HK1186739B - Binding agent - Google Patents

Description

binder

Background of the Invention

The present invention relates to binding agents of the formula A-a':a-S-b:b'-B:X(n), wherein A and B are monovalent binders, wherein a':a and b:b' are binding pairs, wherein a' and a do not interfere with the binding of b to b' and vice versa, wherein S is a spacer of at least 1 nm in length, wherein: X refers to a functional moiety that binds to at least one of a', a, b, b' or S covalently or via a binding pair, wherein (n) is an integer and is at least 1, wherein - represents a covalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm. Methods of producing such binding agents and certain uses thereof are also disclosed.

In general, bispecific antibodies or bispecific binding agents are unique in that they can simultaneously bind to two different epitopes on an antigen or two different antigens. This property enables the development of new therapeutic and diagnostic strategies that are not possible with conventional monoclonal antibodies. A large number of bispecific dual binding agents, such as bispecific antibodies, have been developed and reflect the strong scientific and commercial interest in these molecules.

Monoclonal antibodies (mAbs) directed against a single epitope on an antigen generally bind with a lower affinity than that of polyclonal antisera. However, certain pairs of mAbs directed against different epitopes on the same antigen can bind to the antigen more efficiently and with an affinity greater than the sum of the affinities of the corresponding individual mAbs.

However, the affinity constants of synergistic pairs of monoclonal antibodies or chemically cross-linked bispecific F(ab') ₂ generally only reach 15 times higher than the affinity constants of the individual monoclonal antibodies, which is significantly lower than the theoretical affinity expected for an ideal combination between the reactants (Cheong, HS et al., Biochem. Biophys. Res. Commun. 173 (1990) 795-800). One reason for this may be that the individual epitope/paratope interactions involved in cooperative binding (generating high avidity) must be oriented in a specific manner relative to each other to achieve optimal cooperation.

The generation of bispecific antibodies is recorded in for example WO2004/081051. In this application, a bispecific antibody (BAb) comprising two antibodies is disclosed, each of which has binding specificity to different epitopes located on the target structure surface. In order to achieve the desired improvement in specificity, two monoclonal antibodies each having relatively low binding affinity to its corresponding epitope are used. The BAb produced provides high avidity to the target tissue (due to the cumulative nature of the binding interactions), but has much lower avidity (due to the lower avidity of each monoclonal antibody used to generate them) to the non-target tissue of cross reactivity. The generation of these bispecific antibodies is quite complicated and, for example, requires complex (sophisticated) chemical coupling and purification steps.

Bispecific monoclonal antibodies also represent a very interesting new therapeutic modality. A wide range of bispecific antibody formats have been designed and developed (see, for example, Fischer, N. and Leger, O., Pathobiology 74 (2007) 3-14). Such bispecific therapeutic monoclonal antibodies can be obtained, for example, by chemical cross-linking, interactions of appropriately engineered protein domains, complete recombination, etc. Obviously, recombinant engineering of each conjugate and careful purification of the desired heterodimer from the biochemically similar homodimer represent some of the challenges encountered.

The chelated recombinant antibody (CRAb) originally described by Neri, D. etc. (1995) represents a kind of antibody with very high affinity, wherein two scFvs specific for non-overlapping epitopes on the same antigen molecule are connected by a flexible linker polypeptide.The anti-hen egg lysozyme (HEL) CRAb of initial modeling and design adopted 18 amino acid linker polypeptide to span the distance between two scFv antibodies, and later showed that the resulting affinity enhancement was up to 100 times higher than the higher of the two scFvs, as shown by multiple biophysical methods (Neri, D. etc., J.Mol.Biol.246 (1995)367-373).

Wright M.J. and Deonarain M.P. (Molecular Immunology 44 (2007) 2860-2869) developed a phage display library for generating chelating recombinant antibodies. The library described therein utilizes expression vectors constructed to provide dual binders with linker peptides of varying lengths between the two binding entities. This facilitates the selection of optimal binders, i.e., those with the optimal length for these linkers. However, for each of these chelating recombinant antibodies, a complete library of recombinant expression systems (allowing expression of the "binder 1 - linker (of varying lengths) - binder 2" polypeptides) must be constructed.

As outlined above, the preparation of bispecific dual binding agents remains quite challenging and requires complex techniques to identify, construct, and generate each of those bispecific binding agents separately. The frequent need to derivatize (e.g., label) such bispecific binding agents even further exacerbates the level of complexity.

Surprisingly, it has now been found that the novel bispecific binding agents disclosed in the present invention are able to overcome at least some of the disadvantages known in the prior art.

SUMMARY OF THE INVENTION

The present invention relates to binders of the formula A-a':a-S-b:b'-B:X(n), wherein A and B are monovalent binders, wherein a':a and b:b' are binding pairs, wherein a' and a do not interfere with the binding of b to b' and vice versa, wherein S is a spacer of at least 1 nm in length, wherein: X refers to a functional moiety that binds to at least one of a', a, b, b' or S covalently or via a binding pair, wherein (n) is an integer and is at least 1, wherein - represents a covalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm.

Also disclosed are methods of preparing such binding agents and the use of such preparations, for example, in immunoassay procedures.

Also described and claimed are the uses of the novel binding agents, in particular in immunological detection protocols.

Detailed Description of the Invention

The present invention relates to binding agents of the formula A-a':a-S-b:b'-B:X(n), wherein A and B are monovalent binders, wherein a':a and b:b' are binding pairs, wherein a' and a do not interfere with the binding of b to b' and vice versa, wherein S is a spacer of at least 1 nm in length, wherein: X refers to a functional moiety that binds to at least one of a', a, b, b' or S covalently or via a binding pair, wherein (n) is an integer and is at least 1, wherein - represents a covalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm. Obviously, the binding agent according to the present invention is a binding agent comprising at least two monovalent binders with different specificities. In one embodiment, the binding agent according to the present invention comprises two monovalent binders. In one embodiment, the binding agent according to the present invention is a bivalent or dual binding agent.

As will be appreciated by those skilled in the art, the binding agents described herein can be separated and purified as desired. In one embodiment, the present invention relates to separated binding agents as disclosed herein. An "isolated" binding agent is one that has been identified and separated and/or recovered from, for example, a reagent mixture used to synthesize such a binding agent. Unwanted components of such a reaction mixture are, for example, monovalent binders that are not used up in the desired binding agent. In one embodiment, the binding agent is purified to greater than 80%. In some embodiments, the binding agent is purified to greater than 90%, 95%, 98%, or 99% by weight, respectively. In the case where both monovalent binders of the binding agent according to the present invention are polypeptides, purity is readily determined in protein assays using, for example, Coomassie blue or silver staining by SDS-PAGE under reducing or non-reducing conditions. In the case where purity is assessed at the nucleic acid level, size chromatography is applied to separate the binding agent from by-products and the OD at 260 nm is monitored to assess its purity.

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

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

A "target molecule" is a biomolecule of interest for which an assay or measurement method is sought. Preferred target molecules are lipoproteins, polypeptides, polypeptide complexes, secondary modified polypeptides, and complexes between polypeptides and nucleic acids. In a preferred embodiment, the target molecule is a polypeptide.

According to the present invention, a "monovalent binder" (A and B, respectively, in Formula I) is a molecule that interacts with a target molecule (e.g., a target polypeptide) at a single site, the specific binding site. In the case of using a monovalent antibody or antibody fragment as a binder, this site is called the paratope.

As will be appreciated, monovalent binders A and B each specifically bind to their respective antigens. In a preferred embodiment, the epitopes specifically bound by monovalent binders A and B do not overlap. As will be appreciated by the skilled artisan, the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent being used. Preferably, for a specific binder, the level of binding affinity for biomolecules other than the target molecule results in a binding affinity that is only 10% or less, more preferably only 5% or less, of the affinity it has for the specifically bound target molecule.

Examples of monovalent binders are peptides, peptidomimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, single domain antibodies (see Hey, T. and Fiedler, E. et al., Trends Biotechnol. 23 (2005) 514-522) and monovalent fragments of antibodies.

In certain preferred embodiments, the monovalent binder is a polypeptide. In a preferred embodiment, each of the monovalent binders A and B is a polypeptide.

In certain preferred embodiments, the monovalent binders A and B are each monovalent antibody fragments, preferably monovalent fragments derived from monoclonal antibodies.

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

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

It also represents a preferred embodiment that, in the binding agents disclosed herein, both monovalent binders are derived from monoclonal antibodies and are Fab fragments, or Fab' fragments, or Fab fragments and Fab' fragments. Binding agents comprising two Fab fragments as monovalent binders A and B are also preferred.

Monoclonal antibody technology allows the generation of extremely specific binding agents in the form of specific monoclonal antibodies or fragments thereof. Particularly well known in the art is the technique of creating monoclonal antibodies or fragments thereof by immunizing mice, rabbits, hamsters, or any other mammal with a polypeptide of interest. Another method for creating monoclonal antibodies or fragments thereof is the use of phage libraries of sFv (single-chain variable domains), particularly human sFv (see, for example, Griffiths et al., U.S. Patent No. 5,885,793; McCafferty et al., WO 92/01047; Liming et al., WO 99/06587).

Antibody fragments can be produced by traditional means (such as enzymatic digestion) or by recombinant technology. 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 a complete antigen binding site and lacks a constant region. In one embodiment, the double-chain Fv species consists of a dimer of a heavy chain variable domain and a light chain variable domain in a tight, non-covalent association. In one embodiment of the single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can be combined in a dimer structure similar to the dimer structure in the double-chain Fv species. For a review of scFv, see, for example, Plueckthun, in: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. In general, six hypervariable regions (HVRs) confer antigen-binding specificity to antibodies. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen.

The Fab fragment contains the heavy and light chain variable domains, the light chain constant domain, and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the constant domain cysteine residues bear free thiol groups.

Various techniques have been developed for generating antibody fragments. Traditionally, antibody fragments are derived via proteolytic digestion of intact antibodies (see, for example, Morimoto, K. et al., Journal of Biochemical and Biophysical Methods 24 (1992) 107-117; and Brennan, M. et al., Science 229 (1985) 81-83). For example, papain digestion of an antibody produces two identical antigen-binding fragments, each with a single antigen-binding site, called "Fab" fragments, and a residual "Fc" fragment, whose name reflects its ability to easily crystallize.

Antibody fragments can also be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed and secreted in E. coli, allowing for the 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/Technology 10 (1992) 163-167). Mammalian cell systems can also be used to express and, if desired, secrete antibody fragments.

In certain embodiments, the monovalent binders of the present invention are single-domain antibodies. A single-domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl). In one embodiment, the single-domain antibody consists of all or part of the heavy chain variable domain of an antibody.

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

The term oligonucleotide should be interpreted broadly and includes DNA and RNA, as well as analogs and modifications thereof.

For example, the oligonucleotide may contain substituted nucleotides carrying substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), and deoxyuridine (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines such as 5-methyl dC, aminoallyl dU or dC, 5-(aminoethyl-3-acrylimido)-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-alkyl dG or dA; and 2-substituted dA such as 2-amino dA.

For example, the oligonucleotide may contain substituted nucleotides carrying substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), and deoxyuridine (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines such as 5-methyl dC, aminoallyl dU or dC, 5-(aminoethyl-3-acrylimido)-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-alkyl dG or dA; and 2-substituted dA such as 2-amino dA.

Oligonucleotides may contain nucleotide or nucleoside analogs. That is, naturally occurring nucleobases may be replaced by using nucleobase analogs such as 5-nitroindole d-nucleoside; 3-nitropyrrolyl 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-formycin; pseudo-dU; pseudo-iso-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 strands must be chosen 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 can be modified to contain substituted sugar residues, sugar analogs, modifications to the internucleoside phosphate moiety, and/or be a PNA.

Oligonucleotides may, for example, contain nucleotides with substituted deoxyribose sugars, such as 2'-methoxy, 2'-fluoro, 2'-methylseleno, 2'-allyloxy, 4'-methyldN (wherein N is a nucleobase, e.g., A, G, C, T or U).

Sugar analogs are, for example, xylose; 2',4'-bridged riboses 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, hexitols (oligomers known as HNA); cyclohexenyl (oligomers known as CeNA); altritol (oligomers known as ANA); tricyclic ribose analogs in which the C3' and C5' atoms are linked by an ethylene bridge fused to a cyclopropane ring (oligomers known as tricyclic DNA); glycerol (oligomers known as GNA); glucopyranose (oligomers known as homoDNA); carbaribose (having cyclopentan instead of tetrahydrofuran subunits); and hydroxymethyl-morpholine (oligomers known as morpholino DNA).

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

PNA (which has a backbone without phosphate and d-ribose) can also be used as a DNA analog.

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

The joint L being made up of a-S-b has a length of 6 to 100nm. Preferably, the joint L being made up of a-S-b has a length of 6 to 80nm. Also preferably, the joint has a length of 6 to 50nm or 6 to 40nm. In another preferred embodiment, the joint can have a length of 10nm or longer or 15nm or longer. In one embodiment, the length that the joint has is between 10nm and 50nm. In one embodiment, a and b are respectively in conjunction with pair members, and each has a length of at least 2.5nm.

Theoretically and through a composite approach, the length of a non-nucleoside entity for a given linker (a-S-b) can be calculated using known bond distances and angles from chemically similar compounds. Such bond distances are summarized for several molecules in the standard textbook: CRC Handbook of Chemistry and Physics, 91st edition, 2010-2011, Section 9. However, the exact bond distances vary for each compound. There is also variability in bond angles.

Therefore, it is more practical to use average parameters (easy to understand approximations) in such calculations.

In calculations of spacer or linker lengths, the following approximations apply: a) for calculations of the length of non-nucleoside entities, an average bond length of 130 pm and a bond angle of 180° are used, which are independent of the nature of the attached atoms; b) one nucleotide in a single strand is calculated as 500 pm, and c) one nucleotide in a double strand is calculated as 330 pm.

The value of 130 pm is calculated based on the distance between the two terminal carbon atoms of a C(sp3)-C(sp3)-C(sp3) chain, where the bond angle is 109°28′ and the distance between two C(sp3)s is 153 pm, which assumes a bond angle of 180° and a bond distance of 125 pm between two C(sp3)s, which translates to about 250 pm. Taking into account that heteroatoms such as P and S as well as sp2 and sp1 C atoms can also be part of the spacer, the value of 130 pm is used. 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 the cyclic structure as part of the entire chain of atoms defining the distance.

The spacer S can be constructed as desired, for example, to provide a desired length and other desired properties. The spacer can, for example, be composed entirely or partially of naturally occurring or non-naturally occurring amino acids, of phosphate-sugar units, such as a DNA-like backbone without nucleobases, of a sugar-peptide structure, or at least partially of carbohydrate units or at least partially of polymerizable subunits such as diols or acrylamides.

The length of the spacer S in the binding agent according to the present invention can be varied as desired. In order to easily utilize variable-length spacers (i.e., libraries), it is preferred that spacers of such libraries be easily synthesized. Combinatorial solid phase synthesis of spacers is preferred. Since spacers up to about 100 nm in length have to be synthesized, the synthesis strategy is selected in the following manner so that monomeric synthetic building blocks can be assembled with high efficiency during solid phase synthesis. The synthesis of deoxyoligonucleotides assembled based on phosphoramidites as monomeric building blocks fully meets this requirement. In such spacers, the monomeric units in the spacer are connected in each case via phosphate or phosphate analog modules.

The spacer S may contain free positively and/or negatively charged polyfunctional amino-carboxylic acid groups, such as amino, carboxylate or phosphate groups. 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, α,β-diaminopropionic acid, arginine, aspartic acid and glutamic acid, carboxyglutamic acid and symmetrical trifunctional carboxylic acids such as those described in EP-A-0618192 or US-A-5,519,142. Alternatively, one carboxylate group a) in the trifunctional aminocarboxylic acid may be replaced with a phosphate, sulfonate or sulfate group. An example of such a trifunctional amino acid is phosphoserine.

The spacer S may also contain an uncharged hydrophilic group. Preferred examples of uncharged hydrophilic groups are ethylene oxide or polyethylene oxide groups, preferably having at least 3 ethylene oxide units, sulfoxides, sulfones, carboxylic acid amides, carboxylic acid esters, phosphonic acid amides, phosphonic acid esters, phosphoric acid amides, phosphoric acid esters, sulfonic acid amides, sulfonic acid esters, sulfuric acid amides, sulfuric acid esters, sulfuric acid amides, and sulfuric 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 ester is derived from a hydrophilic alcohol, in particular a C1-C3 alcohol, or a diol or triol.

In one embodiment, the spacer S is composed of one type of monomer. For example, the spacer is composed only of amino acids, sugar residues, diols, phosphate-sugar units, respectively, or it can be a nucleic acid.

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

Spacer building blocks, as the name suggests, can be used to introduce spacer modules into spacer S or to construct spacer S with linkers a-S-b.

There are different numbers and types of non-nucleotide and nucleotide spacer building blocks used to introduce the spacer module.

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 construction affects 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 linked to a phosphoramidite group.

In one embodiment, the bifunctional spacer building block is a non-nucleoside compound. For example, this type of spacer is a C2-C18 alkyl, alkenyl, alkynyl carbon chain, and the alkyl, alkenyl, alkynyl chain can be interrupted to increase the hydrophilicity of the joint with other ethyleneoxy (ethyleneoxy) and/or amide module or quaternized (quarternized) cationic amine module. It is also possible to use a cyclic module such as C5-C6 cycloalkyl, C4N, C5N, C4O, C5O heterocyclic hydrocarbon, phenyl optionally substituted with one or two C1-C6 alkyl groups as a non-nucleoside bifunctional spacer module. Preferred bifunctional building blocks include C3-C6 alkyl modules and three-to six-ethylene glycol chains. Table 1 shows some examples of nucleotide bifunctional spacer building blocks with different hydrophilicity, different rigidity and different charges. An oxygen atom is connected to an acid labile protecting group (preferably dimethoxytrityl), and another is a part for phosphoramidite.

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

A simple way to construct a spacer S or to introduce a spacer module into a 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 change the spacer length (the distance between binding pair members a and b) and the flexibility of the spacer because the length of the duplex is shortened compared to that of the single strand and the duplex is more rigid than the single strand.

In one embodiment, oligonucleotides modified with a functional moiety X are used for hybridization. The oligonucleotides used for hybridization can have one or two terminal extensions that do not hybridize to the spacer and/or be internally branched. These 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 other hybridization events. In one embodiment, the oligonucleotide hybridized to the terminal extension is a labeled oligonucleotide. This labeled oligonucleotide can further contain terminal extensions or be branched to allow for further hybridization, thereby obtaining polynucleotide aggregates or dendrimers. Preferably, poly-oligonucleotide dendrimers are used to generate multiple labels or to achieve higher local concentrations of X.

In one embodiment, the spacer S has a main chain length of 1 to 100 nm. In other words, 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 member of a binding pair, and the spacer S has a main chain length of 1 to 95 nm.

"a':a" and "b:b'" each independently represent a binding pair. In one embodiment, each binding pair member a and b each has a length of at least 2.5 nm.

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

The binding affinity of (within) a:a' or b':b', respectively, is at least 10 ^⁻¹ /mol. These two binding pairs are differentiated. For a binding pair, differentiation is considered if, for example, reciprocal binding, e.g., the binding of a and a' to b or b', is 10% or less of the affinity within the a:a' pair. Reciprocal binding is also preferred, 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 differentiation is so significant that the reciprocal (cross-reactive) binding is 1% or less compared to the specific binding affinity within the binding pair.

In one embodiment, a':a and b:b' are binding pairs, and the members of the binding pairs a':a and b: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.

The term "leucine zipper domain" is used to refer to a generally recognized dimerization domain characterized by the presence of a leucine residue at every seventh residue in a stretch of approximately 35 residues. Leucine zipper domains are peptides that promote the oligomerization of proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz, H.W. et al., Science 240 (1988) 1759-1764) and have since been found in a variety of different proteins. Among the known leucine zippers are 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 WO 94/10308, 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 (bound pairs) held together by an alpha-helical coiled-coil. The coiled-coil has 3.5 residues per turn, meaning every seventh residue occupies an equivalent position relative to the helical axis. The regular arrangement of leucine residues within the coiled-coil stabilizes the structure through hydrophobic and van der Waals interactions.

If a leucine zipper domain forms 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 to b and b'. Leucine zipper domains can be isolated from naturally occurring proteins known to contain such domains, such as transcription factors. For example, one leucine zipper domain can be derived from the transcription factor fos, while the second can be derived from the transcription factor jun. Leucine zipper domains 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 pairs a':a and b:b', i.e., a, a', b, and b', represent leucine zipper domains, and the spacer S consists of amino acids. In this embodiment, it is possible to easily generate the construct a-S-b. It is straightforward for one skilled in the art to vary the length of such spacer S as desired. Such polypeptides can be produced synthetically or recombinantly.

For example, a recombinant fusion protein comprising a spacer polypeptide fused to a leucine zipper peptide at the N-terminus and to a leucine zipper peptide at the C-terminus can be expressed in a suitable host cell according to standard techniques. A DNA sequence encoding the desired peptide spacer can 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 the amino acid sequence motif GGGGS (SEQ ID NO: 13) one or more times. 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', as well as b and b', 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 the binding pair a':a, respectively, do not bind to the sequences in the binding pair b:b', and vice versa. In one embodiment, the present invention relates to at least dual binders of Formula I, wherein the binding pairs a:a' and b:b', respectively, are hybridizing nucleic acid sequences, and wherein the hybridizing nucleic acid sequences of the 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 the hybridization of 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., no interference with b or b') of a binding pair (e.g., a:a') is considered to be specific and non-interfering (e.g., no interference with b or b') if the melting temperature of the a:a' pair is at least 20°C higher than that of any possible combination of b or b' (i.e., a:b; a:b'; a':b; and a':b').

The nucleic acid sequence that forms a binding pair, such as (a:a') or any other nucleic acid sequence-based binding pair, can contain any naturally occurring nucleobase or its analog and can have a modified or unmodified backbone as described above, as long as it is capable of forming a stable duplex via polybase pairing. Stable means that the melting temperature of the duplex is above 37°C. Preferably, the duplex consists of two fully complementary single strands. However, mismatches or insertions are possible, as long as stability at 37°C is achieved.

As the skilled person will appreciate, the nucleic acid duplex can be further stabilized by interstrand cross-linking. Several suitable cross-linking methods are known to the skilled person, for example using psoralen or thionucleoside-based methods.

Preferably, the nucleic acid sequence representing a member of a binding pair will consist of between 12 and 50 nucleotides. It is also preferred that such a nucleic acid sequence 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. Although it is certainly 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 easily designed to provide more than two pairs of orthogonally complementary oligonucleotides, thereby allowing for the simple generation and use of more than two binding pairs. Another advantage of using hybridizing nucleic acid sequences in the binding agents of the present invention is that modifications can be easily introduced into the nucleic acid sequence. Modified building blocks are commercially available, which, for example, allow for the simple synthesis of linkers containing 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 b' and/or S, as long as they represent oligonucleotides.

In a preferred embodiment, the spacer S contained 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':a and b:b', respectively. In the case where the monovalent binders A and B are polypeptides, they can each be easily coupled to the hybridizing nucleic acid sequences a' and b', respectively. The length of the spacer S contained in such constructs can be easily varied in any desired manner. Based on the three constructs a-S-b, A-a' and b'-B, the binders of formula I can be most easily obtained by hybridization between a':a and b:b', respectively, according to standard procedures. When spacers of different lengths are used, the resulting constructs provide binders that are otherwise identical but have different distances between the 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 appropriate spacers is important for optimizing the binding of a binding agent as disclosed herein to its antigen(s).

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 an enantiomeric L-DNA. In a preferred embodiment, a, a', b, and b', and the spacer S are enantiomeric L-DNA or L-RNA. In a preferred embodiment, a, 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 simply constructed by hybridizing these hybridizable ends to each other. The resulting spacer construct comprises an oligonucleotide duplex portion. Obviously, in the case of constructing a spacer by the method described, the sequence of the hybridizable oligonucleotide entity forming the duplex is selected in such a way that hybridization with or interference with the binding pair a:a' and b:b' cannot occur.

As described above, the monovalent specific binders A and B of Formula I can be nucleic acids. In one embodiment of the present invention, a', a, b, 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 procedures and combined by hybridization according to convenient standard procedures. Preferably, the functional moiety X is selected from the group consisting of a binding group, a labeling group, an effector group, and a reactive group.

If more than one functional moiety X is present, each such functional moiety may in each case independently 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, group X is a binding group. As will be apparent to one skilled in the art, the binding group X will be selected so as not to interfere with the a':a and b:b' pairs.

Examples of binding groups are partners of a bioaffine binding pair that specifically interact with the other partner of the bioaffine binding pair. Suitable bioaffine binding pairs are a hapten or antigen and an antibody; biotin or a biotin analog (such as aminobiotin, iminobiotin, or desthiobiotin) and avidin or streptavidin; sugars and lectins, oligonucleotides and complementary oligonucleotides, receptors and ligands (such as 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, b', or S of the compound of formula I. Preferably, the smaller partner of the bioaffine binding pair, such as biotin or its analog, receptor ligand, hapten, or oligonucleotide, is covalently bound to at least one of a', a, S, 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 a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin; an oligonucleotide and a steroid hormone.

In one embodiment, the functional moiety X is a reactive group. The reactive group can 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 maleimido, succinimidyl, dithiopyridyl, nitrophenyl ester, hexafluorophenyl ester.

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

The labeling group can be selected from any known detectable group. In one embodiment, the labeling group is selected from dyes such as luminescent labeling groups, such as chemiluminescent groups (e.g., acridinium esters or dioxetanes or fluorescent dyes such as fluorescein, coumarin, rhodamine, oxazine, resorufin, 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., EP0061888), 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(L ₁ L ₂ L ₃ )] _n -YX _m A (II)

wherein M is a divalent or trivalent metal cation selected from rare earth or transition metal ions, L ₁ , L ₂ and L ₃ are the same or different and represent a ligand having at least two nitrogen-containing heterocycles, wherein L ₁ , L ₂ and L ₃ are bound to the metal cation via a nitrogen atom, X is a reactive functional group covalently bound to at least one of the ligands L ₁ , L ₂ and L ₃ via a linker Y, n is an integer from 1 to 10, preferably from 1 to 4, m is 1 or 2, and preferably 1, and A refers to a counter ion that may be required for charge balance.

Preferably, the metal complex is a luminescent metal complex, i.e., a metal complex that undergoes a detectable luminescence reaction upon appropriate excitation. The luminescence reaction can be detected, for example, by fluorescence or by electrochemiluminescence measurement. 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. Particularly preferred are ruthenium, iridium, rhenium, chromium, and osmium. Most preferred is ruthenium.

Ligands L ₁ , L ₂ and L ₃ are ligands having at least two nitrogen-containing heterocyclic rings. Preferred are aromatic heterocyclic rings such as bipyridyl, bipyrazyl, tripyridyl and phenanthrolyl. Particularly preferably, ligands L ₁ , L ₂ and L ₃ are selected from bipyridine and phenanthroline ring systems.

In addition, the complex may contain one or more counterions A to balance the charge. Examples of suitable negatively charged counterions are halides, OH- ^, carbonates, hydrocarbyl carboxylates such as trifluoroacetates, sulfates, hexafluorophosphates, and tetrafluoroborate groups. Hexafluorophosphates, trifluoroacetates, and tetrafluoroborate groups are particularly preferred. Examples of suitable positively charged counterions are monovalent cations such as alkali metals and ammonium ions.

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

Therapeutically active substances have different ways of being effective, for example in suppressing cancer. They can damage the DNA template by alkylation, by crosslinking, or by double-stranded cleavage of the DNA. Other therapeutically active substances can block RNA synthesis by intercalation. Some agents are spindle poisons, such as vinca alkaloids, or antimetabolites that inhibit enzyme activity, or hormones and anti-hormones. The effector group X can be selected from alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxins, nitrosoureas, hormones and anti-hormones, and toxins.

Currently preferred alkylating agents include cyclophosphamide, chlorambucil, busulfan, melphalan, thiotepa, ifosphamide (ifosfamide), and nitrogen mustard.

Currently preferred antimetabolites include methotrexate, 5-fluorouracil, cytosine arabinoside, 6-thioguanine, and 6-mercaptopurine.

Currently preferred antitumor antibiotics include doxorubicin, daunorubicin, idarubicin, nimitoxantron, dactinomycin, bleomycin, mitomycin, and plicamycin.

Currently preferred spindle poisons can be exemplified by maytansine and maytansinoids, and vinca alkaloids and epipodophyllotoxins can be exemplified by vincristine, vinblastin, vindesine, etoposide, and teniposide.

Currently preferred nitrosoureas include carmustin (carmustine), lomustin (lomustine), semustin (semustine), and streptozocin.

Currently preferred hormones and anti-hormones include adrenocortical steroids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, and antiandrogens.

Other preferred randomized synthetic agents can be exemplified by dacarbazine, hexamethylmelamine, hydroxyurea, mitotane, procarbazide (procarbazine), cisplatin, and carboplatin.

Functional moiety X is covalently bound to at least one of (a'), (a), (b), (b'), or S, or via another binding pair. The functional moiety X may be present once or a number (n) of times. (n) is an integer and is 1 or more than 1. Preferably, (n) is between 1 and 100. Also preferably, (n) is 1-50. In certain embodiments, n is 1 to 10, or 1 to 5. In other embodiments, n is 1 or 2.

For the covalent attachment of the functional moiety X to at least one of a', a, b, b' or S, any suitable coupling chemistry can be used. A skilled person can easily select such coupling chemistries from standard protocols. It is also possible to incorporate the functional moiety during the synthesis of a', a, b, b' or S by using suitable building blocks.

In a preferred embodiment, the functional moiety X is bound to a, b, or S of the binding agent defined in Formula I.

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

In a preferred embodiment, the functional moiety X is covalently bound to a, b or S of the binding agent defined in 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 (WO2007/059816). 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 that are used to introduce 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 are used as CPG for end labeling or as phosphoramidites for internal labeling (see Wojczewski, C. et al., Synlett 10 (1999) 1667-1678).

Bifunctional modifier building blocks

Bifunctional modifier building blocks link a functional moiety or a protected functional moiety (if necessary) to a phosphoramidite group to attach the building block to the terminal hydroxyl group of a growing oligonucleotide chain at the 5' end (regular synthesis) or 3' end (inverted synthesis).

Preferably, the bifunctional modifier building block is a non-nucleoside compound. For example, this type of modified agent building block is a C2-C18 alkyl, alkenyl, or alkynyl carbon chain, and the alkyl, alkenyl, or alkynyl chain can be interrupted by other ethyleneoxy and/or amide modules to increase the hydrophilicity of the spacer and the entire joint structure. Cyclic modules such as C5-C6-cycloalkyl, C4N, C5N, C4O, C5O-heterocycloalkyl, and phenyl (optionally substituted with one or two C1-C6 alkyl groups) can also be used as non-nucleoside bifunctional modification building blocks. Preferred modified bifunctional building blocks include C3-C6 alkyl modules and tri- to hexa-ethylene glycol chains. Non-limiting but preferred examples of bifunctional modifier building blocks are provided in Table II below.

Table II:

Trifunctional modifier building blocks

The trifunctional building block connects (i) a functional module or (if necessary) a protected functional module, (ii) a phosphoramidite group to couple the reporter or functional module or (if necessary) a protected functional module to the hydroxyl group of the growing oligonucleotide chain during oligonucleotide synthesis, and (iii) a hydroxyl group protected with an acid-labile protecting group, preferably a dimethoxytrityl protecting group. Upon removal of this acid-labile protecting group, the hydroxyl group is released and can react with other phosphoramidites. Therefore, the trifunctional building block allows the functional module to be placed at any position in the oligonucleotide. The trifunctional building block is also a prerequisite for the use of solid supports such as controlled pore glass (CPG) for synthesis, which is used for 3' end labeling of oligonucleotides. In this case, the trifunctional building block is linked to a functional moiety or, if necessary, a protected functional moiety via a C2-C18 alkyl, alkenyl, or alkynyl carbon chain, which can be interrupted by further ethyleneoxy and/or amide moieties to increase the hydrophilicity of the spacer and thus the entire 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. Upon removal of this protecting group, the hydroxyl group is released, which can then react with the phosphoramidite.

Trifunctional building blocks can be non-nucleoside or nucleoside.

The trifunctional building blocks of non-nucleosides are C2-C18 alkyl, alkenyl, alkynyl carbon chains, and the alkyl, alkenyl, alkynyl groups are optionally interrupted with additional ethyleneoxy and/or amide moieties to increase the hydrophilicity of the spacer and thus the entire linker structure. Other trifunctional building blocks are cyclic groups such as C5-C6 cycloalkyl, C4N, C5N, C4O, C5O heterocycloalkyl, phenyl, which are optionally substituted with one or two C1-C6 alkyl groups. Cyclic and non-cyclic groups can be substituted with a -(C1-C18)alkyl-O-PG group, and the C1-C18 alkyl group contains (ethyleneoxy)n, (amide)m moieties, where n and M are independently 0-6, and PG is an acid-labile protecting group. Preferred trifunctional building blocks are C3-C6 alkyl, cycloalkyl, C50 heterocycloalkyl modules, optionally containing one amide bond, and substituted with C1-C6 alkylO-PG groups, wherein PG is an acid-labile protecting group, preferably monomethoxytrityl, dimethoxytrityl, pixyl, xanthyl, most preferably dimethoxytrityl.

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

Table III: Examples of non-nucleoside trifunctional modifier building blocks

Nucleoside Modifier Building Blocks:

Whenever it is necessary not to affect the hybridization properties of oligonucleotides compared to non-modified oligonucleotides, internal labeling is performed using nucleoside modifier building blocks. Thus, nucleoside building blocks contain base analogs or bases that are still capable of hybridizing with complementary bases. The general formula of the labeling compound is given in Formula II, which is used to label the nucleic acid sequence of one or more of a, a', b, b' or S contained in the binding agent of Formula I according to the present invention.

Formula II:

wherein PG is an acid-labile protecting group, preferably a monomethoxytrityl, dimethoxytrityl, pixyl, xanthene group, and most preferably a dimethoxytrityl group, wherein Y is a C2-C18 alkyl, alkenyl, or alkynyl group, wherein the alkyl, alkenyl, or alkynyl group may contain an ethyleneoxy and/or amide module, wherein Y is preferably a C4-C18 alkyl, alkenyl, or alkynyl group and contains an amide module, and wherein X is a functional module.

Specific base positions can be selected for such substitutions to minimize the effect 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 analogs: 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 aza amino A substituted at C7; pseudouracil substituted at N1 and pyrimidine substituted at N2.

Non-limiting but preferred examples of nucleoside trifunctional building blocks are given in Table IV.

Table IV:

In Tables II, III and IV, one of the terminal oxygen atoms of the bifunctional module or one of the terminal oxygen atoms of the trifunctional module is part of a phosphoramidite, which is not shown in full detail but is obvious 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 for introducing covalently bound functional modules into linker or spacer molecules. In this approach, amino groups are introduced during solid phase synthesis using bifunctional or trifunctional building blocks. After cleavage from the support and purification, the amino-modified oligonucleotide is reacted with an activated ester of the functional module or a bifunctional reagent in which one of the functional groups is an active ester. Preferred active esters are NHS esters or pentafluorophenyl esters.

Post-synthetic modifications are particularly useful for introducing functional moieties that are unstable during solid-phase synthesis and deprotection. Examples include modification with triphenylphosphincarboxymethyl ester for Staudinger ligation (Wang, C.C. et al., Bioconjugate Chemistry 14 (2003) 697-701), modification with digoxigenin, or introduction of maleimide groups using commercially available sulfo-SMCC.

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

Preferably, the additional binding pair that can bind to the functional moiety X is a leucine zipper domain or a hybridizing nucleic acid. In the case where the functional moiety X binds to at least one of a', a, b, b', or S via another binding pair member, the binding pair member that binds to X and the binding pairs a':a and b:b' are each selected to have different specificities. The binding pairs a:a', b:b', and the binding pair that binds to X each bind to (e.g., hybridize with) their respective partners 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 properties of the conjugate.

In case the conjugate is a naturally occurring protein or recombinant polypeptide of 50 to 500 amino acids, standard procedures for protein conjugate synthetic chemistry are described in textbooks and can be easily followed by the skilled artisan (Hackenberger, C.P. et al., Angew. Chem., Int. Ed., 47 (2008) 10030-10074).

In one embodiment, a maleimide moiety is used to react with cysteine residues within a protein. This is a preferred coupling chemistry when, for example, a Fab or Fab' fragment of an antibody is used as a monovalent conjugate. Alternatively, in one embodiment, conjugation of a binding pair member (a' or b', respectively, of Formula I) to the C-terminal end of the conjugate polypeptide is performed. For example, C-terminal modification of a protein (e.g., a Fab fragment) can be performed as described (Sunbul, Murat and Yin, Jun, Organic & Biomolecular Chemistry 7 (2009) 3361-3371).

Typically, site-specific reactions and covalent coupling of binding pair members to monovalent polypeptide conjugates are based on converting natural amino acids into amino acids with orthogonal reactivity to the reactivity of other functional groups present in the protein. For example, specific cysteines within rare sequence contexts can be enzymatically converted to aldehydes (see Frese, M.-A. et al., ChemBioChem 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification by exploiting the specific enzymatic reactivity of certain enzymes with naturally occurring amino acids in a given sequence context (see, for example: Taki, M. et al., Protein Engineering, Design & Selection 17 (2004) 119-126; Gautier, A. et al., Chemistry & Biology 15 (2008) 128-136; Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403 for protease-catalyzed C-N bond formation, while Mao, H. et al., J. Am Chem Soc. 126 (2004) 2670-2671 for sortase-mediated protein ligation and Proft, T., Biotechnol. Lett 32 (2010) 1-10 for review).

Site-specific reaction and covalent coupling of binding pair members to monovalent polypeptide conjugates can also be achieved by selective reaction of terminal amino acids with appropriate modifying agents.

Site-specific covalent coupling can be achieved using the reactivity of the N-terminal cysteine with benzonitril (Ren, Hongjun, Xiao et al., Angewandte Chemie, International Edition 48 (2009) 9658-9662).

Native chemical ligation can also rely on a C-terminal cysteine residue (Taylor, E. Vogel, Imperiali, B., Nucleic Acids and Molecular Biology 22 (2009) (Protein Engineering) 65-96).

EP1074563 describes a conjugation method based on the faster reaction of cysteines within negatively charged stretches of amino acids with cysteines located in positively charged stretches of amino acids.

Monovalent conjugates can also be synthetic peptides or peptide mimetics. 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 Chemistry 20 (2009) 1281-1295). Since a wide variety of orthogonal functional groups are discussed and can be introduced into synthetic peptides, conjugation of such peptides to linkers is standard chemistry.

To obtain single-labeled proteins, conjugates with a 1:1 stoichiometry can be separated from other conjugated 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, single-conjugated proteins are readily separated from unlabeled proteins and proteins carrying more than one linker, as differences in charge and molecular weight can be exploited for separation. Fluorescent dyes are valuable for purifying binders from unbound components, such as labeled monovalent binders.

Thus, in one embodiment, it is preferred to use binding pair members (a' and/or b', respectively, of Formula I) that are labeled with fluorescent dyes (e.g., synthesized during synthesis using a bifunctional or trifunctional modifier building block in combination with a bifunctional spacer building block) to form the binding agents 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. In a preferred embodiment, the spacer S and the sequences a, a', b, and b' are DNA, and both a' and b' are labeled with different fluorescent dyes.

In one embodiment, the present invention relates to a bispecific binder of formula I: A-a':a-S-b:b'-B:X(n); wherein A and B are monovalent specific binders, wherein a':a and b:b' represent binding pairs, a':a and b:b' have different specificities, wherein S represents a spacer, wherein (:X) refers to a functional module that binds to at least one of a', a, b, b' or S via another binding pair, wherein (n) is an integer and is at least 1, wherein - represents a covalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm.

In a preferred embodiment, the binding pairs a':a and b:b' are hybridizing nucleic acid sequences, the spacer S is a nucleic acid, and the other binding pair that binds to the functional module X is also a nucleic acid. In this embodiment, preferably, the spacer S is constructed to contain, in addition to the two specifically hybridizing sequences a and a' and b and b', one or more other sequences that can also hybridize to their complementary sequences. In this embodiment, the functional module X is bound to the spacer S via the other binding pair that also consists of hybridizing nucleic acid sequences.

Monovalent binders used to construct binding agents as disclosed herein must have a _Kdiss of 10 ⁻² /sec to 10 ⁻⁵ /sec. It is also preferred that monovalent binders used to construct binding agents as disclosed herein must have _{a Kdiss} of 10 ⁻³ /sec to 10 ⁻⁵ /sec.

Preferably, in the binder according to formula I, each of the monovalent binders A and B has _{a Kdiss} of 10 ⁻² /s to 10 ⁻⁵ /s, further preferably 10 ⁻³ /s to 10 ⁻⁵ /s.

Preferably, the binding agent according to formula I has _{a Kdiss of} 10 ⁻⁵ /sec or better, further preferably 10 ⁻⁶ /sec or better. Also preferably, the binding agent according to formula I has _{a Kdiss of} 10 ⁻⁷ /sec or better.

As will be appreciated by the skilled artisan, K is a temperature-dependent _value . Logically, the K value of a _binding agent according to the invention is determined at the same temperature. As will be appreciated, the K _value is preferably determined at the same temperature at which the binding agent will be used (e.g., the assay will be performed). In one embodiment, the K value is established at room temperature, i.e., at 20°C, 21°C, 22°C, 23°C, ₂₄ °C, or 25°C, respectively. In one embodiment, the K _value is established at 4 or 8°C, respectively. In one embodiment, the K _value is established at 25°C. In one embodiment, the K _value is established at 37°C.

As already mentioned above, it is now possible and quite simple to generate binders as defined in formula I. If desired, a complete library of binders according to formula I can be easily provided, analyzed, and the most potent binders of such a library produced on a large scale.

The libraries referred to above refer to a complete collection of binding agents according to Formula I, wherein each of A, a, a', b, b' and B is identical, and wherein the length of the spacer S is adjusted to best meet the requirements listed for the binding agent. A spacer ladder spanning the entire range of 1 to 100 nm with a gradient of approximately 10 nm intervals can be readily used initially. The spacer length is then further optimized around the optimal length identified in the first round.

In one embodiment, the present invention relates to a method for producing a binding agent of formula I: A-a':a-S-b:b'-B:X(n); wherein A and B are monovalent binders, wherein a':a and b:b' are binding pairs, wherein a' and a do not interfere with the binding of b to b' and vice versa, wherein S is a spacer of at least 1 nm in length, wherein (:X) refers to a functional moiety that binds to at least one of a', a, b, b' or S covalently or via a binding pair, wherein (n) is an integer and is at least 1, wherein - represents a covalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm, the method comprising the steps of: a) synthesizing A-a' and b'-B separately, b) synthesizing the linker a-S-b, and c) forming a binding agent of formula I, wherein the functional moiety X that binds to at least one of a', a, b, b' or S is bound in step a), b) or c).

In a preferred embodiment of this method, several linker molecules with spacers of different lengths are synthesized and used to form binders comprising spacers of different lengths according to Formula I, and those binders are selected that have at least a 5-fold improvement in K _off relative to the better of the two monovalent binders. In one embodiment, selection of binders with the desired K _off is performed by BiaCore analysis as disclosed in Example 2.8.

The binding agents according to the present invention can be used, for example, to strongly bind to an analyte in an immunoassay. If, for example, the analyte has at least two non-overlapping epitopes, the binding agents of the present invention are constructed so that the spacer S has an optimal length for cooperative binding of these epitopes by the monovalent binder. This improvement may be extremely useful, for example, in methods for detecting analytes using such binding agents. Therefore, in one embodiment, the present invention relates to the use of a binding agent as disclosed above for detecting an analyte of interest. In certain embodiments, the detection method used is an enzyme-linked immunosorbent assay (ELISA), a direct, indirect, competitive or sandwich immunoassay using any suitable signal detection method, such as electrochemiluminescence, or the binding agent is used in immunohistochemistry.

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 should be understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

Sequence Listing Description

1. Antibody fragments

SEQ ID NO: 1 _VH (mAb 1.4.168):

QCDVKLVESGGGLVKPGGSL KLSCAASGFT FSDYPMSWVR QTPEKRLEWV

ATITTTGGTYT YYPDSIKGRF TISRDNAKNT LYLQMGSLQS EDAAMYYCTR

VKTDLWWGLA YWGQGTLVTV SA

SEQ ID NO: 2 _VL (mAb 1.4.168):

QLVLTQSSSA SFSLGASAKL TCTLSSQHST YTIEWYQQQP LKPPKYVMEL

KKDGSHTGD GIPDRFSGSS SGADRYLSIS NIQPEDESIY ICGVGDTIKE

QFVYVFGGGT KVTVLG

SEQ ID NO: 3 _VH (mAb 8.1.2):

EVQLQQSGPA LVKPGASVKM SCKASGFTFT SYVIHWVKQK PGQGLEWIGY

LNPYNDNTKY NEKFKGKATL TSDRSSSTVY MEFSSLTSED SAVYFCARRG

IYAYDHYFDY WGQGTSLTVS S

SEQ ID NO: 4 V _L (mAb 8.1.2):

QIVLTQSPAI MSASPGEKVT LTCSASSSVN YMYWYQQKPG SSPRLLIYDT

SNLASGVPVR FSGSGSVTSY SLTISRMEAE DAATYYCQQW STYPLTFGAG

TKLELK

2. ssDNA sequence

a) 17-mer ssDNA (covalently bound at the 5' end to Fab' of anti-troponin T monoclonal antibody a or Fab'1.4.168 targeting IGF-1R)

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

b) 19-mer ssDNA (covalently bound at the 3' end to Fab' of anti-troponin T monoclonal antibody b or Fab'8.1.2 targeting phosphorylated IGF-1R)

5’-A GTC TAT TAA TGC TTC TGC-3’ (SEQ ID NO: 6)

c) Complementary 19-mer ssDNA (used as part of the adapter)

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

d) Complementary 17-mer ssDNA (used as part of the adapter)

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

3. Sequence of Troponin T Epitope

SEQ ID NO:9 = ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAE amide, wherein U represents β-alanine. (Epitope "A" of the antibody anti-troponin α)

SEQ ID NO:10 = SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O represents amino-trioxa-octanoic acid. (Epitope "B" of the antibody anti-troponin b)

4. Sequence of IGF-1R/IR epitope

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

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

5. Protein linker and tag sequences

SEQ ID NO: 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)

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Analytical gel filtration experiments evaluating the assembly efficiency of anti-pIGF1-R dual binders. Panels a, b, and c show the elution profiles of the various dual binder components (Fluorescein-ssFab'1.4.168, Cy5-ssFab'8.1.2, and linker DNA (T=0); Fab' indicates the Fab' fragment conjugated to a single-stranded oligonucleotide). Panel d shows the elution profile after the three components required to form the bivalent binder were mixed in a 1:1:1 molar ratio. The thicker (bottom) curve represents absorbance measured at 280 nm and indicates the presence of ssFab' protein or linker DNA, respectively. The thinner top curves (absorbance at 495 nm) in b) and d) indicate the presence of fluorescein, while the thinner top curve in a) and the middle curve in d) (absorbance at 635 nm) indicate the presence of Cy5. Comparison of the elution volumes of the individual dual binder components (VE _{ssFab'1.4.168} - 15 ml; VE _ssFab'8.1.2 - 15 ml; VE _linker - 16 ml) with the elution volume of the reaction mixture (VE _mixture - 12 ml) indicated that the dual binder assembly reaction was successful (yield: -90%). The major peak at 280 nm, representing the eluted dual binder, overlapped well with the major peaks in the 495 nm and 635 nm channels, demonstrating the presence of both ssFab'8.1.2 and ssFab'1.4.168 in the peak representing the bivalent binder.

Figure 2. Schematic diagram of a Biacore ^™ experiment. This schematic and illustrative diagram shows two binding molecules in solution: the T0-T-Dig (linker 16) bivalent binder and the T40-T-Dig (linker 15) bivalent binder. These two bivalent binders differ only in their linker length (a digoxigenin-ylated T in the middle between the two hybridizing nucleic acid sequences, with no additional Ts versus 40 additional Ts (20 on each side of the central T-Dig)). Additionally, ssFab fragments 8.1.2 and 1.4.168 were used.

Figure 3 is a Biacore ^™ sensorgram with an overlay of three kinetics showing the interaction of a 100 nM bivalent binder consisting of ssFab'8.1.2 and ssFab'1.4.168 hybridized on a T40-T-Dig ssDNA linker (i.e., Linker 15) with the immobilized peptide pIGF-1R, compared to the binding characteristics of 100 nM ssFab'1.4.168 or 100 nM ssFab'8.1.2 for the same peptide. The highest binding performance was obtained with the dual binder construct, clearly demonstrating that the cooperative binding effect of the dual binder improves affinity for the target peptide pIGF-1R.

Figure 4 shows a Biacore ^™ sensorgram with an overlay of three kinetics showing the interaction of a bivalent binder 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, clearly demonstrating that the cooperative binding effect of the dual binder improves specificity for the target peptide pIGF-1R compared to, for example, the phosphorylated insulin receptor peptide (pIR).

Figure 5 is a Biacore ^™ sensorgram with an overlay of two kinetics showing the interaction of 100 nM bivalent binder 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 100 nM ssFab'8.1.2 and 100 nM ssFab'1.4.168 without linker DNA. Optimal binding performance was achieved with the bivalent binder alone, while the ssFab' mixture without a linker showed no observable cooperative binding effect, despite the fact that the total concentration of these ssFab's was already 200 nM.

Figure 6 Schematic diagram of the Biacore ^™ sandwich assay. This assay was used to investigate the epitope accessibility of two antibodies on the phosphorylated IGF-1R peptide. <MIgGFcγ>R presents a rabbit anti-mouse antibody used to capture the murine antibody M-1.4.168. M-1.4.168 is then used to capture the pIGF-1R peptide. Finally, M-8.1.2 forms a sandwich consisting of M-1.4.168, the peptide, and M-8.1.2.

Figure 7. Biacore ^™ sensorgram showing the binding signal (thick line) of secondary antibody 8.1.2 to the pIGF-1R peptide after capture by antibody 1.4.168 on a Biacore ^™ chip. Other signals (thin lines) are control signals: from top to bottom, 500 nM 8.1.2, 500 nM 1.4.168; 500 nM target-unrelated antibody <CKMM>M-33-IgG; and 500 nM target-unrelated control antibody <TSH> M-1.20-IgG, respectively. No binding events were detected in any of these controls.

Figure 8 Schematic diagram of the Biacore ^™ assay showing biotinylated dual binders on the sensor surface. Amino-PEO-biotin was captured on flow cell 1 (=FC1) (not shown). Bivalent binders with increasing linker lengths 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 20 Ts upstream and downstream) are shown, respectively). Analyte 1: IGF-1R peptide containing the M-1.4.168ssFab' epitope at the right-hand end of the peptide (top line), without the M-8.1.2ssFab' phospho-epitope because this peptide is not phosphorylated; Analyte 2: pIGF-1R peptide containing the M-8.1.2ssFab' phospho-epitope (P) and the M-1.4.168ssFab' epitope (second line); Analyte 3: pIR peptide containing the cross-reactive M-8.1.2ssFab' phospho-epitope but not the M-1.4.168 epitope (third line).

Figure 9 Kinetic data of a dual binder experiment. The T40-T-Bi linker dual binder (= T40 in the figure) with ssFab'8.1.2 and ssFab'1.4.168 shows a 1300-fold lower off-rate for pIGF-1R (kd = 2.79E-05/s) compared to pIR (kd = 3.70E-02/s).

FIG10 is a Biacore ^™ sensorgram showing the concentration-dependent measurement of pIGF-1R peptide (phosphorylated IGF-1R peptide) by the T40-T-Bi dual binder. The assay setup is as depicted in FIG8 . A concentration series of pIGF-1R peptide was injected at 30 nM, 10 nM, twice at 3.3 nM, 1.1 nM, 0.4 nM, and 0 nM. The corresponding data are presented in the table in FIG9 .

Figure 11 shows a Biacore ^™ sensorgram showing the concentration-dependent measurement of IGF-1R peptide (non-phosphorylated IGF-1R peptide) by the T40-T-Bi dual binder. The assay setup is as depicted in Figure 8. A concentration series of IGF-1R peptide was injected at 300 nM, 100 nM, twice at 33 nM, 11 nM, 4 nM, and 0 nM. The corresponding data are presented in the table in Figure 9.

FIG12 Biacore ^™ sensorgram showing the concentration-dependent measurement of pIR peptide (phosphorylated insulin receptor peptide) by the T40-T-Bi dual binder. The assay setup is as depicted in FIG8 . A concentration series of pIR peptide was injected at 100 nM, then twice at 33 nM, 11 nM, 4 nM, and 0 nM. The corresponding data are given in the table depicted in FIG9 .

Example

Example 1: Bivalent binders to troponin T

1.1 Monoclonal Antibodies and Fab’ Fragments

Two monoclonal antibodies are used that bind to human cardiac troponin T at different, non-overlapping epitopes (epitope A' and epitope B', respectively). These two antibodies are used in the current Roche Elecsys ^™ Troponin T assay, in which troponin T is detected in a sandwich immunoassay format.

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

Purified monoclonal antibodies were protease digested with preactivated papain (anti-epitope A' mAb) or pepsin (anti-epitope B' mAb) to generate F(ab') ₂ fragments, which were subsequently reduced to Fab' fragments (A and B, respectively, in Formula I (A-a':aSb:b'-B:Xn)) using a low concentration of cysteamin at 37°C. The reaction was stopped by separating the cysteamin from the peptide-containing sample fractions on a Sephadex G-25 column (GE Healthcare).

1.2 Conjugation of Fab’ fragments to ssDNA oligonucleotides

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

Prepare Fab' fragment-ssDNA conjugates A'' and B'' respectively:

a) Fab’-anti-troponin T <epitope A’>-ssDNA conjugate (=A’’)

For the preparation of Fab'-anti-troponin T <epitope A'>-ssDNAa-conjugate A'', a derivative of SEQ ID NO: 5 was used, namely 5'-AGT CTA TTA ATG CTT CTG C (=SEQ ID NO: 5)-XXX-Y-Z-3', 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 TFAamino C-6lcaa CPG (ChemGenes), and wherein Z = 4-[N-maleimidomethyl]cyclohexane-1-carboxyl introduced via sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).

b) Fab’-anti-troponin T<epitope B’>-ssDNAb conjugate (=B’’)

For the preparation of Fab'-anti-troponin T<epitope B'>-ssDNA-conjugate (B''), a 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 introduced via phosphoramidite C3 (3-(4,4′-dimethoxytritylamino)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research)), wherein Y = 5′-amino modifier C6 introduced via 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research), and wherein Z = 4-[N-maleimidomethyl]cyclohexane-1-carboxylate introduced via sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Thermo Fischer).

The oligonucleotides of SEQ ID NO: 5 or 6, respectively, have been synthesized by prior art oligonucleotide synthesis methods. Introduction of the maleimido group was 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 construct shown above carries a thiol-reactive maleimido group that reacts with the cysteine residues in the hinge region of the Fab' 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. The singly labeled Fab' fragments (ssDNA:Fab' = 1:1) were purified by anion exchange chromatography (column: MonoQ, GE Healthcare). Efficient labeling and purification were verified by analytical gel filtration chromatography and SDS-PAG.

1.3 Biotinylated linker molecules

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

Linker 1 (= L1), a biotinylated ssDNA linker 1 without a spacer other than biotinylated thymidine,

It has the following composition: 5'-GCA GAA GCA TTA ATA GAC T(biotin-dT)-TGG ACG ACG ATAGAA CT-3'. It contains the ssDNA oligonucleotides of SEQ ID NOs: 7 and 8, respectively, and is biotinylated using biotin-dT (=T-Bi)(5'-dimethoxytrityloxy-5-[N-((4-tert-butylphenylyl)-biotinyl)-aminohexyl)-3-acrylimide]-2'-deoxyuridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch) in the middle of the spacer.

Linker 2 (=L2), a biotinylated ssDNA linker 2 with an 11-mer spacer,

The composition is as follows: 5'-GCA GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5TGG ACGACG ATA GAA CT-3'. It contains the ssDNA oligonucleotides of SEQ ID NOs: 7 and 8, two oligonucleotide segments of 5 thymidines each, and is biotinylated with biotin-dT (5'-dimethoxytrityloxy-5-[N-((4-tert-butylphenylyl)-biotinyl)-aminohexyl)-3-acrylimide]-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, has the following composition:

5'-GCA GAA GCA TTA ATA GAC T T15-(Biotin-dT)-T15TGG ACG ACG ATA GAACT-3'. It contains the ssDNA oligonucleotides of SEQ ID NOs: 7 and 8, two oligonucleotide segments of 15 thymidines each, and is biotinylated using biotin-dT (5'-dimethoxytrityloxy-5-[N-((4-tert-butylphenylyl)-biotinyl)-aminohexyl)-3-acrylimide]-2'-deoxyuridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch).

1.4 Epitopes of Monovalent Troponin T Binders A and B

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:

SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAE amide, wherein U represents β-alanine.

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

SEQ ID NO: 10 = SLKDRIERRRAERAEOOERAEQQRIRAEREKE amide, wherein O represents amino-trioxa-octanoic acid.

As the skilled person will appreciate, it is possible to combine these two epitope-containing peptides in two ways, and two 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 compared to the original epitope on the human cardiac troponin T sequence (P45379/UniProtKB). In these cases, the kinetics of the heterobivalent binding effect can be better visualized, for example, by analyzing the binding affinity using Biacore ^™ technology.

1.5 Analysis of biomolecular interactions

For this experiment, a Biacore ^™ 3000 instrument (GE Healthcare) was used at T = 25° C. with a Biacore ^™ SA sensor embedded in the system. Preconditioning was done with three 1 minute injections of 1 M NaCl in 50 mM NaOH and 1 minute of 10 mM HCl at 100 μl/min.

HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% 20) was used as the system buffer. The sample buffer was the same as the system buffer.

The Biacore ^™ 3000 system was driven using control software V1.1.1. Flow cell 1 was saturated with 7 RU of D-biotin. On flow cell 2, 1063 RU of biotinylated ssDNA adapter L1 was immobilized. On flow cell 3, 879 RU of biotinylated ssDNA adapter L2 was immobilized. On flow cell 4, 674 RU of biotinylated ssDNA adapter 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 conjugates were injected at a flow rate of 2 μl/min for 3 minutes. The conjugates were injected continuously to monitor the corresponding saturation signals of each Fab' fragment DNA conjugate on its corresponding 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 corresponding linker. A stable baseline was generated after the linker had been saturated by the Fab' fragment DNA conjugate, which is a prerequisite for further kinetic measurements.

Artificial peptide analytes TnT-1 and TnT-2 were injected into the system as solutions as analytes to interact with the surface-displayed Fab' fragments.

TnT-1 was injected at 500 nM and TnT-2 was injected at 900 nM analyte concentration. Both peptides were injected at 50 μl/min for a 4-minute binding time. Dissociation was monitored for 5 minutes. Regeneration was completed by injecting 50 mM NaOH at 50 μl/min over all flow cells for 1 minute.

Kinetic data were determined using Biaevaluation software (V.4.1). The dissociation rates (kd) (1/s) of the TnT-1 and TnT-2 peptides from the corresponding surface-displayed Fab' fragment combinations were determined according to a linear Langmuir 1:1 fit model. The complex half-life (in minutes) was calculated by solving the first-order kinetic equation: ln(2)/(60 x kd).

result:

The experimental data presented in Tables 1 and 2 demonstrate increased stability of the complex between the analyte (TnT-1 or TnT-2) and various heterobivalent Fab'-Fab' dimers A''-B'' 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) Connector L1

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

b) Connector L2

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

c) Connector L3

Fab’ fragment DNA conjugate A’’ Fab’ fragment DNA conjugate B’’ kd(1/s) t1/2 dissociation (minutes) 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) Connector L1

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

b) Connector L2

Fab’ fragment DNA conjugate A’’ Fab’ fragment DNA conjugate B’’ kd(1/s) t1/2 dissociation (minutes) x x 4.9E-03 2.3 x - 3.5E-02 0.3 - x 1.3E-01 0.1

c) Connector L3

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

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

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

Together, these data demonstrate that the flexibility in linker length inherent in the approach presented in this invention has great utility and advantage.

Example 2: Bivalent binders to phosphorylated IGF-1R

2.1 Monoclonal Antibody Development (mAb8.1.2 and mAb1.4.168)

a) Immunization of mice

BALB/C mice were immunized at weeks 0, 3, 6, and 9. Each immunization received 100 μg of a conjugate containing the phosphorylated peptide pIGF-1R (1340-1366) (SEQ ID NO: 11). This peptide was phosphorylated at tyrosine 1346 (1346-pTyr) and conjugated to KLH via a C-terminal cysteine (Aoc-Cys-MP-KLH-1340). Immunizations were administered intraperitoneally at weeks 0 and 6, and subcutaneously at various sites on the body at weeks 3 and 9.

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. Approximately ¹ x 108 spleen cells from immunized mice were mixed with ² x 107 myeloma cells (P3X63-Ag8653, ATCC CRL1580) and centrifuged at 250 g for 10 minutes at 37°C. The cells were then washed once with RPMI1640 medium without fetal calf serum (FCS) and centrifuged again at 250 g in a 50 ml conical tube. The supernatant was discarded, the cell pellet was gently loosened by tapping, and 1 ml of PEG (molecular weight 4000, Merck, Darmstadt) was added and mixed by pipetting. After incubation for 1 minute in a 37°C water bath, 5 ml of RPMI1640 without FCS was added dropwise over 4-5 minutes at room temperature. This step was repeated with 10 ml of RPMI1640 without FCS. Then, 25 ml of RPMI1640 containing 10% FCS was added, followed by a 30-minute incubation step at 37°C and 5% _CO₂ . After centrifugation at 250 g for 10 minutes at 4°C, the settled cells were aspirated into RPMI1640 containing 10% FCS and plated in hypoxanthine-diazaserine selection medium (100 mmol/l hypoxanthine, 1 μg/ml azaserine in RPMI1640 with 10% FCS). 100 U/ml of interleukin-6 was added to the culture medium as a growth factor. After 7 days, the medium was replaced with fresh medium. On day 10, the primary culture was tested for specific antibodies. Positive primary cultures were cloned in 96-well cell culture plates using a fluorescence-activated cell sorter.

c) Isolation of immunoglobulins from cell culture supernatant

The obtained hybridoma cells were seeded at a density of ¹ x 107 cells in CELLine 1000CL flasks (Integra). Hybridoma cell supernatants containing IgG were collected twice a week. Yields typically ranged from 400 μg to 2000 μg of monoclonal antibody per 1 ml of supernatant. Antibodies were purified from the culture supernatant using conventional protein chemistry methods (e.g., according to Bruck, C., Methods in Enzymology 121 (1986) 587-695).

2.2 Synthesis of hybridizable oligonucleotides

The following amino-modified precursors were synthesized according to standard methods and contain the sequences given in SEQ ID NOs: 5 and 6, respectively. The oligonucleotides given below contain not only the so-called amino linker but also a fluorescent dye. As the skilled person will readily appreciate, this fluorescent dye is very convenient for facilitating the purification of the oligonucleotides themselves and 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-(βL AGT CTA TTA ATG CTT CTG C)-(Spacer C3)3-C7 Amino Linker-; (βL indicates this is an L-DNA oligonucleotide) and

e) 5'-Amino linker-(Spacer C3) 3-(βL-AGT TCT ATC GTC GTC CA)-Fluorescein-3' (βL indicates this is an L-DNA oligonucleotide).

The synthesis was performed on an ABI394 synthesizer at a 10 μmol scale in trityl-on (for 5' amino modification) or trityl-off (for 3' amino modification) mode using commercial CPG as a solid support and standard dA(bz), dT, dG(iBu), and dC(Bz) phosphoramidites (Sigma Aldrich).

The following amidites, amino modifiers, and CPG supports were used to introduce C3-spacers, dyes, and amino moieties, respectively, during oligonucleotide synthesis:

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

The 5′-amino modifier was introduced by using the 5′-amino modifier C6 (6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research);

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

Cy5 ^™ phosphoramidite 1-[3-(4-monomethoxytrityl)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropylphosphoramidityl]propyl]-3,3,3′,3′-tetramethylindodicarbocyanine chloride (Glen Research);

LightCycler Fluorescein CPG500A (Roche Applied Science); and

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

For Cy5-labeled oligonucleotides, dA(tac), dT, dG(tac), and dC(tac) phosphoramidites (Sigma Aldrich) were used, and deprotection was performed with 33% ammonia water at room temperature for 2 hours.

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

Purification of the fluorescein-modified hybridizable oligonucleotide was performed using a two-step procedure: the oligonucleotide was first purified on reverse-phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3 minutes, 20% B in A, 12 minutes, 20-50% B in A, and 25 minutes, 20% B in A, flow rate 1.0 ml/minute, detection at 260 nm). Fractions containing the desired product (monitored by analytical RP HPLC) were combined and evaporated to dryness. (Oligonucleotides modified with a monomethoxytrityl-protected alkylamino group at the 5' end were detritylated by incubation with 20% acetic acid for 20 minutes). The oligomer containing fluorescein as a label was further purified by IEX chromatography on HPLC [Mono Q column: buffer A: sodium hydroxide (10 mM/l; pH approximately 12); buffer B: 1 M sodium chloride dissolved in sodium hydroxide (10 mM/l; pH approximately 12), gradient: 100% buffer A to 100% buffer B over 30 minutes, flow rate 1 ml/min, detection at 260 nm]. The product was desalted by dialysis.

After initial purification on reverse phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3 minutes, 20% B in A, 12 minutes, 20-50% B in A, and 25 minutes, 20% B in A, flow rate 1.0 ml/minute, detection at 260 nm), the Cy5-labeled oligomer was used. The oligomer was desalted by dialysis and lyophilized on a Speed-Vac evaporator to produce a solid, which was frozen at -24°C.

2.3 Activation of hybridizable oligonucleotides

The amino-modified oligonucleotide from Example 2 was dissolved in 0.1 M sodium borate buffer, pH 8.5 (c = 600 μmol) and reacted with an 18-fold molar excess of sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) (c = 3 mg/100 μl) from Thermo Scientific dissolved in DMF. The reaction product was dialyzed extensively against water to remove the hydrolysis product of sulfo-SMCC, 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

The dialysate was concentrated by evaporation and used directly for conjugation with a monovalent binder containing a thiol group.

2.4 Synthesis of a linker oligonucleotide containing hybridizable oligonucleotides at both ends

Oligonucleotides were synthesized by standard methods on an ABI394 synthesizer at a 10 μmol scale in trityl-on mode using commercial dT-CPG as a solid support and using standard dA(bz), dT, dG(iBu), and dC(Bz) phosphoramidites (Sigma Aldrich).

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

Oligonucleotide purification was performed on reverse phase HPLC as described in Example 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. The acetic acid was removed by evaporation. The residue was dissolved in water and lyophilized.

The following amid(es) and CPG supports were used to introduce C18 spacers, digoxigenin, and biotin groups during oligonucleotide synthesis:

Spacer phosphoramidite 18 (18-O-dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research);

Biotin-dT (5′-dimethoxytrityloxy-5-[N-((4-tert-butylphenylyl)-biotinyl)-aminohexyl)-3-acrylimide]-2′-deoxyuridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch);

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

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

The following bridging constructs or linkers were synthesized:

Connector 1: 5’-G CAG AAG CAT TAA TAG ACT-TGG ACG ACG ATA GAA CT-3’

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

Linker 3: 5’-[B-L]G CAG AAG CAT TAA TAG ACT-(Biotin-dT)-TGG ACG ACG ATAGAA CT-3’

Linker 4: 5'-[B-L]G CAG AAG CAT TAA TAG ACT-T5-(Biotin-dT)-T5-TGG ACG ACGATA GAA CT-3'

Linker 5: 5’-[B-L]G CAG AAG CAT TAA TAG ACT-T20-(Biotin-dT)-T20-TGG ACGACG ATA GAA CT-3’

Linker 6: 5’-[B-L]G CAG AAG CAT TAA TAG ACT-T30-(Biotin-dT)-T30-TGG ACGACG ATA GAA CT-3’

Linker 7: 5’-GCA GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5TG GAC GAC GATAGA ACT-3’

Linker 8: 5’-GCA GAA GCA TTA ATA GAC T T10-(Biotin-dT)-T10TGG ACG ACG ATAGAA CT-3’

Linker 9: 5’-GCA GAA GCA TTA ATA GAC T T15-(Biotin-dT)-T15TGG ACG ACG ATAGAA CT-3’

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

Linker 11: 5'-G CAG AAG CAT TAA TAG ACT-Spacer C18-(Biotin-dT)-Spacer C18-TGG ACG ACG ATA GAA CT-3'

Linker 12: 5'-G CAG AAG CAT TAA TAG ACT-(Spacer C18)2-(Biotin-dT)-(Spacer C18)2-TGG ACG ACG ATA GAA CT-3'

Linker 13: 5'-G CAG AAG CAT TAA TAG ACT-(Spacer C18)3-(Biotin-dT)-(Spacer C18)3-TGG ACG ACG ATA GAA CT-3'

Linker 14: 5'-G CAG AAG CAT TAA TAG ACT-(Spacer C18)4-(Biotin-dT)-(Spacer C18)4-TGG ACG ACG ATA GAA CT-3'

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

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

Linker 17: 5’-G CAG AAG CAT TAA TAG ACT-(Biotin-dT)-TGG ACG ACG ATA GAACT-3’

The above example bridge constructs comprise at least a first hybridizable oligonucleotide and a second hybridizable oligonucleotide. Linkers 3 to 17 each comprise a central biotinylated or digoxigenylated thymidine outside the hybridizable nucleic acid segment, or a spacer consisting of thymidine units of the lengths given above.

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

In the above examples of bridge constructs, [B-L] indicates that the L-DNA oligonucleotide sequence is given; spacer C18, biotin, and biotin-dT refer to the C18 spacer, biotin, and biotin-dT, respectively, as derived from the building blocks given above; and 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 labeling of Fab' fragments with ssDNA

Purified monoclonal antibodies were cleaved with the help of the protease pepsin to produce F(ab') ₂ fragments, which were subsequently reduced to Fab' fragments by treatment with a low concentration of cysteamine at 37°C. The reaction was stopped by separation of the cysteamine on a PD10 column. The Fab' fragments were labeled with an activated oligonucleotide, as generated according to Example 3. This single-stranded DNA (ssDNA) carried a thiol-reactive maleimido group that reacted with the cysteine residues in the Fab' hinge region. To obtain a high percentage of singly labeled Fab' fragments, the relative molar ratio of ssDNA to Fab' fragments was kept low. The singly labeled Fab' fragments (ssDNA:Fab' = 1:1) were purified by ion exchange chromatography (column: Source15Q PE4.6/100, Pharmacia/GE). Efficient purification was confirmed by analytical gel filtration and SDS-PAGE.

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

The dual anti-pIGF-1R binder is based on two Fab' fragments targeting different epitopes in the intracellular domain of IGF-1R: Fab'8.1.2 detects a phosphorylation site (pTyr1346) of the target protein, while Fab'1.4.168 detects a non-phospho-site. The Fab' fragments have been covalently linked to single-stranded DNA (ssDNA): Fab'1.4.168 is linked to a 17-mer ssDNA containing SEQ ID NO:6 and fluorescein as a fluorescent marker, while Fab'8.1.2 is covalently linked to a 19-mer ssDNA containing SEQ ID NO:5 and Cy5 as a fluorescent marker. Hereinafter, these Fab' fragments covalently linked to 17-mer or 19-mer ssDNA are referred to as ssFab'1.4.168 and ssFab'8.1.2, respectively. The assembly of the dual binder 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' fragments. The distance between the two ssFab' fragments of the dual binder can be modified individually by using spacers (e.g., C18 spacers) or DNA of different lengths.

For assembly assessment, the dual binder components ssFab'8.1.2, ssFab'1.4.168, and the linker constructs (I) (= linker 17 from Example 2.4) 5'-G CAG AAG CAT TAA TAG ACT T(-Bi)-TGG ACG ACG ATA GAACT-3' and (II) (= linker 10 from 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 a 1-minute incubation step, the reaction mixture was analyzed on an analytical gel filtration column (Superdex ^™ 200, 10/300GL, GE Healthcare). Comparison of the elution volumes ( _VE ) of the individual dual binder components with the _VE of the reaction mixture indicated that the dual binder had been successfully formed ( FIG1 ). (The biotinylated thymidine (T-(Bi)) in the middle of both linkers was not functional in these experiments.)

2.6 Biacore ^™ Assays to Evaluate Binding of Anti-pIGF-1R Dual Binders to Immobilized IGF-1R and IR Peptides

For this experiment, a Biacore ^™ 2000 instrument (GE Healthcare) was used at T = 25° C. with a Biacore ^™ SA sensor embedded in the system. Preconditioning occurred at 100 μl/min with three 1 minute injections of 1 M NaCl in 50 mM NaOH and 1 minute of 10 mM HCl.

HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% HCl) was used as the system buffer. The sample buffer was the same as the system buffer. The Biacore ^™ 2000 system was driven by control software V1.1.1.

Subsequently, the biotinylated peptides were captured on the SA surface in the corresponding flow cells. 16 RU of IGF-1R (1340-1366) [1346-pTyr; Glu (Bi-PEG-1340] amide (i.e., a peptide of SEQ ID NO: 11 with 1346 tyrosine phosphorylated, 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 RU of IGF-1R (1340-1366); Glu (Bi-PEG-1340] amide (i.e., a peptide of SEQ ID NO: 11 with 1346 tyrosine non-phosphorylated, 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. 20 RU of hIR (1355-1382) [1361-pTyr; Glu (Bi-PEG-1355] amide (i.e., a peptide of SEQ ID NO: 11 with 1361 tyrosine phosphorylated) were captured on flow cell 4. (Peptide No. 12, comprising a PEG linker conjugated via 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 described in Example 2.5 was used. When individual runs were performed with only one of the two ssFab's, the absence or presence of linker DNA did not affect the association or dissociation curves (data not shown).

100 nM analyte (in these experiments, the bivalent dual binder) in solution was injected at 50 μl/min for 240 seconds of binding time, and dissociation was monitored for 500 seconds. Regeneration was achieved using a 1-minute injection step with 80 mM NaOH at 50 μl/min. Flow cell 1 served as a reference. Blank buffer injections were used instead of antigen injections to double-reference the data by buffer signal subtraction.

In each measurement cycle, one of the following analytes was injected in solution over all four flow cells: 100 nM ssFab'8.1.2, 100 nM ssFab'1.4.168, a mixture of 100 nM ssFab'8.1.2 and 100 nM ssFab', 100 nM bivalent binder consisting 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 100 nM consisting 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 15 of Example 2.4)). A bivalent binder consisting of ssFab'8.1.2 and ssFab'1.4.168 hybridized on CT-3' (linker 16 in Example 2.4). (The digoxigeninylation (T(-Dig)) of the central thymidine in the linker was not relevant to these experiments.)

The signal was monitored as a time-dependent BIAcore ^™ 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. Dissociation rates (kd) (1/s) were calculated using a linear 1:1 Langmuir fit using Biacore ^™ Evaluation Software 4.1. Complex half-lives in minutes were calculated using the formula ln(2)/(60 x kd).

Sensorgrams (Figures 2-5) show that both pIGF-1R binding specificity and complex stability are increased when ssFab'1.4.168 and ssFab'1.4.168 are used in a dual binder (= bivalent binder) format, presumably due to an underlying cooperative binding effect. Fab'1.4.168 alone exhibits no cross-reactivity with the pIR peptide but does not discriminate between the phosphorylated and unphosphorylated forms of IGF-1R (T1/2 dissociation = 3 minutes in both cases). However, Fab'8.1.2 binds only to the phosphorylated form of the IGF1-R peptide but exhibits some undesirable cross-reactivity with the phosphorylated insulin receptor. The dual binder fully discriminates between the pIGF-1R peptide and the other two peptides (see Figure 4), thus helping to overcome the problem of nonspecific binding. Note that the increased specificity is lost when both Fab's are used without a linker DNA (Figure 5). The increased affinity of the dual binders for the pIGF-1R peptide was demonstrated by a prolonged dissociation half-life compared to individual Fab's and to a mixture of Fab's with the linker DNA omitted (Figures 3 and 5). Although the dual binders tested with two different DNA linker lengths shared an overall positive effect on target binding specificity and affinity, the longer linker ((III) with T40-T-Dig as a spacer) (i.e., linker 15 of Example 2.4) appeared to be advantageous by both criteria.

2.7 Biacore ^™ Assay of Sandwiches of M-1.4.168-IgG and M-8.1.2-IgG

Biacore ^™ CM5 sensors were embedded in the system using a Biacore ^™ T100 instrument (GE Healthcare).The sensors were preconditioned by injecting 0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mM H3PO4 at 100 μl/min for 1 minute.

The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% 20). The sample buffer was the system buffer.

The Biacore ^™ T100 system was operated using control software V1.1.1. Polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearch Laboratories Inc.) was immobilized at 10,000 RU at 30 μg/ml in 10 mM sodium acetate, pH 4.5, on flow cells 1, 2, 3, and 4, respectively, using EDC/NHS chemistry according to the manufacturer's instructions. Finally, the sensor surface was blocked with 1 M ethanolamine. The entire experiment was operated at 13°C.

500 nM of the primary monoclonal antibody M-1.004.168-IgG was captured on the <IgGFCγM>R surface at 10 μl/min for 1 minute. A 3 μM blocking solution containing a mixture of IgG fragments (IgG classes IgG1, IgG2a, IgG2b, and IgG3) was injected at 30 μl/min for 5 minutes. The peptide IGF-1R (1340-1366) [1346-pTyr;Glu(Bi-PEG-1340]amide] at 300 nM was injected at 30 μl/min for 3 minutes. The secondary antibody M-8.1.2-IgG was injected at 30 μl/min at 30 μl/min. The sensor was regenerated with 10 mM glycine-HCl, pH 1.7, at 50 μl/min for 3 minutes.

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

2.8 Biacore ^™ Assay Dual Binders on Sensor Surfaces

A Biacore ^™ SA sensor was embedded in the system using a Biacore ^™ 3000 instrument (GE Healthcare) at T = 25° C. The system was preconditioned with three 1-min injections of 1 M NaCl in 50 mM NaOH and 1 min of 10 mM HCl at 100 μl/min.

The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% 20). The sample buffer was the system buffer.

The Biacore ^™ 3000 system was driven under control software V4.1.

124 RU of amino-PEO-biotin were captured on reference flow cell 1. 1595 RU of biotinylated 14.6 kDa T0-Bi3 7-mer ssDNA-adapter (i) (5'-G CAG AAG CAT TAA TAGACT-T(-Bi)-TGG ACG ACG ATA GAA CT-3') (= adapter 17 of Example 2.4) and 1042 RU of biotinylated 23.7 kDa T40-Bi7 7-mer ssDNA-adapter (II) (5'-G CAG AAG CAT TAA TAG ACT-T(20)-(biotin-dT)-(T20)-TGG ACG ACG ATA GAA CT-3' = adapter 10 of Example 2.4) were captured on different flow cells.

300nM ssFab'8.1.2 and 300nM ssFab'1.004.168 were injected into the system at 50μl/min for 3 minutes. As a control, only 300nM ssFab'8.1.2 or 300nM ssFab'1.004.168 were injected to test the kinetic contribution of each ssFab. As a control, buffer was injected instead of ssFab. Free peptide pIR(1355-1382)[1361-pTyr]amide and IGF-1R(1340-1366)amide in solution were injected into the system at 50μl/min for 4 minutes 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, a concentration step of 0 nM, 0.4 nM, 1.1 nM, 3.3 nM (twice), 10 nM and 30 nM was used.

Dissociation was monitored for 5.3 min at 50 μl/min. After each concentration step, the system was regenerated with a 12 s pulse of 25 mM NaOH and reloaded with the ssFab' ligand.

Figure 8 schematically depicts the assay setup on a Biacore ^™ instrument. The table presented in Figure 9 shows the quantitative results of this approach. Figures 10, 11 and 12 depict exemplary Biacore ^™ results from this assay setup using the T40 dual binder.

The table in Figure 9 demonstrates the benefits of the dual binder concept. Compared to the T0 dual binder (i.e., dual binder with linker 16 of Example 2.4), which had a half-life of 192 minutes and a binding affinity of 30 pM, the T40 dual binder (i.e., dual binder with linker 10 of Example 2.4, i.e., a linker with a T20-biotin-dT-T20 spacer) produced a 2-fold improvement in antigen complex half-life (414 minutes) and a 3-fold improvement in affinity (10 pM). This supports the necessity of optimizing linker length to produce the best cooperative binding effect.

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

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

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

Furthermore, the fully assembled construct roughly multiplies its off-rate kd (1/s) when compared to the construct hybridized with a single Fab' (Figures 10, 11, 12 and the table in Figure 9). Interestingly, the on-rate ka (1/Ms) was also slightly increased when compared to a single Fab' interaction event, which may be due to the increased molecular flexibility of the construct.

Diagnostic systems using intensive washing protocols should certainly exploit the high performance of the T40 dual binder compared to individual (monovalent) Fab' molecules. The hybridized construct (i.e., the bivalent binder according to the invention) produces specific and quite stable binding events, whereas the monovalent binders dissociate more quickly, i.e., they are washed away more quickly.

Example 3: Bivalent binders to HER2

3.1 Assembly of anti-HER2 bivalent binders

Two monoclonal antibodies are used that bind to human HER2 (ErbB2 or ^p185neu ) at different, non-overlapping epitopes A and B. The first antibody is the anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAbHER2, trastuzumab, or HERCEPTIN; see US Pat. No. 5,821,337, which is incorporated herein by reference in its entirety).

The "4D5 epitope" is a region of the extracellular domain of ErbB2 that is bound by the anti-HER2 antibody 4D5 (ATCC CRL10463). This epitope is close to the transmembrane domain of ErbB2.

The second antibody is the anti-HER2 antibody 2C4 (pertuzumab). Antibody 2C4, particularly its humanized variants, is described in detail in WO 01/00245, which is incorporated herein by reference in its entirety. 2C4 is produced by a hybridoma cell line deposited with the American Type Culture Collection, Manassas, VA, USA, under ATCC HB-12697. An example of a humanized 2C4 antibody is provided in Example 3 of WO 01/00245, which is incorporated herein by reference in its entirety. Humanized anti-HER2 antibody 2C4 is also known as pertuzumab.

Pertuzumab (previously 2C4) is the first in a new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at its dimerization domain, thereby inhibiting its ability to form an active dimeric receptor complex, thereby blocking the downstream signaling cascade that ultimately leads to cell growth and division (see Franklin, M.C., Cancer Cell 5 (2004) 317-328). Pertuzumab is a fully humanized, recombinant monoclonal antibody directed against the extracellular domain of HER2.

Purification of monoclonal antibodies from culture supernatants can be performed using state-of-the-art methods of protein chemistry.

Purified monoclonal antibodies were digested with preactivated papain or pepsin to yield F(ab') ₂ fragments. These fragments were then reduced to Fab' fragments using a low concentration of cysteamine at 37°C. The reaction was terminated by separating the cysteamine from the peptide-containing fraction of the sample on a Sephadex G-25 column (GE Healthcare).

The obtained Fab' fragment is conjugated to the activated ssDNA polynucleotide.

a) Anti-HER2 antibody 4D5Fab’-ssDNA conjugate

To prepare the anti-HER2 antibody 4D5Fab'-ssDNA conjugate, a derivative of SEQ ID NO: 5 was used, i.e., 5'-AGT CTA TTA ATG CTT CTG C (=SEQ ID NO: 5)-XXX-Y-Z-3', wherein X = propylene-phosphate introduced via phosphoramidite C3 (3-(4,4'-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research)), wherein Y = propylene-phosphate introduced via 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research)). Research) and wherein Z = 4-[N-maleimidomethyl]cyclohexane-1-carboxyl introduced via sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).

b) Anti-HER2 antibody 2C4Fab’-ssDNA conjugate

To prepare anti-HER2 antibody 2C4 Fab'-ssDNA conjugate B, a derivative of SEQ ID NO: 6, 5'-Y-Z-XXX-AGT TCT ATC GTC GTC CA-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 = 5'-amino modifier C6 introduced via 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research), and wherein Z = 4-[N-maleimidomethyl]cyclohexane-1-carboxylate introduced via sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).

The polynucleotides of SEQ ID NO: 5 or SEQ ID NO: 6, respectively, have been synthesized by prior art polynucleotide synthesis methods. Introduction of the maleimido group was accomplished via reaction of the amino group of Y with the succinimidyl group of Z incorporated during the solid phase polynucleotide synthesis process.

The single-stranded DNA construct carries a thiol-reactive maleimido group, which reacts with the cysteine residues in the hinge region of the Fab' 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. The singly labeled Fab' fragments (ssDNA:Fab' = 1:1) were purified by anion exchange chromatography (MonoQ column, GE Healthcare). Efficient labeling and purification were verified by analytical gel filtration chromatography and SDS-PAG.

3.2 Biomolecular interaction analysis

For this experiment, a Biacore T100 instrument (GE Healthcare) was used at 25°C, with a Biacore SA sensor embedded in the system. Preconditioning was accomplished with three 1-minute injections of 1 M NaCl in 50 mM NaOH, pH 8.0, followed by a 1-minute injection of 10 mM HCl at 100 μl/min. The system buffer was HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% P20). The sample buffer was the system buffer supplemented with 1 mg/ml CMD (carboxymethyldextrane).

Biotinylated ss-L-DNA linkers were captured on the SA surface in the corresponding flow cell. Flow cell 1 was saturated with amino-PEO-biotin (PIERCE).

40 RU of a biotinylated 37-mer oligonucleotide adapter (Adapter 3 in Example 2.4) was captured on flow cell 2. 55 RU of a biotinylated 77-mer oligonucleotide adapter (Adapter 5 in Example 2.4) was captured on flow cell 3. 60 RU of a biotinylated 97-mer oligonucleotide adapter (Adapter 6 in Example 2.4) was captured on flow cell 4.

250 nM of the anti-HER2 antibody 4D5-Fab'-ss-L-DNA was injected into the system for 3 minutes. 300 nM of the anti-HER2 antibody 2C4-Fab'-ss-L-DNA was injected into the system at 2 μl/min for 5 minutes. DNA-labeled Fab fragments were injected alone or in combination.

As controls, only 250 nM of the anti-HER2 antibodies 4D5-Fab'-ss-D-DNA and 300 nM of the anti-HER2 antibody 2C4-Fab'-ss-D-DNA were injected into the system. Also as a control, buffer was injected instead of the DNA-labeled Fab fragments. After hybridization of the Fab fragments labeled with ss-L-DNA to the corresponding ss-L-DNA bi-linkers, the analyte hHER2-ECD in solution was injected into the system at a concentration series of 24 nM, 8 nM, 3 nM, 1 nM, 0.3 nM, and 0 nM at 100 μl/min for a 3.5-minute association phase. The dissociation phase was monitored at 100 μl/min for 15 minutes. The system was regenerated by injecting 100 mM glycine buffer (glycine pH 11, 150 mM NaCl) at 20 μl/min for 30 seconds, followed by a 1-minute injection of water at 30 μl/min.

The signal was measured as an analyte concentration-dependent, time-resolved sensorgram. The data were evaluated using Biacore Biaevaluation software 4.1. The standard Langmuir two-component binding model was used as the fitting model.

result:

When Fab fragments labeled with ss-D-DNA were injected into the system, no HER2-ECD interaction could be observed because the Fab fragments labeled with ss-D-DNA did not hybridize to the mirror-ferromeric ss-L-DNA linker presented on the sensor surface.

Table 3: Kinetic results of dual binder experiments. Linker: Surface-displayed biotinylated ss-L-DNA polynucleotide linker, such as Oligo_37mer-Bi, Oligo_77mer-Bi, and Oligo_97mer-Bi, varying in linker length, as described above. ss-L-DNA-Fab: 2C4-ss-L-DNA: Anti-HER2 antibody 2C4-Fab'-ss-L-DNA labeled with 19mer-fluorescein. 4D5-ss-L-DNA: Anti-HER2 antibody 4D5-Fab'-ss-L-DNA labeled with 17mer-fluorescein. 4D5-+2C4-ss-L-DNA refers to a surface-bound dual binder comprising a combination of two monovalent anti-HER2 antibody fragments.

The following abbreviations are used in Table 3: LRU: Mass hybridized on the sensor surface in response units. Antigen: 87 kDa HER2-ECD was used as the analyte in solution. ka: Association rate (1/Ms). kd: Dissociation rate (1/s). t1/2dissociation: Half-life of the antigen complex calculated in hours according to the solution of the first-order kinetic equation: ln(2)/kd*3600. KD: Affinity in moles. KD: Affinity in picomoles. _Rmax : Maximum analyte response signal at saturation in response units (RU). MR: Molar ratio, indicating the stoichiometry of the interaction. ^χ2 , U value: Indicator of the measured mass.

Table 3

In the table above, 35-mer, 75-mer and 95-mer should be read as 37-mer, 77-mer and 97-mer respectively.

The biacore data for the 37-mer dual binder HER2-ECD interaction (i.e., for a binder with a linker consisting solely of a hybridization sequence motif to which the binder is attached and a central biotinylated thymidine) indicated that this dual binder showed no improvement in kinetic performance. This is most likely due to the insufficient linker length and lack of flexibility of the 37-mer linker.

The biacore data for the 77-mer dual binder HER2-ECD interaction (i.e., for binders with linkers containing 20 thymidines twice and a central biotinylated thymidine to increase linker length) indicated that this dual binder showed a dramatic improvement in its kinetic performance. This is most likely due to the optimal linker length and flexibility of the 77-mer linker.

The biacore data for the 97-mer dual binder HER2-ECD interaction (i.e., for binders with linkers containing 30 thymidines twice and a central biotinylated thymidine to increase linker length) indicated that this dual binder showed a dramatic improvement in its kinetic performance. This is most likely due to the optimal linker length and flexibility of the 97-mer linker.

The data in Table 3 provide evidence for the existence of cooperative binding events. Although _{the Rmax} of the fully established dual binder is approximately twice the signal height of the single Fab-arm construct, the molar ratio is exactly 1 (MR = 1). This is clear evidence for the existence of simultaneous, cooperative binding events of the two Fab fragments. The dual binder counts as a single molecule with a 1:1 Langmuir binding stoichiometry. Despite having two independent HER2 binding interfaces, no intermolecular binding between one dual binder and the two HER2 domains could be detected.

In general, the affinity constants of cooperative pairs of monoclonal antibodies or chemically cross-linked bispecific F (ab ') ₂ only reach 15 times higher than the affinity constants of the separate monoclonal antibodies, which is significantly lower than the theoretical affinity expected for an ideal combination between the reactants (Cheong, HS et al., Biochem. Biophys. Res. Commun. 173 (1990) 795-800). Without being bound by this theory, one possible reason for this is that the individual epitope/paratope interactions involved in cooperative binding (generating high avidity) must be oriented in a specific manner relative to each other to achieve optimal cooperation.

Furthermore, the data presented in Table 3 provide evidence that the short 37-mer linker (composed solely of the ss-L-DNA hybridization motif) exhibits insufficient flexibility and/or insufficient linker length to produce a cooperative binding effect. The 37-mer linker is a rigid, double-helical L-DNA construct. The hybridization produces a double L-DNA helix that is shorter and less flexible than the ss-L-DNA sequence. This helix exhibits reduced degrees of freedom and can be considered a rigid linker construct. Table 3 shows that the 37-mer linker is unable to produce a cooperative binding event. The fully constructed 37-mer dual binder exhibited the same affinity as the construct with only a single hybridization.

The linker length was extended by highly flexible poly-T ss-L-DNA to form 77-mer and 97-mer, respectively, which provided an increase in affinity and especially in antigen complex stability kd (1/s).

The ^χ² values indicate the high quality of the measurements. All measurements showed extremely small errors. The data could be fitted to a Langmuir 1:1 model with residual deviations of only +/- 1 RU. The ^χ² values were small, and only 10 iterations were required to obtain the data.

Cooperative binding operates according to physical principles (i.e., the sum of the free binding energies ΔG1 and ΔG2). Affinities are multiplied: Kdcooperative = KD1 x KD2. Furthermore, dissociation rates are also multiplied: Kdcooperative = KD1 x KD2. This is clearly observed in experiments with 77-mer and 97-mer linkers. This results in very long complex half-lives of 4146 hours (173 days) and 3942 hours (164 days), respectively. Affinities are in the 100 fmol/l range. Clearly, cooperative binding events are occurring.

All dual binders had faster on-rates compared to the single hybrid constructs. This increased on-rate was despite displaying a higher molecular weight.

Here, we show that trastuzumab and pertuzumab, linked together in the complex reported herein, bind simultaneously to the HER2 extracellular domain (ECD). Both Fab fragments bind to the authentic epitope on the HER2-ECD. Furthermore, the two Fab fragments differ strongly in their binding angles. By using the optimal 77-mer linker (approximately 30 nm in length), ss-L-DNA, and its favorable flexibility and length properties, we were able to demonstrate a cooperative binding event.

Thus, we were able to show cooperative binding between Herceptin Fab and Pertuzumab Fab linked together via a highly flexible ss-L-DNA linker.

Claims (11)

1. A separating binder, having the following formula: A-a’:a-S-b:b’-B:X(n) This binder can bind to one antigen or two different epitopes on two different antigens. The divalent binder comprises exactly two monovalent binders, A and B, with different specificities, connected to each other via a–S–b connector. The epitopes that are specifically bound by the monovalent conjugates A and B do not overlap. Where A and B are monovalent fragments derived from monoclonal antibodies. Here, a':a and b:b' are binding pairs composed of hybrid nucleic acid sequences that form stable double strands through multi-base pairing. The sequence of binding pair a':a does not bind to the sequence of binding pair b:b', and vice versa. The binding affinity within one binding pair of the hybrid nucleic acid sequence is at least ^10⁸ l/mol. Where S is a spacer with a length of at least 1 nm. Where (:X) refers to a functional module that is covalently or via a binding pair to at least one of a’, a, b, b’, or S. Where (n) is an integer and at least 1. Where - represents a covalent bond, and The length of the connector a-S-b is 6 to 100 nm. 2. The binder of claim 1, wherein the spacer S has a length of 1 to 95 nm. 3. The binding agent of any one of claims 1 and 2, wherein the spacer S is a nucleic acid. 4. The binder of any one of claims 1-3, wherein X is a functional module selected from the group consisting of a labeling group, a binding group, and an effector group. 5. The binder of any one of claims 1-4, wherein the functional module X is bound to a, b, or S. 6. The binder of claim 5, wherein the functional module X binds to the spacer S. 7. The binder of claim 6, wherein the functional module X is covalently bound to the spacer S. 8. The binder of claim 6, wherein the functional module X binds the spacer S via hybrid nucleic acid. 9. The binding agent of any one of claims 1-8, wherein the monovalent conjugates A and B are Fab fragments of monoclonal antibodies. 10. Use of the binder according to any one of claims 1-9 in the preparation of a detection reagent or kit for detecting an analyte of interest. 11. Use of the binder according to any one of claims 1-9 in the preparation of a detection reagent or kit for performing an immunoassay.