ribose such as (2 '-O, 4' -C methylene) - (oligomers known as LNA) or (2 '-O, 4' -C ethylene) - (oligomers known as ENA); l-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (altritol) (oligomer known as ANA); tricyclic ribo-saccharide analogs, in which the C3 'and C5' atoms are connected by an ethylene bridge fused to a cyclopropane ring (referred to as oligomers of tricyclic DNA); glycerol (oligomer known as GNA); glucopyranose (oligomers called homo DNA); carbaibose (with cyclopentane (cyclopentane) instead of tetrahydrofuran subunits); hydroxymethyl-morpholine (called oligomer of morpholino DNA).

It is also known that a large number of internucleoside phosphate module modifications do not interfere with hybridization properties, and that such backbone modifications can also be combined with substituted nucleotides or nucleotide analogs. Examples are phosphorothioate, phosphorodithioate, phosphoramidate and methylphosphonate oligonucleotides.

PNAs (with a backbone free of phosphate and d-ribose) can also be used as DNA analogs.

The modified nucleotides, nucleotide analogs, and oligonucleotide backbone modifications mentioned above can be combined as desired to form oligonucleotides within the meaning of the invention.

The terms "polypeptide" and "protein" are used interchangeably. In the sense of the present invention, a polypeptide consists of at least 5 amino acids linked by alpha amino peptide bonds.

A "target polypeptide" is a polypeptide of interest for which an assay or measurement method is sought. The target polypeptides of the invention are polypeptides known or suspected to carry post-translational polypeptide modifications.

According to the invention, a "monovalent binder" is 5x10^-3Second to 10^-4K/sec_DissociationA molecule that interacts with a target polypeptide at a single binding site. Preferably, the biophysical characterization of the kinetic association rate profile, in particular the dissociation rate constant kd (1/s) determined according to the Langmuir model, is analyzed by biosensor-based surface plasmon resonance spectroscopy. Preferably, Biacore is used as detailed in the examples^TMProvided is a technique.

Examples of monovalent binders are peptides, peptide mimetics, aptamers, spiegelmers, darpin, lectins, ankyrin repeats, Kunitz-type domains, single domain antibodies (see Hey, t. et al, Trends Biotechnol23(2005) 514-522) and monovalent antibody fragments.

In certain preferred embodiments, the monovalent binding agent is a monovalent antibody fragment, preferably a monovalent fragment derived from a monoclonal antibody.

Monovalent antibody fragments include, but are not limited to, Fab '-SH (Fab'), single domain antibodies, Fv, and scFv fragments, as provided below.

In a preferred embodiment, at least one monovalent binding agent is a single domain antibody, a Fab fragment of a monoclonal antibody, or a Fab' fragment.

Also representative of a preferred embodiment is the bivalent binding agent disclosed herein wherein both monovalent binding agents are derived from a monoclonal antibody and are a Fab fragment, or a Fab 'fragment or a Fab fragment and a Fab' fragment.

Monoclonal antibody technology allows for the generation of extremely specific binding agents in the form of specific monoclonal antibodies or fragments thereof. Particularly well known in the art are techniques for creating monoclonal antibodies or fragments thereof by immunizing a mouse, rabbit, hamster, or any other mammal with a polypeptide of interest. Another method for creating monoclonal antibodies or fragments thereof is to use phage libraries of sFvs (single chain variable regions), particularly human sFvs (see, e.g., Griffiths et al, U.S. Pat. No.5,885,793; McCafferty et al, WO 92/01047; Liming et al, WO 99/06587).

Antibody fragments may be generated by conventional means (e.g., enzymatic digestion) or by recombinant techniques. For a review of certain antibody fragments, see Hudson, P.J., et al, nat. Med.9(2003) 129-134.

Fv is the smallest antibody fragment that contains the entire antigen binding site and lacks the constant region. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In one embodiment of a single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker, thereby allowing the light and heavy chains to associate in a dimeric structure similar to that in a two-chain Fv species. For a review of scfvs, see, e.g., Plueckthun, in: the Pharmacology of monoclonal Antibodies, Vol 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp 269-315; see also WO 93/16185; and U.S. Pat. nos. 5,571,894 and 5,587,458. In general, the 6 hypervariable regions (HVRs) confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only 3 HVRs specific for an antigen) has the ability to recognize and bind antigen.

The Fab fragment contains both the heavy and light chain variable domains, and also contains the light chain constant domain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domains carry a free thiol group.

Various techniques have been developed for generating antibody fragments. Traditionally, antibody fragments have been derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto, K. et al, Journal of Biochemical and biophysical methods24(1992) 107-117; and Brennan et al, Science 229(1985) 81-83). For example, papain digestion of antibodies produces two identical antigen-binding fragments, each with a single antigen-binding site, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily.

Antibody fragments may also be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from e.coli (e.coli), thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries according to standard procedures. Alternatively, Fab' -SH fragments can be recovered directly from E.coli (Carter, P. et al, Bio/Technology10(1992) 163-. Mammalian cell systems can also be used to express and, if desired, secrete antibody fragments.

In certain embodiments, the monovalent binding agent of the invention is a single domain antibody. Single domain antibodies are single polypeptide chains that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1). In one embodiment, the single domain antibody consists of all or part of the heavy chain variable domain of the antibody.

One of the two monovalent binders, the first monovalent binder, binds to a polypeptide epitope on the target polypeptide.

A "polypeptide epitope" according to the present invention, i.e. a binding site on a target polypeptide to which a corresponding monovalent binder binds, is composed of amino acids. This binding agent binds to a linear epitope, i.e. an epitope consisting of a segment of 5 to 12 consecutive amino acids, or a monovalent binding agent binds to a tertiary structure formed by the spatial arrangement of several short segments of the target polypeptide. Tertiary epitopes recognized by a binding agent, e.g., by an antigen recognition site or paratope of an antibody, can be viewed as three-dimensional surface features of an antigenic molecule; these features precisely fit the respective binding sites of the binding agent and thereby facilitate binding between the binding agent and the target polypeptide.

Whereas in the bivalent binding agent as disclosed herein, the first monovalent binding agent binds to a polypeptide epitope, the second monovalent binding agent binds to a post-translational polypeptide modification.

"post-translational polypeptide modifications" are covalent modifications of amino acids within or at the terminus of a polypeptide (protein). The terms secondary modification and post-translational modification are interchangeable.

Many types of covalent amino acid modifications are known and subject to scientific review articles. By way of mention, the posttranslational modifications described in the review articles by Mann and Jensen (2003) and by Seo and Lee (2004) are included hereby (Mann, M. and Jensen, O.N., nat. Biotechnol.21(2003) 255-.

In a preferred embodiment, the post-translational modification is selected from the group consisting of: acetylation, phosphorylation, acylation, methylation, glycosylation, ubiquitination, sumoylation, sulfation, and nitration.

Acetylation (+ 42 Da) is a fairly stable secondary modification. Examples are acetylation found on the N-terminus of many proteins or acetylation on lysine or serine residues. Typically, acetylation of lysine residues is found at one or more well-defined positions within a polypeptide chain, while other lysine residues are less frequently or not acetylated at all.

It is known that phosphorylation and dephosphorylation of proteins (the net balance of which can be referred to as phosphorylation state) is one of the key elements in the regulation of protein biological activity. A lower percentage of phosphorylated amino acid residues may already be sufficient to trigger a certain biological activity. Phosphorylation results in an increase in mass of 80 Da. The amino acids tyrosine (Y), serine (S), threonine (T), histidine (H) and aspartic acid (D) may be phosphorylated. The more complex the biological function of a polypeptide, the more complex the corresponding pattern of possible phosphorylation sites. This is particularly known and true for membrane-bound receptors, particularly the so-called Receptor Tyrosine Kinases (RTKs). As nomenclature has suggested, at least a portion of the intracellular signaling of RTKs is mediated by the phosphorylation state of a certain tyrosine of the intracellular domain of such RTKs.

Polypeptides may be acylated by farnesyl (farnesyl), myristoyl (myristoyl) or palmitoyl (palmitoyl) groups. Acylation typically occurs on the side chain of a cysteine residue.

Methylation as a secondary modification occurs via the side chain of a lysine residue. It has been shown that the binding properties of regulatory proteins capable of binding nucleic acids can be regulated, for example, via methylation.

Glycosylation is a very important secondary modification. It has a major impact on protein-protein interactions, solubilization of proteins, their stability, etc. Two different types of glycosylation are known: an N-linked (via the amino acid N (asparagine)) side chain and an O-linked side chain (via serine (S) or threonine (T)). Many different polysaccharides (linear or with branched side chains) have been identified, some containing sugar derivatives such as O-Glc-NAc.

Ubiquitination and sumoylation, respectively, are known to affect the half-life of proteins in circulation. Ubiquitination may act as a disruption signal, resulting in cleavage and/or removal of the ubiquitinated polypeptide.

Sulfation via tyrosine residues (Y) appears to be important in the regulation of protein-protein (cell-cell) interactions as well as in protein ligand interactions.

Nitration of tyrosine residues (Y) appears as a marker of oxidative damage (hall-mark), as for example in inflammatory processes.

Preferably, the post-translational modification bound by the second monovalent binder is selected from the group consisting of: phosphorylation, glycosylation and acetylation.

As mentioned above, phosphorylation, dephosphorylation, and phosphorylation states are critical for the regulation of cell signaling and protein activity. This is known and true in particular for membrane bound receptors, in particular the so-called Receptor Tyrosine Kinases (RTKs). As nomenclature has suggested, at least a portion of the intracellular signaling of RTKs is mediated by the phosphorylation state of a certain tyrosine of the intracellular domain of such RTKs. Thus, in a preferred embodiment, the invention relates to a bivalent binding agent that binds a phosphorylated target protein. Clearly, such bivalent binding agents have great utility in the detection of phosphorylated target polypeptides.

In a preferred embodiment, the present invention relates to a bivalent binding agent as disclosed herein above, wherein the target polypeptide is selected from the group consisting of: membrane-bound receptor molecules with intracellular phosphorylation sites and intracellular cell signaling molecules. In such bivalent binding agent, a first monovalent binding agent that binds to a polypeptide epitope on a target protein will specifically bind to the receptor molecule or the intracellular cell signaling molecule, while a second monovalent binding agent that targets phosphorylation need not specifically bind to a phosphorylation site on the target protein. Cross-reactivity with, for example, a phosphorylation site on the relevant receptor does not impair specific detection of the target polypeptide, since significant binding requires both binding of the first monovalent binder and binding of the second monovalent binder.

In some embodiments, the RTK is selected from the group consisting of: ALK, adhesion-related kinase receptors (e.g., Axl), ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB 4), erythropoietin-producing hepatocyte (EPH) receptors (e.g., EphA 1; EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA7, EphB 7, EphB 7, EphB 7, EphB 7, EphB 7, EphB 7), Fibroblast Growth Factor (FGF) receptors (e.g., FGFR 7, FGFR 7, FGFR 7, FGFR 7, FGR, IGK, insulin R, LTK, M-CSFR, MUSK, platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-A, PDGFR-B), PDGFR, 7, VEFR-R, VEGFR, VEGF-like receptor (e) receptors (e.g., VEGF/VEGF) receptor with an endothelial growth factor-like receptor (VEGF/VEGF) receptor such as VEGF/VEGF receptor with an IG receptor tyrosine, VEGF/VEGF-like kinase (e receptor such as VEGF/VEGF) receptor with A receptor, INS-R, IGF-IR, IR-R), Discoidin Domain (DD) receptors (e.g., DDR1, DDR 2), c-Met (MET) receptors, registered' origin navias (RON); also known as macrophage stimulating 1 receptor, Flt3 fins-associated tyrosine kinase 3(Flt3), colony stimulating factor 1(CSF1) receptor, receptor for c-KIT (KIT, or SCFR), and insulin receptor-associated (IRR) receptor.

In some embodiments, the intracellular cell signaling molecule is selected from the group consisting of: AKT, abl, cbl, erbA, ERK, fes, fgr, fms, fos, jun, met, myb, myc, PI3K, raf, ret, ryk, and src. In a preferred embodiment, the present invention relates to a bivalent binding agent binding to a post-translationally modified target polypeptide, said bivalent binding agent consisting of two monovalent binding agents linked to each other via a linker, wherein a) a first monovalent binding agent binds to a polypeptide epitope of said target polypeptide, b) a second monovalent binding agent binds to a post-translational polypeptide modification, c) each monovalent binding agent has a range of 5x10^-3Second to 10^-4K/sec_DissociationD) wherein the bivalent binding agent has 3x10^-5K/sec or less_DissociationAnd wherein the post-translational modification is selected from the group consisting of: phosphorylation, ubiquitination and glycosylation.

In a preferred embodiment, the kinetic rate properties of each monovalent binder and of the divalent binder are characterized by Biacore SPR techniques, as detailed in the examples.

In a preferred embodiment, the bivalent binding agent according to the invention will bind to a target polypeptide with a post-translational modification, wherein the post-translational modification is phosphorylation.

As discussed, the monovalent binders used in the construction of the bivalent binders as disclosed herein must have a 5x10^-3Second to 10^-4K/sec_Dissociation。

Preferably, the first monovalent binding agent specifically binds to a polypeptide epitope. That is, the binding agent binds to an epitope that is not secondary modified or, in the alternative, specifically binds to a native (non-secondary modified) epitope. The binding agent has at least a 20-fold lower K for a non-post-translationally modified polypeptide if compared to the same polypeptide carrying a post-translational modification_DissociationSpecific binding to the polypeptide epitope is then granted. It is also preferred that the K of the first monovalent binder for the unmodified polypeptide is compared to the same polypeptide carrying the post-translational modification in the epitope of the polypeptide bound by the first monovalent binder_DissociationAt least 30, 40, 50, 80, 90, 95, or at least 100 times higher.

Preferably, the second monovalent binding agent specifically binds to a post-translational polypeptide modification, i.e. said binding agent has at least a 20-fold lower K for a polypeptide carrying such a post-translational modification compared to the same non-post-translationally modified polypeptide_Dissociation. It is also preferred that the second monovalent binder pair carries a K of a post-translationally modified polypeptide as compared to the same non-modified polypeptide_DissociationAt least 30, 40, 50, 80, 90, 95, or at least 100 times lower.

As mentioned above, a bivalent binding agent according to the invention will have at most 3x10^-5K/sec or lower (i.e., better)_Dissociation。

In one embodiment, in the bivalent binding agent according to the invention, each monovalent binding agent has 2x10^-3Second to 10^-4K/sec_Dissociation。

In one embodiment, in the bivalent binding agent according to the present invention, each monovalent binding agent has 10^-3Second to 10^-4K/sec_Dissociation。

Automated immunohistochemical stainers distributed by Ventana Medical Systems inc. In thatThe antibodies used on the analyzer series should have up to 5x10^-5K/sec_DissociationTo give reasonable staining intensity. K_DissociationThe better the staining intensity will be. The bivalent binding agent as disclosed herein has at most 3x10^-5K/sec_Dissociation. In another embodiment, the bivalent binding agent as disclosed herein has 2x10^-5Second or less or preferably also 10^-5K/sec or less_Dissociation。

In one embodiment, by Biacore^TMThe SPR technique characterizes the kinetic rate properties of each monovalent and divalent binder, as detailed in the examples.

The bivalent binding agent according to the present invention contains a linker. The linker may covalently link two monovalent binders or the linker and monovalent binder may be bound by two different specific binding pairs a: a 'and b: b'.

The linker may, for example, be composed of suitable monomers that are linked together and to two monovalent binders by a covalent bond. Preferably, the linker will contain a sugar moiety, a nucleotide moiety, a nucleoside moiety and/or an amino acid. In certain preferred embodiments, the linker will consist essentially of nucleotides, nucleotide analogs, or amino acids.

Preferably, the linker covalently linked or bound to the two monovalent binders via a binding pair has a length of 6 to 100 nm. It is also preferred that the linker has a length of 6 to 50nm or 6 to 40 nm. In yet another preferred embodiment, the linker will have a length of 10nm or more or 15nm or more. In one embodiment, the linker comprised in the bivalent binding agent according to the present invention has a length between 10nm and 50 nm.

Theoretically and by a complex approach, the length of the non-nucleoside entity of a given linker (a-S-b) can be calculated by using the known bond distance and bond angle of compounds that are chemically similar to the non-nucleoside entity. Such bond distances are summarized in standard textbooks for some molecules: CRC Handbook of Chemistry and Physics, 91 st edition, 2010-2011, section 9. However, the exact bond distance varies for each compound. There is also variability in key angle.

It is therefore more practical to use averaging parameters (easy to understand approximations) in such calculations.

In the calculation of the spacer or linker length, the following approximation applies: a) to calculate the length of the non-nucleoside entity, an average bond length of 130pm and a bond angle of 180 ° were used, which is independent of the nature of the atoms attached; b) one nucleotide in the single strand was calculated with 500pm, and c) one nucleotide in the double strand was calculated with 330 pm.

The value 130pm is calculated based on the distance of the two terminal carbon atoms of the C (Sp3) -C (Sp3) -C (Sp3) chain, with a bond angle of 109 ° 28', and a distance between the two C (Sp3) of 153pm, assuming that the bond angle of 180 ° and the bond distance 125pm between the two C (Sp3) are converted to about 250 pm. The value of 130pm is used, considering that heteroatoms such as P and S and sp2 and sp1C atoms may also be part of the spacer. If the spacer comprises a cyclic structure such as a cycloalkyl or aryl group, the distance is calculated in a similar manner by counting the bonds of a portion of the entire chain of atoms in the cyclic structure that are the defined distance.

As mentioned above, the linker may covalently link two monovalent binders, or the linker and monovalent binder may be bound by two different specific binding pairs a: a 'and b: b'. Thus, a bivalent binding agent according to the invention that binds to a post-translationally modified target polypeptide can also be depicted by the following formula I:

A-a’:a-S-b:b’-B，

wherein A is a first monovalent binder that binds to a polypeptide epitope of the target polypeptide, wherein B is a second monovalent binder that binds to a post-translational polypeptide modification, wherein each monovalent binder A and B has a range of 5x10^-3Second to 10^-4K/sec_DissociationWherein a 'a and b: b' are independently a binding pair or a 'a and/or b: b' are covalently bound, wherein a 'a and b: b' are different, wherein S is a spacer, wherein-represents a covalent bond, wherein the linker a-S-b has a length of 6 to 100nm, and wherein the divalent binding agent has a length of 3x10^-5K/sec or less_Dissociation。

The linker L consisting of a-S-b has a length of 6 to 100 nm. Preferably, the linker L consisting of a-S-b has a length of 6 to 80 nm. It is also preferred that the linker has a length of 6 to 50nm or 6 to 40 nm. In yet another preferred embodiment, the linker will have a length of 10nm or more or 15nm or more. In one embodiment, the linker has a length between 10nm and 50 nm. In one embodiment, a and b are each a member of a binding pair, and each has a length of at least 2.5 nm.

The spacer S may be constructed as desired, for example to provide a desired length and other desired characteristics. The spacer may, for example, consist entirely or partially of naturally occurring or non-naturally occurring amino acids, of phosphate-sugar units, e.g., nucleobase-free DNA-like backbones, of sugar-peptide structures, or at least partially of sugar units or at least partially of polymerizable subunits, e.g., diols or acrylamides.

The length of the spacer S in the compound according to the invention may vary as desired. To easily utilize spacers of variable length (i.e., libraries), spacers that enable simple synthesis of such libraries are preferred. Combinatorial solid phase synthesis of spacers is preferred. Since spacers up to about 100nm in length have to be synthesized, the synthesis strategy is chosen in such a way that the monomeric synthesis building block is assembled with high efficiency during solid phase synthesis. The synthesis of deoxyoligonucleotides assembled based on phosphoramidites as monomeric building blocks fully satisfies this requirement. In such spacers, the monomer units within the spacer are in each case linked via a phosphate or phosphate analogue module.

The spacer S may contain free positively or/and negatively charged multifunctional amino-carboxylic acid groups, such as amino groups, carboxylates or phosphates. For example, the charge carrier may be derived from a trifunctional aminocarboxylic acid containing a) one amino group and two carboxylate groups or b) two amino groups and one carboxylate group. Examples of such trifunctional aminocarboxylic acids are lysine, ornithine, hydroxylysine, alphｃA, betｃA-diaminopropionic acid, arginine, aspartic acid and glutamic acid, carboxyglutamic acid and symmetrical trifunctional carboxylic acids such as those described in EP-A-0618192 or U.S. Pat. No. 3, 5,519,142. Alternatively, one carboxylate group a) in the trifunctional aminocarboxylic acid can be replaced by a phosphate, sulfonate or sulfate group. An example of such a trifunctional amino acid is phosphoserine.

The spacer S may also contain uncharged hydrophilic groups. Preferred examples of uncharged hydrophilic groups are ethylene oxide or polyethylene oxide groups preferably having at least 3 ethylene oxide units, sulphoxides, sulphones, carboxylic acid amides, carboxylic acid esters, phosphonic acid (phosphonic acid) amides, phosphonic acid (phosphonic acid) esters, phosphoric acid (phosphoric acid) amides, phosphoric acid (phosphoric acid) esters, sulfonic acid amides, sulphonic acid esters, sulphuric acid amides and sulphuric acid ester groups. Preferably, the amide group is a primary amide group, particularly preferably a carboxylic acid amide residue in an amino acid side chain group (e.g., the amino acids asparagine and glutamine). Preferably, the esters are derived from hydrophilic alcohols, in particular C1-C3 alcohols or diols or triols.

In one embodiment, the spacer S is composed of a class of monomers. For example, the spacer is composed solely of amino acids, sugar residues, diols, phosphate-sugar units, respectively, or it may be a nucleic acid.

In one embodiment, the spacer is DNA. In a preferred embodiment, the spacer is an L-stereoisomer of DNA, also known as β -L-DNA, L-DNA or mirrored DNA. L-DNA is characterized by advantages such as orthogonal (orthogonal) hybridization behavior (which means that duplexes are formed only between two complementary single strands of L-DNA, but not between single strands of L-DNA and complementary DNA strands), nuclease resistance and ease of synthesis (even with longer spacers). As noted, ease of synthesis and variability of spacer length are important for spacer libraries. Variable length spacers are extremely useful in identifying bivalent dual binders according to the invention that have spacers of optimal length, thus providing an optimal distance between two monovalent binders.

Spacer building blocks, as the name implies, may be used to introduce spacer modules into the spacer S or to build the spacer S of the joints a-S-b.

There are different numbers and types of non-nucleotides and nucleotide spacer building blocks for introducing spacer modules.

Many different non-nucleotide bifunctional spacer building blocks are known in the literature and many are commercially available. The choice of non-nucleotide bifunctional spacer building influences the charge and flexibility of the spacer molecule.

In the bifunctional spacer building block, a hydroxyl group protected with an acid labile protecting group is attached to a phosphoramidite group.

In one embodiment, the bifunctional spacer building block is a non-nucleoside compound. For example, such spacers are C2-C18 alkyl, alkenyl, alkynyl carbon chains, which may be interrupted with additional ethyleneoxy and/or amide modules or quaternized cationic amine modules to increase the hydrophilicity of the linker. Cyclic moieties such as C5-C6-cyclic hydrocarbyl, C4N, C5N, C4O, C5O-heterocyclic hydrocarbyl, phenyl optionally substituted with one or two C1-C6 hydrocarbyl groups may also be used as non-nucleoside bifunctional spacer moieties. Preferred difunctional building blocks include C3-C6 hydrocarbon-based blocks and tri-to hexa-ethylene glycol chains. Table I shows some examples of nucleotide bifunctional spacer building blocks with different hydrophilicity, different rigidity and different charge. One oxygen atom is attached to an acid labile protecting group (preferably dimethoxytrityl) and the other is part of a phosphoramidite.

Table I: examples of non-nucleotide bifunctional spacer building blocks

One simple way to build the spacer S or to introduce spacer modules into the spacer S is to use standard D or L nucleoside phosphoramidite building blocks. In one embodiment, a single stranded segment of dT is used. This is advantageous because dT does not carry a base protecting group.

Hybridization can be used to vary the spacer length (the distance between binding pair members a and b) and the flexibility of the spacer, since double stranded lengths are shortened compared to single strands and double strands are more rigid than single strands.

In one embodiment, oligonucleotides modified with a functional moiety X are used for hybridization. The oligonucleotide used for hybridization may have one or two terminal extensions that do not hybridize to the spacer and/or be internally branched. Such terminal extensions that do not hybridize to the spacer (and do not interfere with the binding pairs a: a 'and b: b') can be used for additional hybridization events. In one embodiment, the oligonucleotide to which the terminal extension hybridizes is a labeled oligonucleotide. The labeled oligonucleotide may again comprise a terminal extension or be branched to allow further hybridization, whereby polynucleotide aggregates or dendrimers may be obtained. Preferably, a polyigonucleotide dendrimer is used to generate multiple labels or to obtain higher local concentrations of X.

In one embodiment, the spacer S has a backbone length of 1 to 100 nm. In other words herein, the groups a and b of formula I are separated by between 1 and 100 nm. In one embodiment, a and b are each a binding pair member, respectively, and the spacer S has a backbone length of 1 to 95 nm.

"a ': a" and "b: b'" each independently represent a binding pair or respectively represent covalently bound a ': a and/or b: b'.

"a" and "b: b" are different. The term different indicates that the binding of a to a '(binding pair binding or covalent coupling) does not interfere with the binding pair binding or covalent coupling of another pair b to b', and vice versa.

In one embodiment, a 'or b' is covalently bound and the other, b 'or a', represents a binding pair, respectively.

In one embodiment, both a 'a and b' are covalently bound.

The coupling chemistry between a 'and b: b' is different from each other and is selected from standard protocols. Depending on the nature of the binding partner and spacer, the appropriate conjugation chemistry is chosen.

The chemistry used in coupling (a ') to (a), i.e. coupling a- (a') to a linker comprising (a), does not interfere with the chemistry used in coupling (B) to (B '), i.e. coupling (B') -B to a linker comprising (B). As the skilled artisan will appreciate, preferably the reactive sites (a), (a '), (B) and (B') that result in covalent bonds a ': a and B: B', respectively, also do not interfere with any functional groups (a and/or B of formula I) that may be present on the monovalent binder, respectively.

In case at least one monovalent binder is a protein, peptide or peptidomimetic, it may carry one or more OH, COOH, NH2 and/or SH groups, which may potentially react with certain coupling reagents. Such (side) reactions can be avoided by selecting one of the coupling chemistries, e.g. as given in table II.

Table II provides an overview of the reactive groups conventionally used to bind a- (a ') and (B') -B to (a) and (B), respectively, both of which are covalently bound to linker (a-S-B).

Table II:

if at least one monovalent binder is a polypeptide, the aforementioned bioorthogonal coupling chemistry is suitable, for example. If the two binding partners do not carry certain reactive functional groups, respectively, for example in the case of a combination of two aptamers as monovalent binders a and B, there is more freedom in the choice of reactive sites (a '), (a), (B) and (B'), respectively. Thus, in addition to or in combination with the corresponding reactive site pairs given in the above table, amino/active esters (e.g. NHS esters), and SH/SH or SH/maleimide groups can be used for orthogonal coupling.

As is apparent from the above examples, at least one covalent bond between a 'and b' is not an alpha amino peptide bond, respectively. It is also preferred that neither covalent bond is an aminopeptide bond.

In one embodiment, both a 'a and b' are binding pairs. Thus, in one embodiment, the invention relates to a compound of formula I: at least bispecific binding agents of A-a 'a-S-B: B' -B; wherein A is a first monovalent binder that binds to a polypeptide epitope of a target polypeptide and wherein B is a knotA second monovalent binder that binds to a post-translational polypeptide modification on a target polypeptide, wherein each monovalent binder A and B has a range of 5x10^-3Second to 10^-4K/sec_DissociationWherein a 'a and b' are independently a binding pair and are different, wherein S is a spacer, wherein-represents a covalent bond, wherein linker a-S-b has a length of 6 to 100nm, and wherein divalent binding agent has a length of 3x10^-5K/sec or less_Dissociation。

In this embodiment, a and a 'are members of a binding pair a', and b 'are members of a binding pair b, b', respectively. Preferably, each member of the binding pair has a molecular weight of 10kD or less. In other also preferred embodiments, each binding agent of such a binding pair has a molecular weight of 8, 7, 6, 5 or 4kD or less.

In one embodiment, a 'and b' are a binding pair and the members of the binding pair a 'and b' are selected from the group consisting of leucine zipper domain dimers and hybridizing nucleic acid sequences. In one embodiment, both binding pairs represent leucine zipper domain dimers. In one embodiment, both binding pairs are hybridizing nucleic acid sequences.

In case a 'or b' represents a binding pair, the binding affinity of such binding pair (within) is at least 10⁸l/mol. The two binding pairs differ. For binding pairs, a difference is for example accepted if the cross-binding, e.g. a and binding of a ' to b or b ', is 10% or less of the affinity within a ' pair. It is also preferred that the cross-binding, i.e.the binding of a and a 'to b or b', respectively, is 5% or less of the affinity within the a: a 'pair, or if it is 2% or less of the affinity within the a: a' pair. In one embodiment, the difference is so pronounced that the cross-reactive binding is 1% or less compared to the specific binding affinity within the binding pair.

The term "leucine zipper domain" is used to refer to a generally accepted dimerization domain characterized by the presence of one leucine residue at every seventh residue in a segment of about 35 residues. The leucine zipper domain is a peptide that facilitates oligomerization of the protein from which it is found. Leucine zippers were originally identified in several DNA binding proteins (Landshulz, W.H., et al, Science240(1988) 1759-1764) and since then found in a variety of different proteins. Among the known leucine zippers are the naturally occurring peptides and their dimerized or trimerized derivatives. Examples of leucine zipper domains suitable for generating soluble multimeric proteins are described in PCT application WO94/10308, and leucine zippers derived from lung Surfactant Protein D (SPD) are described in Hoppe, H.J. et al, FEBS Lett.344(1994) 191-195.

The leucine zipper domain forms dimers (binding pairs) that are held together by alpha helical coiled coils. The coiled coil has 3.5 residues per turn, which means that every 7 th residue occupies an equivalent position with respect to the helical axis. The regular arrangement of leucine inside the coiled coil stabilizes the structure by hydrophobic and van der waals interactions.

If the leucine zipper domains form a first binding pair (a ': a) and a second binding pair (b: b'), then the two leucine zipper sequences are different, i.e., sequences a and a 'do not bind b and b'. The leucine zipper domain may be isolated from natural proteins known to contain such domains, such as transcription factors. One leucine zipper domain may be derived, for example, from the transcription factor fos, while the second may be derived from the transcription factor jun. The leucine zipper domain can also be artificially designed and synthesized using standard synthesis and design techniques known in the art.

In a preferred embodiment, both members of the binding pair a 'a and b' i.e. a, a ', b and b' all represent the leucine zipper domain and the spacer S consists of amino acids. In this embodiment, it is possible to generate the construct a-S-b easily. It is simple for the person skilled in the art to vary the length of such spacers S as desired. Such polypeptides may be produced synthetically or recombinantly.

For example, a recombinant fusion protein comprising a spacer polypeptide fused at the N-terminus to a leucine zipper peptide and at the C-terminus to a leucine zipper peptide can be expressed in a suitable host cell according to standard techniques. The DNA sequence encoding the desired peptide spacer may be inserted between the sequence encoding a member of the first leucine zipper domain a and the DNA sequence encoding a member of the second leucine zipper domain b in the same reading frame.

In one embodiment, if the linker a-S-b is a polypeptide, the spacer S comprises, once or several times, a GGGGS (SEQ ID NO: 13) amino acid sequence motif. The spacer S may also comprise a tag sequence. The tag sequence may be selected from commonly used protein recognition tags such as YPYDVPDYA (HA tag) (SEQ ID NO: 14) or GLNDIFEAQKIEWHE (Avi tag) (SEQ ID NO: 15).

In a preferred embodiment, both binding pairs (a ': a) and (b: b') are hybridizing nucleic acid sequences.

As the nomenclature already indicates, a and a 'and b', respectively, hybridize to each other. The nucleic acid sequences contained in a and a 'on the one hand and in b and b' on the other hand are different. In other words, the sequences in binding pair a 'do not bind to the sequences of binding pair b', respectively, and vice versa. In one embodiment, the invention relates to at least dual binding agents of formula I wherein both binding pairs a: a 'and b: b' are hybridizing nucleic acid sequences, respectively, and wherein the hybridizing nucleic acid sequences of different binding pairs a 'a and b: b' do not hybridize to each other. In other words, a and a 'hybridize to each other but do not bind to or interfere with either b or b', and vice versa. Hybridization kinetics and hybridization specificity can be easily monitored by melting point analysis. Specific hybridization and non-interference (e.g.non-interference b or b ') of a binding pair (e.g.a: a') is admitted if the melting temperature of the a: a 'pair is at least 20 ℃ higher than any possible combination of b or b' (i.e.a: b; a: b '; a': b and a ': b'), respectively.

The nucleic acid sequence forming a binding pair, e.g. (a: a') or any other nucleic acid sequence based binding pair, may comprise any naturally occurring nucleobase or analogue thereof and may have a modified or unmodified backbone as described above, provided it is capable of forming a stable duplex via a multiple base pairing. By stable is meant that the melting temperature of the duplex is above 37 ℃. Preferably, the double strand consists of two fully complementary single strands. However, mismatches or insertions are possible, as long as stability at 37 ℃ is given.

As the skilled artisan will appreciate, the nucleic acid duplex may be further stabilized by interchain crosslinking. Several suitable crosslinking methods are known to the skilled person, for example using psoralen (psoralen) or thionucleoside-based methods.

Preferably, the nucleic acid sequence representing a member of a binding pair consists of between 12 and 50 nucleotides. It is also preferred that such nucleic acid sequences will consist of between 15 and 35 nucleotides.

Rnases are ubiquitous and special care must be taken to avoid unwanted digestion of RNA-based binding pairs and/or spacer sequences. While it is indeed possible to use, for example, RNA-based binding pairs and/or spacers, DNA-based binding pairs and/or spacers represent a preferred embodiment.

Appropriate hybridizing nucleic acid sequences can be readily designed to provide more than two pairs of orthogonally complementary oligonucleotides, allowing for simple generation and use of more than two binding pairs. Another advantage of using a hybridizing nucleic acid sequence in a dual binding agent of the invention is that modifications can be easily introduced into the nucleic acid sequence. Modified building blocks are commercially available, which for example allow simple synthesis of linkers comprising functional modules. Such functional modules can be easily introduced at any desired position and in any of the structures a and a 'and b' and/or S, as long as they represent oligonucleotides.

In a preferred embodiment, the spacer S comprised in the binding agent according to formula I is a nucleic acid. In a preferred embodiment, both binding pairs are hybridizing nucleic acid sequences and the spacer S is also a nucleic acid. In this embodiment, the linker L consisting of a-S-b is an oligonucleotide.

In the case where the spacer S and the sequences a, a ', b and b' are all oligonucleotide sequences, it is readily possible to provide and synthesize a single oligonucleotide representing a linker L comprising S and the members a and b of the binding pairs a 'and b' respectively. In case the monovalent binders a and B are polypeptides, respectively, they can be easily coupled to the hybridizing nucleic acid sequences a 'and B', respectively. The length of the spacer S comprised in such a construct can easily be varied in any desired way. Based on the three constructs a-S-B, A-a 'and B' -B, the binding agent of formula I can be most easily obtained by hybridization between a 'a and B: B', respectively, according to standard protocols. When spacers of different lengths are used, the resulting construct provides dual binders that are otherwise identical, but have different distances between monovalent binders a and B. This allows for optimal distance and/or flexibility.

In a preferred embodiment, the spacer S and the sequences a, a ', b and b' are DNA.

Enantiomeric L-DNA is known for its orthogonal hybridization behavior, its nuclease resistance and the ease with which oligonucleotides of variable length can be synthesized. This ease of linker length variability via design of suitable spacers is important to optimize binding of a binding agent as disclosed herein to one or more antigens thereof.

In a preferred embodiment, the linker L (= a-S-b) is an enantiomeric L-DNA or L-RNA. In a preferred embodiment, the linker a-S-b is enantiomeric L-DNA. In a preferred embodiment, a ', b and b' and the spacer S are enantiomeric L-DNA or L-RNA. In a preferred embodiment, a ', b and b' and the spacer S are enantiomeric L-DNA.

In one embodiment, the spacer S is an oligonucleotide and is synthesized in two parts comprising ends that are hybridizable to each other. In this case, the spacer S can be constructed simply by hybridizing these hybridizable ends to each other. The resulting spacer construct comprises an oligonucleotide duplex portion. Obviously, in the case of a spacer constructed in the manner described, the sequence of the hybridizable oligonucleotide entities forming the duplex is selected in such a way that no hybridization or interference with the binding pairs a: a 'and b: b' can occur.

As already described above, the monovalent specific binding agents a and B of formula I may be nucleic acids. In one embodiment of the invention, a ', a, b', A, B and S are all oligonucleotide sequences. In this embodiment, the subunits A-a ', a-S-B and B' -B of formula I can be readily and independently synthesized according to standard protocols and combined by hybridization according to convenient standard protocols.

As discussed in detail above, the coupling may be covalent, or it may be via a specific binding pair.

As the skilled artisan will readily appreciate, the bivalent binding agent according to the present invention may be further modified to carry one or more functional modules. Preferably, such functional modules X are selected from the group consisting of: binding groups, labeling groups, effector groups, and reactive groups.

If more than one functional module X is present, each such functional module may independently in each case be a binding group, a labeling group, an effector group or a reactive group.

In one embodiment, preferably, the functional module X is selected from the group consisting of: a binding group, a labeling group, and an effector group.

In one embodiment, the group X is a binding group. As will be apparent to those skilled in the art, the binding group X will be chosen so as to have no interference with a 'and b'.

An example of a binding group is a partner of a bioaffinity (bioaffinity) binding pair that is capable of specifically interacting with another partner of the bioaffinity binding pair. Suitable bioaffinity binding pairs are haptens or antigens and antibodies; biotin or biotin analogues (such as aminobiotin, iminobiotin or desthiobiotin) and avidin or streptavidin; sugars and lectins, oligonucleotides and complementary oligonucleotides, receptors and ligands (e.g., steroid hormone receptors and steroid hormones). In one embodiment, X is a binding group and is covalently bound to at least one of a ', a, b' or S of the compound of formula I. Preferably, a smaller partner of a bioaffinity binding pair, e.g., biotin or an analog thereof, a receptor ligand, a hapten or an oligonucleotide, is covalently bound to at least one of a ', a, L, b or b' as defined above.

In one embodiment, the functional moiety X is a binding group selected from the group consisting of: a hapten; biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin; oligonucleotides and steroid hormones.

In one embodiment, the functional moiety X is a reactive group. The reactive group may be selected from any known reactive group such as amino, sulfhydryl, carboxylate, hydroxyl, azido, alkynyl or alkenyl. In one embodiment, the reactive group is selected from the group consisting of maleimido, succinimidyl, Dithiopyridyl (dithiopyrdyl), nitrophenyl, and hexafluorophenyl.

In one embodiment, the functional moiety X is a labeling group. The labeling group may be selected from any known detectable group. The skilled artisan will select the number of labels suitable for optimal sensitivity and minimal quenching.

The labeling group may be selected from any known detectable group. In one embodiment, the label group is selected from dyes such as luminescent label groups, such as chemiluminescent groups (e.g. acridinium esters or dioxetanes) or fluorescent dyes such as fluorescein, coumarin, rhodamine, oxazidine, resorufin, cyanine (cyanine), and derivatives thereof), luminescent metal complexes (e.g. ruthenium or europium complexes), enzymes such as those used in CEDIA (Cloned Enzyme Donor Immunoassay), e.g. EP 0061888, microparticles or nanoparticles such as latex particles or metal sols, and radioisotopes.

In one embodiment, the labeling group is a luminescent metal complex, and the compound has the structure of formula (II):

[M(L₁L₂L₃)]_n-Y-X_mA (II)

wherein M is a divalent or trivalent metal cation selected from rare earth or transition metal ions, L₁、L₂And L₃Are identical or different and denote a ligand having at least two nitrogen-containing heterocycles, in which L₁、L₂And L₃The metal cation being bound via a nitrogen atom, X being covalently bound to L via a linker Y₁、L₂And L₃A reactive functional group of at least one of the ligands, n is an integer from 1 to 10, preferably from 1 to 4, m is 1 or2, and preferably 1, and a refers to a counter ion which may be required to balance the charge.

Preferably, the metal complex is a luminescent metal complex, i.e. a metal complex that undergoes a detectable luminescent reaction upon suitable excitation. The luminescence reaction may be detected, for example, by fluorescence or by electrochemiluminescence measurements. The metal cation in this complex is, for example, a transition metal or a rare earth metal. Preferably, the metal is ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium, or tungsten. Ruthenium, iridium, rhenium, chromium and osmium are particularly preferred. Ruthenium is most preferred.

Ligand L₁、L₂And L₃Is a ligand having at least two nitrogen-containing heterocycles. Aromatic heterocycles such as bipyridyl, bipyrazinyl (bipyrazyl), tripyridyl and phenanthrenyl (phenanthrolyl) are preferred. Particularly preferably, the ligand L₁、L₂And L₃Selected from bipyridine and phenanthroline (phenanthroline) ring systems.

In addition, the complex may contain one or several counter ions a to balance the charge. Is suitably aExamples of negatively charged counterions are halide, OH^-Carbonate, hydrocarbyl carboxylates such as trifluoroacetate, sulfate, hexafluorophosphate and tetrafluoroborate groups. Hexafluorophosphate, trifluoroacetate and tetrafluoroborate groups are particularly preferred. Examples of suitable positively charged counter ions are monovalent cations such as alkali metal and ammonium ions.

In a further preferred embodiment, the functional moiety X is an effector group. Preferred effector groups are therapeutically active substances.

Therapeutically active substances have different ways in which they can function effectively, for example in the inhibition of cancer. They can damage the DNA template by alkylation, by cross-linking, or by double-stranded cleavage of the DNA. Other therapeutically active substances are capable of blocking RNA synthesis by intercalation. Some agents are spindle poisons, such as vinca alkaloids, or antimetabolites that inhibit enzymatic activity, or hormonal and anti-hormonal agents. The effector group X may be selected from alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxins (epipodophyllotoxins), nitrosoureas, hormonal and anti-hormonal agents, and toxins.

Currently preferred alkylating agents may be exemplified by cyclophosphamide, chlorambucil (chlorambucil), busulfan (busulfan), Melphalan (Melphalan), Thiotepa (Thiotepa), ifosfamide (ifosfamide), mechlorethamine.

The antimetabolites which are presently preferred can be exemplified by methotrexate, 5-fluorouracil, cytarabine (cytarabine), 6-thioguanine, and 6-mercaptopurine (6-mercaptopurine).

Currently preferred antitumor antibiotics may be exemplified by doxorubicin (doxorubicin), daunorubicin (daunorubicin), idarubicin (idarubicin), nimitoxantrone, dactinomycin (dactinomycin), bleomycin (bleomycin), mitomycin, and plicamycin (plicamycin).

Currently preferred spindle poisons may be exemplified by maytansine (maytansine) and maytansinoids (maytansinoids), and vinca alkaloids and epipodophyllotoxins may be exemplified by vincristine (vincristin), vinblastine (vinblastin), vindesine (vindesine), Etoposide (Etoposide), Teniposide (Teniposide).

Presently preferred nitrosoureas are exemplified by carmustine (carmustine), lomustine (lomustine), semustine (semustine), and streptozocin (streptozocin).

Currently preferred hormones and anti-hormones may be exemplified by corticosteroids, estrogens, anti-estrogens, progestins, aromatase inhibitors, androgens, anti-androgens.

Other preferred random synthetic agents may be exemplified by dacarbazine (dacarbazine), hexamethylmelamine (hexamethylmelamine), hydroxyurea, mitotane (mitotane), procarbazine (procarbazine), cisplatin, and carboplatin.

The functional module X is bound to, for example, at least one of (a '), (a), (b') or S, covalently or via another binding pair. The functional module X may be present once or several (n) times. (n) is an integer and is 1 or more than 1. Preferably, (n) is between 1 and 100. It is also preferred that (n) is 1 to 50. In certain embodiments, n is 1 to 10, or1 to 5. In other embodiments, n is 1 or 2.

For covalent binding of the functional moiety X to at least one of a ', a, b' or S, any suitable coupling chemistry may be used. The skilled artisan can readily select such coupling chemistries from standard protocols. It is also possible to incorporate functional modules by using suitable building blocks when synthesizing a ', a, b' or S.

In a preferred embodiment, the functional moiety X binds to a, b, or S of a binding agent defined by formula I.

In a preferred embodiment, the functional moiety X is bound to a spacer S of a binding agent as defined by formula I.

In a preferred embodiment, the functional moiety X is covalently bound to a, b or S of a binding agent as defined by formula I.

If the functional moiety X is located within a hybridizing oligonucleotide representing a, a ', b or b', respectively, then preferably such functional moiety is bound to a modified nucleotide or attached to an internucleoside P atom (WO 2007/059816). Modified nucleotides that do not interfere with oligonucleotide hybridization are incorporated into those oligonucleotides. Preferably, such modified nucleotides are C5 substituted pyrimidines or C7 substituted 7 deazapurines.

Oligonucleotides can be modified internally or at the 5 'or 3' end with non-nucleotide entities for introducing functional modules. Preferably, such non-nucleotide entities are located within the spacer S, i.e. between the two binding pair members a and b.

Many different non-nucleotide modifier building blocks for constructing spacers are known in the literature and many are commercially available. For the introduction of functional modules, non-nucleoside bifunctional modifier building blocks or non-nucleoside trifunctional modifier building blocks were used as CPG for end labeling or as phosphoramidites for internal labeling (see Wojczewski, C. et al, Synlett10(1999) 1667-1678).

Bifunctional modifier building blocks

The bifunctional modifier building block connects the functional module or protected functional module (if necessary) to the phosphoramidite group to attach the building block to the terminal hydroxyl group of the growing oligonucleotide chain at the 5 'end (regular synthesis) or the 3' end (inverted synthesis).

Preferably, the bifunctional modifier building block is a non-nucleoside compound. For example, such modified building blocks are C2-C18 alkyl, alkenyl, alkynyl carbon chains, which may be interrupted by additional ethyleneoxy and/or amide moieties to increase the hydrophilicity of the spacer and thus the overall linker structure. Cyclic modules such as C5-C6-cycloalkyl, C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl (optionally substituted with one or two C1-C6 hydrocarbyl groups) may also be used as non-nucleoside bifunctional modification building blocks. Preferred modified bifunctional building blocks comprise a C3-C6 hydrocarbyl module and a tri-to hexa-ethylene glycol chain. Non-limiting but preferred examples of bifunctional modifier building blocks are given in Table III below.

Table III:

tri-functional modifier building block

The trifunctional building block connects (i) the functional module or, if necessary, the protected functional module, (ii) a phosphoramidite group for coupling the reporter or the functional module or, if necessary, the protected functional module with a hydroxyl group of the growing oligonucleotide chain during oligonucleotide synthesis, and (iii) a hydroxyl group protected with an acid-labile protecting group, preferably with a dimethoxytrityl protecting group. After removal of the acid-labile protecting group, a hydroxyl group is released which can react with another phosphoramidite. Thus, the trifunctional building block allows for placement of the functional module anywhere within the oligonucleotide. Tri-functional building blocks are also a prerequisite for synthesis using solid supports such as Controlled Pore Glass (CPG), which are used for 3' end labeling of oligonucleotides. In this case, the trifunctional building block is linked to the functional module or, if necessary, the protected functional module via a C2-C18 alkyl, alkenyl, alkynyl carbon chain, which may be interrupted with further ethyleneoxy and/or amide modules to increase the hydrophilicity of the spacer and thus of the overall linker structure, and comprises a hydroxyl group attached to the solid phase via a cleavable spacer and a hydroxyl group protected with an acid labile protecting group. After removal of this protecting group, a hydroxyl group is released, which can then be reacted with a phosphoramidite.

The trifunctional building blocks may be non-nucleoside or nucleoside.

Non nucleoside three functional building block is C2-C18 alkyl, alkenyl, alkynyl carbon chain, and the alkyl, alkenyl, alkynyl optionally with other ethylene oxide and/or amide module interruption to increase the spacer and thus the overall structure of the hydrophilic. Other trifunctional building blocks are cyclic groups such as C5-C6-cycloalkyl, C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl, optionally substituted with one or two C1-C6 hydrocarbyl groups. Cyclic and acyclic groups may be substituted with one- (C1-C18) hydrocarbyl-O-PG group, whereas the C1-C18 hydrocarbyl comprises (ethyleneoxy) n, (amide) M moieties, n and M independently of each other =0-6, and PG is an acid labile protecting group. Preferred trifunctional building blocks are C3-C6 hydrocarbyl, cycloalkyl, C5O heterocycloalkyl modules, optionally containing an amide bond, and substituted with a C1-C6 hydrocarbyl O-PG group, wherein PG is an acid labile protecting group, preferably monomethoxytrityl, dimethoxytrityl, pixyl, xanthyl (xanthotyl), most preferably dimethoxytrityl.

For example, non-limiting but preferred examples of non-nucleoside trifunctional building blocks are summarized in Table IV.

Table IV: examples of non-nucleoside trifunctional modifier building blocks

Nucleoside modifier building block:

the nucleoside modifier building blocks are used for internal labeling whenever it is necessary not to affect the hybridization properties of the oligonucleotide compared to the unmodified oligonucleotide. Thus, the nucleoside building block comprises base analogues or bases which are still capable of hybridising to the complementary base. In formula II the general formula of the labeling compound is given, which is used to label the nucleic acid sequence of one or more of a, a ', b' or S comprised in the binding agent of formula I according to the invention.

Formula II:

wherein PG is an acid-labile protecting group, preferably monomethoxytrityl, dimethoxytrityl, pixyl, xanthenyl, most preferably dimethoxytrityl, wherein Y is C2-C18 alkyl, alkenyl, alkynyl, wherein said alkyl, alkenyl, alkynyl may comprise ethyleneoxy and/or amide moieties, wherein Y is preferably C4-C18 alkyl, alkenyl, or alkynyl, and comprises one amide moiety, and wherein X is a functional moiety which may be bound to a label.

Specific base positions can be selected for such substitutions to minimize the impact on hybridization properties. Thus, the following positions are preferred for substitution: a) in the case of natural bases: uracil substituted at C5; cytosine substituted at C5 or N4; adenine substituted at C8 or N6 and guanine substituted at C8 or N2, and b) in the case of base analogues: 7 deaza a and 7 deaza G substituted at C7; 7 deaza 8 aza a and 7 deaza 8 aza G substituted at C7; 7 deaza 2 amino a substituted at C7; pseudouracil substituted at N1 and synomycin substituted at N2.

Non-limiting but preferred examples of nucleoside trifunctional building blocks are given in table V.

Table V:

in tables III, IV and V, one of the terminal oxygen atoms of the bifunctional module or one of the terminal oxygen atoms of the trifunctional module is part of phosphoramidite, which is not shown in full detail but is apparent to the skilled person. The second terminal oxygen atom of the trifunctional building block is protected with an acid-labile protecting group PG, as defined for formula II above.

Post-synthetic modification is another strategy to incorporate covalently bound functional moieties into linker or spacer molecules. In this approach, the amino group is introduced by using a bifunctional or trifunctional building block during solid phase synthesis. After cleavage from the support and purification, the amino-modified oligonucleotide is reacted with an activated ester of a functional moiety or a bifunctional reagent in which one functional group is an active ester. Preferred active esters are NHS esters or pentafluorophenyl esters.

Post-synthesis modifications are particularly useful for introducing functional modules that are unstable during solid phase synthesis and deprotection. Examples are modification with triphenylphosphine carboxymethyl (triphenylphosphine) ester (Wang, C.C. et al, Bioconjugate Chemistry14(2003) 697-701), modification with digoxigenin or introduction of maleimide groups using commercial sulfoSMCC for Staudinger ligation.

In one embodiment, the functional module X is bound to at least one of a ', a, b' or S via another binding pair.

Preferably, the further binding pair to which the functional module X can bind is a leucine zipper domain or a hybrid nucleic acid. In case the functional module X binds to at least one of a ', a, b' or S via a further binding pair member, the binding pair member binding to X and the binding pair a ', a and b, b', respectively, are both selected to have different specificities. Binding pairs a: a ', b: b' and binding pairs that bind to X each bind their respective partner (e.g., hybridize thereto) without interfering with the binding of any other binding pair.

Covalent coupling of binding pair members to monovalent binders

There are different conjugation strategies depending on the biochemical nature of the binding agent.

In the case of binders which are naturally occurring proteins or recombinant polypeptides of 50 to 500 amino acids, standard procedures describing the chemistry of protein conjugate synthesis are available in textbooks, which the skilled artisan can readily follow (Hackenberger, c.p. and Schwarzer, d., angelw.chem., int.ed.,47(2008) 10030-.

In one embodiment, the reaction of maleimide moieties with cysteine residues within proteins is used. This is a preferred coupling chemistry in case for example Fab or Fab' fragments of antibodies are used as monovalent binders. Alternatively, in one embodiment, coupling of a member of the binding pair (a 'or b' of formula I, respectively) to the C-terminal end of the binding agent polypeptide is effected. For example, C-terminal modification of proteins (e.g., Fab fragments) can be performed as described by Sunbul, M. et al, Organic & Biomolecular Chemistry7(2009) 3361-3371.

In general, site-specific reactions and covalent couplings of binding pair members with monovalent polypeptide binding agents are based on the conversion of natural amino acids into amino acids with orthogonal reactivity with other functional groups present in proteins. For example, specific cysteines within an unusual sequence context can be enzymatically converted to aldehydes (see Formyl ethylene aldehyde-protein engineering through a novel post-translational modification (Freese, M. -A. et al, ChemBiochem10(2009) 425-427)). It is also possible to obtain the desired amino acid modifications by utilizing the specific enzymatic reactivity of certain enzymes with the natural amino acids in the context of a given sequence (see, e.g., Taki, M. et al, Protein Engineering, Design & Selection17(2004) 119-.

Site-specific reaction and covalent coupling of the binding pair member to the monovalent polypeptide binding agent may also be achieved by selective reaction of the terminal amino acids with suitable modifying agents.

Site-specific covalent coupling can be achieved using the reactivity of the N-terminal cysteine with benzonitrile (benzonitril) (Ren, Hongjun Xiao et al, Angewandte Chemie, International Edition48(2009) 9658-.

Native chemical ligation may also rely on the C-terminal cysteine residue (Taylor, E.et al, Nucleic acids and Molecular Biology22(2009) 65-96).

EP 1074563 describes a conjugation method based on a faster reaction of cysteines within a negatively charged amino acid segment with cysteines located in a positively charged amino acid segment.

The monovalent binder may also be a synthetic peptide or peptidomimetic. In the case of chemically synthesized polypeptides, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (de Graaf, A.J., et al, bioconjugate chemistry20(2009) 1281-1295). Conjugation of such peptides to linkers is standard chemistry, as a wide variety of orthogonal functional groups are discussed and can be incorporated into synthetic peptides.

To obtain a singly labeled protein, the conjugate with 1:1 stoichiometry can be separated from the other conjugation products by chromatography. This procedure is facilitated by the use of dye-labeled binding pair members and charged spacers. By using such labeled and highly negatively charged binding pair members, singly conjugated proteins are easily separated from unlabeled proteins and proteins carrying more than one linker, since differences in charge and molecular weight can be exploited for separation. Fluorescent dyes are valuable for purifying divalent binders from unbound components (e.g., labeled monovalent binders).

Thus, in one embodiment, it is preferred to use binding pair members (a 'and/or b' of formula I, respectively) labeled with a fluorescent dye (e.g., synthesized using a bifunctional or trifunctional modifier building block in combination with a bifunctional spacer building block during synthesis) to form the bivalent binding agent of the invention. In a preferred embodiment, the spacer S and the sequences a, a ', b and b' are DNA and at least one of a 'or b' is labeled with a fluorescent dye, respectively. In a preferred embodiment, the spacer S and the sequences a, a ', b and b' are DNA and each is labeled with a different fluorescent dye for both a 'and b', respectively.

In one embodiment, a method of generating a bivalent binding agent that specifically binds to a post-translationally modified target polypeptide is disclosed. The method comprises the following steps: a) selecting a first monovalent binder at 5x10^-3Second to 10^-4K/sec_DissociationBinding a polypeptide epitope of the target polypeptide, b) selecting a second monovalent binder at 5x10^-3Second to 10^-4K/sec_DissociationBinding post-translational polypeptide modifications, c) coupling two monovalent binders by a linker, and d) selecting a peptide having 3x10^-5K/sec or less_DissociationBivalent binding agent of value.

As the skilled artisan will appreciate, K_DissociationIs a temperature dependent value. Logically, the K of two monovalent binders and of a bivalent binder according to the invention is determined at the same temperature_DissociationThe value is obtained. As will be appreciated, preferably K is determined for the same temperature at which a divalent binding agent will be used (e.g., an assay will be performed)_DissociationThe value is obtained. In one embodiment, the K is established at room temperature, i.e.at 20 ℃, 21 ℃, 22 ℃, 23 ℃,24 ℃ or 25 ℃ respectively_DissociationThe value is obtained. In one embodiment, K is established at 4 or 8 ℃ respectively_DissociationThe value is obtained. In one embodiment, K is established at 25 ℃_DissociationThe value is obtained. In one embodiment, K is established at 37 ℃_DissociationThe value is obtained. In one embodiment, K is established at 40 ℃_DissociationThe value is obtained. In a preferred embodiment, K is carried out at 37 ℃_DissociationDetermination of those of K per monovalent binder_DissociationDetermination of K and Dual Binder_DissociationAnd (4) measuring.

Using the method as disclosed in the present invention, it is now quite easy to generate a variety of bivalent binding agents each comprising a linker of different length, and to select for having the desired binding properties (i.e., K)_DissociationValue of 3x10^-5Per second or less). As disclosed in example 2.8, by Biacore^TMAnalytical implementation with expected K_DissociationSelection of bivalent binding agent (c).

In one embodiment, the present invention relates to a method of forming a bivalent binding agent according to the present invention, wherein a first monovalent binding agent, a second monovalent binding agent, and a linker are co-incubated, thereby forming a peptide having 10^-5K/sec or less_DissociationA divalent binder of value, said first monovalent binder being at 10^-3Second to 10^-4K/sec_DissociationA polypeptide epitope that binds a target polypeptide and is coupled to a member of a first binding pair, and a second monovalent binder at 10^-3Second to 10^-4K/sec_DissociationBinding to the post-translational polypeptide modification and coupled to a member of a second binding pair, wherein the first and second binding pairs do not interfere with each other, the linker comprising a spacer and a binding pair member complementary to the first and second binding pair members.

In one embodiment, the above method further comprises the step of isolating the bivalent binding agent.

The preferred stoichiometry for assembling the bivalent binding agent according to the invention is 1:1: 1.

In a preferred embodiment, the method of generating a bivalent binding agent according to the invention utilizes an L-DNA linker. In a preferred embodiment, the method of generating a bivalent binding agent according to the invention utilizes two specific binding pairs consisting of DNA, preferably L-DNA, and an L-DNA linker.

The formation and stoichiometry of the formed bivalent binding agent can be analyzed by size exclusion chromatography according to prior art procedures. If desired, the complexes formed can also be analyzed by SDS-PAGE.

If the bivalent binding agent disclosed in the present invention has 3x10^-5K/sec or better_DissociationIt is only significantly bound and not washed away during the various incubation steps of such a procedure when used in an immunohistochemical staining procedure. This K_DissociationThis can only be achieved if both monovalent binders bind to their respective binding sites. No significant staining was found in the presence of only polypeptide epitopes or only post-translational modifications on the molecules in the sample. Thus, and this has the great advantage that immunohistochemical staining will only be observed in the presence of the post-translationally modified target polypeptide (which carries the relevant modification) in the sample.

Thus, in a preferred embodiment, the present invention relates to a method of histological staining, the method comprising the steps of: a) providing a cell or tissue sample, b) incubating the sample with a bivalent binding agent consisting of two monovalent binding agents linked to each other via a linker, wherein one of the two monovalent binding agents binds to a polypeptide epitope of the target polypeptide, one of the two monovalent binding agents binds to a post-translational polypeptide modification, each monovalent binding agent having a range of 5x10^-3Second to 10^-4K/sec_DissociationAnd wherein the bivalent binding agent has 3x10^-5K/sec or less_DissociationAnd c) detecting the divalent binding agent, whereby the sample is stained for the post-translationally modified target polypeptide.

The use of a bivalent binding agent according to the invention for staining a cell or tissue sample by immunohistochemical methods represents a further preferred embodiment.

More generally, the invention relates to a bivalent binding agent consisting of two monovalent binding agents connected to each other via a linkerBinding agents to meet the requirements of (automated) assay systems or better K_DissociationBinding a post-translationally modified target polypeptide, wherein (a) the first monovalent binder requires at least 10-fold higher K than the (automated) assay system_DissociationA polypeptide epitope that binds the target polypeptide, (b) a second monovalent binder with a K at least 10-fold higher than required by the (automated) assay system_DissociationK binding to a post-translational polypeptide modification, and (c) two monovalent binders (a) and (b) thereof_DissociationThe product of the values being at least K required by the (automated) measurement system_DissociationOr smaller.

In general, methods are described for obtaining a bivalent binding agent with a K that at least meets minimum assay requirements or better for (automated) assay systems_DissociationSpecifically binding to a post-translationally modified target polypeptide, the method comprising the steps of: (a) selection of a K which is at least 10 times higher than the minimum assay requirement of the (automated) assay system_DissociationA first monovalent binder that binds to an untranslated modified epitope of said target polypeptide, (b) selecting a K that is at least 10-fold higher than the minimum assay requirement of an (automated) assay system_DissociationA second monovalent binder that binds to a post-translationally polypeptide-modified epitope, wherein K of the two monovalent binders in steps (a) and (b)_DissociationThe product of the values is at least K required by the (automatic) system_DissociationOr smaller, and (c) coupling the two monovalent binders via a linker.

In one embodiment, the automated system is sold by Ventana Medical Systems incAn analyzer.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications to the procedures set forth can be made without departing from the spirit of the invention.

Sequence listing description

1. Antibody fragments

SEQ ID NO:1V_H(monoclonal antibody 1.4.168):

QCDVKLVESG GGLVKPGGSL KLSCAASGFT FSDYPMSWVR QTPEKRLEWV

ATITTGGTYT YYPDSIKGRF TISRDNAKNT LYLQMGSLQS EDAAMYYCTR

VKTDLWWGLA YWGQGTLVTV SA

SEQ ID NO:2V_L(monoclonal antibody 1.4.168):

QLVLTQSSSA SFSLGASAKL TCTLSSQHST YTIEWYQQQP LKPPKYVMEL

KKDGSHTTGD GIPDRFSGSS SGADRYLSIS NIQPEDESIY ICGVGDTIKE

QFVYVFGGGT KVTVLG

SEQ ID NO:3V_H(monoclonal antibody 8.1.2):

EVQLQQSGPA LVKPGASVKM SCKASGFTFT SYVIHWVKQK PGQGLEWIGY

LNPYNDNTKY NEKFKGKATL TSDRSSSTVY MEFSSLTSED SAVYFCARRG

IYAYDHYFDY WGQGTSLTVS S

SEQ ID NO:4V_L(monoclonal antibody 8.1.2):

QIVLTQSPAI MSASPGEKVT LTCSASSSVN YMYWYQQKPG SSPRLLIYDT

SNLASGVPVR FSGSGSVTSY SLTISRMEAE DAATYYCQQW STYPLTFGAG

TKLELK

SEQ ID NO:19V_H(monoclonal antibody 30.4.33):

EVQLQESGPE VAKPGASVKM SCKASGYTFT DYIIHWVKQR PGQDLEWIGY

INPYNDKSKY NEKFKDKATL TSDRSSSTSY MDLSTLTSDD SAVYYCTRHG

YYRSDGFDYW GQGTTLTVSS

SEQ ID NO:20V_L(monoclonal antibody 30.4.33):

DIVLTQSPTI MSASPGEKVT MTCRASSSVS SSSLHWYQQK PGSSPKLWIY

STSTLASGVP ARFSGSGSGT SYSLTISGVE TEDAATYYCQ QYGTSPYTFG

SGTKVDIK

SEQ ID NO:21V_H(monoclonal antibody 7.2.32):

EFEVQLQESG GGLVQPKGSL QLSCAASGFT FNTYAMHWVR QAPGKGLEWV

ARIRTESSDY ATDYADSVKD RFIISRDDSQ NMLYLQMNNL KSEDTAIYYC

VRSSGFDYWG QGTTLTVSSS

SEQ ID NO:22V_L(monoclonal antibody 7.2.32):

DIQMTQSPSL PVSLGDQASI SCRSSQSLVH DNGNTYLHWF LQKPGQSPKL

LIYKVSNRFS GVPDRFGGSG SGTDFTLKIS GVEAEDLGVY FCSQGTHVPT

FGGGTKLEIK

SEQ ID NO:23V_H(monoclonal antibody 4.1.15):

EFEVQLQESG PELVKPGTSV TISCKTSGYA FSNSWMSWVK QRPGQGLEWI

GRIFPGNGDT DYNGNFRAKA TLTADKSSST AFMQLSRLTS VDSAVYFCAR

SRGLRQGAGF AYWGQGTLVT VSA

SEQ ID NO:24V_L(monoclonal antibody 4.1.15):

DIVMTQSPSS LAMSVGQKAT MSCKSSQSLL NSSTQRNYLA WYQQKPGQSP

KLLVYFASTR ESGVPDRFIG SGSGTDFTLT ISSVQAEDLA AYFCQQHYSN

PRTFGGGTKL EIK

sequence of ssDNA

a) 19-mer ssDNA (covalently bound at the 3 ' end to Fab ' of anti-troponin T mab b or Fab ' 8.1.2 for phosphorylated IGF-1R, respectively)

5’-A GTC TAT TAA TGC TTC TGC-3’（SEQ ID NO:5）

b) 17-mer ssDNA (covalently bound at the 5 ' end to Fab ' of anti-troponin T mab a or Fab ' 1.4.168 for IGF-1R, respectively)

5’-AGT TCT ATC GTC GTC CA-3’（SEQ ID NO:6）

c) Complementary 19-mer ssDNA (used as part of a linker)

5’-G CAG AAG CAT TAA TAG ACT-3’（SEQ ID NO:7）

d) Complementary 17-mer ssDNA (used as part of a linker)

5’-TGG ACG ACG ATA GAA CT-3’（SEQ ID NO:8）

3. Sequences of troponin T epitopes

9= ERAEQQREKEUUSKDRIEKRRRAERAE amide, wherein U represents beta-alanine. (epitope "A" represents an antibody anti-troponin antibody a)

10= slkdrierrraaeoeoeraeqqriraereke amide, wherein O represents amino-trioxa-octanoic acid. (epitope "B" represents an antibody anti-troponin antibody B)

Sequences of IGF-1R/IR epitopes

SEQ ID NO:11=FDERQPYAHMNGGRKNERALPLPQSST;IGF-1R(1340-1366)

SEQ ID NO:12=YEEHIPYTHMNGGKKNGRILTLPRSNPS;hIR(1355-1382)

5. Protein linker and tag sequences

13= GGGGS (= G4S) motif (e.g. as part of a polypeptide linker)

SEQ ID NO 14= YPYDVPDYA (HA tag)

SEQ ID NO 15= GLNDIFEAQKIEWHE (Avi tag)

SEQ ID NO 16= LPETGGGSGS (sortase cleavage tag)

Sequence of the HER3 epitope

SEQ ID NO:17=PLHPVPIMPTAGTTPDEDYEYMNRQR;hHER3(1242-1267)

SEQ ID NO:18=PASEQGYEEMRAF;hHER3(1283-1295)

7. ssDNA sequences for sortase-mediated Fab labeling

SEQ ID NO:25=5’-(Gly)₂Amino linker- (spacer C3)3-AGT TCT ATC GTC GTC

CA-fluorescein-3' (17-mer oligomer)

26= 5' -fluorescein-AGT CTA TTA ATG CTT CTG C- (spacer C3) 3-amino acid SEQ ID NO

Base joint- "Gly- (Gly)₂-3' (19-mer oligomer)

Brief Description of Drawings

FIG. 1 analytical gel filtration experiment to evaluate the efficiency of assembly of anti-pIGF 1-R dual binders. Panels a, b and c show the elution profiles of the various dual binder components (fluorescein-ssFab '1.4.168, Cy 5-ssFab' 8.1.2 and linker DNA (T = 0); ssFab 'represents Fab' fragments conjugated to single-stranded oligonucleotides). Figure d shows the elution profile after 3 components required to form a divalent binder have been mixed in a 1:1:1 molar ratio. The thicker (bottom) curve represents the absorbance measured at 280nm, indicating the presence of ssFab' protein or linker DNA, respectively. b) And the thinner top curve in d) (absorbance at 495 nm) indicates the presence of fluorescein, while the thinner top curve in a) and the middle curve in d) (absorbance at 635 nm) indicateThe presence of Cy5 is shown. Elution Volume (VE) of Single Dual Binder component_{ssFab’1.4.168}About 15 ml; VE_{ssFab’8.1.2}About 15 ml; VE_JointAbout 16 ml) and elution Volume (VE) of the reaction mixture_{Mixture of}About 12 ml) showed that the dual binder assembly reaction was successful (yield: about 90%). The 280nm main peak representing the eluted dual binder overlaps well with the main peaks in the 495nm and 635nm channels, demonstrating the presence of both ssFab '8.1.2 and ssFab' 1.4.168 in the peak representing the bivalent binder.

FIG. 2 Biacore^TMSchematic representation of the experiment. Two binding molecules in solution are shown schematically and exemplarily: T0-T-Dig (linker 16) bivalent binding agent and T40-T-Dig (linker 15) bivalent binding agent. The two bivalent binding agents differ only in their linker length (central digoxigenylated T between the two hybridizing nucleic acid sequences, no additional T to 40 additional T (20 on each side of the central T-Dig)). In addition, ssFab' fragments 8.1.2 and 1.4.168 were used.

FIG. 3 Biacore with overlay of 3 kinetics^TMSensorgram showing the interaction of 100nM bivalent binding agent consisting of ssFab '8.1.2 and ssFab' 1.4.168 hybridized on T40-T-Dig ssDNA linker (i.e., linker 15) with immobilized peptide pIGF-1R compared to the binding characteristics of 100nM ssFab '1.4.168 or 100nM ssFab' 8.1.2 for the same peptide. The highest binding performance was obtained with the dual binder construct, which clearly shows that the synergistic binding effect of the dual binders increases the affinity for the target peptide pIGF-1R.

FIG. 4 Biacore with overlay of 3 kinetics^TMSensorgram showing the interaction of a bivalent binding agent consisting of ssFab '8.1.2 and ssFab' 1.4.168 hybridized on a T40-T-Dig ssDNA linker (i.e., linker 15) with immobilized peptides pIGF-1R (phosphorylated IGF-1R), IGF-1R, or pIR (phosphorylated insulin receptor). The highest binding performance was obtained with the pIGF-1R peptide, which clearly shows a synergistic binding of the dual binders compared to, for example, the phosphorylated insulin receptor peptide (pIR)The resultant effect enhances specificity for the target peptide pIGF-1R.

FIG. 5 Biacore with overlay of 2 kinetics^TMA sensorgram showing the interaction of 100nM of a bivalent binding agent consisting of ssFab '8.1.2 and ssFab' 1.4.168 hybridized on a T40-T-Dig ssDNA linker (i.e., linker 15), and a mixture of 100nM ssFab '8.1.2 and 100nM ssFab' 1.4.168 without linker DNA. Optimal binding performance was obtained with only the bivalent binding agent, while ssFab 'mixtures without linker did not show observable synergistic binding effects, despite the fact that the total concentration of these ssFab' was already 200 nM.

FIG. 6 Biacore^TMSchematic representation of a sandwich assay. This assay has been used to investigate epitope accessibility of both antibodies on phosphorylated IGF-1R peptides.<MIgGFcy>R presents rabbit anti-mouse antibody for capturing the murine antibody M-1.4.168. M-1.4.168 was then used to capture the pIGF-1R peptide. Finally, M-8.1.2 formed a sandwich consisting of M-1.4.168, the peptide and M-8.1.2.

FIG. 7 Biacore^TMSensorgram showing that the secondary antibody 8.1.2 is coupled to pIGF-1R peptide in this is Biacore^TMBinding signal after capture of antibody 1.4.168 on the chip (bold line). The other signals (thin lines) are control signals: respectively giving 500nM8.1.2 and 500nM1.4.168 from top to bottom; 500nM target-independent antibody<CKMM>M-33-IgG; and 500nM target independent control antibody<TSH>Line of M-1.20-IgG. No binding events could be detected in any of these controls.

FIG. 8 Biacore^TMSchematic of the assay presenting a biotinylated dual binding agent on the sensor surface. amino-PEO-biotin was captured on flow cell 1 (= FC 1) (not shown). Bivalent binders with increasing linker length were immobilized on FC2, FC3, and FC4 (dual binders on FC2 (T0-Bi = only 1 middle T-Bi) and FC4 (T40-Bi =1 middle T-Bi and 20T upstream and downstream, respectively) are shown, respectively). Analyte 1: IGF-1R peptide containing the M-1.4.168 ssFab' epitope at the right hand end of the peptide (top line), absentAt the M-8.1.2 ssFab' phosphoepitope, since this peptide is not phosphorylated; analyte 2: pIGF-1R peptide containing the M-8.1.2ssFab 'phospho epitope (P) and the M-1.4.168 ssFab' epitope (second line); analyte 3: pIR peptide containing the cross-reactive M-8.1.2 ssFab' phosphoepitope but not the M-1.4.168 epitope (third line).

Figure 9 kinetic data for the dual binder experiment. T40-T-Bi linker dual binding agents with ssFab '8.1.2 and ssFab' 1.4.168 (= T40 in the figure) showed 1300-fold lower off-rates to pIGF-1R (kd = 2.79E-05/s) when compared to pIR (kd = 3.70E-02/s).

FIG. 10 Biacore^TMSensorgram showing concentration-dependent measurement of pIGF-1R peptide (phosphorylated IGF-1R peptide) by T40-T-Bi dual binders. Assay setup was as depicted in figure 8. Concentration series of pIGF-1R peptide were injected at 30nM, 10nM, 2 times 3.3nM, 1.1nM, 0.4nM, 0 nM. The corresponding data are given in the table of fig. 9.

FIG. 11 Biacore^TMSensorgram showing concentration-dependent measurement of the T40-T-Bi dual binder on IGF-1R peptide (non-phosphorylated IGF-1R peptide). Assay setup was as depicted in figure 8. IGF-1R peptide was injected at 300nM, 100nM, 2 times 33nM, 11nM, 4nM, 0nM in concentration series. The corresponding data are given in the table of fig. 9.

FIG. 12 Biacore^TMSensorgram showing the concentration-dependent measurement of the pIR peptide (phosphorylated insulin receptor peptide) by T40-T-Bi dual binders. Assay setup was as depicted in figure 8. pIR peptide concentration series were injected at 100nM, 2 times 33nM, 11nM, 4nM, 0 nM. The corresponding data are given in the table of fig. 9.

Fig. 13(a) Western blot experiment with 3T3 cell lysate used to generate formalin fixed, paraffin embedded (FFPE)3T3 cell pellet. Each lysate was treated with 5. mu.g total protein by SDS-PAGE and Western blotting. Detection occurred with an anti-phosphotyrosine antibody (Millipore, clone 4G 10). Asterisks (#) or asterisk pairs (#) indicate the band positions of phosphorylated IGF-1R or phosphorylated IR proteins. (B) Results of IHC experiments with FFPE3T3 cell pellets. The detector molecule consisting of the 8xC18 linker molecule (linker 14 of example 2.4) and ssFab '1.4.168 only or ssFab' 30.4.33 only did not produce staining on any of the FFPE3T3 cell aggregates tested (rows 1 and 2). In contrast, detection with the intact dual binder molecule (consisting of two ssFab' fragments +8xC18 linkers) produced staining, but only on IGF-1R overexpressing cells stimulated with IGF-1 (row 3). Even when phosphorylation of IR has been induced, no cross-reactivity was observed on IR overexpressing cells. (C) IHC experiments comparing the performance of anti-pIGF-1R dual binders with different linker lengths (linkers containing 2xC18,4xC18,6xC18 or 8xC18 spacers, see example 2.4) on IGF-1R overexpressing FFPE3T3 cells that have been stimulated with IGF-1 to induce IGF-1R phosphorylation.

FIG. 14H 322M immunostaining of xenograft sections. 10 μ g/ml of each ssFab ' fragment (ssFab ' 30.4.33 or/and ssFab ' 1.4.168, respectively) and an equimolar amount of 8xC18 linker molecule were used for detection. The biotin label within the linker molecule serves as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit.

Figure 15 schematic of Biacore assay presenting biotinylated dual binding agents on the sensor surface. Biotinylated 8xC18 linker molecules were immobilized, which were used to capture ssFab '1.4.168 and/or ssFab' 30.4.33, respectively. The analyte was a pIGF-1R peptide containing an M-1.4.168ssFab epitope at one end of the peptide and an M-30.4.33ssFab phospho epitope at the other end.

Figure 16 summarizes the table of kinetic data for the dual binder experiment. Dual binders containing both ssFab '30.4.33 and ssFab' 1.4.168 showed 230-fold lower dissociation rates (kd = 3.22E-03/s) than ssFab '1.4.168 and 110-fold lower dissociation rates (kd = 1.39E-05/s) than ssFab' 30.4.33 alone (kd = 1.57E-03/s).

FIG. 17 Biacore sensorgram showing the concentration-dependent measurement of phosphorylated IGF-1R peptide by a monovalent binder consisting of an 8xC18 linker molecule and ssFab' 30.4.33. Assay setup was as depicted in figure 15. The concentration series of the synthesized phosphorylated pIGF-1R peptide of SEQ ID NO. 11 was injected at 30nM, 10nM, 2 times 3.3nM, 1.1nM, 0.4nM, 0 nM. The corresponding kinetic data are given in fig. 16.

FIG. 18 Biacore sensorgram showing the concentration-dependent measurement of phosphorylated IGF-1R peptide by a monovalent binder consisting of an 8xC18 linker molecule and ssFab' 1.4.168. Assay setup was as depicted in figure 15. Concentration series of pIGF-1R peptide were injected at 30nM, 10nM, 2 times 3.3nM, 1.1nM, 0.4nM, 0 nM. The corresponding kinetic data are given in fig. 16.

FIG. 19 Biacore sensorgram showing the concentration-dependent measurement of phosphorylated IGF-1R peptide by a bivalent binding agent consisting of an 8xC18 linker molecule, ssFab '30.4.33 and ssFab' 1.4.168. Assay setup was as depicted in figure 15. The concentration series of the synthesized phosphorylated pIGF-1R peptide of SEQ ID NO. 11 was injected at 30nM, 10nM, 2 times 3.3nM, 1.1nM, 0.4nM, 0 nM. The corresponding kinetic data are given in fig. 16.

Fig. 20(a) Western blot experiment with Hek293 cell lysate used to generate formalin fixed, paraffin embedded (FFPE)293 cell pellet. Each lysate was treated with 5. mu.g total protein by SDS-PAGE and Western blotting. Detection occurred with an anti-phosphotyrosine antibody (Millipore, clone 4G 10). (B) Results of IHC experiments with Hek293 cell pellets. The detector molecule consisting of the 4xC18 linker molecule (linker 12 of example 2.4) and ssFab '4.1.15 only or ssFab' 7.2.32 only did not produce staining on any of the FFPE Hek293 cell pellets tested (rows 1 and 2). In contrast, detection with the intact dual binder molecule (consisting of two ssFab' fragments +4xC18 linkers) produced staining but only on wild-type HER3 overexpressing cells stimulated with NRG1- β 1 (row 3; column 2). No staining was observed on unstimulated cells (row 3; column 1) and cells over-expressing mutant HER3(Y > F) (lacking the Tyr1289 phosphorylation site) stimulated with NRG1- β 1, but not wild-type HER3 (row 3; column 3), respectively.

Examples

Example 1

Bivalent binding agents against troponin T

1.1 monoclonal antibodies and Fab' fragments

Two monoclonal antibodies that bind human cardiac troponin T at different, non-overlapping epitopes (epitope a 'and epitope B', respectively) were used. At present Roche Elecsys^TMBoth antibodies are used in the troponin T assay, wherein troponin T is detected in the form of a sandwich immunoassay.

Purification of monoclonal antibodies from culture supernatants was performed using prior art methods of protein chemistry.

Carrying out protease digestion on the purified monoclonal antibody by using preactivated papain (anti-epitope A ' monoclonal antibody) or pepsin (anti-epitope B ' monoclonal antibody) to obtain F (ab ')₂The fragment was subsequently reduced to Fab ' fragments, i.e.A and B in formula I (A-a ': a-S-B: B ' -B), respectively, with a low concentration of cysteamine (cysteamin) at 37 ℃. The reaction was stopped by separating the cysteamine from the sample fraction containing the polypeptide on a Sephadex G-25 column (GE Healthcare).

1.2 conjugation of Fab' fragments to ssDNA-oligonucleotides

The Fab' fragments were conjugated to activated ssDNAa and ssDNAb oligonucleotides, respectively, described below.

Fab fragment-ssDNA conjugates a "and B" were prepared separately:

a) fab '-anti-troponin T < epitope A' > -ssDNA conjugate (= A ")

For the preparation of the Fab '-antimyconin T < epitope A' > -ssDNA-conjugate A ", a derivative of SED ID NO:5, i.e. 5 '-AGT CTA TTA ATG CTT CTG C (= SEQ ID NO:5) -XXX-Y-Z-3', was used, wherein X = propylene-phosphate introduced via phosphoramidite C3(3- (4,4 '-dimethoxytrityloxy) propyl-1- [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research)), wherein Y = 3' -amino modifier C6 introduced via 3 '-amino modifier TFA amino C-6 lcCPaa G (mChegenes), and wherein Z = 4' -amino modifier C6 introduced via sulfosuccinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (ThermoFischer) [ N-Maleimidomethyl ] cyclohexane-1-carboxy.

b) Fab ' -anti-troponin T < epitope B ' > -ssDNA conjugate (= B ')

For the preparation of the Fab-anti-troponin T < epitope B ' > -ssDNAb-conjugate (B ' ') the derivative of SEQ ID NO:6 was used, i.e. 5 ' -Y-Z-XXX-AGT TCT ATC GTC GTC CA-3 ', wherein X = propylene-phosphate ester introduced via phosphoramidite C3(3- (4,4 ' -dimethoxytrityloxy) propyl-1- [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (GlenResearch)), wherein Y =5 ' -amino modifier C6 introduced via 6- (4-Monomethoxytritylamino) hexyl- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite (GlenResearch)), and wherein Z = 4[ N-maleimidomethyl ] cyclohexane-1-carboxy introduced via sulfosuccinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (ThermoFischer).

Oligonucleotides of SEQ ID NO 5 or 6, respectively, have been synthesized by prior art oligonucleotide synthesis methods. The introduction of the maleimide group is accomplished via reaction of the amino group of Y with the succinimidyl group of Z incorporated during the solid phase oligonucleotide synthesis process.

The single stranded DNA constructs shown above carry a thiol-reactive maleimide group which reacts with the cysteine of the Fab' hinge region generated by cysteamine treatment. To obtain a high percentage of singly labeled Fab 'fragments, the relative molar ratio of ssDNA to Fab' fragments was kept low. Purification of the singly labeled Fab 'fragments (ssDNA: Fab' =1: 1) takes place by anion exchange chromatography (column: MonoQ, GE Healthcare). Validation of efficient labeling and purification was achieved by analytical gel filtration chromatography and SDS-PAG.

1.3 biotinylated linker molecules

The oligonucleotides used in ssDNA linkers L1, L2, and L3, respectively, have been synthesized by prior art oligonucleotide synthesis methods and using biotinylated phosphoramidite reagents for biotinylation.

Linker 1 (= L1), a biotinylated ssDNA linker 1 without a spacer, having the following composition:

5 '-GCA GAA GCA TTA ATA GAC T (Biotin-dT) -TGG ACG ACG ATA GAA CT-3'. It comprises ssDNA oligonucleotides of SEQ ID NO 7 and 8, respectively, and was biotinylated by using biotin-dT (5 ' -dimethoxytrityloxy-5- [ N- ((4-tert-butylbenzoyl) -biotinyl) -aminohexyl) -3-acryloylimino ] -2 ' -deoxyuridine-3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research).

Linker 2 (= L2), a biotinylated ssDNA linker 2 with an 11-mer spacer, having the following composition:

5 '-GCA GAA GCA TTA ATA GAC T T5- (biotin-dT) -T5TGG ACG ACG ATA GAA CT-3'. It comprises ssDNA oligonucleotides of SEQ ID NO 7 and 8, respectively, an oligonucleotide segment of 5 thymidine each 2 times, and biotinylated by using biotin-dT (= T-Bi) (5 ' -dimethoxytrityloxy-5- [ N- ((4-tert-butylbenzoyl) -biotinyl) -aminohexyl) -3-acryloylimino ] -2 ' -deoxyuridine-3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research) in the middle of the spacer.

Linker 3 (= L3), a biotinylated ssDNA linker 3 with a 31-mer spacer, having the following composition:

5 '-GCA GAA GCA TTA ATA GAC T T15- (biotin-dT) -T15TGG ACG ACG ATA GAACT-3'. It comprises ssDNA oligonucleotides of SEQ ID NO 7 and 8, respectively, an oligonucleotide segment of 15 thymidine each 2 times, and biotinylated by using biotin-dT (5 ' -dimethoxytrityloxy-5- [ N- ((4-tert-butylbenzoyl) -biotinyl) -aminohexyl) -3-acrylamido ] -2 ' -deoxyuridine-3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research) in the middle of the spacer.

1.4 epitopes of monovalent troponin T binding Agents A and B, respectively

Synthetic peptides have been constructed which individually have only moderate affinity for the corresponding Fab' fragments derived from anti-troponin T antibodies a and b, respectively.

a) Epitope a' of antibody a is contained in:

9= ERAEQQREKEUUSKDRIEKRRRAERAE amide, wherein U represents beta-alanine.

b) Epitope B' of antibody B is contained in:

10= slkdrierrraaeoeoeraeqqriraereke amide, wherein O represents amino-trioxa-octanoic acid.

As the skilled artisan will appreciate, it is possible to combine the two epitope-containing peptides in two ways, and both variants have been designed and prepared by linearly combining epitopes a 'and B'. The sequences of the two variants, i.e. the linear sequences of epitopes a '-B' (= TnT-1) and B '-a' (= TnT-2), have been prepared separately by prior art peptide synthesis methods.

The sequences of epitopes a 'and B' have been modified to reduce the binding affinity of each Fab thereto, respectively, compared to the original epitope on the human cardiac troponin T sequence (P45379/UniProtKB). In these cases, the kinetics of the heterobivalent binding effect are better visible, for example by using Biacore^TMThe binding affinity implementation was analyzed technically.

1.5 biomolecular interaction analysis

For this experiment, Biacore was used at T =25 ℃^TM3000 Instrument (GE Healthcare), Biacore^TMThe SA sensor is embedded in the system. The pre-conditioning was done with 100. mu.l/min 31 min injections of 1M NaCl in 50mM NaOH and 1 min 10mM HCl.

HBS-ET (10 mM HEPES pH7.4,150mM NaCl,1mM EDTA, 0.05%)) As a system buffer. The sample buffer was the same as the system buffer.

Biacore driven under control software V1.1.1^TM3000 System. Flow cell 1 was saturated with 7RU D-biotin. On flow cell 2, 1063 RUs of biotinylated ssDNA linker L1 were immobilized. On flow cell 3, 879 RUs of biotinylated ssDNA linker L2 was immobilized. On flow cell 4, 674 RU of biotinylated ssDNA linker L3 was captured.

Then, Fab' fragment DNA conjugate a "was injected at 600 nM. Fab' fragment DNA conjugate B "was injected into the system at 900 nM. The conjugate was injected at a flow rate of 2 μ Ι/min for 3 minutes. The conjugates were injected sequentially to monitor the respective saturation signal of each Fab' fragment DNA conjugate on its respective linker. The Fab 'combination was driven with a single Fab' fragment DNA conjugate a ", a single Fab 'fragment DNA conjugate B", and two Fab' fragment DNA conjugates a "and B" present on the respective linkers. A stable baseline is generated after the linker has been saturated by the Fab' fragment DNA conjugate, a prerequisite for further kinetic measurements.

The artificial peptide analytes TnT-1 and TnT-2 were injected as analytes in solution into the system to interact with the Fab' fragments presented on the surface.

TnT-1 was injected at 500nM and TnT-2 was injected at an analyte concentration of 900 nM. Both peptides were injected at 50. mu.l/min for a binding time of 4 min. Dissociation was monitored for 5 min. Regeneration was accomplished by injecting 50mM NaOH1 min at 50. mu.l/min on all flow cells.

Kinetic data were determined using Biaevaluation software (v.4.1). The dissociation rates kd (1/s) of combinations of TnT-1 and TnT-2 peptides from the corresponding surface-presented Fab' fragments were determined according to a linear Langmuir 1:1 fitting model. According to a first order kinetic equation: the solution of ln (2)/(60xkd) was used to calculate the half-life of the complex in minutes.

As a result:

the experimental data given in tables 1 and 2 show the increased stability of the complex between the analyte (TnT-1 or TnT-2) and various hetero-bivalent Fab ' -Fab ' dimer A "-B", respectively, compared to the monovalent dsDNA Fab ' A "or B" conjugates, respectively. This effect is seen in row 1 compared to rows 2 and 3 in each table.

Table 1: analytical data Using TnT-1 and linkers of various lengths

a) Joint L1

Fab' fragment DNA conjugate A "	Fab' fragment DNA conjugate B "	kd(1/s)	t1/2 dissociation (minute)
				x	x	6.6E-03	1.7
x	-	3.2E-02	0.4
				-	x	1.2E-01	0.1

b) Joint L2

Fab' fragment DNA conjugate A "	Fab' fragment DNA conjugate B "	kd(1/s)	t1/2 dissociation (minute)
				x	x	4.85E-03	2.4
x	-	2.8E-02	0.4
				-	x	1.3E-01	0.1

c) Joint L3

Fab' fragment DNA conjugate A "	Fab' fragment DNA conjugate B "	kd(1/s)	t1/2 dissociation (minute)
				x	x	2.0E-03	5.7
x	-	1.57E-02	0.7
				-	x	1.56E-02	0.7

Table 2: analytical data Using TnT-2 and linkers of various lengths

a) Joint L1

Fab' fragment DNA conjugate A "	Fab' fragment DNA conjugate B "	kd(1/s)	t1/2 dissociation (minute)
				x	x	1.4E-02	0.8
x	-	4.3E-02	0.3
				-	x	1.4E-01	0.1

b) Joint L2

Fab' fragment DNA conjugate A "

Fab' fragment DNA conjugate B "

kd (1/s)

t1/2 dissociation (minute)

x	x	4.9E-03	2.3
				x	-	3.5E-02	0.3
-	x	1.3E-01	0.1

c) Joint L3

Fab' fragment DNA conjugate A "	Fab' fragment DNA conjugate B "	kd(1/s)	t1/2 dissociation (minute)
				x	x	8.0E-03	1.5
x	-	4.9E-02	0.2
				-	x	3.2E-01	0.04

The affinity effect also depends on the length of the linker. In the sub-table shown below table 1, i.e. for the artificial analyte TnT-1, linker L3 comprising a thymidine-based 31-mer spacer showed the lowest off-rate or the highest complex stability.

In the sub-table shown below table 2, linker L2 comprising a thymidine-based 11-mer spacer showed the lowest dissociation rate or the highest complex stability for the artificial analyte TnT-2.

Together, these data demonstrate the great utility and advantage of the inherent linker length flexibility of the method presented in the present invention.

Example 2

Bivalent binding agents against phosphorylated IGF-1R

2.1 monoclonal antibody development (monoclonal antibody 8.1.2, monoclonal antibody 1.4.168 and monoclonal antibody 30.4.33)

a) Immunization of mice

BALB/C mice were immunized at weeks 0, 3, 6, and 9, respectively. Each immunization used 100. mu.g of a conjugate comprising the phosphorylated peptide pIGF-1R (1340-1366) (SEQ ID NO: 11). This peptide has been phosphorylated at tyrosine 1346 (= 1346-pTyr) and coupled via the C-terminal cysteine to KLH (= Aoc-Cys-MP-KLH-1340) to give a conjugate for immunization. Immunization was performed intraperitoneally at week 0 and week 6, and subcutaneously at each site of the mouse at week 3 and week 9, respectively.

b) Fusion and cloning

Spleen cells from immunized mice were fused with myeloma cells according to Galfre G. and Milstein C, Methods in Enzymology 73 (1981) 3-46. In this process, about 1x10 from immunized mice⁸Spleen cells and 2x10⁷Individual myeloma cells a (P3X 63-Ag8653, ATCC CRL 1580) were mixed and centrifuged (at 250g and at 37 ℃ for 10 minutes). Then, the cells were washed once with RPMI1640 medium without Fetal Calf Serum (FCS) and centrifuged again at 250g in a 50ml conical tube. The supernatant was discarded, the cell sediment was gently loosened by tapping, 1ml of peg (molecular weight 4000, Merck, Darmstadt) was added, and mixed by pipetting. After incubation in a 37 ℃ water bath for 1 minute, 5ml of FCS-free RPMI1640 was added dropwise at room temperature over a period of 4-5 minutes. This procedure was repeated with 10ml of RPMI1640 without FCS. Then, 25ml of RPMI1640 containing 10% FCS was added, followed by 5% CO at 37 ℃₂An incubation step lasting 30 minutes. After centrifugation at 250g and at 4 ℃ for 10 minutes, the settled cells were aspirated in RPMI1640 medium containing 10% FCS and inoculated in hypoxanthine-azaserine selection medium (100 mmol/l hypoxanthine, 1. mu.g/ml azaserine in RPMI1640+10% FCS). 100U/ml of interleukin 6 was added to the medium as a growth factor. After 7 days, the medium was replaced with fresh medium. On day 10, primary cultures were tested for specific antibodies. Positive primary cultures were cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter.

c) Isolation of immunoglobulins from cell culture supernatants

The hybridoma cells obtained were cultured at 1 × 10⁷The density of each cell is inCELLine1000CL flasks (Integra) were inoculated. Hybridoma cell supernatants containing IgG were collected twice a week. The yield range is typically between 400 and 2000. mu.g of monoclonal antibody per 1ml of supernatant. Purification of antibodies from culture supernatants is carried out using conventional Methods of protein chemistry (e.g. according to Bruck, C., Methods in enzymology121(1986) 587-596).

2.2 Synthesis of hybridizable oligonucleotides

The following amino-modified precursors comprising the sequences given in SEQ ID NO 5 and 6, respectively, were synthesized according to standard methods. The oligonucleotides given below contain not only the so-called amino linker but also a fluorescent dye. As the skilled artisan will readily appreciate, this fluorescent dye is very convenient to facilitate purification of the oligonucleotides themselves, as well as the components comprising them.

a) 5' -fluorescein-AGT CTA TTA ATG CTT CTG C- (spacer C3)3-C7 amino linker-;

b) 5' -Cy5AGT CTA TTA ATG CTT CTG C- (spacer C3)3-C7 amino linker-;

c)5 '-amino linker- (spacer C3)3-AGT TCT ATC GTC GTC CA-fluorescein-3';

d) 5' -fluorescein- (. beta.L AGT CTA TTA ATG CTT CTG C) - (spacer C3)3-C7 amino linker-; (beta.L indicates that this is an L-DNA oligonucleotide) and

e)5 '-amino linker- (spacer C3)3- (. beta.L-AGT TCT ATC GTC GTC CA) -fluorescein-3' (betaL indicates this is an L-DNA oligonucleotide).

The synthesis was performed on a10 μmol scale in either the trityl on (for 5 'amino modification) or trityl off (for 3' amino modification) mode on an ABI394 synthesizer using commercial CPG as a solid support and standard da (bz), dT, dG (iBu) and dc (bz) phosphoramidites (Sigma Aldrich).

The C3-spacer, dye and amino module were introduced during oligonucleotide synthesis using the following imide (amidite), amino modifier and CPG support, respectively:

spacer phosphoramidite C3(3- (4, 4' -dimethoxytrityloxy) propyl-1- [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research);

introduction of 5 'amino modifier by using 5' -amino modifier C6(6- (4-monomethoxytritylamino) hexyl- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite (Glen Research);

5 ' -fluorescein phosphoramidite 6- (3 ', 6 ' -ditivalofluorosceinyl) -6-carboxamido) -hexyl-1-O- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite (GlenResearch);

Cy5^TMphosphoramidite 1- [3- (4-monomethoxytrityloxy) propyl]-1' - [3- [ (2-cyanoethyl) - (N, N-diisopropylphosphoramidite)]Propyl radical]-3,3,3 ', 3' -tetramethylindodicarbocyanine chloride (Glen Research);

LightCycler fluorescein CPG500A (Roche Applied Science); and

3' -amino modifier TFA amino C-6lcaa CPG500A (Chemsenes),

for Cy5 labeled oligonucleotides, dA (tac), dT, dG (tac), dC (tac) phosphoramidite (SigmaAldrich) was used, and deprotection was performed with 33% ammonia at room temperature for 2 hours.

L-DNA oligonucleotides were synthesized by using β -L-dA (bz), dT, dG (iBu) and dC (Bz) phosphoramidites (Chemsenes).

Purification of fluorescein-modified hybridizable oligonucleotides was performed by a two-step procedure: the oligonucleotides were first purified on reverse phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system [ A: 0.1M (Et3NH) OAc (pH7.0)/MeCN95: 5; B: MeCN ]: 3 min, 20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A at a flow rate of 1.0 ml/min, detection at 260 nm). Fractions containing the desired product (monitored by analytical RP HPLC) were combined and evaporated to dryness. (oligonucleotides modified at the 5' end with a monomethoxytrityl protected hydrocarbylamino group were detritylated by incubation with 20% acetic acid for 20 minutes). The oligomers containing fluorescein as label were repurified by IEX chromatography on HPLC [ Mono Q column: and (3) buffer solution A: sodium hydroxide (10 mM/l; pH about 12); and (3) buffer solution B: 1M sodium chloride in sodium hydroxide (10 mM/l; pH about 12), gradient: flow 1 ml/min from 100% buffer A to 100% buffer B in 30 min, detection at 260nm ]. The product was desalted by dialysis.

The Cy 5-labeled oligomer was used after the first purification on reverse phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system [ A: 0.1M (Et3NH) OAc (pH7.0)/MeCN95: 5; B: MeCN ]: 3 min, 20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A at a flow rate of 1.0 ml/min, detection at 260 nm). The oligos were desalted by dialysis and lyophilized on a Speed-Vac evaporator to yield a solid, which was frozen at-24 ℃.

2.3 activation of hybridizable oligonucleotides

The amino-modified oligonucleotide from example 2 was dissolved in 0.1M sodium borate buffer pH8.5 buffer (c = 600. mu. mol) and reacted with a 18-fold molar excess of sulfoSMCC (sulfosuccinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (c =3 mg/100. mu.l) dissolved in DMF from Thermo Scientific.

The dialysate is concentrated by evaporation and used directly for conjugation with monovalent binders containing thiol groups.

2.4 Synthesis of adaptor oligonucleotides containing hybridizable oligonucleotides at both ends

Oligonucleotides were synthesized by standard methods on an ABI394 synthesizer in a trityl-on mode on a10 μmol scale using commercial dT-CPG as a solid support and standard da (bz), dT, dG (iBu) and dc (bz) phosphoramidites (sigmaaaldrich).

L-DNA oligonucleotides were synthesized by using commercially available β L-dT-CPG as a solid support and β -L-dA (bz), dT, dG (iBu) and dC (Bz) phosphoramidites (Chemmenes).

Oligonucleotide purification was performed on reverse phase HPLC as described under example 2.3. Fractions containing the desired product (analyzed/monitored by analytical RP HPLC) were combined and evaporated to dryness. Detritylation was performed by incubation with 80% acetic acid for 15 minutes. Acetic acid was removed by evaporation. The residue was dissolved in water and lyophilized.

The C18 spacer, digoxigenin and biotin groups were introduced during oligonucleotide synthesis using the following imide (ester) and CPG supports:

spacer phosphoramidite 18 (18-O-dimethoxytrityl hexaethylene glycol), 1- [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research);

biotin-dT (5 ' -dimethoxytrityloxy-5- [ N- ((4-tert-butylbenzoyl) -biotinyl) -aminohexyl) -3-acryloylimino ] -2 ' -deoxyuridine-3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (glenreach);

biotin phosphoramidite 1-dimethoxytrityloxy-2- (N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite and

5 ' -Dimethoxytrityl-5- [ N- (trifluoroacetylaminohexyl) -3-acryloylimino ] -2 ' -deoxyuridine, 3 ' - [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite, for amino modification and post-labeling with digoxigenin-N-hydroxy-succinimidyl ester (postlabeling).

The following bridging constructs or linkers were synthesized:

the joint 1: 5 '-G CAG AAG CAT TAA TAG ACT-TGG ACG ACG ATA GAA CT-3'

And (3) joint 2: 5 '-G CAG AAG CAT TAA TAG ACT- (T40) -TGG ACG ACG ATA GAA CT-3'

And (3) a joint: 5 '- [ B-L ] G CAG AAG CAT TAA TAG ACT- (biotin-dT) -TGG ACG ACG ATAGAA CT-3'

And (4) connecting the joint: 5 '- [ B-L ] G CAG AAG CAT TAA TAG ACT-T5- (biotin-dT) -T5-TGG ACG ACGATA GAA CT-3'

And (5) a joint: 5 '- [ B-L ] G CAG AAG CAT TAA TAG ACT-T20- (biotin-dT) -T20-TGG ACGACG ATA GAA CT-3'

And (6) a joint: 5 '- [ B-L ] G CAG AAG CAT TAA TAG ACT-T30- (biotin-dT) -T30-TGG ACGACG ATA GAA CT-3'

And (3) a joint 7: 5 '-GCA GAA GCA TTA ATA GAC T T5- (biotin-dT) -T5TG GAC GAC GATAGA ACT-3'

And (3) a joint 8: 5 '-GCA GAA GCA TTA ATA GAC T T10- (biotin-dT) -T10TGG ACG ACG ATAGAA CT-3'

A joint 9: 5 '-GCA GAA GCA TTA ATA GAC T T15- (biotin-dT) -T15TGG ACG ACG ATAGAA CT-3'

The joint 10: 5 '-GCA GAA GCA TTA ATA GAC T T20- (Biotin-dT) -T20TGG ACG ACGATA GAA CT-3'

The joint 11: 5 '-G CAG AAG CAT TAA TAG ACT-spacer C18- (biotin-dT) -spacer C18-TGG ACG ACG ATA GAA CT-3'

The joint 12: 5 '-G CAG AAG CAT TAA TAG ACT- (spacer C18)2- (biotin-dT) - (spacer C18)2-TGG ACG ACG ATA GAA CT-3'

A joint 13: 5 '-G CAG AAG CAT TAA TAG ACT- (spacer C18)3- (biotin-dT) - (spacer C18)3-TGG ACG ACG ATA GAA CT-3'

The joint 14: 5 '-G CAG AAG CAT TAA TAG ACT- (spacer C18)4- (biotin-dT) - (spacer C18)4-TGG ACG ACG ATA GAA CT-3'

A joint 15: 5 '-G CAG AAG CAT TAA TAG ACT-T20- (Dig-dT) -T20-TGG ACG ACG ATAGAA CT-3'

A joint 16: 5 '-G CAG AAG CAT TAA TAG ACT- (Dig-dT) -TGG ACG ACG ATA GAA CT-3'

A joint 17: 5 '-G CAG AAG CAT TAA TAG ACT- (biotin-dT) -TGG ACG ACG ATA GAACT-3'

The bridging construct examples described above comprise at least a first hybridizable oligonucleotide and a second hybridizable oligonucleotide. Linkers 3 to 17 comprise a central biotinylated or digoxigenylated thymidine outside the hybridizable nucleic acid segment, or a spacer consisting of thymidine units of the length given above, respectively.

The 5 'hybridizable oligonucleotide corresponds to SEQ ID NO. 7, and the 3' hybridizable oligonucleotide corresponds to SEQ ID NO. 8, respectively. The oligonucleotide of SEQ ID NO. 7 will readily hybridize to the oligonucleotide of SED ID NO. 5. The oligonucleotide of SEQ ID NO 8 will readily hybridize to the oligonucleotide of SED ID NO 6.

In the above bridging construct examples, [ B-L ] indicates the L-DNA oligonucleotide sequence; spacers C18, biotin and biotin dT refer to the C18 spacer, biotin and biotin-dT, respectively, as derived from the building blocks given above; whereas the numbered T indicates the number of thymidine residues incorporated into the linker at a given position.

2.5 Assembly of Dual Binder constructs

A) Cleavage of IgG and labelling of Fab' fragments with ssDNA

Cleavage of the purified monoclonal antibody with the aid of the protease pepsin, producing F (ab')₂Fragments which are subsequently reduced to Fab' fragments by treatment with a low concentration of cysteamine at 37 ℃. The reaction was stopped via separation of cysteamine on a PD10 column. The Fab' fragments were labeled with activated oligonucleotides as generated according to example 3. This single-stranded DNA (= ssDNA) carries a thiol-reactive maleimide group, which reacts with cysteines of the Fab' hinge region. To obtain a heightPercentage of singly-labeled Fab 'fragments, keeping the relative molar ratio of ssDNA to Fab' fragments low. Purification of the singly labeled Fab 'fragments (ssDNA: Fab' =1: 1) takes place by ion exchange chromatography (column: Source15Q PE4.6/100, Pharmacia/GE). Confirmation of effective purification was achieved by analytical gel filtration and SDS-PAGE.

B) Assembly of anti-pIGF-1R dual binders

anti-pIGF-1R dual binders are based on two Fab' fragments targeting different epitopes of the IGF-1R intracellular domain: fab '8.1.2 detects a phosphorylated site (pTyr 1346) of the target protein, while Fab' 1.4.168 detects a non-phosphorylated site. The Fab' fragment has been covalently linked to single-stranded dna (ssdna): fab '1.4.168 was covalently linked to a 17-mer ssDNA comprising SEQ ID NO 6 and containing fluorescein as the fluorescent marker, while Fab' 8.1.2 was covalently linked to a 19-mer ssDNA comprising SEQ ID NO 5 and containing Cy5 as the fluorescent marker. Hereinafter, these fabs ' to which 17-mer or 19-mer ssDNA is covalently bound are referred to as ssFab ' 1.4.168 and ssFab ' 8.1.2, respectively. The dual binder assembly is mediated by a linker (i.e., a bridging construct comprising two complementary ssDNA oligonucleotides (SEQ ID NOs: 7 and 8, respectively)) that hybridizes to the corresponding ssDNA of the ssFab' fragment. The distance between the two ssFab' fragments of the dual binder can be modified separately by using spacers (e.g., C18-spacer) or DNA of different lengths.

For assembly evaluation, the dual binder components ssFab '8.1.2, ssFab' 1.4.168 and linker construct (I) (= linker 17 of example 2.4) 5 '-G CAG AAG CAT TAA TAG ACT T (-Bi) -TGG ACG ACG ATA GAACT-3' and (II) (= linker 10 of example 2.4) 5 '-G CAG AAG CAT TAA TAG ACT- (T20) -T (-Bi) - (T20) -TGG ACG ACG ATA GAA CT-3' were mixed in equimolar amounts at room temperature. After an incubation step of 1 minute, on an analytical gel filtration column (Superdex)^TM200,10/300GL, GE Healthcare). Elution volume (V) of a Single Dual Binder component_E) V with reaction mixture_EThe comparison of (a) shows that a dual binder has been successfully formed (figure 1). (in both types of linkersPartial biotinylated thymidine (T- (Bi)) was not functional in these experiments. )

2.6 Biacore to assess binding of anti-pIGF-1R Dual Binders to immobilized IGF-1R and IR peptides^TMExperiment of

For this experiment, Biacore was used at T =25 ℃^TM2000 apparatus (GE Healthcare), Biacore^TMThe SA sensor is embedded in the system. Pre-conditioning occurred with 100. mu.l/min 31 min injections of 1M NaCl in 50mM NaOH and 1 min 10mM HCl.

HBS-ET (10 mM HEPES pH7.4,150mM NaCl,1mM EDTA, 0.05%)) As a system buffer. The sample buffer was the same as the system buffer. Biacore driven under control software V1.1.1^TM2000 systems.

Subsequently, biotinylated peptides were captured on the SA surface in the corresponding flow cell. 16 RUs of IGF-1R (1340-1366) [1346-pTyr; Glu (Bi-PEG-1340] amide (i.e., peptide of 1346 tyrosine phosphorylated SEQ ID NO:11 comprising a PEG linker bound via glutamic acid corresponding to position 1340 and the other end of the linker being biotinylated) were captured on flow cell 2 18 RUs of IGF-1R (1340-1366), Glu (Bi-PEG-1340] amide (i.e., peptide of 1346 tyrosine non-phosphorylated SEQ ID NO:11 comprising a PEG linker bound via glutamic acid corresponding to position 1340 and the other end of the linker being biotinylated) were captured on flow cell 3. hIR of 20 RUs (1355-1382) [1361-pTyr; Glu (Bi-PEG-1355] amide (i.e., peptide of 1361 tyrosine phosphorylated SEQ ID NO: 12) were captured on flow cell 4, which comprises a PEG linker bound via the glutamic acid corresponding to position 1355 of the human insulin receptor, and the other end of the linker is biotinylated). Finally, all flow cells were saturated with d-biotin.

For dual binder formation, the assembly protocol as described in example 2.5 was used. When individual runs were performed with only one of the two ssFab', the presence or absence of linker DNA did not affect the binding or dissociation curves (data not shown).

100nM analyte in solution (which in these experiments was the bivalent dual binder) was injected at 50. mu.l/min for 240 seconds of binding time and dissociation was monitored for 500 seconds. Efficient regeneration was accomplished by injection at 50 μ l/min with 80mM NaOH using a1 minute injection procedure. Flow cell 1 serves as a reference. Blank buffer injection was used instead of antigen injection to double reference the data by buffer signal subtraction.

In each measurement cycle, one of the following analytes in solution was injected on all 4 flow cells: 100nM ssFab '8.1.2, 100nM ssFab' 1.4.168, a mixture of 100nM ssFab '8.1.2 and 100nM ssFab', 100nM of the combination of ssFab '8.1.2 and ssFab' 1.4.168 hybridized on linker III (5 '-G CAG AAG CAT TAA TAG ACT-T (20) -T (-Dig) - (T20) -TGGACG ACG ATA GAA CT-3' (= linker 15 of example 2.4)), and 100nM of the combination of ssFab '8.1.2 and ssFab' 1.4.168 hybridized on linker (IV) (5 '-G CAG AAG CAT TAA TAG ACT-T (-Dig) -TGGACG ACG ATA GAA CT-3' (= linker 16 of example 2.4)), respectively. (the digoxigenylation of the central thymidine (T (-Dig)) in the above linker was not relevant for these experiments.)

Biacore with time-dependent signal^TMAnd (5) monitoring a sensorgram.

Reporting points were set at the end of the analyte binding phase (late binding, BL) and the end of the analyte dissociation phase (late stability, SL) to monitor the response unit signal height for each interaction. Biacore was used according to a linear 1:1 Langmuir fit^TMThe evaluation software 4.1 calculates the dissociation rate kd (1/s). The half-life of the complex in minutes was calculated according to the formula ln (2)/(60x kd).

The sensorgrams (fig. 2-5) show that when ssFab '1.4.168 and ssFab' 1.4.168 are used as dual binders (= bivalent binders), both the specificity of pIGF-1R binding and the complex stability are increased, presumably due to a fundamental synergistic binding effect. Fab' 1.4.168 alone showed no cross-linking to pIR peptideCross-reactivity, but without distinguishing between phosphorylated and non-phosphorylated forms of IGF-1R (T1/2 in both cases)_Dissociation=3 minutes). However, Fab' 8.1.2 only binds to the phosphorylated form of IGF1-R peptide, but exhibits some undesirable cross-reactivity with phosphorylated insulin receptor. The dual binders completely distinguished the pIGF-1R peptide from the other two peptides (see fig. 4) and thus helped overcome the problem of non-specific binding. It was noted that the specificity increase was lost when both fabs' without linker DNA were applied (figure 5). The increased affinity of the dual binders for the pIGF-1R peptide was shown to increase dissociation half-life compared to individual Fab 'and Fab' mixtures omitting the linker DNA (FIGS. 3 and 5). Although test dual binders with two different DNA linker lengths share an overall positive impact on target binding specificity and affinity, the longer linker (with T40-T-Dig as spacer (III)) (i.e. linker 15 of example 2.4) appeared to be advantageous for both criteria.

2.7Biacore^TMSandwich for assays M-1.4.168-IgG and M-8.1.2-IgG

Using Biacore^TMT100 Instrument (GE Healthcare), Biacore^TMCM5 sensors are embedded in the system. The sensors were pre-conditioned by injecting with 0.1% SDS, 50mM NaOH, 10mM HCl and 100mM H3PO4 for 1 minute at 100. mu.l/min.

The system buffer was HBS-ET (10 mM HEPES pH7.4,150mM NaCl,1mM EDTA,0.05%). The sample buffer is the system buffer.

Biacore driven under control software V1.1.1^TMT100 system. 10mM sodium acetate pH4.5 in 30. mu.g/ml polyclonal rabbit IgG antibody immobilized in 10000 RUs on flowcells 1, 2, 3 and 4, respectively, via EDC/NHS chemistry according to the manufacturer's instructions<IgGFCγM>R (Jackson ImmunoResearch Laboratories Inc.). Finally, the sensor surface was blocked with 1M ethanolamine. Driving the whole nut at 13 ℃And (6) testing.

500nM primary mAb M-1.004.168-IgG was captured for 1 min at 10. mu.l/min on the < IgGFC. gamma.M > R surface. mu.M blocking solution containing a mixture of IgG fragments (of IgG classes IgG1, IgG2a, IgG2b, IgG 3) was injected at 30. mu.l/min for 5 min. The peptide IGF-1R (1340) -1366) [1346-pTyr; Glu (Bi-PEG-1340] amide was injected at 300nM at 30. mu.l/min for 3 minutes, 300nM of the secondary antibody M-8.1.2-IgG was injected at 30. mu.l/min, and the sensor was regenerated at 50. mu.l/min for 3 minutes using 10mM glycine-HCl pH 1.7.

Figure 6 depicts the assay setup. The measurement results are given in fig. 7. This measurement clearly indicates that both monoclonal antibodies are able to bind simultaneously to two different, unrelated epitopes on their respective target peptides. This is a prerequisite for any subsequent experiments aimed at generating a cooperative binding event.

2.8 Biacore on sensor surface^TMAssay Dual Binders

Biacore was used at T =25 ℃^TM3000 Instrument (GE Healthcare), Biacore^TMThe SA sensor is embedded in the system. The system was preconditioned with 100. mu.l/min 31 minute injections of 1M NaCl in 50mM NaOH and 1 minute 10mM HCl.

The system buffer was HBS-ET (10 mM HEPES pH7.4,150mM NaCl,1mM EDTA,0.05%). The sample buffer is the system buffer.

Driving Biacore under control software V4.1^TM3000 System.

124 RU of amino-PEO-biotin were captured on reference flow cell 1. 1595 RUs of biotinylated 14.6kDa T0-Bi37 mer ssDNA-linker (I) (5 '-G CAG AAG CAT TAA TAGACT-T (-Bi) -TGG ACG ACG ATA GAA CT-3') (= linker 17 of example 2.4) and 1042 RUs of biotinylated 23.7kDa T40-Bi77 mer ssDNA-linker (II) (5 '-G CAG AAG CAT TAA TAG ACT-T (20) - (biotin-dT) - (T20) -TGG ACG ACG ATA GAA CT-3' = linker 10 of example 2.4) were captured on different flowcells.

300nM ssFab '8.1.2 and 300nM ssFab' 1.004.168 were injected into the system at 50. mu.l/min for 3 minutes. As a control, only 300nM ssFab '8.1.2 or 300 nMssFab' 1.004.168 was injected to test the kinetic contribution of each ssFab. As a control, buffer was injected instead of ssFab. The free peptides pIR (1355-pTyr 1382) [1361-pTyr ] amide and IGF-1R (1340-1366) amide in solution were injected into the system at 50. mu.l/min for 4 min at concentration steps of 0nM, 4nM, 11nM, 33nM (twice), 100nM and 300nM, respectively. In another set of experiments, to measure the affinity for the peptide pIGF-1R (1340-1366) [1346-pTyr ] amide, concentration steps of 0nM, 0.4nM, 1.1nM, 3.3nM (twice), 10nM and 30nM were used.

Dissociation was monitored at 50. mu.l/min for 5.3 min. The system was regenerated with a 12 second 25mM NaOH pulse after each concentration step and the ssFab' ligands were reloaded.

FIG. 8 schematically depicts Biacore^TMOn-board assay setup. The table given in fig. 9 shows the quantification results of this approach. Figures 10, 11 and 12 depict exemplary Biacore from this assay setup using T40 dual binders^TMAnd (6) obtaining the result.

The table in figure 9 demonstrates the benefit of the dual binder concept. The T40 dual binder (dual binder with linker 10 of example 2.4, i.e., linker with T20-biotin-dT-T20 spacer) resulted in a 2-fold improvement in antigen complex half-life (414 minutes) and a 3-fold improvement in affinity (10pM) compared to the T0 dual binder with 192 minutes and 30pM (i.e., dual binder with linker 16 of example 2.4). This supports the necessity of optimizing the length of the linker to produce the best synergistic binding effect.

The T40 dual binder (i.e., the dual binder comprising the T40-Bi linker (linker 10 of example 2.4)) exhibited 10pM affinity for phosphorylated IGF-1R peptide (table in fig. 9, fig. 10). This is a 2400-fold improvement in affinity for phosphorylated insulin receptor peptide (24nM) and a 100-fold improvement in non-phosphorylated IGF-1R peptide.

Thus, the goal of improving specificity and affinity by combining two distinct and separate binding events is achieved.

The synergistic binding effect becomes particularly evident from the off-rate against phosphorylated IGF-1R peptide, where the dual binder showed a 414 minute antigen complex half-life relative to 0.5 minutes for monovalent binder 8.1.2 alone and 3 minutes for monovalent binder 1.4.168 alone.

Furthermore, the fully assembled construct roughly multiplies its off-rate kd (1/s) when compared to a single Fab' hybridized construct (tables in figures 10, 11, 12 and 9). Interestingly, the binding rate ka (1/Ms) was also slightly increased when compared to the single Fab' interaction event, probably due to the increased molecular flexibility of the construct.

Indeed, diagnostic systems using intensive washing protocols should employ the high performance of the T40 dual binder, as compared to the formation of individual (monovalent) Fab' molecules. The hybridized constructs (i.e. the bivalent binding agent according to the invention) give rise to specific and rather stable binding events, whereas monovalent binding agents dissociate faster, e.g. they are washed away faster.

2.9 evaluation of anti-pIGF-1R Dual Binder molecules in Immunohistochemistry (IHC) experiments

The IHC experiments described herein were performed on a BenchMark XT platform from Ventana. For this assay, an anti-pIGF-1R dual binding agent consisting of ssFab '1.4.168 (a non-phospho epitope that binds the IGF-1R intracellular domain), ssFab' 30.4.33 (a pTyr1346 phospho epitope that binds the IGF-1R intracellular domain), and a flexible linker was used. The production of antibody 1.4.168 has been described in example 2.1, and antibody 30.4.33 (variable region heavy chain shown in SEQ ID NO:19 and variable region light chain shown in SEQ ID NO: 20) has been produced using the same protocol as described herein. The Fab ' fragment of 30.4.33 has a higher affinity for the phosphorylation site of pTyr1346IGF-1R than the Fab ' fragment of antibody 8.1.2 used previously (about 0.5 min for T1/2 and about 7 min for T1/2 of ssFab ' 30.4.33).

Flexible linkers with spacers of different lengths (= linkers 11, 12, 13, 14 of example 2.4) were used in this assay. The biotin label within the linker molecule serves as a detection tag for the streptavidin-based Ventana iviewda detection kit.

To test the specificity of the anti-pIGF-1R dual binder molecules, a sophisticated test system based on formalin-fixed, paraffin-embedded (FFPE)3T3 cells was used. 3T3 cells have been stably transfected with IGF-1R or IR expression vectors. Cells were fixed with formalin according to standard protocols and embedded in paraffin. Cells were stimulated with 100ng/ml IGF-1 or insulin to induce IGF1-R or IR phosphorylation or remained untreated prior to fixation. Western blot experiments (fig. 13A) demonstrated successful stimulation of receptor phosphorylation.

Each ssFab 'fragment of either ssFab' 1.4.168 only or ssFab '30.4.33 only, at 0.5 μ g/ml, and an equimolar amount of 8xC18 linker, and a mixture of both ssFab' fragments of ssFab '1.4.168 and ssFab' 30.4.33 (both 0.5 μ g/ml) and an equimolar amount of 8xC18 linker were used for detection. The biotin label within the linker molecule serves as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit. Reference scheme details: pretreatment with cell conditioning buffer 1(CC1) occurred with an incubation time of the binding molecules of 32 minutes and an incubation temperature of 37 ℃.

The detector molecule consisting of the 8xC18 linker molecule (linker 14 of example 2.4) and ssFab '1.4.168 only or ssFab' 30.4.33 only did not produce staining on any of the FFPE3T3 cell aggregates tested (fig. 13B, lines 1 and 2). In comparison, detection with the intact dual binder molecule (consisting of two ssFab' fragments +8xC18 linkers) resulted in staining, but only on IGF-1R overexpressing cells stimulated with IGF-1 (FIG. 13B, line 3). No cross-reactivity was observed on IR overexpressing cells even when IR phosphorylation had been induced. This experiment demonstrates the high specificity of the dual binders for phosphorylated IGF-1R.

To evaluate the effect of linker length on staining performance, 2xC18,4xC18,6xC18 and 8xC18 linker molecules (linkers 11, 12, 13, 14 of example 2.4) were used in the same IHC setup. Of the dual binders tested, the dual binder with the longest linker (8 xC 18) showed excellent staining results (fig. 13C). This indicates that at least in this case the long flexible linker promotes simultaneous binding of two different epitopes on the pIGF-1R by two dual binder arms.

The dual binding agent consisting of ssFab '1.4.168, ssFab' 30.4.33, and 8xC18 linker molecules (linker 14 of example 2.4) was further tested on FFPE H322M xenograft tissue. Reference scheme details: pretreatment with cell conditioning buffer 1(CC1) occurred with an incubation time of the binding molecules of 32 minutes and an incubation temperature of 25 ℃. Again, no pIGF-1R staining was observed with detector molecules consisting of the 8xC18 linker molecule and only one of ssFab '1.4.168 or ssFab' 30.4.33. However, detection with intact dual binder molecules (consisting of two ssFab' fragments +8xC18 linkers) resulted in characteristic pIGF-1R membrane staining (fig. 14).

2.10 Biacore assay Dual Binders on sensor surface

To obtain kinetic data, additional Biacore experiments were also performed on the optimized version of the anti-pIGF-1R dual binder of example 2.9.

A Biacore SA sensor was embedded in the system using a Biacore3000 instrument (GE Healthcare) at T =25 ℃. The system was preconditioned with 100. mu.l/min with 31 minute injections of 1M NaCl in 50mM NaOH and 1 minute of 10mM HCl.

The system buffer was HBS-ET (10 mM HEPES pH7.4,150mM NaCl,1mM EDTA,0.05%). The sample buffer is the system buffer.

The Biacore3000 system is driven under control software V4.1.

89 RU of amino-PEO-biotin were captured on the reference flow cell 1. 595 RUs of biotinylated 8xC 18-linker (I) (5 '-G CAG AAG CAT TAA TAG ACT- (spacer C18)4- (biotin-dT) - (spacer C18)4-TGG ACG ACG ATA GAA CT-3') (= linker 14 of example 2.4) were captured on the second flow cell.

300nM ssFab' 30.4.33 and 300nM ssFab1.004.168 were injected into the system at 50. mu.l/min for 3 min. As a control, only 300nM ssFab ' 30.4.33 or 300nM ssFab ' 1.004.168, respectively, was injected to test the kinetic contribution of each ssFab '. The free peptide IGF-1R (1340) -1366) [1346-pTyr ] amide (peptide of 1346 tyrosine phosphorylated SEQ ID NO:11 = synthetic analyte) in solution was injected into the system at 50. mu.l/min for 4 minutes at concentration steps 0nM, 0.4nM, 1.1nM, 3.3nM (twice), 10nM and 30 nM. Dissociation was monitored at 50. mu.l/min for 5.3 min. The system was regenerated with a 12 second pulse of 250mM NaOH after each concentration step and reloaded with ssFab ligand.

As additional controls, a) injection buffer was substituted for ssFab', and b) flowcells immobilized with amino-PEO-biotin have been used (data not shown). In these experiments, no non-specific binding of "analyte" was observed.

Figure 15 schematically depicts the assay setup on a Biacore instrument. The table given in table 16 shows the quantification results of this approach. Figures 17, 18 and 19 plot the Biacore results for this assay setup.

As seen in fig. 16, the dual binding molecule exhibited an affinity of about 10pM for the phosphorylated, synthetic IGF-1R analyte. This is a 200-fold or 300-fold improvement in affinity compared to binding molecules consisting of ssFab '30.4.33 or ssFab' 1.4.168 alone. The measured off-rates were 830 minutes for the dual binder, 7.3 minutes for the monovalent binder ssFab '30.4.33, and 3.5 minutes for the monovalent binder ssFab' 1.4.168. These data clearly demonstrate the synergistic binding effect of the dual binder molecules used.

Example 3

Bivalent binding agents against phosphorylated HER3

The HER protein receptor tyrosine kinase family consists of 4 members: HER1, HER2, HER3 and HER 4. Upon ligand binding, the receptor dimerizes in various ways, either as homo-or heterodimers, to trigger different signal transduction pathways, depending on the ligand and the expression level of each of the 4 family members. For example, HER3 undergoes a conformational change when it binds to its ligand neuregulin 1(NRG1) or neuregulin 2(NRG2), and the HER3 dimerization domain is exposed, and it can interact with other HER receptors. Upon dimerization, HER3 becomes phosphorylated. In this example, we developed a dual binding agent for detecting the phosphorylated form of HER 3.

3.1 monoclonal antibody development (monoclonal antibody 7.2.32 and monoclonal antibody 4.1.15)

a) Immunization of mice

Balb/c and NMRI mice were immunized with HER3 (1243-. The initial immunization dose was 100. mu.g. Mice were further immunized with 100 μ g of immunogen after 6 and 10 weeks.

b) Fusion and cloning

Performing the fusion and cloning steps as described in 2.1b)

c) Isolation of immunoglobulins from cell culture supernatants

Isolation of immunoglobulins as described in 2.1c)

d) Biophysical characterization of monoclonal antibodies

Using Biacore^TMTechniques for investigating monoclonal antibodies to HER3 or pHER3 by surface plasmon resonance kinetic screeningKinetics of the interaction between phosphorylated forms.

Using Biacore under control of software version V1.1^TMA100 instrument. Biacore^TMA CM5 chip is embedded in the instrument and is hydrodynamically addressed and conditioned according to the manufacturer's instructions. As a running buffer, HBS-EP buffer (10 mM HEPES (pH7.4),150mM NaCl,1mM EDTA,0.05% (w/v) P20) was used. Polyclonal rabbit anti-mouse IgG Fc capture antibodies were immobilized at 10,000 RUs in 10mM sodium acetate buffer (ph4.5) at 30 μ g/ml at spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4. Antibodies were covalently immobilized via NHS/EDC chemistry. Thereafter, the sensor was deactivated with a 1M ethanolamine solution. The measurements were performed using points 1 and 5, and using points 2 and 4 as reference. Hybridoma supernatants containing the mabs were diluted 1:2 in HBS-EP buffer before application to the sensor chip. The diluted solution was applied at a flow rate of 30. mu.l/min for 1 minute. Immediately thereafter, the analytes human _ HER3 (1242. sup. -]) PEG2-EDA-Btn (SEQ ID NO: 18) or human _ HER3(1283-1295) -PEG2-EDA-Btn (SEQ ID NO: 18) for 2 minutes. After this time, the signal was recorded for 5 minutes dissociation time. The sensor was regenerated by injecting 10mM glycine-HCl solution (pH1.7) at a flow rate of 30. mu.l/min for 2 minutes. The dissociation rate constant kd (1/s) was calculated according to the langmuir model using evaluation software according to the manufacturer's instructions. The selected monoclonal antibodies interact with either the HER3 epitope comprising amino acids 1242-1267 or the phosphorylated (pTyr1289) HER3 epitope comprising amino acids 1283-1295 with an off-rate constant lying within the boundaries of the present patent claims. Antibodies that bound the unphosphorylated form of the epitope HER3(1283-1295) were excluded from further studies.

The selected antibody against HER3(1242-1267) was designated 7.2.32 (variable region heavy chain shown in SEQ ID NO:21 and variable region light chain shown in SEQ ID NO: 22) and its dissociation rate constant was determined to be 2.3X10^-31/s and therefore within the necessary range required for the dual binder approach. Selected against pHER3(1283-1295)[pTyr1289]) The antibody of (2) was named 4.1.15 (variable region heavy chain shown in SEQ ID NO:23 and variable region light chain shown in SEQ ID NO: 24), and its dissociation rate constant was 2.5x10^-31/s, and thus also within the limits required for the dual binder approach.

e) Sequencing of variable regions of selected antibodies

The variable regions of the selected antibodies were sequenced using standard molecular biology methods. The sequences are shown in SEQ ID NO 21-24.

3.2 development of a Dual Binder that recognizes phosphorylated HER3(pTyr1289)

a) Recombinant expression of Fab fusion proteins

Fab fragments 7.2.32 and 4.1.15 were expressed in Hek293F cells as fusion proteins carrying an 8XHIS tag and a sortase cleavage recognition sequence (SEQ ID NO: 16). Use of293fectin ^TM Transfection reagent(Invitrogen) 1L1X10 was transfected with plasmids encoding the heavy and light chains of 7.2.32 or 4.1.15 in a 1:1 ratio according to the manufacturer's instructions⁶The viability of the HEK293 cells is more than 90 percent/ml. After transfection, HEK293F cells were transfected at 130rpm, 37 ℃ and 8% CO₂Incubate for 7 days. Then, the cells were centrifuged at 8000rpm for 20 minutes at 4 ℃. The supernatant containing the recombinant protein was further filtered using a 0.22 μm Steriflip (Millipore) vacuum filtration system. Using standard purification methods, usingFPLC system, Fab fragment purified by nickel affinity column chromatography and preparative gel filtration. Purity was assessed by SDS-PAGE and analytical gel filtration.

b) DNA-oligomer conjugation using enzyme sortases in transpeptidase reactions

The enzyme sortase is a prokaryotic proteolytic enzyme That also has transpeptidase activity (Ton-That et al, PNAS 1999). Here, the enzyme catalyzes the interaction between LPXTG (a sortase cleavage motif) and glycine residues attached to DNA-oligosIs subjected to transpeptidase reaction. Oligomers of 17-mer (oligomer shown in SEQ ID NO:25 for 4.1.15 labeling) and 19-mer (oligomer shown in SEQ ID NO:26 for 7.2.32 labeling) were used for the labeling reaction. In 20mM Tris pH8,200mM NaCl,5mM CaCl₂Was labeled overnight at 37 ℃ with 20. mu.M recombinant sortase, 50. mu.M Fab fragment and 200. mu.M oligo. Next, the labeling reaction was diluted 10-fold in 20mM Tris pH8.0 and applied to a Resource Q ion exchange column (GE Healthcare) equilibrated in 20mM Tris pH8.0. The strongly negatively charged oligo and oligo-Fab fragments were eluted with a high salt gradient of 20mM Tris pH8.0 and 1M NaCl and thus separated from the sortase and unlabeled Fab fragments which eluted at lower salt concentrations. The elution was monitored by tracking the absorbance at 495nm and the oligomer was detected for fluorescein labeling. The elution fractions containing oligo and Fab-oligo were combined and Fab-oligo was separated from unconjugated oligo by preparative gel filtration on a HiLoad16/60 Superdex200 column (GE Healthcare) using 20mM Tris8.0,200mM NaCl as equilibration and running buffer. The purity of the final product was assessed using analytical gel filtration and SDS-PAGE, and only more than 90% pure final product was used in the assembly of the dual binder. Hereinafter, Fab-oligomers are referred to as "ssFab".

c) Assembly of anti-pHER 3 dual binders

The anti-pHER 3 dual binders are based on ssDNA linker molecules and two ssFab fragments targeting different epitopes of the HER3 endodomain: ssfab4.1.15 detects a phosphorylation site (pTyr1289) of the target protein, while ssfab7.2.32 detects a non-phosphorylation site. Assembly evaluation was performed as described in 2.5. B. Experiments show efficient assembly of the dual binder molecules.

3.3 evaluation of anti-pHER 3 Dual Binder molecules in Immunohistochemistry (IHC) experiments

IHC experiments were performed on a BenchMark XT platform from Ventana. For this assay, an anti-pHER 3 dual binder consisting of ssfab7.2.32 (a non-phospho epitope that binds the intracellular domain of HER 3), ssfab4.1.15 (a pTyr1289 phospho epitope that binds the intracellular domain of HER 3) and a flexible linker was used. A flexible linker with a 4xC18 spacer (= linker 12 of example 2.4) was used in this assay. The biotin label within the linker molecule serves as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit.

To test the specificity of the anti-pHER 3 dual binder molecules, a sophisticated test system based on formalin fixed, paraffin embedded (FFPE) Hek293 cells was used. Hek293 cells have been transiently transfected with both HER2 and HER3 expression vectors. In one case, a HER3 expression vector encoding a mutated version of HER3 was used in which 14 tyrosines in the intracellular domain that serve as phosphorylation sites were replaced with phenylalanine (Y975F, Y1054F, Y1132F, Y1159F, Y1197F, Y1199F, Y1222F, Y1224F, Y1260F, Y1262F, Y1276F, Y1289F, Y1307F, Y1328F). Cells were fixed with formalin and embedded in paraffin according to standard protocols. Cells were stimulated with 20nM NRG 1-beta 1(Peprotech) for 15 min at 37 ℃ to induce HER3 phosphorylation or remained untreated prior to fixation. Western blot experiments (fig. 20A) demonstrated successful stimulation of receptor phosphorylation.

Each ssFab fragment of 1 μ g/ml ssfab7.2.32 only or ssfab4.1.15 only and an equimolar amount of 4xC18 linker, and a mixture of both ssFab fragments of ssfab7.2.32 and ssfab4.1.15 (both 1 μ g/ml) and an equimolar amount of 4xC18 linker were used for the detection. The biotin label within the linker molecule serves as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit. Reference scheme details: pretreatment with cell conditioning buffer 1(CC1) occurred with an incubation time of the binding molecules of 32 minutes and an incubation temperature of 37 ℃.

The detector molecule consisting of the 4xC18 linker molecule (linker 12 of example 2.4) and ssfab7.2.32 only or ssfab4.1.15 only did not produce staining on any of the FFPE cell pellets tested (fig. 20B, lines 1 and 2). In comparison, detection with the intact dual binder molecule (consisting of two ssFab fragments +4xC18 linkers) resulted in staining, but only on cells stimulated with NRG1- β 1 and expressing wild-type HER3 (fig. 20B, line 3). No staining was observed on NRG1- β 1 stimulated cells overexpressing mutant HER3 lacking the Tyr1289 phosphorylation site. This experiment demonstrates the high specificity of the dual binders for phosphorylated HER 3.

Claims (5)

1. An isolated bivalent binding agent of formula I capable of binding a post-translationally phosphorylated target polypeptide,

a-a '-a-S-B: B' -B (formula I)

The bivalent binding agent consists of two monovalent binding agents a and B of different specificities linked to each other via a linker, wherein:

a) -represents a covalent bond,

b) a-S-b is a linker having a length between 10nm and 50nm, wherein S is a spacer,

c) a 'a and b: b' are binding pairs consisting of hybridizing nucleic acid sequences each forming a stable duplex via a multiple base pair, wherein the sequences in binding pair a 'a do not bind to the sequences of binding pair b: b', respectively, and vice versa,

d) a first monovalent binder A is a molecule that interacts with a target polypeptide at a single binding site, wherein the first monovalent binder binds a polypeptide epitope of the target polypeptide, and wherein the polypeptide epitope is not post-translationally modified,

e) a second monovalent binder B binds to post-translational phosphorylation, wherein the second monovalent binder binds to K bearing a phosphorylated polypeptide compared to the same non-post-translational phosphorylated polypeptide_DissociationAt least a factor of 20 lower than the average,

f) each monovalent binder is selected from the group consisting of: single chain antibodies, Fab fragments of monoclonal antibodies, and Fab' fragments of monoclonal antibodies,

g) each monovalent binder has a range of 5x10^-3Second to 10^-4K/sec_DissociationAnd is and

h) the bivalent binding agent has 3x10^-5K/sec or less_Dissociation。

2. The isolated divalent binding agent according to claim 1, wherein the divalent binding agent has 10^-5K/sec or less_Dissociation。

3. The isolated divalent binding agent according to claim 1 or2, wherein the phosphorylated target polypeptide is a membrane bound receptor or an intracellular cell signaling molecule.

4. A method of histological staining, the method comprising the steps of:

a) providing a sample of a cell or tissue,

b) incubating the sample with an isolated bivalent binding agent according to any one of claims 1 to 3 and

c) detecting the divalent binding agent, thereby staining the sample for the post-translationally modified target polypeptide.

5. Use of an isolated bivalent binding agent according to any one of claims 1 to 3 in staining of a cell or tissue sample.