US20130288266A1 - Detection of a posttranslationally modified polypeptide by a bi-valent binding agent - Google Patents

Detection of a posttranslationally modified polypeptide by a bi-valent binding agent Download PDF

Info

Publication number
US20130288266A1
US20130288266A1 US13/923,618 US201313923618A US2013288266A1 US 20130288266 A1 US20130288266 A1 US 20130288266A1 US 201313923618 A US201313923618 A US 201313923618A US 2013288266 A1 US2013288266 A1 US 2013288266A1
Authority
US
United States
Prior art keywords
binding agent
linker
polypeptide
sec
valent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/923,618
Other languages
English (en)
Inventor
Michael Gerg
Dieter Heindl
Christian Klein
Alfred Mertens
Volker Schmid
Michael Schraeml
Monika Soukupova
Michael Tacke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics Operations Inc
Original Assignee
Roche Diagnostics Operations Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics Operations Inc filed Critical Roche Diagnostics Operations Inc
Assigned to ROCHE DIAGNOSTICS GMBH reassignment ROCHE DIAGNOSTICS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KLEIN, CHRISTIAN, GERG, MICHAEL, HEINDL, DIETER, SCHMID, VOLKER, SCHRAEML, MICHAEL, SOUKUPOVA, MONIKA, TACKE, MICHAEL, MERTENS, ALFRED
Assigned to ROCHE DIAGNOSTICS OPERATIONS, INC. reassignment ROCHE DIAGNOSTICS OPERATIONS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROCHE DIAGNOSTICS GMBH
Publication of US20130288266A1 publication Critical patent/US20130288266A1/en
Priority to US15/465,439 priority Critical patent/US10982007B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the primary structure of a polypeptide i.e. its sequence, is determined by the nucleic acid coding for it. However, knowing the primary structure of a polypeptide is only part of the story. Many polypeptides—estimates range from 50 to 90%—undergo secondary modifications. Dependent e.g. on the type of secondary modification, the percentage of modified polypeptides and/or e.g. on the exact position/location of a secondary modification, a polypeptide with one and the same primary structure can assume quite different biological functions.
  • posttranslational modifications finely tune the cellular functions of each protein. Understanding the relationship between post-translational modifications and functional changes (“posttranslatomics”) is enormous effort going on all around the world, not unlike to the human genome project. Proteomics, combined with separation technology and mass spectrometry, makes it possible to dissect and characterize the individual parts of post-translational modifications and provide a systemic analysis.
  • the immunological detection of a posttranslationally modified polypeptide has consistently turned out to be rather difficult.
  • Various types of problems may be encountered. It may be difficult to obtain a required immunogen in sufficient purity and quantity.
  • the antibodies obtained according to standard immunization and screening methods may not have the required specificity and/or affinity.
  • Such antibody would have to bind strongly to an epitope consisting of the secondary modification and parts of the polypeptide carrying it.
  • many binding agents generated by routine procedures show cross-reactions to other polypeptides with the same kind of posttranslational modification, do not exhibit the required affinity to the epitope recognized and/or show cross-reactivity to the non-modified polypeptide.
  • polypeptides even comprise several sites for one type of posttranslational modification to occur. There may be e.g. several threonine residues that are glycosylated in a statistical manner. Assessing the glycosylation status of such polypeptide might require several different antibodies with specificity for each of the positions potentially carrying the posttranslational modification.
  • the present disclosure relates to a bi-valent binding agent consisting of a first monovalent binder that binds to a polypeptide epitope of a target polypeptide, a second monovalent binder that binds to a posttranslational polypeptide modification on the target polypeptide and a linker. Further disclosed is a method for the detection of a posttranslationally modified target polypeptide by aid of such bi-valent binding agent, a method of making such bi-valent binding agent and the use of such bi-valent agent in histological staining procedures.
  • the present disclosure provides a binding agent that binds to a posttranslationally modified polypeptide with high affinity, and can be produced reproducibly in virtually unlimited quantity and uncompromised quality.
  • Posttranslational polypeptide modifications are crucial for modulating and/or regulating the property and/or activity of a polypeptide.
  • One advantageous method for use in the detection of a certain type of secondary modification on a target polypeptide would be by means of a specific binding agent.
  • the present embodiment relates to a bi-valent binding agent binding a posttranslationally modified target polypeptide consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to a polypeptide epitope of said target polypeptide, wherein the second monovalent binder binds to a posttranslational polypeptide modification, wherein each monovalent binder has a kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, and wherein the bi-valent binding agent has a kdiss of 3 ⁇ 10 ⁇ 5 /sec or less.
  • Also disclosed is a method for obtaining a bi-valent binding agent that specifically binds a posttranslationally modified target polypeptide comprising the steps of selecting a first monovalent binder that binds to a non-posttranslationally modified epitope of said target polypeptide with a kdiss of between 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, selecting a second monovalent binder that binds to a posttranslational polypeptide modification with a Kdiss of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, coupling both monovalent binders by a linker, and selecting a bi-valent binding agent having a Kdiss-value of 3 ⁇ 10 ⁇ 5 /sec or less.
  • FIG. 1A presents an analytical gel filtration experiments assessing efficiency of the anti-pIGF1-R dual binder assembly.
  • the thicker (bottom) curve represents absorbance measured at 280 nm indicating the presence of the ssFab′ proteins or the linker DNA, respectively.
  • FIG. 1B presents an analytical gel filtration experiments assessing efficiency of the anti-pIGF1-R dual binder assembly.
  • the thicker (bottom) curve represents absorbance measured at 280 nm indicating the presence of the ssFab′ proteins or the linker DNA, respectively.
  • the thinner top curve in b) and d) indicates the presence of fluorescein and the thinner top curve in a) and the middle curve in d) (absorbance at 635 nm) indicates the presence of Cy5.
  • Comparison of the elution volumes of the single dual binder components VE ssFab′ 1.4.168 ⁇ 15 ml; VE ssFab′ 8.1.2 ⁇ 15 ml; VE linker ⁇ 16 ml
  • VE mix ⁇ 12 ml demonstrates that the dual binder assembly reaction was successful (rate of yield: ⁇ 90%).
  • FIG. 1C presents an analytical gel filtration experiments assessing efficiency of the anti-pIGF1-R dual binder assembly.
  • the thicker (bottom) curve represents absorbance measured at 280 nm indicating the presence of the ssFab′ proteins or the linker DNA, respectively.
  • the thinner top curve in b) and d) indicates the presence of fluorescein and the thinner top curve in a) and the middle curve in d) (absorbance at 635 nm) indicates the presence of Cy5.
  • Comparison of the elution volumes of the single dual binder components VE ssFab′ 1.4.168 ⁇ 15 ml; VE ssFab′ 8.1.2 ⁇ 15 ml; VE linker ⁇ 16 ml
  • VE mix ⁇ 12 ml demonstrates that the dual binder assembly reaction was successful (rate of yield: ⁇ 90%).
  • FIG. 1D presents an analytical gel filtration experiments assessing efficiency of the anti-pIGF1-R dual binder assembly and shows the elution profile after the 3 components needed to form the bi-valent binding agent had been mixed in a 1:1:1 molar ratio.
  • the thicker (bottom) curve represents absorbance measured at 280 nm indicating the presence of the ssFab′ proteins or the linker DNA, respectively.
  • the thinner top curve in b) and d) (absorbance at 495 nm) indicates the presence of fluorescein and the thinner top curve in a) and the middle curve in d) (absorbance at 635 nm) indicates the presence of Cy5.
  • FIG. 2 shows a scheme of the BiacoreTM experiment. Schematically and exemplarily, two binding molecules in solution are shown: The T0-T-Dig (linker 16), bi-valent binding agent and the T40-T-Dig (linker 15), bi-valent binding agent. Both these bi-valent binding agents only differ in their linker-length (a central digoxigenylated T with no additional T versus 40 additional Ts (20 on each side of the central T-Dig), between the two hybridizing nucleic acid sequences). Furthermore, ssFab′ fragments 8.1.2 and 1.4.168 were used.
  • FIG. 3 shows a BiacoreTM sensorgram with overlay plot of three kinetics showing the interaction of 100 nM bi-valent binding agent (consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on the 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 to the same peptide.
  • Highest binding performance is obtained with the dual binder construct, clearly showing, that the cooperative binding effect of the dual binder increases affinity versus the target peptide pIGF-1R
  • FIG. 4 is a BiacoreTM sensorgram with overlay plot of three kinetics showing the interactions of the bi-valent binding agent consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on the T40-T-Dig ssDNA-linker, i.e. linker 15, with immobilized peptides pIGF-1R (phosphorylated IGF-1R), IGF-1R or pIR (phosphorylated insulin receptor). Highest binding performance is obtained with the pIGF-1R peptide, clearly showing, that the cooperative binding effect of the dual binder increases specificity versus the target peptide pIGF-1R as compared to e.g. the phosphorylated insulin receptor peptide (pIR).
  • pIGF-1R phosphorylated IGF-1R
  • IGF-1R phosphorylated insulin receptor peptide
  • pIR phosphorylated insulin receptor peptide
  • FIG. 5 is a BiacoreTM sensorgram with overlay plot of two kinetics showing the interactions of 100 nM bi-valent binding agent consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on the 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. Best binding performance is only obtained with the bi-valent binding agent, whereas the mixture of the ssFab′s without linker doesn't show an observable cooperative binding effect, despite the fact that the total concentration of these ssFab′s had been at 200 nM.
  • FIG. 6 is a schematic drawing of a BiacoreTM sandwich assay. This assay has been used to investigate the epitope accessibility for both antibodies on the phosphorylated IGF-1R peptide.
  • ⁇ MIgGFcy>R presents a rabbit anti-mouse antibody used to capture the murine antibody M-1.4.168.
  • M-1.4.168 then is used to capture the pIGF-1R peptide.
  • M-8.1.2 finally forms the sandwich consisting of M-1.4.168, the peptide and M-8.1.2
  • FIG. 7 is a BiacoreTM sensorgram showing the binding signal (thick line) of the secondary antibody 8.1.2. to the pIGF-1R peptide after this was captured by antibody 1.4.168 on the BiacoreTM chip.
  • the other signals (thin lines) are control signals: given are the lines 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 event could be detected in any of these controls
  • FIG. 8 is a schematic drawing of the BiacoreTM assay, presenting the biotinylated dual binders on the sensor surface.
  • FC1 Flow Cell 1
  • Analyte 1 IGF-1R-peptide containing the M-1.4.168 ssFab′ epitope at the right hand end of the peptide (top line)—the M-8.1.2 ssFab′ phospho-epitope is not present, because this peptide is not phosphorylated; analyte 2: pIGF-1R peptide containing the M-8.1.2 ssFab′ phospho-epitope (P) and the M-1.4.168 ssFab′ epitope (second line); analyte 3: pIR peptide, containing the cross reacting M-8.1.2 ssFab′ phospho-epitope, but not the epitope for M-1.4.168 (third line)
  • FIG. 9 is kinetic data of the dual binder experiment.
  • FIG. 10 is a BiacoreTM sensorgram, showing concentration dependent measurement of the T40-T-Bi dual binding agent vs. the pIGF-1R peptide (the phosphorylated IGF-1R peptide).
  • the assay setup was as depicted in FIG. 8 .
  • a concentration series of the pIGF-1R peptide was injected at 30 nM, 10 nM, 2 ⁇ 3.3 nM, 1.1 nM, 0.4 nM, 0 nM.
  • the corresponding data are given in the table of FIG. 9
  • FIG. 11 is a BiacoreTM sensorgram, showing concentration dependent measurement of the T40-T-Bi dual binding agent vs. the IGF-1R peptide (the non-phosphorylated IGF-1R peptide).
  • the assay setup was as depicted in FIG. 8 .
  • a concentration series of the IGF-1R peptide was injected at 300 nM, 100 nM, 2 ⁇ 33 nM, 11 nM, 4 nM, 0 nM.
  • the corresponding data are given in the table of FIG. 9
  • FIG. 12 is a BiacoreTM sensorgram, showing concentration dependent measurement of the T40-T-Bi dual binding agent vs. the pIR peptide (the phosphorylated insulin receptor peptide).
  • the assay setup was as depicted in FIG. 8 .
  • a concentration series of the pIR peptide was injected at 100 nM, 2 ⁇ 33 nM, 11 nM, 4 nM, 0 nM.
  • the corresponding data are given in the table of FIG. 9
  • FIG. 13 A presents Western Blotting experiment with lysates of 3T3 cells that were used for the generation of formalin-fixed paraffin-embedded (FFPE) 3T3 cell pellets. 5 ⁇ g total protein of each lysate was subjected to SDS-PAGE and Western Blotting. Detection occurred with an anti-phosphotyrosine antibody (Millipore, clone 4G10). The asterisk (*) or the pair of asterisks (**) indicate the position of the bands for phosphorylated IGF-1R or phosphorylated IR proteins.
  • FFPE formalin-fixed paraffin-embedded
  • FIG. 13 B presents Results from IHC experiments with FFPE 3T3 cell pellets.
  • the detection molecule composed of an 8 ⁇ C18 linker molecule (linker 14 of example 2.4) and only ssFab′ 1.4.168 or only ssFab′ 30.4.33 did not produce a staining on any of the tested FFPE 3T3 cell pellets (rows 1&2).
  • detection with the full dual binder molecule led to a staining—but only on IGF-1R overexpressing cells that were stimulated with IGF-1 (row 3). No cross-reactivity was observed on cells overexpressing IR even when phosphorylation of IR had been induced.
  • FIG. 13 C presents IHC experiment comparing the performance of anti-pIGF-1R dual binders with different linker length (linkers contained 2 ⁇ C18, 4 ⁇ C18, 6 ⁇ C18 or 8 ⁇ C18 spacers, see example 2.4) on IGF-1R-overexpressing FFPE 3T3 cells that had been stimulated with IGF-1 to induce IGF-1R phosphorylation
  • FIG. 14 is an immunostaining of H322M xenograft sections. 10 ⁇ g/ml per ssFab′ fragment (ssFab′ 30.4.33 or/and ssFab′ 1.4.168, respectively) and an equimolar amount of 8 ⁇ C18 linker molecule were used for detection. A biotin label within the linker molecule served as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit
  • FIG. 15 is a schematic drawing of the Biacore assay, presenting the biotinylated dual binders on the sensor surface
  • An biotinylated 8 ⁇ C18 linker molecule was immobilized that was used to capture ssFab′ 1.4.168 and/or ssFab′ 30.4.33, respectively.
  • the analyte was a pIGF-1R-peptide containing the M-1.4.168 ssFab epitope at one end of the peptide and the M-30.4.33 ssFab phospho-epitope on the other end
  • FIG. 16 is a table summarizing the kinetic data of the dual binder experiment.
  • FIG. 18 is a Biacore sensorgram, showing concentration dependent measurement of a monovalent binding agent composed of an 8 ⁇ C18 linker molecule and ssFab′ 1.4.168 versus the phosphorylated IGF-1R peptide.
  • the assay setup was as depicted in FIG. 15 .
  • a concentration series of the pIGF-1R peptide was injected at 30 nM, 10 nM, 2 ⁇ 3.3 nM, 1.1 nM, 0.4 nM, 0 nM.
  • the corresponding kinetic data are given in FIG. 16
  • FIG. 20 A presents Western Blotting experiment with lysates of Hek293 cells that were used for the generation of formalin-fixed paraffin-embedded (FFPE) 293 cell pellets. 5 ⁇ g total protein of each lysate was subjected to SDS-PAGE and Western Blotting. Detection occurred with an anti-phosphotyrosine antibody (Millipore, clone 4G10).
  • FFPE formalin-fixed paraffin-embedded
  • FIG. 20 B presents Results from IHC experiments with Hek293 cell pellets.
  • the detection molecule composed of an 4 ⁇ C18 linker molecule (linker 12 of example 2.4) and only ssFab 4.1.15 or only ssFab 7.2.32 did not produce a staining on any of the tested FFPE Hek293 cell pellets (rows 1 &2).
  • detection with the full dual binder molecule led to a staining—but only on wild-type HER3 overexpressing cells that were stimulated with NRG1- ⁇ 1 (row 3; column 2).
  • V H (mAb 1.4.168): QCDVKLVESG GGLVKPGGSL KLSCAASGFT FSDYPMSWVR QTPEKRLEWV ATITTGGTYT YYPDSIKGRF TISRDNAKNT LYLQMGSLQS EDAAMYYCTR VKTDLWWGLA YWGQGTLVTV SA SEQ ID NO: 2 V L (mAb 1.4.168): QLVLTQSSSA SFSLGASAKL TCTLSSQHST YTIEWYQQQP LKPPKYVMEL KKDGSHTTGD GIPDRFSGSS SGADRYLSIS NIQPEDESIY ICGVGDTIKE QFVYVFGGGT KVTVLG SEQ ID NO: 3 V H (mAb 8.1.2): EVQLQQSGPA LVKPGASVKM SCKASGFTFT SYVIHWVKQK PGQGLEWIGY LNPYNDNTKY NEK
  • the present embodiment relates to a bi-valent binding agent binding a posttranslationally modified target polypeptide the binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein a) the first monovalent binder binds to a polypeptide epitope of said target polypeptide, b) the second monovalent binder binds to a posttranslational polypeptide modification, c) each monovalent binder has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, and d) wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or less.
  • a posttranslationally modified target polypeptide can be detected by a bi-valent binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to a polypeptide epitope of said target polypeptide, the second monovalent binder binds to a posttranslational polypeptide modification, wherein each monovalent binder has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, and wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or less
  • the bi-valent binding agent according to the present disclosure is a binding agent comprising exactly two monovalent binders of different specificity.
  • each monovalent binder and of the bivalent binding agent are characterized by BiacoreTM SPR technology as described in detail in the examples.
  • the bi-valent binding agent described in the present embodiment can be isolated and purified as desired.
  • the present embodiment relates to an isolated bi-valent binding agent as disclosed herein.
  • An “isolated” bi-valent binding agent is one which has been identified and separated and/or recovered from e.g. the reagent mixture used in the synthesis of such bi-valent binding agent. Unwanted components of such reaction mixture are e.g. monovalent binders that did not end up in the desired bi-valent binding agent.
  • the bi-valent binding agent is purified to greater than 80%. In some embodiments, the bi-valent binding agent is purified to greater than 90%, 95%, 98% or 99% by weight, respectively.
  • an antibody means one antibody or more than one antibody.
  • oligonucleotide or “nucleic acid sequence” as used herein, generally refers to short, generally single stranded, polynucleotides that comprise at least 8 nucleotides and at most about 1000 nucleotides. In an exemplary embodiment an oligonucleotide will have a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In an exemplary embodiment an oligonucleotide will have a length of no more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. The description given below for polynucleotides is equally and fully applicable to oligonucleotides.
  • oligonucleotide is to be understood broadly and includes DNA and RNA as well as analogs and modification thereof.
  • An oligonucleotide may for example contain a substituted nucleotide carrying a substituent at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), deoxythymidine (dT), deoxyuracil (dU).
  • dA deoxyadenosine
  • dG deoxyguanosine
  • dC deoxycytosine
  • dT deoxythymidine
  • deoxyuracil deoxyuracil
  • substituted nucleobases examples include: 5-substituted pyrimidines like 5 methyl dC, aminoallyl dU or dC, 5-(aminoethyl-3-acrylimido)-dU, 5-propinyl-dU or -dC, 5 halogenated-dU or -dC; N substituted pyrimidines like N4-ethyl-dC; N substituted purines like N6-ethyl-dA, N2-ethyl-dG; 8 substituted purines like 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 like 2 amino dA.
  • An oligonucleotide may contain a nucleotide or a nucleoside analog.
  • nucleobase analogs like 5-Nitroindol d riboside; 3 nitro pyrrole d riboside, deoxyinosine (dI), deoyxanthosine (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-d
  • nucleobase in the complementary strand has to be selected in such manner that duplex formation is specific. If, for example, 5-methyl-iso-dC is used in one strand (e.g. (a)) iso dG has to be in the complementary strand (e.g. (a′)).
  • the oligonucleotide backbone may be modified to contain substituted sugar residues, sugar analogs, modifications in the internucleoside phosphate moiety, and/or be a PNA.
  • An oligonucleotide may for example contain a nucleotide with a substituted deoxy ribose like 2′-methoxy, 2′-fluoro, 2′-methylseleno, 2′-allyloxy, 4′-methyl dN (wherein N is a nucleobase, e.g., A, G, C, T or U).
  • Sugar analogs are for example Xylose; 2′,4′ bridged Ribose like (2′-O, 4′-C methylene)—(oligomer known as LNA) or (2′-O, 4′-C ethylene)—(oligomer known as ENA); L-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (oligomer known as ANA); a tricyclic ribose analog where C3′ and C5′ atoms are connected by an ethylene bridge that is fused to a cyclopropane ring (oligomer known as tricycloDNA); glycerin (oligomer known as GNA); Glucopyranose (oligomer known as Homo DNA); carbaribose (with a cyclopentan instead of a tetrahydrofuran subunit); hydroxymethyl-morpholin (
  • a great number of modification of the internucleosidic phosphate moiety are also known not to interfere with hybridization properties and such backbone modifications can also be combined with substituted nucleotides or nucleotide analogs. Examples are phosphorthioate, phosphordithioate, phosphoramidate and methylphosphonate oligonucleotides.
  • PNA having a backbone without phosphate and d-ribose
  • PNA can also be used as a DNA analog.
  • modified nucleotides, nucleotide analogs as well as oligonucleotide backbone modifications can be combined as desired in an oligonucleotide in the sense of the present embodiment.
  • polypeptide and “protein” are used inter-changeably.
  • a polypeptide in the sense of the present embodiment consists of at least 5 amino acids linked by alpha amino peptidic bonds.
  • a “target polypeptide” is a polypeptide of interest for which a method for determination or measurement is sought.
  • the target polypeptide of the present embodiment is a polypeptide known or suspected to carry a posttranslational polypeptide modification.
  • a “monovalent binder” is a molecule interacting with the target polypeptide at a single binding site with a Kdiss of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec.
  • the biophysical characterization of kinetic binding rate properties, respectively the determination of the dissociation rate constant kd(1/s) according to a Langmuir model is, according to some embodiments, analyzed by biosensor-based surface plasmon resonance spectroscopy.
  • the BiacoreTM technology as described in detail in the Examples section is used.
  • monovalent binders are peptides, peptide mimetics, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptines, ankyrin repeat proteins, Kunitz type domains, single domain antibodies, (see: Hey, T. et al., Trends Biotechnol 23 (2005) 514-522) and monovalent fragments of antibodies.
  • the monovalent binder is a monovalent antibody fragment, for example a monovalent fragment derived from a monoclonal antibody.
  • Monovalent antibody fragments include, but are not limited to Fab, Fab′-SH (Fab′), single domain antibody, Fv, and scFv fragments, as provided below.
  • At least one of the monovalent binders is a single domain antibody, an Fab-fragment or an Fab′-fragment of a monoclonal antibody.
  • both the monovalent binders are derived from monoclonal antibodies and are Fab-fragments, or Fab′-fragments or an Fab-fragment and an Fab′-fragment.
  • Monoclonal antibody techniques allow for the production of extremely specific binding agents in the form of specific monoclonal antibodies or fragments thereof.
  • Particularly well known in the art are techniques for creating monoclonal antibodies, or fragments thereof, by immunizing mice, rabbits, hamsters, or any other mammal with a polypeptide of interest.
  • Another method of creating monoclonal antibodies, or fragments thereof is the use of phage libraries of sFv (single chain variable region), specifically human sFv. (See e.g., Griffiths et al., U.S. Pat. No. 5,885,793; McCafferty et al., WO 92/01047; Liming et al., WO 99/06587).
  • Antibody fragments may be generated by traditional means, such as enzymatic digestion or by recombinant techniques. For a review of certain antibody fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134.
  • HVRs hyper variable regions
  • antibody fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto, K. et al., Journal of Biochemical and Biophysical Methods 24 (1992) 107-117; and Brennan et al., Science 229 (1985) 81-83).
  • papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily.
  • Antibody fragments can also be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli , thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries according to standard procedures. Alternatively, Fab′-SH fragments can be directly recovered from E. coli (Carter, P. et al., Bio/Technology 10 (1992) 163-167). Mammalian cell systems can be also used to express and, if desired, secrete antibody fragments.
  • One of the two monovalent binders binds to a polypeptide epitope on the target polypeptide.
  • a “polypeptide epitope” is composed of amino acids.
  • This binder either binds to a linear epitope, i.e. an epitope consisting of a stretch of 5 to 12 consecutive amino acids, or the monovalent binder binds to a tertiary structure formed by the spatial arrangement of several short stretches of the target polypeptide.
  • Tertiary epitopes recognized by a binder e.g.
  • the antigen recognition site or paratope of an antibody can be thought of as three-dimensional surface features of an antigen molecule; these features fit precisely (in)to the corresponding binding site of the binder and thereby binding between binder and target polypeptide is facilitated.
  • the first monovalent binder binds to a polypeptide epitope
  • the second monovalent binder binds to a posttranslational polypeptide modification.
  • posttranslational polypeptide modification is a covalent modification of an amino acid within or at the end of a polypeptide (protein).
  • the terms secondary modification and post-translational modification are inter-changeable.
  • the posttranslational modification is selected from the group consisting of acetylation, phosphorylation, acylation, methylation, glycosylation, ubiquitinylation, sumoylation, sulfatation and nitration.
  • Acetylation (+42 Da) is a rather stable secondary modification. Examples are the acetylation which is found on the N-termini of many proteins or the acetylation on lysine or serine residues. Usually acetylation of a lysine residue is found at one or more well-defined position(s) within a polypeptide chain, while other lysine residues are acetylated less frequently or not at all.
  • Phosphorylation and de-phosphorylation (the net balance of which may be referred to as phosphorylation status) of a protein is known to be one of the key elements in regulating a proteins biological activity.
  • a low percentage of phosphorylated amino acid residues may already be sufficient to trigger a certain biological activity.
  • Phosphorylation results in a mass increase of 80 Da.
  • the amino acids tyrosine (Y), serine (S), threonine (T), histidine (H), and aspartic acid (D) can be phosphorylated.
  • the more complex the biological function of a polypeptide the more complex the corresponding pattern of possible sites of phosphorylation.
  • RTKs receptor tyrosine kinases
  • Polypeptides may be acylated by farnesyl, myristoyl or palmitoyl groups. Acylation usually occurs on the side chain of a cysteine residue.
  • Methylation as a secondary modification occurs via the side chain of a lysine residue. It has been shown that the binding properties of regulatory proteins that are able to bind to a nucleic acid can e.g. be modulated via methylation.
  • Glycosylation is a very important secondary modification. It has a major influence on protein-protein interactions, on solubilization of proteins, their stability, aso.
  • Two different types of glycosylation are known: the N-linked (via the amino acid N (asparagine)) side chains and the O-linked side chains (via serine (S) or threonine (T)).
  • S amino acid
  • T threonine
  • Many different polysaccharides linear or with branched side chains, some containing sugar derivatives like O-Glc-NAc, have been identified.
  • Ubiquitinylation and sumoylation are known to influence the half-life of proteins in the circulation. Ubiquitinylation may serve as a destruction signal, resulting in cleavage and/or removal of ubiquitinylated polypeptides.
  • Sulfatation via a tyrosine residue appears to be important in the modulation of protein-protein (cell-cell) interaction as well as in protein ligand-interaction.
  • Nitration of tyrosine residues appears to be a hall-mark of oxidative damage as e.g. in inflammatory processes.
  • the posttranslational modification bound by the second monovalent binder may be selected from the group consisting of phosphorylation, glycosylation and acetylation.
  • phosphorylation, de-phosphorylation and phosphorylation statuses are key to the regulation of cell signaling and protein activity. This is especially known and true for membrane-bound receptors, especially the so-called receptor tyrosine kinases (RTKs).
  • RTKs receptor tyrosine kinases
  • the present embodiment thus relates to a bi-valent binding agent binding to a phosphorylated target protein. Obviously such bi-valent binding agent is of great utility in the detection of a phosphorylated target polypeptide.
  • the present embodiment relates to a bi-valent binding agent as disclosed herein above, wherein the target polypeptide is selected from the group consisting of membrane-bound receptor molecules having an intracellular phosphorylation site and intracellular cell signaling molecules.
  • the first monovalent binder, binding a polypeptide epitope on the target protein will be specifically binding said receptor molecule or said intracellular cell signaling molecule, whereas the second monovalent binder targeting phosphorylation does not need to specifically bind a phosphorylation site on said target protein.
  • Cross-reactivity with a phosphorylation site on e.g. a related receptor would not impair the specific detection of the target polypeptide, because significant binding requires the both, the binding of the first and the binding of the second monovalent binder.
  • the RTK is selected from the group consisting of: ALK, adhesion related kinase receptor (e.g., Axl), ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4), erythropoietin-producing hepatocellular (EPH) receptors (e.g., EphA1; EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB5, EphB6), fibroblast growth factor (FGF) receptors (e.g., FGFR1, FGFR2, FGFR3, FGFR4, FGFR5), Fgr, IGFIR, Insulin R, LTK, M-CSFR, MUSK, platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-A, PDGFR-B), RET, ROR1, ROR
  • the intracellular cell signaling molecule is selected from the group consisting of: AKT, abl, cbl, erbA, ERK, fes, fgr, fms, fos, jun, met, myb, myc, PI3K, raf, ret, ryk, and src.
  • the present embodiment relates to a bi-valent binding agent binding a posttranslationally modified target polypeptide consisting of two monovalent binders that are linked to each other via a linker, wherein a) the first monovalent binder binds to a polypeptide epitope of said target polypeptide, b) the second monovalent binder binds to a posttranslational polypeptide modification, c) each monovalent binder has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, d) wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or less and wherein the posttranslational modification is selected from the group consisting of phosphorylation, ubiquitinylation and glycosylation.
  • each monovalent binder and of the bivalent binding agent are characterized by Biacore SPR technology as described in detail in the examples.
  • the bi-valent binding agent according to the present embodiment will bind to a target polypeptide having a posttranslational modification, wherein the posttranslational modification is phosphorylation.
  • a monovalent binder for use in the construction of a bi-valent binding agent as disclosed herein has to have a Kdiss from 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec.
  • the first monovalent binder is specifically binding to a polypeptide epitope. I.e. this binder binds to an epitope that is either not subject to a secondary modification or in the alternative it specifically binds to the native (non-secondarily modified) epitope. Specific binding to a polypeptide epitope is acknowledged if said binder has a Kdiss that is at least 20 times lower for the non-posttranslationally modified polypeptide as compared to the same polypeptide carrying a posttranslational modification.
  • the Kdiss of the first monovalent binder to the non-modified polypeptide is at least 30-, 40-, 50-, 80-, 90-, 95- or at least 100-fold higher as compared to the same polypeptide carrying a posttranslational modification in the polypeptide epitope bound by the first monovalent binder.
  • the second monovalent binder is specifically binding to a posttranslational polypeptide modification, i.e., said binder has a Kdiss that is at least 20 times lower for a polypeptide carrying this posttranslational modification as compared to the same non-posttranslationally modified polypeptide.
  • the Kdiss of the second monovalent binder to the polypeptide carrying a posttranslational modification is at least 30-, 40-, 50-, 80-90-, 95- or at least 100-fold lower as compared to same non-modified polypeptide.
  • the bi-valent binding agent according to the present embodiment will have a Kdiss of at most 3 ⁇ 10 ⁇ 5 /sec or lower, i.e. better.
  • each monovalent binder has a Kdiss from 2 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec.
  • each monovalent binder has a Kdiss from 10 ⁇ 3 /sec to 10 ⁇ 4 /sec.
  • An antibody used on the BenchMark® analyzer series should have a Kdiss of at most 5 ⁇ 10 ⁇ 5 /sec in order to give a reasonable staining intensity. The better the Kdiss, the better the staining intensity will be.
  • the bi-valent binding agent as disclosed herein has a Kdiss of at most 3 ⁇ 10 ⁇ 5 /sec. In a further embodiment the bi-valent binding agent as disclosed herein has a Kdiss of 2 ⁇ 10 ⁇ 5 /sec or less or also in some cases of 10 ⁇ 5 /sec or less.
  • each monovalent binder and of the bivalent binding agent are characterized by BiacoreTM SPR technology as described in detail in the examples.
  • the bi-valent binding agent according to the present embodiment contains a linker.
  • the linker can either covalently link the two monovalent binders or the linker and the monovalent binders can be bound by two different specific binding pairs a:a′ and b:b′.
  • the linker may for example be composed of appropriate monomers, linked together and to the two monovalent binders by co-valent bonds.
  • the linker will contain sugar moieties, nucleotide moieties, nucleoside moieties and/or amino acids.
  • the linker will essentially consist of nucleotides, nucleotide analogues or amino acids.
  • the linker covalently linking, or binding the two monovalent binders via binding pairs has a length of 6 to 100 nm. Also in some embodiments the linker has a length of 6 to 50 nm or of 6 to 40 nm. In an exemplary embodiment the linker will have a length of 10 nm or longer or of 15 nm or longer. In one embodiment the linker comprised in a bi-valent binding agent according to the present embodiment has between 10 nm and 50 nm in length.
  • the length of non-nucleosidic entities of a given linker (a-S-b) in theory and by complex methods can be calculated by using known bond distances and bond angles of compounds which are chemically similar to the non-nucleosidic entities.
  • Such bond distances are summarized for some molecules in standard text books: CRC Handbook of Chemistry and Physics, 91st edition, 2010-2011, section 9. However, exact bond distances vary for each compound. There is also variability in the bond angles.
  • the value of 130 pm is based on calculation of the distance of the two terminal carbonatoms of a C(sp3)-C(sp3)-C(sp3) chain with a bond angle of 109° 28′ and a distance of 153 pm between two C(sp3) which is approx 250 pm which translates with an assumed bond angle of 180° to and bond distance between two C(Sp3) with 125 pm. Taking in account that heteroatoms like P and S and sp2 and sp1 C atoms could also be part of the spacer the value 130 pm is taken. If a spacer comprises a cyclic structure like cycloalkyl or aryl the distance is calculated in analogous manner, by counting the number of the bonds of said cyclic structure which are part of the overall chain of atoms that are defining the distance
  • A is a first monovalent binder, binding to a polypeptide epitope of said target polypeptide
  • B is a second monovalent binder, binding to a posttranslational polypeptide modification
  • each monovalent binder A and B has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec
  • a′:a as well as b:b′ independently are a binding pair or a′:a and/or b:b′ are covalently bound, wherein a′:a and b:b′ are different
  • S is a spacer
  • - represents a covalent bond
  • the linker a-S-b has a length of 6 to 100 nm and wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or less.
  • the linker L consisting of a-S-b has a length of 6 to 100 nm. In some embodiments the linker L consisting of a-S-b has a length of 6 to 80 nm. In some embodiments the linker has a length of 6 to 50 nm or of 6 to 40 nm. In some embodiments the linker will have a length of 10 nm or longer or of 15 nm in length or longer. In one embodiment the linker has between 10 nm and 50 nm in length. In one embodiment a and b, respectively, are binding pair members and have a length of at least 2.5 nm each.
  • the spacer S can be construed as required to e.g. provide for the desired length as well as for other desired properties.
  • the spacer can e.g. be fully or partially composed of naturally occurring or non-naturally occurring amino acids, of phosphate-sugar units e.g. a DNA like backbone without nucleobases, of glyco-peptidic structures, or at least partially of saccharide units or at least partially of polymerizable subunits like glycols or acryl amide.
  • the length of spacer S in a compound according to the present embodiment may be varied as desired.
  • some embodiments may have a simple synthetic access to the spacers of such library.
  • a combinatorial solid phase synthesis of a spacer is possible. Since spacers have to synthesized up to a length of about 100 nm, the synthesis strategy is chosen in such a manner that the monomeric synthetic building blocks are assembled during solid phase synthesis with high efficiency. The synthesis of deoxy oligonucleotides based on the assembly of phosphoramidite as monomeric building blocks perfectly meet this requirements. In such spacer monomeric units within a spacer are linked in each case via a phosphate or phosphate analog moiety.
  • the spacer S is composed of one type of monomer.
  • the spacer is composed exclusively of amino acids, of sugar residues, of diols, of phospho-sugar units or it can be a nucleic acid, respectively.
  • the spacer is DNA.
  • the spacer is the L -stereoisomer of DNA also known as beta- L -DNA, L -DNA or mirror image DNA.
  • L -DNA features advantages like orthogonal hybridization behaviour, which means that a duplex is formed only between two complementary single strands of L-DNA but no duplex is formed between a single strand of L-DNA and the complementary DNA strand, nuclease resistance and ease of synthesis even of a long spacer.
  • ease of synthesis and variability in spacer length are important for a spacer library. Spacers of variable length are extremely utile in identifying the bi-valent dual binder according to the present embodiment having a spacer of optimal length thus providing for the optimal distance between the two monovalent binders.
  • Spacer building blocks can be used to introduce a spacing moiety into the spacer S or to build the spacer S of the linker a-S-b.
  • non nucleotidic bifunctional spacer building blocks are known in literature and a great variety is commercially available. The choice of the non nucleotidic bifunctional spacer building is influencing the charge and flexibility of the spacer molecule.
  • Bifunctional spacer building blocks in one embodiment are non-nucleosidic compounds.
  • such spacers are C2-C18 alkyl, alkenyl, alkinyl carbon chains, whereas said alkyl, alkenyl, alkinyl chains may be interrupted by additional ethyleneoxy and/or amide moieties or quarternized cationic amine moieties in order to increase hydrophilicity of the linker.
  • Exemplary bifunctional building blocks comprise C3-C6 alkyl moieties and tri- to hexa-ethyleneglycol chains.
  • Table I shows some examples of nucleotidic bifunctional spacer building blocks with different hydrophilicity, different rigidity and different charges.
  • One oxygen atom is connected to an acid labile protecting group such as dimethoxytrityl and the other is part of a phosphoramidite.
  • Hybridization can be used in order to vary the spacer length (distance between the binding pair members a and b) and the flexibility of the spacer, because the double strand length is reduced compared to the single strand and the double strand is more rigid than a single strand.
  • oligonucleotides modified with a functional moiety X are used.
  • the oligonucleotide used for hybridization can have one or two terminal extentions not hybridizing with the spacer and/or is branched internally. Such terminal extensions that are not hybridizing with the spacer (and not interfering with the binding pairs a:a′ and b:b′) can be used for further hybridization events.
  • an oligonucleotide hybridizing with a terminal extension is labeled oligonucleotide.
  • This labeled oligonucleotide again may comprise terminal extensions or being branched in order to allow for further hybridization, thereby a polynucleotide aggregate or dendrimer can be obtained.
  • a poly-oligonucleic acid dendrimer may be used in order to produce a polylabel or in order to get a high local concentration of X.
  • the spacer S has a backbone length of 1 to 100 nm.
  • the groups a and b of Formula I are between 1 and 100 nm apart.
  • a and b, respectively, each are a binding pair member and the spacer S has a backbone length of 1 to 95 nm.
  • a′:a as well as “b:b′” are different.
  • the term different indicates that the binding of a to a′ (intra-binding pair-binding or covalent coupling) does not interfere with the intra-binding pair-binding or covalent coupling of the other pair b to b′, and vice versa.
  • the chemistry used in coupling (a′) to (a), i.e. in coupling A-(a′) to a linker comprising (a) does not interfere with the chemistry used in coupling (b) to (b′), i.e. in coupling (b′)-B to a linker comprising (b).
  • the reactive sites (a), (a′), (b) and (b′), respectively, leading to the covalent bond a′:a as well as b:b′, respectively, may also not interfere with any functional group that might be present on a monovalent binder (A and/or B of Formula I).
  • the monovalent binders is a protein, a peptide or a peptide mimic, it likely carries one or more OH, COOH, NH2 and/or SH groups, which could potentially react with certain coupling reagents.
  • Such (side-)reaction can be avoided by selecting e.g. one of the coupling chemistries given in Table II.
  • Table II provides an overview over routinely used reactive groups for binding A-(a′) and (b′)-B, respectively, to (a) and (b), respectively, both being covalently bound to the linker (a-S-b).
  • both a′:a and b:b′ are a binding pair. Consequently, in one embodiment the present embodiment relates to an at least bispecific binding agent of the Formula I: A-a′:a-S-b:b′-B; wherein A is a first monovalent binder, binding to a polypeptide epitope of a target polypeptide, wherein B is a second monovalent binder, binding to a posttranslational polypeptide modification on a target polypeptide, wherein each monovalent binder A and B has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, wherein a′:a as well as b:b′ independently are a binding pair and are different, wherein S is a spacer, wherein - represents a covalent bond, wherein the linker a-S-b has a length of 6 to 100 nm and wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec
  • the binding affinity for (within) such binding pair is at least 10 8 1/mol. Both binding pairs are different.
  • a binding pair difference is e.g. acknowledged if the affinity for the reciprocal binding, e.g. binding of a as well as a′ to b or b′ is 10% of the affinity within the pair a:a′ or lower.
  • the reciprocal binding i.e. binding of a as well as a′ to b or b′, respectively, is 5% of the affinity within the pair a:a′ or lower, or if it is 2% of the affinity within the pair a:a′ or lower.
  • the difference is so pronounced that the reciprocal (cross-reactive) binding is 1% or less as compared to the specific binding affinity within a binding pair.
  • leucine zipper domain is used to denote a commonly 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 oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz, W. H. 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 derivatives thereof that dimerize or trimerize.
  • Leucine zipper domains form dimers (binding pairs) held together by an alpha-helical coiled coil.
  • a coiled coil has 3.5 residues per turn, which means that every seventh residue occupies an equivalent position with respect to the helix axis.
  • the regular array of leucines inside the coiled coil stabilizes the structure by hydrophobic and Van der Waals interactions.
  • both binding pairs (a′:a) and (b:b′) are hybridizing nucleic acid sequences.
  • a and a′ as well as b and b′ hybridize to one another, respectively.
  • the nucleic acid sequences comprised in a and a′ one the one hand and in b and b′ on the other hand are different. With other words the sequences of in the binding pair a′:a do not bind to the sequences of the binding pair b:b′, respectively, and vice versa.
  • the present embodiment relates to an at least dual binding agent of Formula I, wherein the binding pairs a:a′ and b:b′, respectively, both 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 with one another.
  • a and a′ hybridize to each other but do not bind to any of b or b′ or interfere with their hybridization and vice versa.
  • Hybridization kinetics and hybridization specificity can easily be monitored by melting point analyses.
  • Specific hybridization of a binding pair (e.g. a:a′) and non-interference (e.g. with b or b′) is acknowledged, if the melting temperature for the pair a:a′ as compared to any possible combination with b or b′, respectively, (i.e. a:b; a:b′; a′:b and a′:b′) is at least 20° C. higher.
  • the nucleic acid sequences forming a binding pair may compromise any naturally occurring nucleobase or an analogue thereto and may have a modified or an un-modified backbone as described above, provided it is capable of forming a stable duplex via multiple base pairing.
  • Stable means that the melting temperature of the duplex is higher than 37° C.
  • the double strand may consist of two fully complementary single strands. However mismatches or insertions are possible as long as the a stability at 37° C. is given.
  • RNAses are ubiquitous and special care has to be taken to avoid unwanted digestion of RNA-based binding pairs and/or spacer sequences. While it certainly is possible to use, e.g. RNA-based binding pairs and/or spacers, binding pairs and/or spacers based on DNA represent exemplary embodiments.
  • the spacer S comprised in a binding agent according to Formula I is a nucleic acid. In some embodiments both binding pairs are hybridizing nucleic acid sequences and the spacer S also is a nucleic acid.
  • the linker L consisting of a-S-b is an oligonucleotide.
  • the spacer S as well as the sequences a, a′, b and b′ all are oligonucleotide sequences it is easily possible to provide for and synthesize a single oligonucleotide representing the linker L comprising S and the members a and b of the binding pairs a′:a and b:b′, respectively.
  • the monovalent binders A and B, respectively are polypeptides, they can each be coupled easily to the hybridizing nucleic acid sequences a′ and b′, respectively.
  • the length of the spacer S comprised in such construct can easily be varied in any desired manner.
  • the binding agent of Formula I can be most easily obtained according to standard procedures by hybridization between a′:a and b:b′, respectively.
  • the resulting constructs provide for otherwise identical dual binding agents, yet having a different distance in between the monovalent binders A and B. This allows for optimal distance and/or flexibility.
  • the spacer S as well as the sequences a, a′, b and b′ are DNA.
  • the enantiomeric L-DNA is known for its orthogonal hybridization behavior, its nuclease resistance and for ease of synthesis of oligonucleotides of variable length. This ease of variability in linker length via designing appropriate spacers is important for optimizing the binding of a binding agent as disclosed herein to its antigen or antigens.
  • linker a-S-b is enantiomeric L-DNA.
  • a′, b and b′ as well as the spacer S are enantiomeric L-DNA or L-RNA.
  • a′, b and b′ as well as the spacer S are enantiomeric L-DNA.
  • the spacer S is an oligonucleotide and is synthesized in two portions comprising ends hybridizable with each other.
  • the spacer S can be simply constructed by hybridization of these hybridizable ends with one another.
  • the resulting spacer construct comprises an oligonucleotide duplex portion.
  • the sequence of the hybridizable oligonucleotide entity forming said duplex is chosen in such a manner that no hybridization or interference with the binding pairs a:a′ and b:b′ can occur.
  • the monovalent specific binders A and B of Formula I may be nucleic acids.
  • a′, a, b, b′, A, B and S all are oligonucleotide sequences.
  • the sub-units A-a′, a-S-b and b′-B of Formula I can easily and independently be synthesized according to standard procedures and combined by hybridization according to convenient standard procedures.
  • the coupling can be either co-valent or it can be via specific binding pairs.
  • the bi-valent binding agent according to the present embodiment may be further modified to carry one or more functional moieties.
  • Such functional moiety X may be selected from the group consisting of a binding group, a labeling group, an effector group and a reactive group.
  • each such functional moiety can in each case be independently a binding group, a labeling group, an effector group or a reactive group.
  • the functional moiety X may be selected from the group consisting of a binding group, a labeling group and an effector group.
  • the group X is a binding group.
  • the binding group X will be selected to have no interference with the pairs a′:a and b:b′.
  • binding groups are the partners of a bioaffine binding pair which can specifically interact with the other partner of the bioaffine binding pair.
  • Suitable bioaffine binding pairs are hapten or antigen and antibody; biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin and avidin or streptavidin; sugar and lectin, oligonucleotide and complementary oligonucleotide, receptor and ligand, e.g., steroid hormone receptor and steroid hormone.
  • 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.
  • the smaller partner of a bioaffine binding pair e.g. biotin or an analogue thereto, a receptor ligand, a hapten or an oligonucleotide is covalently bound to at lest one of a′, a, L, b or b′ as defined above.
  • functional moiety X is a binding group selected from hapten; biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin; oligonucleotide and steroid hormone.
  • the functional moiety X is a reactive group.
  • the reactive group can be selected from any known reactive group, like Amino, Sulfhydryl, Carboxylate, Hydroxyl, Azido, Alkinyl or Alkenyl.
  • the reactive group is selected from Maleinimido, Succinimidyl, Dithiopyridyl, Nitrophenylester, Hexafluorophenylester.
  • the functional moiety X is a labeling group.
  • the labeling group can be selected from any known detectable group. The skilled artisan will choose the number of labels as appropriate for best sensitivity with least quenching.
  • the labeling group can be selected from any known detectable group.
  • the labeling group is selected from dyes like luminescent labeling groups such as chemiluminescent groups e.g. acridinium esters or dioxetanes or fluorescent dyes e.g. fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof, luminescent metal complexes such as ruthenium or europium complexes, enzymes as used for CEDIA (Cloned Enzyme Donor Immunoassay, e.g. EP 0 061 888), microparticles or nanoparticles e.g. latex particles or metal sols, and radioisotopes.
  • chemiluminescent groups e.g. acridinium esters or dioxetanes
  • fluorescent dyes e.g. fluorescein, coumarin, rhodamine, oxa
  • the labeling group is a luminescent metal complex and the compound has a structure of the general formula (II):
  • 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 denote ligands with at least two nitrogen-containing heterocycles in which L 1 , L 2 and L 3 are bound to the metal cation via nitrogen atoms
  • X is a reactive functional group which is 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, for example 1 to 4
  • m is 1 or 2 and
  • A denotes the counter ion which may be required to equalize the charge.
  • the metal complex may be a luminescent metal complex i.e. a metal complex which undergoes a detectable luminescence reaction after appropriate excitation.
  • the luminescence reaction can for example be detected by fluorescence or by electrochemiluminescence measurement.
  • the metal cation in this complex is for example a transition metal or a rare earth metal.
  • the metal may be ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium or tungsten. Ruthenium, iridium, rhenium, chromium and osmium are utilized according to some embodiments.
  • the ligands L 1 , L 2 and L 3 are ligands with at least two nitrogen-containing heterocycles. Aromatic heterocycles such as bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl may be utilized.
  • the ligands L 1 , L 2 and L 3 may be selected from bipyridine and phenanthroline ring systems.
  • the complex can additionally contain one or several counter ions A to equalize the charge.
  • suitable negatively charged counter ions are halogenides, OH ⁇ , carbonate, alkylcarboxylate, e.g. trifluoroacetate, sulphate, hexafluorophosphate and tetrafluoroborate groups. Hexafluorophosphate, trifluoroacetate and tetrafluoroborate groups may be used.
  • suitable positively charged counter ions are monovalent cations such as alkaline metal and ammonium ions.
  • the functional moiety X is an effector group.
  • An effector group is a therapeutically active substance.
  • Therapeutically active substances have different ways in which they are effective, e.g. in inhibiting cancer. They can damage the DNA template by alkylation, by cross-linking, or by double-strand cleavage of DNA. Other therapeutically active substances can block RNA synthesis by intercalation. Some agents are spindle poisons, such as vinca alkaloids, or anti-metabolites that inhibit enzyme activity, or hormonal and anti-hormonal agents.
  • the effector group X may be selected from alkylating agents, antimetabolites, antitumor antibiotics, vinca alkaloids, epipodophyllotoxins, nitrosoureas, hormonal and antihormonal agents, and toxins.
  • alkylating agents may be exemplified by cyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa, ifosphamide, Nitrogen mustard.
  • antimetabolites may be exemplified by methotrexate, 5-Fluorouracil, cytosine arabinoside, 6-thioguanine, 6-mercaptopurin.
  • spindle poisons may be exemplified by maytansine and maytansinoids
  • vinca alkaloids and epipodophyllotoxins may be exemplified by vincristin, vinblastin, vindestin, Etoposide, Teniposide.
  • nitrosoureas may be exemplified by carmustin, lomustin, semustin, streptozocin.
  • hormonal and antihormonal agents may be exemplified by adrenocorticorticoids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, antiandrogens.
  • Additional random synthetic agents may be exemplified by dacarbazin, hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplastin, carboplatin.
  • a functional moiety X is bound either covalently or via an additional binding pair, e.g., to at least one of (a′), (a), (b), (b′) or S.
  • the functional moiety X can occur once or several (n) times.
  • (n) is an integer and 1 or more than one. In some embodiments (n) is between 1 and 100, for example (n) being 1-50 or in certain embodiments n is 1 to 10, or 1 to 5. In further embodiments n is 1 or 2.
  • any appropriate coupling chemistry can be used.
  • the skilled artisan can easily select such coupling chemistry from standard protocols. It is also possible to incorporate a functional moiety by use of appropriate building blocks when synthesizing a′, a, b, b′ or S.
  • a functional moiety X is located within the a hybridizing oligonucleotide representing a, a′, b or b′, respectively, in some embodiments such functional moiety is bound to a modified nucleotide or is attached to the internucleosidic P atom (WO 2007/059816).
  • Modified nucleotides which do not interfere with the hybridization of oligonucleotides are incorporated into those oligonucleotides.
  • Such modified nucleotides may be C5 substituted pyrimidines or C7 substituted 7deaza purines.
  • Oligonucleotides can be modified internally or at the 5′ or 3′ terminus with non-nucleotidic entities which are used for the introduction of functional moiety.
  • non-nucleotidic entities are located within the spacer S, i.e. between the two binding pair members a and b.
  • non-nucleotidic modifier building blocks for construction of a spacer are known in literature and a great variety is commercially available.
  • non-nucleosidic bifunctional modifier building blocks or non-nucleosidic trifunctional modified building blocks are either used as CPG for terminal labeling or as phosphroamidite for internal labeling (see: Wojczewski, C. et al., Synlett 10 (1999) 1667-1678).
  • Bifunctional modifier building blocks connect a functional moiety or a—if necessary—a protected functional moiety to a phosphoramidite group for attaching the building block at the 5′ end (regular synthesis) or at the 3′ end (inverted synthesis) to the terminal hydroxyl group of a growing oligonucleotide chain.
  • Bifunctional modifier building blocks may be non-nucleosidic compounds.
  • such modified building blocks are C2-C18 alkyl, alkenyl, alkynyl carbon chains, whereas said alkyl, alkenyl, alkynyl chains may be interrupted by additional ethyleneoxy and/or amide moieties in order to increase hydrophilicity of the spacer and thereby of the whole linker structure.
  • Cyclic moieties like C5-C6-cycloalkyl, C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl which are optionally substituted with one or two C1-C6 alkyl groups can also be used as non-nucleosidic bifunctional modified building blocks.
  • modified bifunctional building blocks comprise C3-C6 alkyl moieties and tri- to hexa-ethyleneglycol chains.
  • Table III Non-limiting, examples of bifunctional modifier building blocks are given in Table III below.
  • Trifunctional building blocks connect (i) a functional moiety or a—if necessary—a protected functional moiety, (ii) a phosphoramidite group for coupling the reporter or the functional moiety or a—if necessary—a protected functional moiety, during the oligonucleotide synthesis to a hydroxyl group of the growing oligonucleotide chain and (iii) a hydroxyl group which is protected with an acid labile protecting group, for example, with a dimethoxytrityl protecting group. After removal of this acid labile protecting group a hydroxyl group is liberated which can react with further phosphoramidites. Therefore trifunctional building blocks allow for positioning of a functional moiety to any location within an oligonucleotide.
  • Trifunctional building blocks are also a prerequisite for synthesis using solid supports, e.g. controlled pore glass (CPG), which are used for 3′ terminal labeling of oligonucleotides.
  • CPG controlled pore glass
  • the trifunctional building block is connected to a functional moiety or a—if necessary—a protected functional moiety via an C2-C18 alkyl, alkenyl, alkinyl carbon chains, whereas said alkyl, alkenyl, alkyinyl chains may be interrupted by additional ethyleneoxy and/or amide moieties in order to increase hydrophilicity of the spacer and thereby of the whole linker structure and comprises a hydroxyl group which is attached via a cleavable spacer to a solid phase and a hydroxyl group which is protected with an acid labile protecting group. After removal of this protecting group a hydroxyl group is liberated which could then react with a phosphoramidite.
  • Trifunctional building blocks may be non-nucleosidic or nucleosidic.
  • Non-nucleosidic trifunctional building blocks are C2-C18 alkyl, alkenyl, alkynyl carbon chains, whereas said alkyl, alkenyl, alkynyl are optionally interrupted by additional ethyleneoxy and/or amide moieties in order to increase hydrophilicity of the spacer and thereby of the whole linker structure.
  • Other trifunctional building blocks are cyclic groups like C5-C6-cycloalkyl, C4N, C5N, C4O, C5O heterocycloalkyl, phenyl which are optionally substituted with one ore two C1-C6 alkyl groups.
  • Exemplary trifunctional building blocks are C3-C6 alkyl, cycloalkyl, C5O heterocycloalkyl moieties optionally comprising one amide bond and substituted with a C1-C6 alkyl O-PG group, wherein PG is an acid labile protecting group, for example monomethoxytrityl, dimethoxytrityl, pixyl, xanthyl.
  • PG is an acid labile protecting group, for example monomethoxytrityl, dimethoxytrityl, pixyl, xanthyl.
  • Non-limiting, examples for non-nucleosidic trifunctional building blocks are e.g. summarized in Table IV.
  • Nucleosidic modifier building blocks are used for internal labeling whenever it is necessary not to influence the oligonucleotide hybridization properties compared to a non-modified oligonucleotide. Therefore nucleosidic building blocks comprise a base or a base analog which is still capable of hybridizing with a complementary base.
  • the general formula of a labeling compound for labeling a nucleic acid sequence of one or more of a, a′, b, b′ or S comprised in a binding agent according to Formula I of the present embodiment is given in Formula II.
  • PG is an acid labile protecting group, such as monomethoxytrityl, dimethoxytrityl, pixyl, xanthyl
  • Y is C2-C18 alkyl, alkenyl alkinyl, wherein said alkyl, alkenyl, alkinyl may comprise ethyleneoxy and/or amide moieties, wherein Y may be C4-C18 alkyl, alkenyl or alkinyl and contains one amide moiety and wherein X is a functional moiety to which a label can be bound.
  • positions of the base may be chosen for such substitution to minimize the influence on hybridization properties. Therefore, in some embodiments the following positions for substitution may be: a) with natural bases: Uracil substituted at C5; Cytosine substituted at C5 or at N4; Adenine substituted at C8 or at N6 and Guanine substituted at C8 or at N2 and b) with 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 Aza 2 amino A substituted at C7; Pseudouridine substituted at N1 and Formycin substituted at N2.
  • nucleosidic trifunctional building blocks are given in Table V.
  • one of the terminal oxygen atom of a bifunctional moiety or one of the terminal oxygen atoms of a trifunctional moiety is part of a phosphoramidite that is not shown in full detail but obvious to the skilled artisan.
  • the second terminal oxygen atom of 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 a covalently bound functional moiety into a linker or a spacer molecule.
  • an amino group is introduced by using bifunctional or trifunctional building block during solid phase synthesis. After cleavage from the support and purification of the amino modified oligonucleotide is reacted with an activated ester of a functional moiety or with a bifunctional reagent wherein one functional group is an active ester.
  • active esters include NHS ester or pentafluor phenyl esters.
  • Post-synthetic modification is especially useful for introducing a functional moiety which is not stable during solid phase synthesis and deprotection.
  • Examples are modification with triphenylphosphincarboxymethyl ester for Staudinger ligation (Wang, C. C. et al., Bioconjugate Chemistry 14 (2003) 697-701), modification with digoxigenin or for introducing a maleinimido group using commercial available sulfo SMCC.
  • the functional moiety X in one embodiment is bound to at least one of a′, a, b, b′ or S via an additional binding pair.
  • the additional binding pair to which a functional moiety X can be bound is may be a leucine zipper domain or a hybridizing nucleic acid.
  • the binding pair member to which X is bound and the binding pairs a′:a and b:b′, respectively, all are selected to have different specificity.
  • the binding pairs a:a′, b:b′ and the binding pair to which X is bound each bind to (e.g. hybridize with) their respective partner without interfering with the binding of any of the other binding pairs.
  • binder is a naturally occurring protein or a recombinat polypeptide of between 50 to 500 amino acids
  • the reaction of a maleinimido moiety with a cystein residue within the protein is used.
  • This is an exemplary coupling chemistry in case e.g. an Fab or Fab′-fragment of an antibody is used a monovalent binder.
  • coupling of a member of a binding pair (a′ or b′, respectively, of Formula I) to the C-terminal end of the binder polypeptide is performed.
  • C-terminal modification of a protein, e.g. of an Fab-fragment can e.g. be performed as described by Sunbul, M. et al., Organic & Biomolecular Chemistry 7 (2009) 3361-3371).
  • site specific reaction and covalent coupling of a binding pair member to a monovalent polypeptidic binder is based on transforming a natural amino acid into an amino acid with a reactivity which is orthogonal to the reactivity of the other functional groups present in a protein.
  • a specific cystein within a rare sequence context can be enzymatically converted in an aldehyde (see Formylglycine aldehyde tag-protein engineering through a novel post-translational modification (Frese, M.-A. et al., ChemBioChem 10 (2009) 425-427).
  • Site specific reaction and covalent coupling of a binding pair member to a monovalent polypeptidic binder can also be achieved by the selective reaction of terminal amino acids with appropriate modifying reagents.
  • EP 1 074 563 describes a conjugation method which is based on the faster reaction of a cystein within a stretch of negatively charged amino acids with a cystein located in a stretch of positively charged amino acids.
  • the monovalent binder may also be a synthetic peptide or peptide mimic.
  • a polypeptide is chemically synthesized, 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 great variety of orthogonal functional groups is at stake and can be introduced into a synthetic peptide, conjugation of such peptide to a linker is standard chemistry.
  • the conjugate with 1:1 stoichiometry may be separated by chromatography from other conjugation products. This procedure is facilitated by using a dye labeled binding pair member and a charged spacer.
  • a dye labeled binding pair member By using this kind of labeled and highly negatively charged binding pair member, mono conjugated proteins are easily separated from non labeled protein and proteins which carry more than one linker, since the difference in charge and molecular weight can be used for separation.
  • the fluorescent dye is valuable for purifying the bi-valent binding agent from un-bound components, like a labeled monovalent binder.
  • a binding pair member may be used (a′ and/or b′, respectively of Formula I) which is labeled with a fluorescent dye (e.g. synthesized using a bifunctional or trifunctional modifier building block in combination with bifunctional spacer building blocks during synthesis) for forming the bi-valent binding agent of the present embodiment.
  • a fluorescent dye e.g. synthesized using a bifunctional or trifunctional modifier building block in combination with bifunctional spacer building blocks during synthesis
  • the spacer S as well as the sequences a, a′, b and b′ are DNA and at least one of a′ or b′, respectively, is labeled with a fluorescent dye.
  • the spacer S as well as the sequences a, a′, b and b′ are DNA and both a′ and b′, respectively, are labeled each with a different fluorescent dye.
  • a method of producing a bi-valent binding agent that specifically binds a posttranslationally modified target polypeptide comprises the steps of (a) selecting a first monovalent binder that binds to a polypeptide epitope of said target polypeptide with a Kdiss of between 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, (b) selecting a second monovalent binder that binds to a posttranslational polypeptide modification with a Kdiss of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec, c) coupling both monovalent binders by a linker, and d) selecting a bi-valent binding agent having a Kdiss-value of 3 ⁇ 10 ⁇ 5 /sec or less.
  • the Kdiss is a temperature-dependent value.
  • the Kdiss-values of both the monovalent binders as well as of the bi-valent binding agent according to the present embodiment are determined at the same temperature.
  • a Kdiss-value may be determined at the same temperature at which the bi-valent binding agent shall be used, e.g., an assay shall be performed.
  • the Kdiss-values are established at room temperature, i.e. at 20° C., 21° C., 22° C., 23° C., 24° C. or 25° C., respectively.
  • the Kdiss-values are established at 4 or 8° C., respectively.
  • the Kdiss-values are established at 25° C. In one embodiment the Kdiss-values are established at 37° C. In one embodiment the Kdiss-values are established at 40° C. In an embodiment, Kdiss determinations, i.e. those for each monovalent binder and the Kdiss determination for the dual binder are made at 37° C.
  • bi-valent binding agents each comprising a linker of different length and to select those bi-valent binding agents having the desired binding properties, i.e. a Kdiss-value of 3 ⁇ 10 ⁇ 5 /sec or less. Selection of a bivalent binding agent with the desired Kdiss is performed by BiacoreTM-analysis as disclosed in Example 2.8.
  • the present embodiment relates to a method of forming a bi-valent binding agent according to the present embodiment, wherein a first monovalent binder that binds to a polypeptide epitope of a target polypeptide with a Kdiss of between 10 ⁇ 3 /sec to 10 ⁇ 4 /sec that is coupled to a member of a first binding pair, a second monovalent binder that binds to a posttranslational polypeptide modification with a Kdiss of 10 ⁇ 3 /sec to 10 ⁇ 4 /sec that is coupled to a member of a second binding pair, wherein the first and the second binding pair do not interfere with each other and a linker comprising a spacer and the complementary binding pair member to the first and the second binding pair member are co-incubated, whereby a bi-valent binding agent having a Kdiss-value of 10 ⁇ 5 /sec or less is formed.
  • the above method further comprises the step of isolating the bi-valent binding agent.
  • Exemplary stoichiometry for assembling the bi-valent binding agent according to the present embodiment is 1:1:1.
  • the method of producing a bi-valent binding reagent according to the present embodiment makes use of an L-DNA-linker. In an embodiment the method of producing a bi-valent binding reagent according to the present embodiment makes use of two specific binding pairs consisting of DNA, for example L-DNA, and of an L-DNA-linker.
  • the formation and stoichiometry of the formed bi-valent binding agent can be analyzed by Size Exclusion Chromatography according to state of the art procedures. If desired, the formed complexes can also be analyzed by SDS-PAGE.
  • the bi-valent binding agent disclosed in this embodiment if used in an immunohistochemical staining procedure only significantly binds and is not washed off during the various incubation steps of such procedure if it has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or better. This Kdiss can only be achieved, if both monovalent binder bind to their corresponding binding site. In case only the polypeptide epitope or only a posttranslational modification is present on a molecule in the sample no significant staining will be found. Thus, and this is of great advantage, immunohistochemical staining will be only observed if the posttranslationally modified target polypeptide—carrying the relevant modification—is present in the sample.
  • the instant disclosure relates to a histological staining method the method comprising the steps of (a) providing a cell or tissue sample, (b) incubating said sample with a bi-valent binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein one of the two monovalent binders binds to a polypeptide epitope of said target polypeptide, one of the two monovalent binders binds to a posttranslational polypeptide modification, each monovalent binder has a Kdiss in the range of 5 ⁇ 10 ⁇ 3 /sec to 10 ⁇ 4 /sec and wherein the bi-valent binding agent has a Kdiss of 3 ⁇ 10 ⁇ 5 /sec or less, and (c) detecting the bi-valent binding agent, thereby staining said sample for a posttranslationally modified target polypeptide.
  • a bi-valent binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein one of the
  • the present embodiment relates to a bi-valent binding agent consisting of two monovalent binders that are linked to each other via a linker, which binding agent binds a posttranslationally modified target polypeptide with a Kdiss meeting the requirements of an (automated) assay system or better, wherein (a) the first monovalent binder that binds to a polypeptide epitope of said target polypeptide with a Kdiss of at least 10-fold above the requirements of the (automated) assays system, (b) the second monovalent binder that binds to a posttranslational polypeptide modification with a Kdiss of at least 10-fold above the requirements of the (automated) assays system, and (c) wherein the product of the Kdiss-values of the two monovalent binders (a) and (b) is at least the Kdiss required by the (automated) system or less.
  • a method for obtaining a bi-valent binding agent that specifically binds a posttranslationally modified target polypeptide with a Kdiss at least meeting the minimal assay requirements of an (automated) assay system or better, the method comprising the steps of (a) selecting a first monovalent binder that binds to a non-posttranslationally modified epitope of said target polypeptide with a Kdiss of at least 10-fold above the minimal assay requirements of the (automated) assays system, (b) selecting a second monovalent binder that binds to a posttranslational polypeptide modification with a Kdiss of at least 10-fold above the minimal assay requirements of the (automated) assays system, wherein the product of the Kdiss-values of the two monovalent binders in steps (a) and (b) is at least the Kdiss required by the (automated) system or less and (c) coupling both monovalent binders by a linker
  • the automated system is the Benchmark® analyzer as distributed by Ventana Medical Systems Inc., Arlington.
  • the purified monoclonal antibodies are protease digested with either pre-activated papain (anti-epitope A′ MAb) or pepsin (anti-epitope B′ MAb) yielding F(ab′)2 fragments that are subsequently reduced to Fab′-fragments with a low concentration of cysteamin at 37° C., i.e. A and B, respectively, in Formula I (A-a′:a-S-b:b′-B).
  • the reaction is stopped by separating the cysteamin on a Sephadex G-25 column (GE Healthcare) from the polypeptide-containing part of the sample.
  • the Fab′-fragments are conjugated with the below described activated ssDNAa and ssDNAb oligonucleotides, respectively.
  • the oligonucleotides of SEQ ID NO:5 or 6, respectively, have been synthesized by state of the art oligonucleotide synthesis methods.
  • the introduction of the maleinimido group was done via reaction of the amino group of Y with the succinimidyl group of Z which was incorporated during the solid phase oligonucleotide synthesis process.
  • the single-stranded DNA constructs shown above bear a thiol-reactive maleimido group that reacts with a cysteine of the Fab′ hinge region generated by the cysteamine treatment.
  • a thiol-reactive maleimido group that reacts with a cysteine of the Fab′ hinge region generated by the cysteamine treatment.
  • the relative molar ratio of ssDNA to Fab′-fragment is kept low.
  • oligonucleotides used in the ssDNA linkers L1, L2 and L3, respectively have been synthesized by state of the art oligonucleotide synthesis methods and employing a biotinylated phosphoramidite reagent for biotinylation.
  • Biotin-dT 5-GCA GAA GCA TTA ATA GAC T (Biotin-dT)-TGG ACG ACG ATA GAA CT-3′. It comprises ssDNA oligonucleotides of SEQ ID NO:7 and 8, respectively, and was biotinylated by using Biotin-dT (5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research).
  • 5′-GCA GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5 TGG ACG ACG ATA GAA CT-3′. It comprises ssDNA oligonucleotides of SEQ ID NO:7 and 8, respectively, twice oligonucleotide stretches of five thymidines each and was biotinylated by using Biotin-dT ( T—Bi) (5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research) in the middle of the spacer.
  • Biotin-dT 5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biot
  • Synthetic peptides have been construed that individually only have a moderate affinity to the corresponding Fab′-fragment derived from the anti-Troponin T antibodies a and b, respectively.
  • the epitope A′ for antibody a is comprised in:
  • SEQ ID NO:9 ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U represents ⁇ -Alanin.
  • the epitope B′ for antibody b is comprised in:
  • SEQ ID NO:10 SKKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O represents Amino-trioxa-octanoic-acid
  • both variants have been designed and prepared by linear combining the epitopes A′ and B′.
  • BiacoreTM 3000 instrument GE Healthcare
  • Preconditioning was done at 100 ⁇ l/min with 3 ⁇ 1 min injection of 1 M NaCl in 50 mM NaOH and 1 min 10 mM HCl.
  • HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20 was used as system buffer.
  • the sample buffer was identical to the system buffer.
  • the BiacoreTM 3000 System was driven under the control software V1.1.1.
  • Flow cell 1 was saturated with 7 RU D-biotin.
  • 1063 RU biotinylated ssDNA linker L1 was immobilized.
  • flow cell 3,879 RU biotinylated ssDNA linker L2 was immobilized.
  • flow cell 4 674 RU biotinylated ssDNA linker L3 was captured.
  • 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 for 3 min at a flow rate of 2 ⁇ l/min.
  • the conjugates were consecutively injected to monitor the respective saturation signal of each Fab′ fragment DNA conjugate on its respective linker.
  • Fab′ combinations were driven with a single Fab′ fragment DNA conjugate A′′, a single Fab′ fragment DNA conjugate B′′ and both Fab′ fragment DNA conjugates A′′ and B′′ present on the respective linker. Stable baselines were generated after the linkers have been saturated by the Fab′ fragment DNA conjugates, which was a prerequisite for further kinetic measurements.
  • the artificial peptidic analytes TnT-1 and TnT-2 were injected as analytes in solution into the system in order to interact with the surface presented Fab′ fragments.
  • TnT-1 was injected at 500 nM
  • TnT-2 was injected at 900 nM analyte concentration. Both peptides were injected at 50 ⁇ l/min for 4 min association time. The dissociation was monitored for 5 min. Regeneration was done by a 1 min injection at 50 ⁇ l/min of 50 mM NaOH over all flow cells.
  • the avidity effect is further dependent on the length of the linker.
  • the linker L3 comprising a thymidine-based 31 mer spacer shows the lowest dissociation rate or highest complex stability.
  • the linker L2 comprising an thymidine-based 11 mer spacer exhibits the lowest dissociation rate or highest complex stability for the artificial analyte TnT-2.
  • BALB/C mice are immunized at week 0, 3, 6 and 9, respectively.
  • the immunization is carried out intraperitoneally and at weeks 3 and 9, respectively, subcutanuosly at various parts of the mouse body.
  • Spleen cells of immunized mice are fused with myeloma cells according to Galfre G., and Milstein C., Methods in Enzymology 73 (1981) 3-46. In this process ca 1 ⁇ 10 8 spleen cells of an immunized mouse are mixed with 2 ⁇ 10 7 myeloma cells a(P3 ⁇ 63-Ag8653, ATCC CRL1580) and centrifuged (10 min at 250 g and 37° C.). The cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 250 g in a 50 ml conical tube.
  • FCS fetal calf serum
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and seeded out in hypoxanthine-azaserine selection medium (100 mmol/l hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640+10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor. After 7 days the medium is exchanged with fresh medium. On day 10, the primary cultures are tested for specific antibodies. Positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter.
  • hybridoma cells obtained are seeded out at a density of 1 ⁇ 10 7 cells in CELLine 1000 CL flasks (Integra).
  • Hybridoma cell supernatants containing IgGs are collected twice a week. Yields typically range between 400 ⁇ g and 2000 ⁇ g of monoclonal antibody per 1 ml supernatant. Purification of the antibody from culture supernatant was carried out using conventional methods of protein chemistry (e.g. according to Bruck, C., Methods in Enzymology 121 (1986) 587-596).
  • the following amino modified precursors comprising the sequences given in SEQ ID NOs: 5 and 6, respectively, were synthesized according to standard methods.
  • the below given oligonucleotides not only comprise the so-called aminolinker, but also a fluorescent dye. As the skilled artisan will readily appreciate, this fluorescent dye is very convenient to facilitate purification of the oligonucleotide as such, as well as of components comprising them.
  • Synthesis was performed on an ABI 394 synthesizer at a 10 ⁇ mol scale in the trityl on (for 5′ amino modification) or trityl off mode (for 3′ amino modification) using commercially available CPGs as solid supports and standard dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (Sigma Aldrich).
  • Spacer Phosphoramidite C3 (3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research);
  • 5′ amino modifier is introduced by using 5′-Amino-Modifier C6 (6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research);
  • dA(tac), dT, dG(tac) dC(tac) phosphoramidites (Sigma Aldrich) were used and deprotection with 33% ammonia was performed for 2 h at room temperature.
  • L-DNA oligonucleotides were synthesized by using beta-L-dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (Chemgenes)
  • oligonucleotides Purification of fluorescein modified hybridizable oligonucleotides was performed by a two step procedure: First the oligonucleotides were purified on reversed-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 min, 20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A with a flow rate of 1.0 ml/min, detection at 260 nm. The fractions (monitored by analytical RP HPLC) containing the desired product were combined and evaporated to dryness.
  • reversed-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 min, 20% B in A, 12 min,
  • Cy5 labeled oligomers were used after the first purification on reversed-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 min, 20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A with a flow rate of 1.0 ml/min, detection at 260 nm.
  • the oligomers were desalted by dialysis and lyophilized on a Speed-Vac evaporator to yield solids which were frozen at ⁇ 24° C.
  • the dialysate was concentrated by evaporation and directly used for conjugation with a monovalent binder comprising a thiol group.
  • Oligonucleotides were synthesized by standard methods on an ABI 394 synthesizer at a 10 ⁇ mol scale in the trityl on mode using commercially available dT-CPG as solid supports and using standard dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (Sigma Aldrich).
  • L-DNA oligonucleotides were synthesized by using commercially available beta L-dT-CPG as solid support and beta-L-dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (Chemgenes)
  • oligonucleotides Purification of the oligonucleotides was performed as described under Example 2.3 on a reversed-phase HPLC. The fractions (analyzed/monitored by analytical RP HPLC) containing the desired product were combined and evaporated to dryness. Detriylation was performed by incubating with 80% acetic acid for 15 min) The acetic acid was removed by evaporation. The reminder was dissolved in water and lyophilized.
  • amidites and CPG supports were used to introduce the C18 spacer, digoxigenin and biotin group 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-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research);
  • Linker 1 5′-G CAG AAG CAT TAA TAG ACT-TGG ACG ACG ATA GAA CT-3′
  • Linker 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 ATA GAA CT-3′
  • Linker 4 5′-[B-L]G CAG AAG CAT TAA TAG ACT-T5-(Biotin-dT)- T5-TGG ACG ACG ATA GAA CT-3′
  • Linker 5 5′-[B-L]G CAG AAG CAT TAA TAG ACT-T20-(Biotin-dT)- T20-TGG ACG ACG ATA GAA CT-3′
  • Linker 6 5′-[B-L] G CAG AAG CAT TAA
  • the above bridging construct examples comprise at least a first hybridizable oligonucleotide and a second hybridizable oligonucleotide.
  • Linkers 3 to 17 in addition to the hybridizable nucleic acid stretches comprise a central biotinylated or digoxigenylated thymidine, respectively, or a spacer consisting of thymidine units of the length given above.
  • the 5′-hybridizable oligonucleotide corresponds to SEQ ID NO:7 and the 3′-hybridizable oligonucleotide corresponds to SEQ ID NO:8, respectively.
  • the oligonucleotide of SEQ ID NO:7 will readily hybridize with the oligonucleotide of SED ID NO:5.
  • the oligonucleotide of SEQ ID NO:8 will readily hybridize with the oligonucleotide of SED ID NO:6.
  • bridging construct examples [B-L] indicates that an L-DNA oligonucleotide sequence is given; spacer C 18, Biotin and Biotin dT respectively, refer to the C18 spacer, the Biotin and the Biotin-dT as derived from the above given building blocks; and T with a number indicates the number of thymidine residues incorporated into the linker at the position given.
  • the anti-pIGF-1R dual binder is based on two Fab′ fragments that target different epitopes of the intracellular domain of IGF-1R: Fab′ 8.1.2 detects a phosphorylation site (pTyr 1346) and Fab′ 1.4.168 a non-phospho site of the said target protein.
  • the Fab′ fragments have been covalently linked to single-stranded DNA (ssDNA): Fab′ 1.4.168 to a 17 mer ssDNA comprising SEQ ID NO:6 and containing fluorescein as an fluorescent marker and Fab′ 8.1.2 to a 19 mer ssDNA comprising SEQ ID NO:5 and containing Cy5 as fluorescent marker.
  • Dual binder assembly is mediated by a linker (i.e. a bridging construct comprising two complementary ssDNA oligonucleotides (SEQ ID NOs:7 and 8, respectively) that hybridize to the corresponding ssDNAs of the ssFab′ fragments.
  • the distance between the two ssFab′ fragments of the dual binder can be modified by using spacers, e.g. C18-spacer or DNAs of different length, respectively.
  • HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20 was used as system buffer.
  • the sample buffer was identical with the system buffer.
  • the BiacoreTM 2000 System was driven under the control software V1.1.1.
  • biotinylated peptides were captured on the SA surface in the respective flow cells.
  • 16 RU of IGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amid i.e. the -1346 tyrosine phosphorylated-peptide of SEQ ID NO:11 comprising a PEG-linker bound via glutamic acid corresponding to position 1340 and being biotinylated at the other end of the linker
  • 18 RU of IGF-1R(1340-1366); Glu(Bi-PEG-1340]amid i.e.
  • the signals were monitored as time-dependent BiacoreTM sensorgrams.
  • Fab′ 8.1.2 binds only to the phosphorylated version of the IGF1-R peptide but exhibits some undesired cross reactivity with phosphorylated Insulin Receptor.
  • a BiacoreTM T100 instrument (GE Healthcare) was used with a BiacoreTM CM5 sensor mounted into the system.
  • the sensor was preconditioned by a 1 min injection at 100 ⁇ l/min of 0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mM H3PO4.
  • the system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20).
  • the sample buffer was the system buffer.
  • the BiacoreTM T100 System was driven under the control software V1.1.1.
  • Polyclonal rabbit IgG antibody ⁇ IgGFC ⁇ M>R Jackson ImmunoResearch Laboratories Inc.
  • 10 mM Na-Acetate pH 4.5 was immobilized at 10 000 RU on the flow cells 1, 2, 3, and 4, respectively, via EDC/NHS chemistry according to the manufacturer's instructions.
  • the sensor surface was blocked with 1M ethanolamine. The complete experiment was driven at 13° C.
  • 500 nM primary mAb M-1.004.168-IgG was captured for 1 min at 10 ⁇ l/min on the ⁇ IgGFC ⁇ M>R surface.
  • 3 ⁇ M of an IgG fragment mixture (of IgG classes IgG1, IgG2a, IgG2b, IgG3) containing blocking solution was injected at 30 ⁇ l/min for 5 min.
  • the peptide IGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amid was injected at 300 nM for 3 min at 30 ⁇ l/min.
  • 300 nM secondary antibody M-8.1.2-IgG was injected at 30 ⁇ l min.
  • the sensor was regenerated using 10 mM Glycine-HCl pH 1.7 at 50 ⁇ l/min for 3 min.
  • FIG. 6 describes the assay setup.
  • FIG. 7 the measurement results are given. The measurements clearly indicate, that both monoclonal antibodies are able to simultaneously bind two distinct, unrelated epitopes on their respective target peptide. This is a prerequisite to any latter experiments with the goal to generate cooperative binding events.
  • the system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20).
  • the sample buffer was the system buffer.
  • the peptides pIR(1355-1382)[1361-pTyr]amid and IGF-1R(1340-1366)amid, respectively, were injected into system at 50 ⁇ l/min for 4 min, free in solution, in concentration steps of 0 nM, 4 nM, 11 nM, 33 nM (twice), 100 nM and 300 nM.
  • concentration steps of 0 nM, 0.4 nM, 1.1 nM, 3.3 nM (twice), 10 nM and 30 nM were used.
  • the dissociation was monitored at 50 ⁇ l/min for 5.3 min.
  • the system was regenerated after each concentration step with a 12 sec pulse of 250 mM NaOH and was reloaded with ssFab′ ligand.
  • the table in FIG. 9 demonstrates the benefits of the dual binder concept.
  • the T40 dual binding agent (a dual binding agent with linker 10 of example 2.4, i.e. a linker with a spacer of T20-Biotin-dT-T20) results in a 2-fold improved antigen complex halftime (414 min) and a 3-fold improved affinity (10 pM) as compared to the T0 dual binding agent (i.e. a dual binding agent with linker 16 of example 2.4) with 192 min and 30 pM, respectively.
  • This underlines the necessity to optimize the linker length to generate the optimal cooperative binding effect.
  • the fully assembled construct roughly multiplies its dissociation rates kd (1/s), when compared to the singly Fab′ hybridized constructs ( FIGS. 10 , 11 , 12 and table in FIG. 9 ).
  • the association rate ka (1/Ms) slightly increases when compared to the single Fab′ interaction events, this may be due to an increase of the construct's molecular flexibility.
  • a biotin label within the linker molecule served as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit.
  • 3T3 cells had been stably transfected with either IGF-1R or IR expression vectors. Cells were fixed with formalin and embedded in paraffin according to standard protocols. Prior to fixation cells were stimulated with 100 ng/ml of either IGF-1 or Insulin to induce IGF1-R or IR phosphorylation or were left untreated. Western blotting experiments ( FIG. 13 A) proved successful stimulation of receptor phosphorylation.
  • the system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20).
  • the sample buffer was the system buffer.
  • the dissociation was monitored at 50 ⁇ l/min for 5.3 min.
  • the system was regenerated after each concentration step with a 12 sec pulse of 250 mM NaOH and was reloaded with ssFab ligand.
  • the receptor tyrosine kinase family of HER proteins consists of four members: HER1, HER2, HER3 and HER4.
  • the receptors dimerize as homo- or heterodimers in various ways to trigger different signal transduction pathways, depending on the ligand and the expression levels of each of the four family members.
  • HER3 undergoes a conformational shift when it is bound to its ligands Neuregulin1 (NRG1) or Neuregulin2 (NRG2) and the HER3 dimerization domain is exposed and it can interact with other HER receptors.
  • NSG1 Neuregulin1
  • NSG2 Neuregulin2
  • HER3 dimerization domain is exposed and it can interact with other HER receptors.
  • HER3 becomes phosphorylated.
  • mice are immunized with HER3(1243-1267)[KLH-MP-Cys-UZU-1243]amide or pHER3(1283-1295)[pTyr1289; KLH-MP-Cys-UZU-1283]amide.
  • the initial immunization dose is 100 ⁇ g.
  • the mice are further immunized with 100 ⁇ g of the immunogen after 6 and 10 weeks.
  • the kinetic properties of the interaction between the monoclonal antibodies and HER3 or the phosphorylated form of pHER3 are investigated by surface plasmon resonance kinetic screening using BiacoreTM technology.
  • human_HER3(1242-1267)-Bi-PEG-amide (SEQ ID NO:17), human pHER3(1283-1295[pTyr1289])-PEG2-EDA-Btn (SEQ ID NO:18) or human_HER3(1283-1295)-PEG2-EDA-Btn (SEQ ID NO:18), singly grafted on streptavidin, is injected at a flow rate of 30 ⁇ l/min for 2 min. Thereafter, the signal is recorded for 5 min dissociation time. The sensor is regenerated by injecting a 10 mM glycine-HCl solution (pH 1.7) for 2 min, at a flow rate of 30 ⁇ l/min.
  • the dissociation rate constant kd (1/s) is calculated according to a Langmuir model using the evaluation software according to the manufacturer's instructions.
  • the selected monoclonal antibodies interact with the HER3 epitope comprising amino acids 1242-1267 or with the phosphorylated (pTyr1289) HER3 epitope comprising amino acids 1283-1295 with a dissociation rate constant that lies within the boundaries of the patent claim.
  • Antibodies that bound the unphosphorylated form of epitope HER3(1283-1295) were rejected from further studies.
  • variable regions of the selected antibodies were sequenced using standard molecular biology methods. Sequences are shown in SEQ ID NO:21-24.
  • Fab-fragments 7.2.32 and 4.1.15 were expressed in Hek293F cells as fusion proteins bearing an 8 ⁇ HIS-Tag and a sortase cleavage recognition sequence (SEQ ID NO:16).
  • 1 L 1 ⁇ 10 6 HEK 293 cells/ml with a viability of >90% were transfected in a ratio of 1:1 with the plasmids encoding the heavy chain and light chain of 7.2.32 or 4.1.15 using 293FectinTM Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After transfection, the HEK293F cells were incubated for 7 days at 130 rpm, 37° C. and 8% CO 2 .
  • Fab fragments were purified by Nickel affinity-column chromatography and preparative gel filtration using the ⁇ KTA explorer FPLC system using standard purification methods. Purity was assessed by SDS-PAGE and analytical gel filtration.
  • the strongly negatively charged Oligo and the Oligo-Fab fragments are eluted with a high salt gradient of 20 mM Tris pH 8.0 and 1M NaCl, and thus separated from the Sortase and the unlabeled Fab fragment that elute at a low salt concentration.
  • the elution is monitored following the absorbance at 495 nm, detecting the fluorescein-label of the Oligo.
  • Fab-Oligo The eluted fractions containing Oligo and Fab-Oligo are pooled and the Fab-Oligo is separated from the unconjugated Oligo by preparative gel filtration on a HiLoad 16/60 column Superdex 200 column (GE Healthcare) using 20 mM Tris 8.0, 200 mM NaCl as equilibration and running buffer. The purity of the final product is assessed using analytical gel filtration and SDS-PAGE and only >90% pure end product will be used in the assembly of dual binders. In the following, a Fab-Oligo is referred to as “ssFab”.
  • the IHC experiments were performed on the BenchMark XT platform from Ventana.
  • an anti-pHER3 dual binder was used that consisted of ssFab 7.2.32 (binding a non-phospho epitope of the intracellular domain of HER3), ssFab 4.1.15 (binding the pTyr1289 phospho-epitope of the intracellular domain of HER3) and a flexible linker.
  • a flexible linker with a 4 ⁇ C18 spacer was used in this assay.
  • a biotin label within the linker molecule served as a detection tag for the streptavidin-based Ventana iVIEW DAB detection kit.
  • Hek293 cells had been transiently transfected with both HER2 and HER3 expression vectors.
  • an HER3 expression vector was used that encodes an mutated version of HER3, in which 14 tyrosines of the intracellular domain that serve as phosphorylation sites are replaced with phenylalanines (Y975F, Y1054F, Y1132F, Y1159F, Y1197F, Y1199F, Y1222F, Y1224F, Y1260F, Y1262F, Y1276F, Y1289F, Y1307F, Y1328F).
  • Cells were fixed with formalin and embedded in paraffin according to standard protocols. Prior to fixation, cells were stimulated with 20 nM NRG1- ⁇ 1 (Peprotech) for 15 min at 37° C. to induce HER3 phosphorylation or were left untreated.
  • Western blotting experiments FIG. 20 A proved successful stimulation of receptor phosphorylation.
  • a detection molecule composed of a 4 ⁇ C18 linker molecule (linker 12 of example 2.4) and only ssFab 7.2.32 or only ssFab 4.1.15 did not produce a staining on any of the tested FFPE cell pellets ( FIG. 20 B, rows 1 &2).
  • detection with the full dual binder molecule led to a staining ⁇ but only on cells that were stimulated with NRG1- ⁇ 1 and express wild-type HER3 ( FIG. 20 B, row 3).
  • the experiment proves high specificity of the dual binder for phosphorylated HER3.
US13/923,618 2010-12-23 2013-06-21 Detection of a posttranslationally modified polypeptide by a bi-valent binding agent Abandoned US20130288266A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/465,439 US10982007B2 (en) 2010-12-23 2017-03-21 Detection of a posttranslationally modified polypeptide by a bivalent binding agent

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP10196687 2010-12-23
EP10196687.7 2010-12-23
EP11173832.4 2011-07-13
EP11173832 2011-07-13
PCT/EP2011/073560 WO2012085064A1 (fr) 2010-12-23 2011-12-21 Détection d'un polypeptide à modification post-translationnelle par un agent de liaison bivalent

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2011/073560 Continuation WO2012085064A1 (fr) 2010-12-23 2011-12-21 Détection d'un polypeptide à modification post-translationnelle par un agent de liaison bivalent

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/465,439 Continuation US10982007B2 (en) 2010-12-23 2017-03-21 Detection of a posttranslationally modified polypeptide by a bivalent binding agent

Publications (1)

Publication Number Publication Date
US20130288266A1 true US20130288266A1 (en) 2013-10-31

Family

ID=45491546

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/923,618 Abandoned US20130288266A1 (en) 2010-12-23 2013-06-21 Detection of a posttranslationally modified polypeptide by a bi-valent binding agent
US15/465,439 Active 2032-06-01 US10982007B2 (en) 2010-12-23 2017-03-21 Detection of a posttranslationally modified polypeptide by a bivalent binding agent

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/465,439 Active 2032-06-01 US10982007B2 (en) 2010-12-23 2017-03-21 Detection of a posttranslationally modified polypeptide by a bivalent binding agent

Country Status (7)

Country Link
US (2) US20130288266A1 (fr)
EP (1) EP2659269B1 (fr)
JP (1) JP6046049B2 (fr)
CN (1) CN103384825B (fr)
CA (1) CA2817928C (fr)
ES (1) ES2605493T3 (fr)
WO (1) WO2012085064A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG191153A1 (en) 2010-12-23 2013-07-31 Hoffmann La Roche Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
RU2015100656A (ru) 2012-06-27 2016-08-20 Ф. Хоффманн-Ля Рош Аг Способ получения конъюгатов fc-фрагмента антитела, включающих по меньшей мере одну связывающую группировку, которая специфически связывается с мишенью, и их применения
BR112014029888A2 (pt) * 2012-06-27 2020-05-12 Hoffmann La Roche Métodos de produção de um anticorpo, de determinação de uma combinação de sítios de ligação e de tratamento de um indivíduo com câncer, formulação farmacêutica, anticorpo e uso de um anticorpo
WO2014001324A1 (fr) 2012-06-27 2014-01-03 Hoffmann-La Roche Ag Méthode de sélection et de production d'entités de ciblage personnalisées fortement sélectives et multi-spécifiques renfermant au moins deux entités de liaison différentes et leurs utilisations
JP6274570B2 (ja) * 2013-11-12 2018-02-07 国立大学法人群馬大学 新規ヌクレオシド誘導体、それを含むポリヌクレオチドならびに該ポリヌクレオチドを用いたボトムアップ的三次元細胞培養方法および核酸アプタマーの選択方法
AU2016218055B2 (en) * 2015-02-09 2019-05-16 Ventana Medical Systems, Inc. Materials and methods for standardizing diffusion of a fluid into tissues
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
CN107098934B (zh) * 2017-04-07 2019-03-12 厦门大学 一种邻苯二醛衍生物亚磷酰胺单体、合成方法及dna快速偶联蛋白质的方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015682A1 (fr) * 1991-02-27 1992-09-17 Creative Biomolecules, Inc. Segments de liaison peptidique riches en serine
US6153190A (en) * 1995-06-07 2000-11-28 Young; Peter Ronald Erythropoietin receptor antibodies
US6569619B1 (en) * 1998-07-29 2003-05-27 Tularik, Inc. High-throughput in vitro screening assays for modulators of nucleic acid helicases
US20030219827A1 (en) * 2002-02-20 2003-11-27 Comb Michael J. Phospho-specific antibodies to Flt3 and uses thereof
US20080280778A1 (en) * 2007-05-03 2008-11-13 Urdea Michael S Binding reagents that contain small epitope binding molecules
US20080312103A1 (en) * 2004-10-15 2008-12-18 Japan Science And Technology Agency Linker For Constructing Mrna-Puromycin-Protein Conjugate
US20090137782A1 (en) * 2001-05-11 2009-05-28 Old Lloyd J Specific binding proteins and uses thereof
US20100266617A1 (en) * 2007-06-18 2010-10-21 N.V. Organon Antibodies to human programmed death receptor pd-1

Family Cites Families (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2095818B (en) 1981-03-27 1985-10-02 Exxon Research Engineering Co Staged adsorption/resorption heat pump
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4868105A (en) 1985-12-11 1989-09-19 Chiron Corporation Solution phase nucleic acid sandwich assay
IL86164A0 (en) 1987-04-28 1988-11-15 Tamir Biotechnology Ltd Improved dna probes
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
PT88550A (pt) 1987-09-21 1989-07-31 Ml Tecnology Ventures Lp Processo para a preparacao de reagentes de ligacao nao nucleotidicos para sondas nucleotidicas
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
FR2632955B1 (fr) 1988-06-20 1991-12-27 Oris Ind Derives de nucleosides utilisables pour la synthese d'oligonucleotides marques, oligonucleotides obtenus a partir de ces derives et leur synthese
KR0184860B1 (ko) 1988-11-11 1999-04-01 메디칼 리써어치 카운실 단일영역 리간드와 이를 포함하는 수용체 및 이들의 제조방법과 이용(법)
FR2642074B1 (fr) 1989-01-20 1994-04-29 Oris Ind Derives de molecules polyhydroxylees permettant l'introduction d'au moins une ramification dans un oligonucleotide
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US5290925A (en) 1990-12-20 1994-03-01 Abbott Laboratories Methods, kits, and reactive supports for 3' labeling of oligonucleotides
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
WO1993002093A1 (fr) 1991-07-15 1993-02-04 La Jolla Pharmaceutical Company Intermediaires phosphoreux modifies produisant des groupes fonctionnels sur la terminaison 5' des oligonucleotides
DE59209203D1 (de) 1991-08-28 1998-03-26 Boehringer Mannheim Gmbh Primer für matrizenabhängige enzymatische nukleinsäuresynthesen
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
DK1024191T3 (da) 1991-12-02 2008-12-08 Medical Res Council Fremstilling af autoantistoffer fremvist på fag-overflader ud fra antistofsegmentbiblioteker
AU675929B2 (en) 1992-02-06 1997-02-27 Curis, Inc. Biosynthetic binding protein for cancer marker
WO1994004550A1 (fr) 1992-08-21 1994-03-03 Triplex Pharmaceutical Corporation Oligonucleotides modifies par cholesterol formant une triple helice, et utilisation desdits oligonucleotides
AU678787B2 (en) 1992-10-23 1997-06-12 Immunex Corporation Methods of preparing soluble, oligomeric proteins
DE4310141A1 (de) 1993-03-29 1994-10-06 Boehringer Mannheim Gmbh Homobidentale trifunktionelle Linker
US5635602A (en) 1993-08-13 1997-06-03 The Regents Of The University Of California Design and synthesis of bispecific DNA-antibody conjugates
US5849879A (en) 1994-11-03 1998-12-15 The Regents Of The University Of California Methods for the diagnosis of glaucoma
WO1997005156A1 (fr) 1995-07-27 1997-02-13 Carsten Behrens Nouveau reactif de liaison achiral pour incorporer de multiples groupes amines dans des oligonucleotides
US5736626A (en) 1996-01-29 1998-04-07 The Perkin-Elmer Corporation Solid support reagents for the direct synthesis of 3'-labeled polynucleotides
ATE252158T1 (de) 1996-05-15 2003-11-15 Biogenex Lab Nicht-nukleotid verknüpfende reagentien
CA2297070A1 (fr) 1997-08-01 1999-02-11 Morphosys Ag Nouvelle methode et nouveau phage d'identification d'une sequence d'acide nucleique
US6117986A (en) 1998-06-10 2000-09-12 Intergen Company, L.P. Pyrimidines linked to a quencher
IL140851A0 (en) * 1998-07-27 2002-02-10 Applied Spectral Imaging Ltd In situ method of analyzing cells
CA2270795A1 (fr) 1999-05-05 2000-11-05 Gestilab Inc. Compositions neuroprotectrices et utilisations de ces compositions
JP2000325086A (ja) * 1999-05-21 2000-11-28 Igaku Seibutsugaku Kenkyusho:Kk リン酸化酵素活性の測定方法
EP1074563A1 (fr) 1999-08-02 2001-02-07 F. Hoffmann-La Roche Ag Polypeptides chimériques augmentant la formation de dimères par des interactions électrostatiques et des ponts disulfures, méthode pour les produire et leurs utilisations
US6727356B1 (en) 1999-12-08 2004-04-27 Epoch Pharmaceuticals, Inc. Fluorescent quenching detection reagents and methods
AU2001281248A1 (en) 2000-08-11 2002-03-13 Nanosphere Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
DE10044373A1 (de) 2000-09-08 2002-03-21 Roche Diagnostics Gmbh Neues Reagenz zur Markierung von Nukleinsäuren
DE60237282D1 (de) 2001-06-28 2010-09-23 Domantis Ltd Doppelspezifischer ligand und dessen verwendung
US20100081792A1 (en) 2001-06-28 2010-04-01 Smithkline Beecham Corporation Ligand
US20030082547A1 (en) 2001-08-27 2003-05-01 Ewing Gregory J. Non-fluorescent quencher compounds and biomolecular assays
KR100527334B1 (ko) 2002-06-07 2005-11-09 (주)넥스젠 올리고뉴클레오타이드의 신규한 링커
ATE319699T1 (de) 2002-12-20 2006-03-15 Hoffmann La Roche Mannitol- und glucitolderivate
GB0305702D0 (en) 2003-03-12 2003-04-16 Univ Birmingham Bispecific antibodies
US7238792B2 (en) 2003-03-18 2007-07-03 Washington State University Research Foundation Foldable polymers as probes
WO2005035753A1 (fr) 2003-10-10 2005-04-21 Chugai Seiyaku Kabushiki Kaisha Anticorps a double specificite remplaçant une proteine fonctionnelle
AU2003271186A1 (en) 2003-10-14 2005-04-27 Chugai Seiyaku Kabushiki Kaisha Double specific antibodies substituting for functional protein
CA2494571C (fr) 2003-12-02 2010-02-09 F.Hoffmann-La Roche Ag Oligonucleotides renfermant des tiges moleculaires
US7705150B2 (en) 2004-02-04 2010-04-27 Biosearch Technologies, Inc. Cyanine dyes
EP1842226B2 (fr) 2004-11-03 2014-07-02 Iris International, Inc. Detection homogene de substance a analyser
US20060199207A1 (en) 2005-02-24 2006-09-07 Matysiak Stefan M Self-assembly of molecules using combinatorial hybridization
WO2008157379A2 (fr) * 2007-06-21 2008-12-24 Macrogenics, Inc. Di-anticorps covalents et leurs utilisations
US7795009B2 (en) 2005-06-15 2010-09-14 Saint Louis University Three-component biosensors for detecting macromolecules and other analytes
ES2804129T3 (es) * 2005-11-23 2021-02-03 Ventana Med Syst Inc Conjugado anticuerpo-enzima
ES2332139T3 (es) 2005-11-23 2010-01-27 F. Hoffmann-La Roche Ag Polinucleotidos con mimetico de fosfato.
ES2435420T3 (es) 2005-12-15 2013-12-19 Centre National De La Recherche Scientifique - Cnrs Oligonucleótidos catiónicos, procedimientos automáticos para preparar los mismos y sus usos
JP5164971B2 (ja) 2006-04-21 2013-03-21 ピープルバイオ，アイ エヌ シー 三次元的相互作用を用いてマルチマー形成ポリペプチドのモノマーからマルチマーを分別検出する方法
GB0614780D0 (en) * 2006-07-25 2006-09-06 Ucb Sa Biological products
US20110143953A1 (en) 2006-10-16 2011-06-16 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Synthetic Antibodies
EP2061901A2 (fr) 2006-10-31 2009-05-27 Noxxon Pharma AG Procédé de détection d'un acide nucléique simple brin ou double brin
KR101072900B1 (ko) * 2007-12-04 2011-10-17 주식회사 파나진 고정화된 펩티드핵산 프로브를 사용한 표적핵산의 선택적 표지 및 검출 방법
TW201007669A (en) 2008-08-01 2010-02-16 Integrated Solutions Technology Inc A display driving device and the driving method thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015682A1 (fr) * 1991-02-27 1992-09-17 Creative Biomolecules, Inc. Segments de liaison peptidique riches en serine
US6153190A (en) * 1995-06-07 2000-11-28 Young; Peter Ronald Erythropoietin receptor antibodies
US6569619B1 (en) * 1998-07-29 2003-05-27 Tularik, Inc. High-throughput in vitro screening assays for modulators of nucleic acid helicases
US20090137782A1 (en) * 2001-05-11 2009-05-28 Old Lloyd J Specific binding proteins and uses thereof
US20030219827A1 (en) * 2002-02-20 2003-11-27 Comb Michael J. Phospho-specific antibodies to Flt3 and uses thereof
US20080312103A1 (en) * 2004-10-15 2008-12-18 Japan Science And Technology Agency Linker For Constructing Mrna-Puromycin-Protein Conjugate
US20080280778A1 (en) * 2007-05-03 2008-11-13 Urdea Michael S Binding reagents that contain small epitope binding molecules
US20100266617A1 (en) * 2007-06-18 2010-10-21 N.V. Organon Antibodies to human programmed death receptor pd-1

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Colman et al. (Research in Immunology, 1994; 145(1): 33-36) *
Harlow & Lane (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 23-26) *
Lederman et al. ("A single amino acid substitution in a common African allele of the CD4 molecule ablates binding of the monoclonal antibody, OKT4" Mol Immunol. 1991 Nov;28(11):1171-81) *

Also Published As

Publication number Publication date
CN103384825A (zh) 2013-11-06
JP6046049B2 (ja) 2016-12-14
JP2014504298A (ja) 2014-02-20
CN103384825B (zh) 2017-05-24
ES2605493T3 (es) 2017-03-14
CA2817928A1 (fr) 2012-06-28
EP2659269A1 (fr) 2013-11-06
EP2659269B1 (fr) 2016-10-26
US20170275381A1 (en) 2017-09-28
WO2012085064A1 (fr) 2012-06-28
CA2817928C (fr) 2019-10-01
US10982007B2 (en) 2021-04-20

Similar Documents

Publication Publication Date Title
US10982007B2 (en) Detection of a posttranslationally modified polypeptide by a bivalent binding agent
US20200277406A1 (en) Binding agent
US20170275380A1 (en) Detection of a polypeptide dimer by a bivalent binding agent
AU2023201547A1 (en) Nucleic acid sequencing using affinity reagents
US10301383B2 (en) Antibody specifically binding to insulin-like growth factor-1
US11123440B2 (en) Compositions and methods for making antibody conjugates
Shen et al. Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies
Nevoltris et al. Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers
JP2022133350A (ja) 分析目的のための多価単一または二重特異性組換え抗体
KR102495650B1 (ko) 알데히드 함유 표적 분자의 라벨링 방법
US20220354962A1 (en) Rapid production of bispecific antibodies from off-the-shelf iggs with high yield and purity

Legal Events

Date Code Title Description
AS Assignment

Owner name: ROCHE DIAGNOSTICS GMBH, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GERG, MICHAEL;HEINDL, DIETER;SCHMID, VOLKER;AND OTHERS;SIGNING DATES FROM 20130703 TO 20130819;REEL/FRAME:031221/0167

Owner name: ROCHE DIAGNOSTICS OPERATIONS, INC., INDIANA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ROCHE DIAGNOSTICS GMBH;REEL/FRAME:031221/0188

Effective date: 20130905

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION