Patent Application
20170275380
A paper copy of the Sequence Listing and a computer readable form of the Sequence Listing containing the file named “27514_US_2. ST25.txt, which is 19,032 bytes in size (as measured in MICROSOFT WINDOWS EXPLORER), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs:1-28.
Many proteins, especially many membrane-bound receptor molecules are inactive as monomers and activated by homo- and/or heterodimerization. The specific detection of homo- or heterodimers of such receptor molecules against the background of single, non-dimerized molecules is a tremendous challenge.
The present disclosure relates to a bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to an epitope of a first target polypeptide comprised in said dimer, wherein the second monovalent binder binds to an epitope of a second target polypeptide comprised in said dimer, wherein each monovalent binder has a Kdiss in the range of 5×10−3/sec to 10−4/sec, and wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less. A method of making such bivalent binding agent and the use of such bivalent agent in histological staining procedures are also disclosed.
In one embodiment the present disclosure relates to a bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to an epitope of a first target polypeptide comprised in said dimer, wherein the second monovalent binder binds to an epitope of a second target polypeptide comprised in said dimer, wherein each monovalent binder has a kdiss in the range of 5×10−3/sec to 10−4/sec, and wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less.
Also disclosed is a method for obtaining a bivalent binding agent that specifically binds a polypeptide dimer, the method comprising the steps of selecting a first monovalent binder that binds to a first target polypeptide with a Kdiss of between 5×10−3/sec to 10−4/sec, selecting a second monovalent binder that binds to a second target polypeptide with a Kdiss of 5×10−3/sec to 10−4/sec, coupling both monovalent binders by a linker, and selecting a bivalent binding agent having a Kdiss-value of 3×10−5/sec or less.
The use of the novel bivalent binding agent, especially in an immunohistochemical procedure is also described and claimed.
The features of this disclosure, and the manner of attaining them, will become more apparent and the disclosure itself will be better understood by reference to the following description of embodiments of the disclosure taken in conjunction with the accompanying drawing.
Although the drawings represent embodiments of the present disclosure, the drawings are not necessarily to scale and certain features may be exaggerated in order to better illustrate and explain the present disclosure. The exemplifications set out herein illustrate an exemplary embodiment of the disclosure, in one form, and such exemplifications are not to be construed as limiting the scope of the disclosure in any manner.
1. Antibody Fragments
2. Sequences of ssDNA
a) 19mer ssDNA (covalently bound with 3′ end to Fab′ of anti-TroponinT MAB b or Fab′ 8.1.2 to phosphorylated IGF-1R, respectively): 5′-A GTC TAT TAA TGC TTC TGC-3′(SEQ ID NO:5)
b) 17mer ssDNA (covalently bound with 5′ end to Fab′ of anti-TroponinT MAB a or Fab′ 1.4.168 to IGF-1R, respectively): 5′-AGT TCT ATC GTC GTC CA-3′(SEQ ID NO:6)
c) complementary 19mer ssDNA (used as part of a linker): 5′-G CAG AAG CAT TAA TAG ACT-3′(SEQ ID NO:7)
d) complementary 17mer ssDNA (used as part of a linker): 5′-TGG ACG ACG ATA GAA CT-3′(SEQ ID NO:8)
3. Sequences of Troponin T Epitopes:
SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U represents β-Alanine. (The epitope “A” for antibody anti-Troponin antibody a.)
SEQ ID NO:10=SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein 0 represents Amino-trioxa-octanoic-acid. (The epitope “B” for antibody anti-Troponin antibody b.)
4. Sequences of IGF-1R/IR Epitopes
5. Protein Linker and Tag-Sequences
6. Sequences Used for Compiling the Bivalent Binding Agent to a Polypeptide Homodimer.
SEQ ID NO:16=ACCTGCTGCT ATCTTGA; this oligonucleotide was FAM-labeled at the 5′ end and for coupling modified to carry a maleimido at the 3′ end.
SEQ ID NO:17=GCAGAAGCATTAATAGACYT(Biotin)-GGACGACGATAGAACT; the linker oligonucleotide
SEQ ID NO:18=GTCTATTAATGCTTCTGCCTGGACGACGATAGAACT; the adaptor oligonucleotide
7. Sequences Used in a Dual-Binder to HER2/HER3 Heterodimers for Use in Immunohistochemistry.
Although the sequence listing represents an embodiment of the present disclosure, the sequence listing is not to be construed as limiting the scope of the disclosure in any manner and may be modified in any manner as consistent with the instant disclosure and as set forth herein.
The embodiments disclosed herein are not intended to be exhaustive or limit the disclosure to the precise form disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may utilize their teachings.
As disclosed herein, it surprisingly has now been found that a bivalent binding agent can be provided that is capable of binding to a homo- or heterodimer and at the same time exhibiting no significant binding to the monomeric form of the one polypeptide comprised in the homodimer or to any of the two polypeptides comprised in the heterodimer.
In one embodiment the present disclosure relates to a bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker, a) wherein the first monovalent binder binds to an epitope of a first target polypeptide comprised in said dimer, b) wherein the second monovalent binder binds to an epitope of a second target polypeptide comprised in said dimer, c) wherein each monovalent binder has a Kdiss in the range of 5×10−3/sec to 10−4/sec, and d) wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less.
Sometimes a single polypeptide is sufficient for the protein to be active. As indicated above, often, however, two or more polypeptides need to interact to allow a protein to perform its particular function. If this is the case, one talks e.g. of a protein or polypeptide dimer in case two polypeptide chains associate with one another. The dimer formation results from interactions between two polypeptide chains and/or and can be triggered by binding of a ligand. Dimers are held together by hydrogen bonds, ionic bonds and, less commonly, hydrophobic interfaces and inter-chain disulphide bonds.
Receptor homo- and/or heterodimerization is an extremely important mechanism in the regulation of cellular activities and physiological and/or pathological processes.
A dimer in the sense of the present disclosure is present if two polypeptide chains are associated and form a biologically relevant complex. The dimer may be a membrane-bound dimer or it may be a dimer present in the circulation, i.e. a complex of two polypetides that is stable under physiological conditions. In one embodiment the two polypeptide chains in a dimer (as they occur in nature) are not covalently linked but held together by protein-protein interactions, e.g. based on ion bridges or on van der Wals forces.
The bivalent binding agent according to the present disclosure is a binding agent comprising exactly two monovalent binders.
In some embodiments the kinetic rate properties of each monovalent binder and of the bivalent binding agent are characterized by BIACORE™ SPR technology as described in detail in the examples.
As the skilled artisan will appreciate the bivalent binding agent described in the present disclosure can be isolated and purified as desired. In one embodiment the present disclosure relates to an isolated bivalent binding agent as disclosed herein. An “isolated” bivalent 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 bivalent binding agent. Unwanted components of such reaction mixture are e.g. monovalent binders that did not end up in the desired bivalent binding agent. In one embodiment, the bivalent binding agent is purified to greater than 80%. In some embodiments, the bivalent binding agent is purified to greater than 90%, 95%, 98% or 99% by weight, respectively. In case both monovalent binders are polypeptides purity is e.g. easily determined by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain in protein detection. In case purity is assessed on the nucleic acid level, size exclusion chromatography is applied to separate the bivalent binding agent from side products and the OD at 260 nm is monitored to assess its purity.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an antibody” means one antibody or more than one antibody.
The term “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 some embodiments an oligonucleotide will have a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In some embodiments 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.
The term 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). Examples of such substituted nucleobases are: 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. I.e. the naturally occurring nucleobases can be exchanged by using nucleobase analogs like 5-Nitroindol d riboside; 3 nitro pyrrole d riboside, deoxyinosine (dl), deoyxanthosine (dX); 7 deaza-dG, -dA, -dl or -dX; 7-deaza-8-aza-dG, -dA, -dl or -dX; 8-aza-dA, -dG, -dl or -dX; d Formycin; pseudo dU; pseudo iso dC; 4 thio dT; 6 thio dG; 2 thio dT; iso dG; 5-methyl-iso-dC; N8-linked 8-aza-7-deaza-dA; 5,6-dihydro-5-aza-dC; and etheno-dA or pyrollo-dC. As obvious to the skilled artisan, the 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 (oligomers known as morpholino DNA).
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) can also be used as a DNA analog.
The above mentioned modified nucleotides, nucleotide analogs as well as oligonucleotide backbone modifications can be combined as desired in an oligonucleotide in the sense of the present disclosure.
The terms “polypeptide” and “protein” are used inter-changeably. A polypeptide in the sense of the present disclosure 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 disclosure is a polypeptide known or suspected to form a homo- or a heterodimeric polypeptide complex.
A “monovalent binder” according to the present disclosure 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 may be analyzed by biosensor-based surface plasmon resonance spectroscopy. For example, the BIACORE™ technology as described in detail in the Examples section may be used.
Examples of monovalent binders are peptides, peptide mimetics, aptamers, spiegelmers, darpins, lectines, 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.
As obvious to the skilled artisan, each monovalent binder according to the present disclosure only binds to a single epitope on a target polypeptide.
In some embodiments 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.
In some embodiments at least one of the monovalent binders is a single domain antibody, an Fab-fragment or an Fab′-fragment of a monoclonal antibody.
It also represents embodiments that in the bivalent binding agent disclosed herein 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.
An Fv is a minimum antibody fragment that contains a complete antigen-binding site and is devoid of constant region. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In one embodiment of a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a dimeric structure analogous to that in a two-chain Fv species. For a review of scFv, see, e.g., Plueckthun, In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Generally, six hyper variable regions (HVRs) confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen.
An Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
Various techniques have been developed for the production of antibody fragments. Traditionally, 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). For example, 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.
In certain embodiments, a monovalent binder of the present disclosure is a single-domain antibody. A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.
One of the two monovalent binders, the first monovalent binder, binds to an epitope on a first (target) polypeptide.
An “epitope” according to the present disclosure—the binding site on the target polypeptide bound by the corresponding monovalent binder—is composed of amino acids. The amino acids are naturally occurring amino acids and may carry one or more secondary modification. In one embodiment the amino acids are not secondarily modified. In one embodiment the monovalent 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 the target polypeptide. Tertiary epitopes recognized by a binder, e.g. by 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.
In the bivalent binding agent as disclosed herein the first monovalent binder binds to an epitope on a first polypeptide and the second monovalent binder binds to a epitope on a second polypeptide. The first and the second polypeptide in a polypeptide dimer can have an identical sequence, i.e. this dimer is a homodimer, or the first and the second polypeptide can be different, i.e. this dimer is a heterodimer.
As mentioned above, for many receptor molecules the formation of a polypeptide homo- or heterodimer, respectively, is 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). As the nomenclature already suggests, at least part of the intracellular signaling of the RTKs is mediated by the phosphorylation status of certain tyrosine of the intracellular domain of such RTKs. In some embodiments the present disclosure thus relates to a bivalent binding agent binding to a receptor polypeptide dimer. Obviously such bivalent binding agent is of great utility in the detection of active homo- or hetrodimeric receptor polypeptides.
In some embodiments the present disclosure relates to a bivalent binding agent as disclosed herein above, wherein the target polypeptide dimer is selected from the group consisting of membrane-bound receptor molecules, in some cases from homo- or heterodimers formed by association of two receptor tyrosine kinase polypeptides.
In some embodiments, the RTK polypeptide being part of a homo- or heterodimer 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, ROR2, ROS, RYK, vascular endothelial growth factor (VEGF) receptors (e.g., VEGFR1/FLT1, VEGFR2/FLK1, VEGF3), tyrosine kinase with immunoglobulin-like and EGF-like domains (TIE) receptors (e.g., TIE-1, TIE-2/TEK), Tec, TYRO10, insulin-like growth factor (IGF) receptors (e.g., INS-R, IGF-IR, IR-R), Discoidin Domain (DD) receptors (e.g., DDR1, DDR2), receptor for c-Met (MET), recepteur d′origine nantais (RON); also known as macrophage stimulating 1 receptor, Flt3 fins-related tyrosine kinase 3 (Flt3), colony stimulating factor 1(CSF1) receptor, receptor for c-kit (KIT, or SCFR) and insulin receptor related (IRR) receptors.
In one embodiment, the RTK being part of a homo- or heterodimer is selected from the group consisting of: ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-A, PDGFR-B) and vascular endothelial growth factor (VEGF) receptors and co-receptors (e.g., VEGFR1/FLT1, VEGFR2/FLK1, VEGF3, neuropilin-1, neuropilin-2), insulin-like growth factor (IGF) receptors (e.g., INS-R, IGF-IR, IR-R), and insulin receptor related (IRR) receptors.
In one embodiment, the RTK being part of a homo- or heterodimer is selected from the group consisting of: ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-A, PDGFR-B) and vascular endothelial growth factor (VEGF) receptors and co-receptors (e.g., VEGFR1/FLT1, VEGFR2/FLK1, VEGF3, neuropilin-1, neuropilin-2), insulin-like growth factor (IGF) receptors (e.g., INS-R, IGF-IR, IR-R), and insulin receptor related (IRR) receptors.
In one embodiment the present disclosure relates to a bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to an epitope of a first target polypeptide comprised in said dimer, wherein the second monovalent binder binds to an epitope of a second target polypeptide comprised in said dimer, wherein each monovalent binder has a Kdiss in the range of 5×10−3/sec to 10−4/sec, wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less and wherein said dimer is a receptor polypeptide dimer selected from the group consisting of ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4) and vascular endothelial growth factor (VEGF) receptors and co-receptors (e.g., VEGFR1/FLT1, VEGFR2/FLK1, VEGF3, neuropilin-1, neuropilin-2).
In one embodiment receptor homo- and/or heterodimerization relates to the polypeptides involved in VEGF-signaling. It includes the vascular endothelial growth factors and their corresponding receptors as one specific example (for full details see Otrock, Z. et al., Blood Cells, Molecules and Diseases: 38 (2007) 258-268).
VEGF signaling often represents a critical rate-limiting step in physiological angiogenesis. Angiogenesis is the sprouting of new blood vessels from the pre-existing ones. This process is important for the growth of new blood vessels during fetal development and tissue repair; however, uncontrolled angiogenesis promotes neoplastic diseases and other disorders. The successful implementation of this process depends upon the balance of growth promoting factors and growth inhibitory factors. One of the most specific and crucial regulators of angiogenesis is VEGF.
The VEGF family comprises seven secreted glycoproteins that are designated VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and VEGF-F. The VEGF family members bind their cognate receptors. The receptors identified so far are designated VEGFR-1, VEGFR-2, VEGFR-3 and the neuropilins (NP-1 and NP-2).
VEGF-A exerts its biologic effect through interaction with cell-surface receptors. These receptors are transmembrane tyrosine kinase receptors and they include VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (kinase insert domain-containing receptor/Flk-1), selectively expressed on vascular endothelial cells, and the neuropilin receptors (NP-1 and NP-2), expressed on vascular endothelium and neurons. Upon binding of VEGF-A to the extracellular domain of the receptor, a cascade of downstream proteins is activated after the dimerization and autophosphorylation of the intracellular receptor tyrosine kinases. VEGFR-2 appears to be the main receptor responsible for mediating the proangiogenic effects of VEGF-A.
VEGFR-1 (fms-like tyrosine kinase; Flt-1) is composed of seven extracellular immunoglobulin (Ig) homology domains, a single transmembrane region and an intracellular tyrosine kinase domain. VEGFR-1 binds VEGF-A, VEGF-B and PIGF with high affinity.
VEGFR-2 (KDR, human; Flk-1, mouse) was first isolated in 1991 and was named Kinase-insert domain containing receptor (KDR). Like VEGFR-1, VEGFR-2 bears an extracellular region with seven immunoglobulin (Ig)-like domains, a transmembrane domain and a tyrosine kinase domain with about 70-amino-acid insert. VEGFR-2 binds VEGF-A, VEGF-C, VEGF-D and VEGF-E.
VEGFR-3 (fins-like tyrosine kinase 4, Flt4), a member of the endothelial cells receptor tyrosine kinases, has only six Ig-homology domains [121]. VEGFR-3 preferentially binds VEGF-C and VEGF-D.
The VEGF receptors 1, 2 and 3 upon ligand binding can form homo-dimers that are active and exert their effects via the intracellular tyrosine kinase domain. However, homodimer formation is not the only way of action and the so-called neuropilins play an important role as co-receptors.
Neuropilin NP-1 was identified initially as a 130- to 140-kDa cell-surface glycoprotein that served as a receptor for the semaphorin/collapsins, a large family of secreted and transmembrane proteins that serve as repulsive guidance signals in axonal and neuronal development. NP-1 binds VEGF-A, VEGF-B and PIGF while NP-2 binds VEGF-A, VEGF-C and PIGF.
NP-1 acts as a co-receptor enhancing VEGF-A-VEGFR-2 interactions, forming complexes with VEGFR-1 and augmenting tumor angiogenesis in vivo. In order to investigate this pathway the specific detection of a VEGFR-2/NP-1 heterodimer is required.
Recent studies have linked NP-2 to lymphatic vessel development.
As has been pointed out above the members of VEGF family of receptors as long as they are monomeric are not active, however, upon ligand binding and homo- and/or heterodimerization an array of different biochemical pathways, leading to different modes of action is induced. The capability to differentiate between (inactive) monomers and the various homo- and/or heterodimers is of utmost importance in elucidating the physiological mode of action of the VEGF/VEGF receptor system and the effect of drugs targeting one or more members of this system. In one embodiment the present disclosure relates to a bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binder binds to an epitope of a first target polypeptide comprised in said dimer, wherein the second monovalent binder binds to an epitope of a second target polypeptide comprised in said dimer, wherein each monovalent binder has a Kdiss in the range of 5×10−3/sec to 10−4/sec, wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less and wherein the homo- and/or heterodimer is part of the VEGF/VEGF receptor system. In such binder in one embodiment one monovalent binder binds to a VEGF selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and VEGF-F and the other monovalent binder binds to a receptor selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3 and the neuropilins (NP-1 and NP-2). Or, such binder in one embodiment has one monovalent binder that binds to a to a receptor polypeptide selected from the group consisting of VEGFR-1, VEGFR-2 and VEGFR-3 and the other monovalent binder binds to a neuropilin (NP-1 or NP-2).
As discussed a monovalent binder for use in the construction of a bivalent binding agent as disclosed herein has to have a kdiss from 5×10−3/sec to 10−4/sec.
In some exemplary embodiments, the first monovalent binder is specifically binding to an epitope on the first polypeptide. I.e. this binder binds to an epitope that is either not present on the second target polypeptide as is the case for a heterodimer bivalent binding agent or it binds to an epitope that overlaps with or is identical to an epitope present on the second target polypeptide as is the case for a homodimer bivalent binding agent.
Specific binding to an epitope is acknowledged if said binder has a Kdiss that is at least 20 times lower for the epitope on the target polypeptide as compared to any (similar or completely un-related) epitope on any other peptide (e.g. on the second target polypeptide if the first and the second target polypeptide are different). Polypeptides in a heterodimer are acknowledged to be different if a monovalent binder specifically binds to only one of the polypeptides. Also in some embodiments the Kdiss of the first monovalent binder to the epitope on the first target polypeptide is at least 30-, 40-, 50-, 80-, 90-, 95- or at least 100-fold lower as compared to any epitope on any other polypeptide.
In some embodiments the bivalent binding agent disclosed herein binds to a heterodimer, comprising two different polypeptides. In this case the second monovalent binder may be selected to have a Kdiss that is at least 20 times lower for the first polypeptide as compared to the Kdiss for its binding to the second target polypeptide. Also, in some embodiments the Kdiss of the second monovalent binder to the second target polypeptide is at least 30-, 40-, 50-, 80-90-, 95- or at least 100-fold lower as compared to the Kdiss for any epitope on the first target polypeptide.
As mentioned above the bivalent binding agent according to the present disclosure will have a Kdiss of at most 3×10−5/sec or lower, i.e. better. In one embodiment in the bivalent binding agent according to this disclosure each monovalent binder has a Kdiss from 2×10−3/sec to 10−4/sec. In one embodiment in the bivalent binding agent according to this disclosure each monovalent binder has a Kdiss from 10−3/sec to 10−4/sec.
The automatic immunohistochemistry staining machines distributed by Ventana Medical Systems Inc. Tucson employ rather stringent washing conditions. 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 lower the Kdiss, the better the staining intensity will be. The bivalent binding agent as disclosed herein has a Kdiss of at most 3×10−5/sec. In a further embodiment the bivalent binding agent as disclosed herein has a Kdiss of 2×10−5/sec or less or also in some embodiments of 10−5/sec or less.
In one embodiment the kinetic rate properties of each monovalent binder and of the bivalent binding agent are characterized by BIACORE™ SPR technology as described in detail in the examples.
The bivalent binding agent according to the present disclosure 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. In some embodiments the linker will contain sugar moieties, nucleotide moieties, nucleoside moieties and/or amino acids. In certain embodiments the linker will essentially consist of nucleotides, nucleotide analogues or amino acids.
In some embodiments the linker covalently linking, or binding the two monovalent binders via binding pairs has a length of 6 to 100 nm. Also the linker may have 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 or longer. In some embodiments the linker comprised in a bivalent binding agent according to the present disclosure 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.
It is therefore more practical to use an average parameter (an easy to understand approximation) in such calculation.
In the calculation of a spacer or a linker length the following approximations apply: a) for calculating lengths of nonnucleosidic entities an average bond length of 130 pm with an bond angle of 180° independently of the nature of the linked atoms is used; b) one nucleotide in a single strand is calculated with 500 pm and c) one nucleotide in a double strand is calculated with 330 pm.
The value of 130 pm is based on calculation of the distance of the two terminal carbon atoms 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
As mentioned above, 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′. Therefore, the bivalent binding agent according to the present disclosure, binding to a polypeptide dimer, can be also depicted by the below Formula I:
A-a′:a-S-b:b′-B,
wherein A is a first monovalent binder, binding to an epitope of said first target polypeptide, wherein B is a second monovalent binder, binding to an epitope of said second 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 or a′:a and/or b:b′ are covalently bound, wherein a′:a and b:b′ are different, wherein S is a spacer, wherein—represents a covalent bond, wherein the linker a-S-b has a length of 6 to 100 nm and wherein the bivalent 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. Also the linker may have 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 some embodiments the linker has between 10 nm and 50 nm in length. In some embodiments, 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 disclosure may be varied as desired. In order to easily make available spacers of variable length, a library, some embodiments may have a simple synthetic access to the spacers of such library. A combinatorial solid phase synthesis of a spacer is also present in some embodiments. 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 these requirements. In such spacer monomeric units within a spacer are linked in each case via a phosphate or phosphate analog moiety.
The spacer S can contain free positively or/and negatively charged groups of polyfunctional amino-carboxylic acids, e.g. amino, carboxylate or phosphate. For example the charge carriers can be derived from trifunctional aminocarboxylic acids which contain a) an amino group and two carboxylate groups or b) two amino groups and one carboxylate group. Examples of such trifunctional aminocarboxylic acids are lysine, ornithine, hydroxylysine, α,β-diamino propionic acid, arginine, aspartic acid and glutamic acid, carboxy glutamic acid and symmetric trifunctional carboxylic acids like those described in EP-A-0 618 192 or U.S. Pat. No. 5,519,142. Alternatively one of the carboxylate groups in the trifunctional aminocarboxylic acids a) can be replaced by a phosphate, sulphonate or sulphate group. An example of such a trifunctional amino acid is phosphoserine.
The spacer S can also contain uncharged hydrophilic groups. Examples of uncharged hydrophilic groups include ethylene oxide or polyethylene oxide groups with at least three ethylene oxide units, sulphoxide, sulphone, carboxylic acid amide, carboxylic acid ester, phosphonic acid amide, phosphonic acid ester, phosphoric acid amide, phosphoric acid ester, sulphonic acid amide, sulphonic acid ester, sulphuric acid amide and sulphuric acid ester groups. The amide groups may be primary amide groups, and in some embodiments carboxylic acid amide residues in amino acid side groups e.g. the amino acids asparagine and glutamine. The esters may be derived from hydrophilic alcohols, in particular C1-C3 alcohols or diols or triols.
In one embodiment the spacer S is composed of one type of monomer. For example, 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.
In one embodiment, the spacer is DNA. In some embodiments 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. As pointed out ease of synthesis and variability in spacer length are important for a spacer library. Spacers of variable length are extremely utile in identifying the bivalent dual binder according to the present disclosure having a spacer of optimal length thus providing for the optimal distance between the two monovalent binders.
Spacer building blocks, as the name says, can be used to introduce a spacing moiety into the spacer S or to build the spacer S of the linker a-S-b.
Different numbers and kinds of non-nucleotidic as well nucleotidic spacer building blocks are at hand for introducing spacing moieties.
Many different 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.
In bifunctional spacer building blocks a hydroxyl group which is protected with an acid labile protecting group is connected to a phosphoramidite group.
Bifunctional spacer building blocks in one embodiment are non-nucleosidic compounds. For example, 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. Cyclic moieties like C5-C6-cycloalkyl, C4N, CSN, C40,C50-heterocycloalkyl, phenyl which are optionally substituted with one or two C1-C6 alkyl groups can also be used as nonnucleosidic bifunctional spacer moieties. 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 nably dimethoxytrityl and the other is part of a phosphoramidite.
A simple way to build the spacer S or to introduce spacing moieties into the spacer S is to use standard D or L nucleoside phosphoramidite building blocks. In one embodiment a single strand stretch of dT is used. This is advantageous, because dT does not carry a base protecting group.
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.
For hybridization in one embodiment oligonucleotides modified with a functional moiety X are used. The oligonucleotide used for hybridization can have one or two terminal extensions 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. In one embodiment 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 is may be used in order to produce a polylabel or in order to get a high local concentration of X.
In one embodiment the spacer S has a backbone length of 1 to 100 nm. With other words here the groups a and b of Formula I are between 1 and 100 nm apart. In one embodiment 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′” each independently represent a binding pair or represent covalently bound a′:a and/or b:b′, respectively.
“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.
In one embodiment either a′:a or b:b′ are bound covalently and the other, i.e., b:b′ or a′:a, respectively, represents a binding pair.
In one embodiment both a′:a and b:b′ are bound covalently.
The coupling chemistry between a′:a and b:b′ is different from one another and selected from standard protocols. Depending on the nature of the binding partner and of the spacer, appropriate conjugation chemistries are chosen.
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). As the skilled artisan will appreciate, the reactive sites (a), (a′), (b) and (b′), respectively, leading to the covalent bond a′:a as well as b:b′, respectively, preferably also do not to interfere with any functional group that might be present on a monovalent binder (A and/or B of Formula I).
In case at least one of 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).
The above bi-orthogonal coupling chemistries are e.g. appropriate if at least one of the monovalent binders is a polypeptide. If the two binding partners are not carrying certain reactive functional groups, e.g. in the case of combination of two aptamers, as the monovalent binders A and B, respectively, there is more freedom in selection of the reactive sites (a′), (a), (b) and (b′), respectively. Therefore in addition or in combination with the pairs of corresponding reactive sites given in the above table, amino/active ester (e.g. NHS ester), and SH/SH or SH/maleinimido can be used for orthogonal coupling.
As obvious from the above examples at least one of the covalent bonds between a′:a and between b:b′, respectively is not an alpha amino peptide bond. Also in some embodiments both covalent bonds are not alpha amino peptide bonds.
In one embodiment both a′:a and b:b′ are a binding pair. Consequently, in one embodiment the present disclosure 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 an epitope of a first target polypeptide, wherein B is a second monovalent binder, binding to an epitope on a second 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 bivalent binding agent has a Kdiss of 3×10−8/sec or less.
In this embodiment a and a′ are the members of the binding pair a′:a and b and b′ are the members of the binding pair b:b′, respectively. In some embodiments each member of a binding pair is of a molecular weight of 10 kD or below. In further embodiments the molecular weight of each binder of such binding pair is 8, 7, 6, 5 or 4 kD or below.
In one embodiment a′:a and b:b′ are binding pairs and the members of the binding pairs a′:a and b:b′ are selected from the group consisting of leucine zipper domain dimers and hybridizing nucleic acid sequences. In one embodiment both binding pairs represent leucine zipper domain dimers. In one embodiment both binding pairs are hybridizing nucleic acid sequences.
In case a:a′ or b′:b represents a binding pair, the binding affinity for (within) such binding pair is at least 108 l/mol. Both binding pairs are different. For 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. Also, in some embodiments 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. In one embodiment 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.
The term “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. Examples of leucine zipper domains suitable for producing soluble multimeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe, H. J. et al., FEBS Lett. 344 (1994) 191-195.
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.
If leucine zipper domains form the first binding pair (a′:a) and the second binding pair (b:b′), both leucine zipper sequences are different, i.e. sequences a and a′ do not bind to b and b′. Leucine zipper domains may be isolated from natural proteins known to contain such domains, such as transcription factors. One leucine zipper domain may e.g. come from the transcription factor fos and a second one from the transcription factor jun. Leucine zipper domains may also be designed and synthesized artificially, using standard techniques for synthesis and design known in the art.
In some embodiments both members of the binding pairs a′:a and b:b′, i.e. a, a′, b and b′ represent leucine zipper domains and the spacer S consists of amino acids. In this embodiment production of the construct a-S-b is easily possible. Varying the length of such spacer S as desired is straightforward for a person skilled in the art. Such polypeptide can be synthesized or recombinantly produced.
E.g., recombinant fusion proteins comprising a spacer polypeptide fused to a leucine zipper peptide at the N-terminus and to a leucine zipper peptide at the C-terminus can be expressed in suitable host cells according to standard techniques. A DNA sequence coding for a desired peptide spacer can be inserted between a sequence coding for a member of a first leucine zipper domain a and in the same reading frame a DNA sequence coding for a member of a second leucine zipper domain b.
The spacer S, if the linker a-S-b is a polypeptide in one embodiment comprises once or several times a GGGGS (G4S) (SEQ ID NO:13) amino acid sequence motif. The spacer S may also comprise a tag sequence. The tag sequence may be selected from commonly used protein recognition tags such as YPYDVPDYA (HA-Tag) (SEQ ID NO:14) or GLNDIFEAQKIEWHE (Avi-Tag) (SEQ ID NO:15).
In some embodiments both binding pairs (a′:a) and (b:b′) are hybridizing nucleic acid sequences.
As indicated already by nomenclature, 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. In one embodiment the present disclosure 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. With other words 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, e.g. (a:a′) or any other nucleic acid sequence-based 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. In some embodiments the double strand consists of two fully complementary single strands. However mismatches or insertions are possible as long as the a stability at 37° C. is given.
As the skilled artisan will appreciate a nucleic acid duplex can be further stabilized by interstrand crosslinking. Several appropriate cross-linking methods are known to the skilled artisan, e.g. methods using psoralen or based on thionucleosides.
The nucleic acid sequences representing the members of a binding pair may consist of between 12 and 50 nucleotides. Also such nucleic acid sequences may consist of between 15 and 35 nucleotides.
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 an exemplary embodiment.
Appropriate hybridizing nucleic acid sequences can easily be designed to provide for more than two pairs of orthogonal complementary oligonucleotides, allowing for an easy generation and use of more than two binding pairs. Another advantage of using hybridizing nucleic acid sequences in a dual binding agent of the present disclosure is that modifications can be easily introduced into a nucleic acid sequences. Modified building blocks are commercially available which e.g. allow for an easy synthesis of a linker comprising a functional moiety. Such functional moiety can be easily introduced at any desired position and in any of the structures a and a′ as well as b and b′ and/or S, provided they represent an oligonucleotide.
In some 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. In this embodiment the linker L consisting of a-S-b is an oligonucleotide.
In case 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. In case 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 Based on the three constructs a-S-b, A-a′ and b′-B the binding agent of Formula I can be most easily obtained according to standard procedures by hybridization between a′:a and b:b′, respectively. When spacers of different length are used, 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.
In one embodiment 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.
In one embodiment the linker L (=a-S-b) is enantiomeric L-DNA or L-RNA. In some embodiments linker a-S-b is enantiomeric L-DNA. In some embodiments a, a′, b and b′ as well as the spacer S are enantiomeric L-DNA or L-RNA. In some embodiments a, a′, b and b′ as well as the spacer S are enantiomeric L-DNA.
In one embodiment the spacer S is an oligonucleotide and is synthesized in two portions comprising ends hybridizable with each other. In this case 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. As obvious, in case the spacer is construed that way, 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.
As indicated above the bivalent binding agent according to the present disclosure in one embodiment binds to a polypeptide homodimer. In case the two target polypeptides form a homodimer, in some embodiments the same monovalent binder may be used twice. An exemplary embodiment of the present disclosure relates to a bivalent binding agent capable of binding to a protein homodimer according to Formula I, wherein S is a polynucleotide spacer wherein the monovalent binder A binds to a first target polypeptide, wherein the second monovalent binder—usually B—is also A and binds to a second target polypeptide, wherein a′:a and b:b′ both represent a polynucleotide binding pair. As obvious to the skilled artisan the epitope bound by the monovalent binder A must be only present once on the target polypeptide or the epitopes recognized by two different monovalent binders A and B, respectively, must be present only once and must be overlapping to avoid binding of the two monovalent binders on a single monomeric target polypeptide. In one embodiment the bivalent binding agent binds to a homodimer and both monovalent binders bind to an overlapping epitope. In one embodiment the bivalent binding agent binds to a homodimer and both monovalent binders bind to the same epitope.
In case the bivalent binding agent according to the present disclosure binds to a polypeptide homodimer, a simplified way can also be used to construct such bivalent binding agent. In this embodiment the monovalent binder A can be coupled to only a single species of a hybridizable polynucleotide (a′) and the linker can be construed to provide for two ends, each hybridizable to the construct A-a′. In this embodiment the linker may be of the form a-S-a. In the example given herein such special linker has been termed adaptor.
As mentioned, in one embodiment the spacer S is an oligonucleotide and is synthesized in two portions comprising ends hybridizable with each other. In this case the spacer S can be simply constructed by hybridization these hybridizable ends with one another and the resulting spacer construct comprises an oligonucleotide duplex portion.
As indicated above the bivalent binding agent according to the present disclosure in one embodiment binds to a polypeptide heterodimer. In case the two target polypeptides form a heterodimer, in some embodiments two different binding pairs a′:a and b:b′ may be used, respectively. In one embodiment the present disclosure relates to a bivalent binding agent according to Formula I, herein S is a polynucleotide spacer wherein the monovalent binder A binds to a first target polypeptide, wherein the second monovalent binder B binds to a second target polypeptide, wherein A and B specifically bind to the first and the second target polypeptide, respectively, wherein a′:a and b:b′ both represent a polynucleotide binding pair, wherein a′:a and b:b′ do not bind to or interfere with one another.
As already described above the monovalent specific binders A and B of Formula I may be nucleic acids. In one embodiment of the present disclosure a′, a, b, b′, A, B and S all are oligonucleotide sequences. In this embodiment 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.
As discussed in detail above, the coupling can be either co-valent or it can be via specific binding pairs.
As the skilled artisan will readily appreciate, the bivalent binding agent according to the present disclosure 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.
If more than one functional moiety X is present, each such functional moiety can in each case be independently a binding group, a labeling group, an effector group or a reactive group.
In one embodiment the functional moiety X may be selected from the group consisting of a binding group, a labeling group and an effector group.
In one embodiment the group X is a binding group. As obvious to a person skilled in the art, the binding group X will be selected to have no interference with the pairs a′:a and b:b′.
Examples of 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. In one embodiment X is a binding group and is covalently bound to at least one of a′, a, b, b′ or S of the compound of Formula I. In some embodiments 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 least one of a′, a, S, b or b′ as defined above.
In one embodiment functional moiety X is a binding group selected from hapten; biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin; oligonucleotide and steroid hormone.
In one embodiment 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. In one embodiment the reactive group is selected from Maleinimido, Succinimidyl, Dithiopyridyl, Nitrophenylester, Hexafluorophenylester.
In one embodiment 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. In one embodiment 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.
In one embodiment the labeling group is a luminescent metal complex and the compound has a structure of the general formula (II):
[M(L1L2L3)]n-Y—XmA (II)
are the same or different and denote ligands with at least two nitrogen-containing heterocycles in which L1, L2 and L3 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 L1, L2 and L3 via a linker Y, n is an integer from 1 to 10, for example, in some exemplary embodiments 1 to 4, m is 1 or 2 and, for example, in some exemplary embodiments 1, 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, for example, may be ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium or tungsten. Ruthenium, iridium, rhenium, chromium and osmium are examples of illustrative embodiments.
The ligands L1, L2 and L3 are ligands with at least two nitrogen-containing heterocycles. Aromatic heterocycles such as bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl are found within exemplary embodiments. The ligands L1, L2 and L3 may also be selected from bipyridine and phenanthroline ring systems.
The complex can additionally contain one or several counter ions A to equalize the charge. Examples of suitable negatively charged counter ions are halogenides, OH—, carbonate, alkylcarboxylate, e.g. trifluoroacetate, sulphate, hexafluorophosphate and tetrafluoroborate groups. Examples of suitable positively charged counter ions are monovalent cations such as alkaline metal and ammonium ions.
In a further illustrative embodiment the functional moiety X is an effector group. A exemplary 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.
Illustrative alkylating agents may be exemplified by cyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa, ifosphamide, Nitrogen mustard.
Illustrative antimetabolites may be exemplified by methotrexate, 5-Fluorouracil, cytosine arabinoside, 6-thioguanine, 6-mercaptopurin.
Illustrative antitumor antibiotics may be exemplified by doxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin, bleomycin, mitomycin, and plicamycin.
Illustrative spindle poisons may be exemplified by maytansine and maytansinoids, vinca alkaloids and epipodophyllotoxins may be exemplified by vincristin, vinblastin, vindestin, Etoposide, Teniposide.
Illustrative nitrosoureas may be exemplified by carmustin, lomustin, semustin, streptozocin.
Illustrative hormonal and antihormonal agents may be exemplified by adrenocorticorticoids, estrogens, antiestrogens, progestins, aromatase inhibitors, androgens, antiandrogens.
Additional exemplary 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) may be 1-50. In certain embodiments n may be 1 to 10, or 1 to 5. In further embodiments n is 1 or 2.
For covalent binding of the functional moiety X to at least one of a′, a, b, b′ or S 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.
In some embodiments functional moiety X is bound to a, b, or S of the binding agent as defined by Formula I.
In some embodiments functional moiety X is bound to the spacer S of the binding agent as defined by Formula I.
In some embodiments functional moiety X is covalently bound to a, b, or S of the binding agent as defined by Formula I.
If 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 are, in some embodiments, 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. In some embodiments, such non-nucleotidic entities are located within the spacer S, i.e. between the two binding pair members a and b.
Many different non-nucleotidic modifier building blocks for construction of a spacer are known in literature and a great variety is commercially available. For the introduction of a functional moiety either 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
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. For example, 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, CSN, 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. Example modified bifunctional building blocks comprise C3-C6 alkyl moieties and tri- to hexa-ethyleneglycol chains. Non-limiting, yet exemplary bifunctional modifier building blocks are given in Table III below.
Trifunctional Modifier Building Blocks
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. In this case, 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, alkylnyl 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, CSN, C40, C50 heterocycloalkyl, phenyl which are optionally substituted with one or two C1-C6 alkyl groups. Cyclic and acyclic groups may be substituted with one—(C1-C18)alkyl-O-PG group, whereas said C1-C18 alkyl comprises (Ethyleneoxy)n, (Amide)m moieties with n and m independently from each other=0-6 and PG is an acid labile protecting group. Example trifunctional building blocks are C3-C6 alkyl, cycloalkyl, C50 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, and xanthyl.
Non-limiting, examples for non-nucleosidic trifunctional building blocks are e.g. summarized in Table IV.
Nucleosidic Modifier Building Blocks:
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 disclosure is given in Formula II.
wherein PG is an acid labile protecting group such as monomethoxytrityl, dimethoxytrityl, pixyl, and xanthyl, wherein Y is C2-C18 alkyl, alkenyl alkinyl, wherein said alkyl, alkenyl, alkinyl may comprise ethyleneoxy and/or amide moieties, wherein Y is, in some embodiments 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.
Specific positions of the base may be chosen for such substitution to minimize the influence on hybridization properties. Therefore the following positions may be used for substitution: 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.
Non-limiting examples for nucleosidic trifunctional building blocks are given in Table V.
In Tables III, IV and 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. In this approach 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. Exemplary active esters are 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, for example, a leucine zipper domain or a hybridizing nucleic acid. In case the functional moiety X is bound to at least one of a′, a, b, b′ or S via an additional binding pair member, 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.
Covalent coupling of a member of a binding pair to a monovalent binder
Depending on the biochemical nature of the binder different conjugation strategies are at hand.
In case the binder is a naturally occurring protein or a recombinat polypeptide of between 50 to 500 amino acids, there are standard procedures in text books describing the chemistry for synthesis of protein conjugates, which can be easily followed by the skilled artisan (Hackenberger, C. P. and Schwarzer, D., Angew. Chem., Int. Ed., 47 (2008) 10030-10074).
In one embodiment 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. Alternatively in one embodiment 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).
In general 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. For example, 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). It is also possible to obtain a desired amino acid modification by utilizing the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see e.g.: Taki, M. et al., Protein Engineering, Design & Selection 17 (2004) 119-126; Gautier, A. et al., Chemistry & Biology 15 (2008) 128-136; Protease-catalyzed formation of C—N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403; and Sortase-mediated protein ligation is used by Mao, H. et al., in J. Am. Chem. Soc. 126 (2004) 2670-2671 and reviewed by Proft, T., in Biotechnol. Lett 32 (2010) 1-10).
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.
The reactivity of an N-terminal cystein with benzonitrils (Ren, Hongjun et al., Angewandte Chemie, International Edition 48 (2009) 9658-9662) can be used to achieve a site-specific covalent coupling.
Native chemical ligation can also rely on C-terminal cystein residues (Taylor, E. et al., Nucleic Acids and Molecular Biology 22 (2009) 65-96).
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. In case 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.
In order to obtain a mono-labeled protein 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. 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 bivalent binding agent from un-bound components, like a labeled monovalent binder.
Therefore in one embodiment a binding pair member (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 bivalent binding agent of the present disclosure may be used. In an embodiment 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. In an embodiment 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.
In one embodiment a method of producing a bivalent binding agent that specifically binds a polypeptide dimer is disclosed. The method comprises the steps of (a) selecting a first monovalent binder that binds to a first 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 second target polypeptide with a Kdiss of between 5×10−3/sec to 10−4/sec, c) coupling both monovalent binders by a linker, and d) selecting a bivalent binding agent having a Kdiss-value of 3×10−5/sec or less.
Using a method as disclosed in the present disclosure it is now rather easy to produce various bivalent binding agents each comprising a linker of different length and to select those bivalent 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 in one embodiment is performed by BIACORE™-analysis as disclosed in Example 2.8.
In one embodiment the present disclosure relates to a method of forming a bivalent binding agent according to the present disclosure, wherein a first monovalent binder that binds to a first target polypeptide with a Kdiss of between 10−3/sec to 10−4/sec and is coupled to a member of a first binding pair, a second monovalent binder that binds to a second target polypeptide with a Kdiss of 10−3/sec to 10−4/sec and 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 members to the first and the second binding pair member, respectively are co-incubated, whereby a bivalent binding agent having a Kdiss-value of 3×10−5/sec or less is formed. As the skilled artisan will appreciate the Kdiss is a temperature-dependent value. Logically, the Kdiss-values of both the monovalent binders as well as of the bivalent binding agent according to the present disclosure are determined at the same temperature. As will be appreciated, a Kdiss-value may be determined at the same temperature at which the bivalent binding agent shall be used, e.g., an assay shall be performed. In one embodiment 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. In one embodiment the Kdiss-values are established at 4 or 8° C., respectively. In one embodiment 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 exemplary embodiment Kdiss determinations, i.e. those for each monovalent binder and the Kdiss determination for the dual binder are made at 37° C.
In one embodiment the above method further comprises the step of isolating the bivalent binding agent.
An exemplary stoichiometry for forming or assembling the bivalent binding agent according to the present disclosure is 1:1:1.
In some embodiments the method of producing a bivalent binding reagent according to the present disclosure makes use of an L-DNA-linker. In some embodiments the method of producing a bivalent binding reagent according to the present disclosure 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 bivalent 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 bivalent binding agent disclosed in this disclosure, 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 one epitope on one of the two target polypeptides is present, i.e. in case the polypeptides are monomeric and not dimeric no significant staining will be found. Thus, and this is of great advantage, immunohistochemical staining will be only observed if a polypeptide dimer is present in the sample
In some embodiments the present disclosure therefore 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 bivalent binding agent consisting of two monovalent binders that are linked to each other via a linker, wherein the first monovalent binders binds to a first polypeptide the second monovalent binder binds to a second polypeptide, each monovalent binder has a Kdiss in the range of 5×10−3/sec to 10−4/sec and wherein the bivalent binding agent has a Kdiss of 3×10−5/sec or less, and (c) detecting the bivalent binding agent, thereby staining said sample for a polypeptide dimer of interest.
Use of a bivalent binding agent according to the present disclosure in the staining of a cell or tissue sample by an immunohistochemical method represents a further illustrative embodiment.
In more general terms the present disclosure relates to a bivalent binding agent consisting of two monovalent binders that are linked to each other via a linker, which binding agent binds a target polypeptide dimer with a Kdiss meeting the requirements of an (automated) assay system or better, wherein (a) the first monovalent binder that binds to a first target polypeptide with a Kdiss of at least 10-fold above (worse than) the requirements of the (automated) assays system, (b) the second monovalent binder binds to a second target polypeptide 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) has at least the Kdiss required by the (automated) system or less.
In general terms a method for obtaining a bivalent binding agent is described that specifically binds a polypeptide dimer, comprising two target polypeptides, 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 first 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 second target polypeptide 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.
In one embodiment the automated system is the BENCHMARK® analyzer as distributed by Ventana Medical Systems Inc., Tucson.
The following examples, sequence listing, and figures are provided for the purpose of demonstrating various embodiments of the instant disclosure and aiding in an understanding of the present disclosure, the true scope of which is set forth in the appended claims. These examples are not intended to, and should not be understood as, limiting the scope or spirit of the instant disclosure in any way. It should also be understood that modifications can be made in the procedures set forth without departing from the spirit of the disclosure.
The following comprises a list of illustrative embodiments according to the instant disclosure which represent various embodiments of the instant disclosure. These illustrative embodiments are not intended to be exhaustive or limit the disclosure to the precise forms disclosed, but rather, these illustrative embodiments are provided to aide in further describing the instant disclosure so that others skilled in the art may utilize their teachings.
1. A bivalent binding agent capable of binding a polypeptide dimer the binding agent consisting of two monovalent binders that are linked to each other via a linker,
1.1 Monoclonal Antibodies and Fab′-Fragments
Two monoclonal antibodies binding to human cardiac Troponin T at different, non-overlapping epitopes, epitope A′ and epitope B′, respectively, were used. Both these antibodies are used in the current Roche ELECSYS™ Troponin T assay, wherein Troponin T is detected in a sandwich immuno assay format.
Purification of the monoclonal antibodies from culture supernatant was carried out using state of the art methods of protein chemistry.
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, i.e. A and B, respectively, in Formula I (A-a′:a-S-b:b′-B), with a low concentration of cysteamin at 37° C. The reaction is stopped by separating the cysteamin on a SEPHADEX® G-25 column (GE Healthcare) from the polypeptide-containing part of the sample.
1.2 Conjugation of Fab′-Fragments to ssDNA-Oligonucleotides
The Fab′-fragments are conjugated with the below described activated ssDNAa and ssDNAb oligonucleotides, respectively.
Preparation of the Fab′-fragment-ssDNA conjugates A″ and B″, respectively:
a) Fab′-anti-Troponin T<epitope A′>-ssDNA-conjugate (=A″)
For preparation of the Fab′-anti-Troponin T<epitope A′>-ssDNAa-conjugate A″ a derivative of SED ID NO:5 is used, i.e. 5′-AGT CTA TTA ATG CTT CTG C(=SEQ ID NO:5)-XXX-Y-Z-3′, wherein X=propylene-phosphate introduced via Phosphoramidite C3 (3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropy)]-phosphoramidite (Glen Research), wherein Y=3″-Amino-Modifier C6 introduced via 3′-Amino Modifier TFA Amino C-6 lcaa CPG (ChemGenes) and wherein Z=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced via Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).
b) Fab′-anti-Troponin T<epitope B′>-ssDNAb-conjugate (=B”)
For the preparation of the Fab′-anti-Troponin T<epitope B′>-ssDNA-conjugate (B″) a derivative of SEQ ID NO:6 is used, i.e. 5′-Y-Z-XXX-AGT TCT ATC GTC GTC CA-3′, wherein X=propylene-phosphate introduced via Phosphoramidite C3 (3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research), wherein Y=5′-Amino-Modifier C6 introduced via (6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research), and wherein Z=4 [N-maleinimidomethyl]cyclohexane-1-carboxy introduced via Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (ThermoFischer).
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. In order to obtain a high percentage of single-labeled Fab′-fragments the relative molar ratio of ssDNA to Fab′-fragment is kept low. Purification of single-labeled Fab′-fragments (ssDNA:Fab′=1:1) occurs via anion exchange chromatography (column: MONO Q®, GE Healthcare). Verification of efficient labeling and purification is achieved by analytical gel filtration chromatography and SDS-PAGE.
1.3 Biotinylated Linker Molecules
The 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.
Linker 1 (=L1), a biotinylated ssDNA linker 1 with no spacer has the following composition:
5-GCA GAA GCA TTA ATA GAC T (Biotin-dT)-TGG ACG ACG ATA GAA CT-3′. It comprises ssDNA oligonucleotides of SEQ ID NO:7 and 8, respectively, and was biotinylated by using Biotin-dT (=T-Bi) (5′-Dimethoxytrityloxy-5-[N-[(4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3 [(2-cyanoethyl)-(N,N-diisopropyyl)]-phosphoramidite (Glen Research) in the middle of the spacer.
Linker 2 (=L2), a biotinylated ssDNA linker 2 with a 11mer thymidine-based spacer has the following composition: 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 (5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-ph osphoramidite (Glen Research) in the middle of the spacer.
Linker 3 (=L3), a biotinylated ssDNA linker 3 with a 31 mer thymidine-based spacer has the following composition:
5-GCA GAA GCA TTA ATA GAC T T15-(Biotin-dT)-T15 TGG ACG ACG ATA GAA CT-3′. It comprises ssDNA oligonucleotides of SEQ ID NO:7 and 8, respectively, twice oligonucleotide stretches of fifteen thymidines each 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).
1.4 Epitopes for Monovalent Troponin T Binders A and B, Respectively
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.
a) The epitope A′ for antibody a is comprised in:
SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U represents β-Alanin.
As the skilled artisan will appreciate it is possible to combine these two epitope-containing peptides in two ways and both variants have been designed and prepared by linear combining the epitopes A′ and B′. The sequences of both variants, the linear sequences of epitopes A′-B′ (=TnT-1) and B′-A′ (=TnT-2), respectively have been prepared by state of the art peptide synthesis methods.
The sequences for epitopes A′ and B′, respectively, had been modified compared to the original epitopes on the human cardiac Troponin T sequence (P45379/UniProtKB) in order to reduce the binding affinity for each of the Fabs thereto. Under these circumstances the dynamics of the effect of hetero-bivalent binding is better visible, e.g. by analyzing binding affinity with the BIACORE™ Technology.
1.5 Biomolecular Interaction Analysis
For this experiment a BIACORE™ 3000 instrument (GE Healthcare) was used with a BIACORE™ SA sensor mounted into the system at T=25° C. Preconditioning was done at 100 μl/min with 3×1 min injection of 1 M NaCl in 50 mM NaOH and 1 mM 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 BIACORE™ 3000 System was driven under the control software V1.1.1. Flow cell 1 was saturated with 7 RU D-biotin. On flow cell 2, 1063 RU biotinylated ssDNA linker L1 was immobilized. On flow cell 3, 879 RU biotinylated ssDNA linker L2 was immobilized. On flow cell 4, 674 RU biotinylated ssDNA linker L3 was captured.
Thereafter, 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 μl/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 μl/min for 4 min association time. The dissociation was monitored for 5 min Regeneration was done by a 1 min injection at 50 μl/min of 50 mM NaOH over all flow cells.
Kinetic data was determined using the Biaevaluation software (V.4.1). The dissociation rate kd (1/s) of the TnT-1 and TnT-2 peptides from the respective surface presented Fab′ fragment combinations was determined according to a linear Langmuir 1:1 fitting model. The complex halftime in min were calculated according to the solution of the first order kinetic equation:
Results:
The experimental data given in Tables 1 and 2, respectively demonstrate an increase in complex stability between analyte (TnT-1 or TnT-2), respectively, and the various hetero bivalent Fab′-Fab′ dimers A″-B″ as compared to the monovalent dsDNA Fab′ A″ or B″ conjugate, respectively. This effect is seen in each Table in line 1 compared to lines 2 and 3.
The avidity effect is further dependent on the length of the linker. In the sub-tables shown under Table 1, i.e. for the artificial analyte TnT-1, the linker L3 comprising a 31 mer spacer shows the lowest dissociation rate or highest complex stability.
In the sub-tables shown under Table 2 the linker L2 comprising an 11 mer spacer exhibits the lowest dissociation rate or highest complex stability for the artificial analyte TnT-2.
These data taken together demonstrate that the flexibility in linker length as inherent to the approach given in the present disclosure is of great utility and advantage.
2.1 Monoclonal Antibody Development (mAb 8.1.2 and mAb 1.4.168)
a) Immunization of Mice
BALB/C mice are immunized at week 0, 3, 6 and 9, respectively. Per immunization 100 μg of the conjugate comprising the phosphorylated peptide pIGF-1R (1340-1366) (SEQ ID NO:11) is used. This peptide had been phosphorylated at tyrosine 1346 (=1346-pTyr) and coupled to KLH via the C-terminal cysteine (=Aoc-Cys-MP-KLH-1340) to yield the conjugate used for immunization. At weeks 0 and 6, respectively, the immunization is carried out intraperitoneally and at weeks 3 and 9, respectively, subcutanuosly at various parts of the mouse body.
b) Fusion and Cloning
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 1×108 spleen cells of an immunized mouse are mixed with 2×107 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. The supernatant is discarded, the cell sediment is gently loosened by tapping, 1 ml PEG (molecular weight 4000, Merck, Darmstadt) is added and mixed by pipetting. After 1 min incubation in a water bath at 37° C., 5 ml RPMI 1640 without FCS is added drop-wise at room temperature within a period of 4-5 min. This step is repeated with additional 10 ml RPMI 1640 without FCS. Afterwards 25 ml RPMI 1640 containing 10% FCS is added followed by an incubation step at 37° C., 5% CO2 for 30 minutes. After centrifugation for 10 min at 250 g and 4° C. the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and seeded out in hypoxanthine-azaserine selection medium (100 mmol/1 hypoxanthine, 1 μg/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.
c) Immunoglobulin Isolation from the Cell Culture Supernatants
The hybridoma cells obtained are seeded out at a density of 1×107 cells in CELLINE™ 1000 CL flasks (Integra). Hybridoma cell supernatants containing IgGs are collected twice a week. Yields typically range between 400 μg and 2000 μg 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) 587-695).
2.2 Synthesis of Hybridizable Oligonucleotides
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.
d) 5′-Fluorescein-(beta L AGT CTA TTA ATG CTT CTG C)-(Spacer C3)3C7-Aminolinker-; (beta L indicates that this is an L-DNA oligonucleotide) and
e) 5′-Aminolinker-(Spacer C3)3-(beta L-AGT TCT ATC GTC GTC CA)-Fluorescein-3′ (beta L indicates that this is an L-DNA oligonucleotide).
Synthesis was performed on an ABI 394 synthesizer at a 10 μmol 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).
The following amidites, amino modifiers and CPG supports were used to introduce the C3-spacer, a dye and amino moieties, respectively, during oligonucleotide synthesis:
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);
5′-Fluorescein Phosphoramidite 6-(3′,6′-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research); CY5™ Phosphoramidite 1-[3-(4-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropyl phosphoramidityl]propyl]-3,3,3′,3′-tetramethylindodicarbocyanine chloride (Glen Research);
3′-Amino Modifier TFA Amino C-6 lcaa CPG 500 A (Chemgenes).
For CY5™ labeled oligonucleotides, 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)
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. (Oligonucleotides modified at the 5′ end with monomethoxytrityl protected alkylamino group are detriylated by incubating with 20% acetic acid for 20 min). The oligomers containing fluorescein as label were purified again by IEX chromatography on a HPLC [MONO Q® column: Buffer A: Sodium hydroxide (10 mM/I; pH ˜12) Buffer B 1M Sodium chloride dissolved in Sodium hydroxide (10 mM/I; pH ˜12) gradient: in 30 minutes from 100% buffer A to 100% buffer B flow 1 ml/min detection at 260 nm]. The product was desalted via dialysis.
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 SPEEDVAC® evaporator to yield solids which were frozen at −24° C.
2.3 Activation of Hybridizable Oligonucleotides
The amino modified oligonucleotides from Example 2 were dissolved in 0.1 M sodium borate buffer pH 8.5 buffer (c=600 μmol) and reacted with a 18-fold molar excess of Sulfo SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate dissolved in DMF (c=3 mg/100 μl) from Thermo Scientific, The reaction product was thoroughly dialyzed against water in order to remove the hydrolysis product of sulfoSMCC 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
The dialysate was concentrated by evaporation and directly used for conjugation with a monovalent binder comprising a thiol group.
2.4 Synthesis of Linker Oligonucleotides Comprising Hybridizable Oligonucleotides at Both Ends
Oligonucleotides were synthesized by standard methods on an ABI 394 synthesizer at a 10 μmol 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)
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
The following 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)]-ph osphoramidite (Glen Research);
Biotin Phosphoramiditel-Dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite and
5′-Dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxy uridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite for amino modification and postlabeling with Digoxigenin-N-Hydroxyl-succininimidyl ester.
The following bridging constructs or linkers were synthesized:
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.
In the above bridging construct examples [B-L] indicates that an L-DNA oligonucleotide sequence is given; spacer C18, 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.
2.5 Assembly of Dual Binder Construct
A) Cleavage of IgGs and Labeling of Fab′ Fragments with ssDNA
Purified monoclonal antibodies were cleaved with the help of pepsin protease yielding F(ab′)2 fragments that are subsequently reduced to Fab′ fragments by treatment with low concentrations of cysteamine at 37° C. The reaction is stopped via separation of cysteamine on a PD 10 column. The Fab′ fragments are labeled with an activated oligonucleotide as produced according to Example 3. This single-stranded DNA (=ssDNA) bears a thiol-reactive maleimido group that reacts with the cysteines of the Fab′ hinge region. In order to obtain high percentages of single-labeled Fab′ fragments the relative molar ratio of ssDNA to Fab′-fragment is kept low. Purification of single-labeled Fab′ fragments (ssDNA: Fab′=1:1) occurs via ion exchange chromatography (column: SOURCE™ 15 Q PE 4.6/100, Pharmacia/GE). Verification of efficient purification is achieved by analytical gel filtration and SDS-PAGE.
B) Assembly of an Anti-pIGF-1R Dual Binder.
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 17mer ssDNA comprising SEQ ID NO:6 and containing fluorescein as an fluorescent marker and Fab′ 8.1.2 to a 19mer ssDNA comprising SEQ ID NO:5 and containing CY5™ as fluorescent marker. In the following, these Fab's with covalently bound 17mer or 19mer ssDNA are named ssFab′ 1.4.168 and ssFab′ 8.1.2 respectively. 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.
For assembly evaluation the dual binder components ssFab′ 8.1.2, ssFab′ 1.4.168 and the linker constructs (I) (=linker 17 of example 2.4) 5′-G CAG AAG CAT TAA TAG ACT T(-Bi)-TGG ACG ACG ATA GAA CT-3′ and (II) (=linker 10 of example 2.4) 5′-G CAG AAG CAT TAA TAG ACT-(T20)-T(-Bi)-(T20)-TGG ACG ACG ATA GAA CT-3′ were mixed in equimolar quantities at room temperature. After a 1 minute incubation step the reaction mix was analyzed on an analytical gel filtration column (SUPERDEX™ 200, 10/300 GL, GE Healthcare). Comparison of the elution volumes (VE) of the single dual binder components with the VE of the reaction mix demonstrates that the dual binder has been formed successfully (
2.6 BIACORE™ Experiment Assessing Binding of Anti-pIGF-1R Dual Binder To Immobilized IGF-1R and IR Peptides
For this experiment a BIACORE™ 2000 instrument (GE Healthcare) was used with a BIACORE™ SA sensor mounted into the system at T=25° C.
Preconditioning occurred at 100 μl/min with 3×1 min injection of 1 M NaCl in 50 mM NaOH and 1 mM 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 with the system buffer.
The BIACORE™ 2000 System was driven under the control software
V1.1.1.
Subsequently 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) was captured on flow cell 2. 18 RU of IGF-1R(1340-1366); Glu(Bi-PEG-1340]amid (i.e. the—1346 tyrosine non-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) was captured on flow cell 3. RU of hIR(1355-1382)[136]-pTyr; Glu(Bi-PEG-1355]amid (i.e. the—1361 tyrosine phosphorylated—peptide of SEQ ID NO:12 comprising a PEG-linker bound via glutamic acid corresponding to position 1355 of human insulin receptor and being biotinylated at the other end of the linker) was captured on flow cell 4. Finally all flow cells were saturated with d-biotin.
For the Dual Binder formation the assembly protocol as described in Example 2.5 was used. When individual runs with only one of the two ssFab's were performed, the absence or presence of linker DNA did not affect the association or dissociation curves (data not shown).
100 nM of analyte (i.e. in these experiments a bivalent dual binding agent) in solution was injected at 50 μl/min for 240 sec association time and dissociation was monitored for 500 sec. Efficient regeneration was achieved by using a 1 min injection step at 50 μl/min with 80 mM NaOH. Flow cell 1 served as a reference. A blank buffer injection was used instead of an antigen injection to double reference the data by buffer signal subtraction.
In each measurement cycle one of the following analytes in solution was injected over all 4 flow cells: 100 nM ssFab′ 8.1.2, 100 nM ssFab′ 1.4.168, a mixture of 100 nM ssFab′ 8.1.2 and 100 nM ssFab′, 100 nM bivalent binding agent consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on linker (III) (5′-G CAG AAG CAT TAA TAG ACT-T(20)-T(-Dig)-(T20)-TGG ACG ACG ATA GAA CT-3′(=linker 15 of example 2.4)), and 100 nM bivalent binding agent consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on linker (IV) (5′-G CAG AAG CAT TAA TAG ACT-T(-Dig)-TGG ACG ACG ATA GAA CT-3′(=linker 16 of example 2.4)), respectively. (The digoxigenylation of the middle thymidine (T(-Dig)) in the above linkers is without relevance to these experiments.)
The signals were monitored as time-dependent BIACORE™ sensorgrams.
Report points were set at the end of the analyte association phase (Binding Late, BL) and at the end of the analyte dissociation phase (Stability Late, SL) to monitor the response unit signal heights of each interaction. The dissociation rates kd (1/s) were calculated according to a linear 1:1 Langmuir fit using the BIACORE™ evaluation software 4.1. The complex halftimes in minutes were calculated upon the formula ln(2)/(60*kd).
The sensorgrams (
2.7 BIACORE™ Assay Sandwich of M-1.4.168-IgG and M-8.1.2-IgG
A BIACORE™ T100 instrument (GE Healthcare) was used with a BIACORE™ CMS sensor mounted into the system. The sensor was preconditioned by a 1 min injection at 100 μl/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 BIACORE™ T100 System was driven under the control software V1.1.1. Polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearch Laboratories Inc.) at 30 μg/ml in 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. Finally, 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 μl/min on the <IgGFCγM>R surface. 3 μM of an IgG fragment mixture (of IgG classes IgG1, IgG2a, IgG2b, IgG3) containing blocking solution was injected at 30 μl/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 μl/min 300 nM secondary antibody M-8.1.2-IgG was injected at 30 μl min. The sensor was regenerated using 10 mM Glycine-HCl pH 1.7 at 50 μl/min for 3 min.
2.8 BIACORE™ Assay Dual Binder on Sensor Surface
A BIACORE™ 3000 instrument (GE Healthcare) was used with a BIACORE™ SA sensor mounted into the system at T=25° C. The system was preconditioned at 100 μl/min with 3×1 min injection of 1 M NaCl in 50 mM NaOH and 1 min 10 mM HCl.
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 BIACORE™ 3000 System was driven under the control software V4.1.
124 RU amino-PEO-biotin were captured on the reference flow cell 1. 1595 RU biotinylated 14.6 kDa T0-Bi 37-mer ssDNA-Linker (I) (5′-G CAG AAG CAT TAA TAG ACT-T(-Bi)-TGG ACG ACG ATA GAA CT-3′) (=linker 17 of example 2.4) and 1042 RU biotinylated 23.7 kDa T40-Bi 77-mer ssDNA-Linker (II) (5′-G CAG AAG CAT TAA TAG ACT-T(20)-(Biotin-dT)-(T20)-TGG ACG ACG ATA GAA CT-3′=linker 10 of example 2.4) were captured on different flow cells.
300 nM ssFab′ 8.1.2 and 300 nM ssFab′ 1.004.168 were injected into the system at 50 μl/min for 3 min. As a control only 300 nM ssFab′ 8.1.2 or 300 nM ssFab′ 1.004.168 was injected to test the kinetic contribution of each ssFab. As a control, buffer was injected instead of the ssFabs. The peptides pIR(1355-1382)[1361-pTyr]amid and IGF-1R(1340-1366)amid, respectively, were injected into system at 50 μl/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. In another set of experiments to measure the affinities versus the peptide pIGF-1R(1340-1366)[1346-pTyr]amid the 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 μl/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
The T40 dual binding agent (i.e. the dual binding agent comprising the T40-T-Bi linker (linker 10 of example 2.4)) exhibits a 10 pM affinity versus the phosphorylated IGF-1R peptide (table in
Therefore, the goal to increase specificity and affinity by the combination of two distinct and separated binding events is achieved.
The cooperative binding effect especially becomes obvious from the dissociation rates against the phosphorylated IGF-1R peptide, where the dual binder shows 414 min antigen complex halftime, versus 0.5 min with the monovalent binder 8.1.2 alone and versus 3 min with the monovalent binder 1.4.168 alone, respectively.
Furthermore, the fully assembled construct roughly multiplies its dissociation rates kd (1/s), when compared to the singly Fab′ hybridized constructs (
A diagnostic system using an intense washing procedure should definitely foster the high performance of the T40 dual binding agent, in contrast to individual (monovalent) Fab′ molecules. The hybridized construct, i.e. a bivalent binding agent according to the present disclosure, generates a specific and quite stable binding event, while the monovalent binders more rapidly dissociate, e.g. they are more rapidly washed away.
The insulin-like growth factor 1 receptor (IGF1-R) belongs to the family of tyrosin kinase receptors and it is activated by IGF1. The receptor is homodimeric.
An IGF1-R monomer consists of one extracellular a subunit which is covalently linked to a transmembrane β subunit via a disulfide bridge. Two monomers are covalently linked by a disulfite bridge between the a subunits of two monomers. The IGF1 receptor plays an important role in cancer, aging and insulin signalling
The dimer-binder, we developed, comprises two identical Fab′-fragments. Both these Fab′-fragments—when used individually—bind to their target peptide (IGF1-R) only with low complex stability. In order to produce a bivalent binding agent the Fab′-fragment is conjugated to a hybridizable oligonucleotide. An adaptor DNA capable of hybridizing with both the linker DNA as well as with this oligonucleotide is used as a bridge to hybridize with the Fab′-DNA and the Linker-DNA, to provide for a stable bivalent binding agent also featuring the appropriate distance for the to monovalent binders employed (a schematic is given in
3.1 Development of a Low-Affinity Monoclonal Antibody Against IGF1 Receptor
a) Immunization of Mice
Balb/c mice were immunized with a peptide of IGF1R(1340-1366)[1346-pTyr; Aoc-Dys-MP-KLH-1340] amide. (The non-phosphorylated amino cid sequence is given as SEQ ID NO:11.) The initial immunization dosis was 100 μg. The mice were further immunized with 100 μg immunogen after 4, 8 and 12 weeks.
b) Fusion and Cloning
Spleen cells of aforementioned mice were fused with myeloma cells according to Galfre, G. and Milstein, C. Methods in Enzymology 73 (1981) 3-46. In this process, 1×108 spleen cells of the immunized mouse were mixed with 2×107 myeloma cells (P3×63-Ag8-653, ATCC CRL1580) and centrifuged for 10 min at 250 g and 37° C. The supernatant was discarded and the cell pellet was loosened by tapping. 1 mL PEG (molecular weight 4000, Merck, Darmstadt) was added drop by drop within 1 minute at 37° C. in a water bath. Afterwards, 5 mL RPMI 1640 without FCS was added drop by drop within 5 min, followed by further 10 mL RPMI 1640 without FCS, which were added drop by drop within 10 minutes. As a next step, 25 mL RPMI 1640 with 10% FCS, 100 mmol/1 hypoxanthine, 1 μg/ml azaserine and 50 u IL-6 were added and the cells were incubated at 37° C., 5% CO2 for 60 min and then centrifuged at 250 g for 10 min. The cell pellet was resuspended in 30 mL RPMI 1640 with 10% FCS, 100 mmol/1 hypoxanthine, 1 μg/ml azaserine and 50 u IL-6, and incubated at 37° C., 5% CO2.
c) Immunoglobulin Isolation from the Cell Culture Supernatants
After ca. 10 days the primary cultures were tested for antigen-specificity. The positive primary cultures were cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. After subsequent grow in a 24-well plate, a 6-well plate and a T75 flask (Corning) in HYCLONE™ medium (Theromo Scientific) with Nutridoma supplements (Roche), the hybridoma cells expressing the desired low-affinity antibody were eventually cultured in a CELLINE™ bioreactor (Integra biosciences) according to manufacturer's instructions. The most promising antibody was named mAb 1.4.168.
d) Biophysical Characterization of mAb 1.4.168
The kinetic properties of the interaction between mAB 1.4.168 and the IGF1 receptor were investigated by surface plasmon resonance using BIACORE™ technology.
A BIACORE™ T100 instrument (GE Healthcare) was used with a BIACORE™ CMS sensor mounted into the system at T=25° C. and was preconditioned by a 1 min injection at 100 μl/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 BIACORE™ T100 System was driven under the control software V1.1.1. The polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearch Laboratories Inc.) at 30 μg/ml in 10 mM Na-Acetate pH 4.5 was immobilized at 8000 RU on the flow cells 1,2,3,4 via EDC/NHS chemistry according to the manufacturer's instructions. Finally, the sensor surface was blocked with 1M ethanolamine
250 nM mAb M-1.004.168-IgG were captured for 1 min at 10 μl/min on the <IgGFCγM>R surface.
A concentration series of 900 nM, 300 nM, 150 nM, 50 nM, 17 nM, 6 nM, and 0 nM of the peptide IGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amide was used as analyte in solution and was injected at 100 μl/min for 3 min association time. The dissociation was monitored for 10 mM at 100 μl/min. As a control, one concentration step was analyzed twice to ensure the reproducibility of the assay. Flow cell 1 served as a reference cell. A blank buffer injection was used instead of an antigen injection to double reference the data by buffer signal subtraction. The capture system was regenerated using a 3 min injection at 10 μl/min with 10 mM glycine pH 1.7.
The data was evaluated according to a 1:1 binary Langmuir interaction model in order to calculate the association rate constant ka [1/Ms], the dissociation rate constant kd [1/s] and the resulting affinity constant KD [M] using the BIACORE™ T100 evaluation software V.1.1.1. The dissociation rate constant for mAb 1.4.168 was 3.6×10−3 1/s and, consequently, within the range of antibodies with low complex stability, required for our dual-binder approach.
Further, surface plasmon resonance was used to determine the antigen specificity. A BIACORE™ 2000 instrument (GE Healthcare) was used with a BIACORE™ SA sensor mounted into the system at T=25° C. and was preconditioned at 100 μl/min with 3×1 min injection of 1 M NaCl in 50 mM NaOH and 1 min 10 mM HCl. 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+1 mg/ml CMD (Carboxymethyldextrane, Sigma).
Subsequently, biotinylated peptides were captured on the SA surface in the respective flow cells. The flow cells finally were saturated with 2 μM d-biotin. Hybridoma Clone Culture supernatant was diluted 1:2 and was injected for 2 min at 50 μl/min, the dissociation was monitored for 5 min Regeneration at 30 μl/min with 1.5 min injection with 10 mM Gly/HCl, pH 1.7. Flow cell 1 served as a reference. A blank buffer injection was used instead of an antigen injection to double reference the data by buffer signal subtraction. The data was evaluated according to the BIACORE™ evaluation software 1.1.1. The signal height was measured at the end of the injection time, Binding Late (RU). The below Table 3 clearly shows, that mAb 1.4.168 recognized the phosphorylated and unphosphorylated IGF-1R peptide (SEQ ID NO:11) equally well, but it did not interact with the phosphorylated insulin receptor peptide (SEQ ID NO:12).
3.2 FC=flow cell, ligand=biotin and biotinylated peptides, respectively, captured on the SA coated chip, RU=resonace unit (correlates to the mass of bi-peptides captured as ligand on the SA surface), analyte=mAb 1.4.168 in solution, binding late (RU)=signal height at the end of the analyte injection phase.
a) Purification
The mAb 1.4.168 was first purified by acid precipitation with 2 N acetic acid to a final pH 4.75. After 15 min incubation time and centrifugation at 4° C. and 13,000 rpm, the supernatant was buffered to pH 7.0 with 1 M K3PO4. Afterwards, the antibody was further purified by ammonium sulphate precipitation (30.5 g/100 mL). After centrifugation at 9000 rpm for 30 min at 4° C., the supernatant was discarded and the pellet was resuspended in 10 mM Na-citrate, 20 mM NaCl, pH 5.5. The antibody was then dialyzed against 10 mM Na-citrate, 20 mM NaCl, pH 5.5 and centrifuged at 4° C., 13,000 rpm for 30 min and the cell pellet was discarded. The antibody was further purified by ion exchange chromatography on a SP SEPHAROSE® column (GE healthcare), equilibrated in 10 mM Na-citrate, 20 mM NaCl, pH 5.5. The antibody was eluted with a gradient of NaCl from 20 mM to 250 mM over 6 column volumes. The eluate was collected with a fraction collector and fractions containing protein were analyzed by HPLC and SDS-PAGE to positively identify the antibody. The fractions containing the antibody were pooled and concentrated by ultrafiltration (AMICON® Ultra 10 kDa MWCO) according to manufacturer's instructions and dialyzed against 50 mM potassium phosphate, 150 mM NaCl, pH 7.5.
b) Papain Digestion to Generate Fab′2 Molecules
The purified antibody was cleaved with 25 mU Papain/mg IgG at 37° C. for 80 min. The reaction was quenched by the addition of 0.1 times the reaction volume of 270 mM iodine acetamide, 75 mM potassium phosphate, 150 mM NaCl and 2 mM EDTA. Afterwards, the reaction mix was dialyzed against 10 mM potassium phosphate, 20 mM NaCl, pH 7.0. The Fab′2 was then purified by DEAE ion exchange chromatography. The DEAE column (GE healthcare) was equilibrated with 10 mM potassium phosphate, 20 mM NaCl, pH 7.0 and, after loading the dialyzed reaction mix, the Fab′2 was eluted with 10 mM potassium phosphate, 1 M NaCl, pH 7.0. Further, the Fab′2 was purified by immunoaffinity chromatography as the flow-through of a column loaded with polyclonal anti-papain antibodies and subsequently a column loaded with polyclonal antibodies against M-Fcγ. PBS was used as a running buffer. The final flow-through was concentrated with an AMICON® Ultra 10 kDa MWCO centrifugational filter and the purity was determined by SDS-PAGE.
c) Reduction of Fab′2 and Generation of Fab′-Fragments
The Fab′2 was reduced at 37° C. in a thermomixer (Eppendorf) in 15 mM cysteamine, 25 mM potassium phosphate, 5 mM EDTA, pH 6.5 for 60 min. The reaction was stopped by separation of the Fab′ from the cysteamine in a size exlusion chromatography gel filtration on a SEPHADEX® G25 P10 column (GE Healthcare) equilibrated in 100 mM potassium phosphate, 2 mM EDTA, pH 6.3. The Fab′ was eluted with 25 mM KPO4, 2 mM EDTA, pH 6.5.
3.3 DNA-Labeling of Fab′-Fragments
The Fab′-fragment obtained in Example 3.2 was conjugated with the ssDNA-maleimide derivative (SEQ ID NO:16) in a 1:1 molar ratio for 20 min. This ssDNA sequences is capable of hybridizing to the complementary sequence found on the linker and the adaptor oligonucleotide, respectively. The reaction was quenched by the addition of excess cysteine-HCl (for quenching of unconjugated DNA-maleimide) followed by the addition of excess N-methylmaleimide (quenching of free Cysteine-SH groups). Afterwards, the Fab′ was dialyzed against 20 mM Tris pH 7.6. The mono-labeled Fab′ was then separated from poly-labeled Fab′ in an ion exchange chromatography with a SOURCE Q column (GE Healthcare). The column was equilibrated in 20 mM Tris pH 7.6 and the Fab′-DNA conjugates were eluted with a NaCl gradient. The flow-rate was 2 ml/min and the Fab′-DNA conjugates eluted at a salt concentration between 400 and 700 mM NaCl. Mono-labelled Fab′-DNA conjugates were collected with a fraction collector and concentrated by ultrafiltration (AMICON® Ultra 10 kDa MWCO) and dialyzed against 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% TWEEN® 20, pH 7.4.
3.4 Formation of the Bivalent Binding Agent Comprising Two Anti-IGF1-R Fab′-Fragments
The Fab′ fragment labeled with the ssDNA linker from Example 3.3, the linker DNA (SEQ ID NO:17) and the adaptor DNA (SEQ ID NO:18) were mixed in equimolar quantities at room temperature to allow for the formation of the bivalent homodimer binder (cf.
3.5 Analytical Size Exclusion Chromatography to Investigate Dual Binder formation
Analytical size exclusion chromatography was performed using a GE Healthcare SUPERDEX™ 200 10/300GL analytical size exclusion chromatography column with a flow rate of 0.5 ml/min PBS was used as a running buffer and 100 μL of sample were injected in each run. The formation of the dual binder via DNA hybridization was established by differences in retention time between the Fab′-DNA conjugate and the ternary complex of Fab′-DNA, Linker-DNA and adaptor DNA (Table 4). The most efficient dual-binder formation was observed at a 2:1:1 ratio of Fab′-DNA, linker DNA and adaptor DNA.
The retention time was monitored by a change in absorbance at 280 nm (aromatic absorbance) and 495 nm (fluorescein absorbance).
The retention time was measured as the maximum absorbance (280 nm) of the resulting peak in the elution profile of each run. A ternary complex of Fab′-DNA conjugate, linker DNA and adaptor DNA eluted with a shorter retention time than the individual components alone and the complex of Fab′-DNA and linker-DNA, indicating a higher molecular weight and, consequently, a ternary complex formation.
3.6 Immunhistochemical Staining
NIH3T3 cells were stably transfected to overexpress human IGF1-receptor. The transfected cells were fixed and embedded in paraffin blocks according to standard procedures. 3 μm cuts of the block were produced with a microtome (HM355S, Thermo Scientific) and transferred onto a microscope slide for automated IHC staining in the Ventana BENCHMARK® XT system. The staining procedure was performed using commercially available reagents from Ventana. The cells were deparaffinized and treated with standard CC1 buffer (Ventana) for antigen retrieval. 100 μL of 1 μg/ml dual-binder, or dual-binder without adaptor DNA, in antibody diluent buffer (Ventana) were manually titrated onto the slides and incubated for 32 min. The cells were counter-stained with 100 μL hematoxylin II (Ventana) for 4 min and 100 μL bluing reagent (Ventana) for 8 min. The dual-binder detection was based on the XT IVIEW DAB detection kit (Ventana) with the exception that the IVIEW DAB biotinylated secondary antibody of the kit was substituted with reaction buffer (Ventana), and therefore the streptavidin-HRP conjugate of the kit only recognized the biotinylated linker-DNA. The staining was analyzed by light microscopy. Only the dual-binder stained the cells whereas the Fab′-DNA/DNA linker complex without adaptor DNA (which cannot hybridize into a dual binder) was washed off and did not stain the IGF1-receptor positive cells (
Discussion: Mice were immunized with an IGF1 receptor antigen for antibody development. The most promising antibody, mAb 1.4.168, was characterized by surface plasmon resonance for kinetic properties and specificity. The antibody was then purified, cleaved and labeled with a 17-mer DNA sequence. The hybridization between Fab′-DNA, linker-DNA and adaptor DNA was confirmed by analytical size exclusion chromatography and the dual binder was then used in automated IHC staining (Ventana BENCHMARK® XT) on stably transfected cells. Only the dual binder positively stained the cells whereas the Fab′-DNA/linker DNA complex alone (without adaptor DNA) was washed off and therefore did not stain. Cooperative binding of both binding elements of the dual binder to the homodimeric receptor most probably resulted in a sufficiently high complex stability to resist the stringent washing on the IHC instrument, which was not the case for the single binding element alone (cf.
The receptor tyrosine kinase family of Her receptors consist of four members: HER1, HER2, HER3 and HER4. Upon ligand binding, 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. For example, HER3 undergoes a conformational shift when it is bound to its ligands Neuregulin1 (NRG1) or Neuregulin2 (NRG2), respectively, and the HER3 dimerization domain is exposed and it can interact with other Her receptors. The dimerization domain is constitutively exposed in HER2HER2 and thus, it does not need to be activated by a specific ligand to induce its dimerization. Consequently, stimulation of HER3 by NRG1 or NRG2 can trigger its oligomerisation with HER2HER2. Upon dimerization, the constitutively active tyrosine kinase domain of HER2 phosphorylates HER3 which lacks a functional tyrosine kinase domain.
A dual binder to detect the heterodimer of HER2/HER3 can be provided. Monoclonal antibodies against HER3 and HER2 are developed with a dissociation rate constant within the range defined in the attached claims. Further, the anitbodies are validated in immunohistochemistry for target recognition in cancer cell lines, xenograft and tumor tissue. The selected antibodies are sequenced using standard molecular biology methods and a library of dual-binders is generated using recombinant protein expression. The dual-binders, with different length of linkers, are then screened on formalin-fixed and paraffin embedded cancer cells. Prior to embedding, the cells either have been starved, or they have been stimulated with human NRG1 to induce HER2/HER3 heterodimerization. The detection of heterodimerization is assessed in an automated immunohistochemistry staining system (Ventana BENCHMARK® XT). A positive hit in the screening is defined as a dual binder that stains stimulated cells (HER2/HER3 dimers present) but not the starved cells (no heterodimers).
Development of Monoclonal Antibodies Against HER3 and HER2 within a Defined Range of the Dissociation Rate Constant
a) Immunization of Mice
SJL, Balb/c and NMRI mice are immunized with HER3(1242-1267)[KLH-MP-Cys-UZU-1243]amide and HER2(1223-1236)[KLH-MP-Cys-UZU]amide. The initial immunization dose is 100 μg. The mice are further immunized with 100 μg of the immunogen after 6 and 10 weeks.
b) Fusion and Cloning
Spleen cells of aforementioned mice are fused with myeloma cells according to Galfre, G. and Milstein, C. Methods in Enzymology 73 (1981) 3-46. In this process, 1×108 spleen cells of the immunized mouse are mixed with 2×107 myeloma cells (P3×63-Ag8-653, ATCC CRL1580) and centrifuged for 10 min at 250 g and 37° C. The supernatant is discarded and the cell pellet is loosened by tapping. 1 mL PEG (molecular weight 4000, Merck, Darmstadt) is added drop by drop within 1 minute at 37° C. in a water bath. Afterwards, 5 mL RPMI 1640 without FCS is added drop by drop within 5 min, followed by further 10 mL RPMI 1640 without FCS, which is added drop by drop within 10 minutes. As a next step, 25 mL RPMI 1640 with 10% FCS, 100 mmol/1 hypoxanthine, 1 μg/ml azaserine and 50 u IL-6 are added and the cells are incubated at 37° C., 5% CO2 for 60 min and then centrifuged at 250 g for 10 min. The cell pellet is resuspended in 30 mL RPMI 1640 with 10% FCS, 100 mmol/1 hypoxanthine, 1 μg/ml azaserine and 50 u IL-6, and incubated at 37° C., 5% CO2. The cell suspension is then transferred to 96-well plates and incubated at 37° C., 5% CO2.
c) Immunoglobulin Isolation from the Cell Culture Supernatants
After ca. 10 days the primary cultures are tested for antigen-specificity. The positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. After subsequent growth in a 24-well plate, a 6-well plate and a T75 flask (Corning) in HYCLONE™ medium (Theromo Scientific) with NUTRIDOMA supplements (Roche), the hybridoma cells expressing the desired low-affinity antibody are eventually cultured in a CELLINE® bioreactor (Integra biosciences) according to manufacturer's instructions.
d) Biophysical Characterization of Monoclonal Antibodies
The kinetic properties of the interaction between the monoclonal antibodies and HER2 or HER3 are investigated by surface plasmon resonance kinetic screening using BIACORE™ technology.
A BIACORE™ A100 instrument under control of the software version V1.1 is used. A BIACORE™ CMS chip is mounted into the instrument and is hydrodynamically addressed conditioned according to the manufacturer's instructions. As a running buffer an HBS-EP buffer is used (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). A polyclonal rabbit anti-mouse IgG Fc capture antibody is immobilized at 30 μg/ml in 10 mM sodium acetate buffer (pH 4.5) to spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4 at 10,000 RU. The antibody is covalently immobilized via NHS/EDC chemistry. The sensor is deactivated thereafter with a 1 M ethanolamine solution. Spots 1 and 5 are used for the determination and spots 2 and 4 are used as reference. Prior to application to the sensor chip the hybridoma supernatants containing mAbs are diluted 1:2 in HBS-EP buffer. The diluted solution is applied at a flow rate of 30 μl/min for 1 min. Immediately thereafter the analyte, human HER3(1242-1267)-Bi-PEG-amide (SEQ ID NO:26) or human HER2(1223-1236)-Bi-PEG-amide (SEQ ID NO:25), singly grafted on streptavidin, is injected at a flow rate of 30 μl/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 μl/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 HER2, respectively HER3, with a dissociation rate constant that lies within the boundaries of the patent claim. The antibodies have to bind their epitope specifically, i.e., the anti-HER2 antibody does not interact with HER3 and vice versa.
The selected antibody directed against HER3 was called 7.2.32 (variable region heavy chain shown in SEQ ID NO:19 and variable region light chain shown in SEQ ID NO:20, respectively) and its dissociation rate constant was determined as 2.3×10−3 1/s and, consequently, is within the range required for the dualbinder approach. The selected antibody directed against HER2 was called 4.1.43 (variable region heavy chain shown in SEQ ID NO:21 and variable region light chain shown in SEQ ID NO:22, respectively) and the dissociation rate constant was 9.63×10−4 l/s, and thus, also within the range required for the dual binder approach.
Characterization of Selected Monoclonal Antibodies in Immunohistochemistry.
Not only do the selected monoclonal antibodies against HER2 and HER3 have to bind their target within a certain range of dissociation rate constant, but they also have to be well characterized in immunohistochemistry to be suitable as the foundation of the dual binder. First, it has to be ensured that both monoclonal antibodies still recognize their antigens in immunohistochemistry. This can be achieved by staining formal-fixed and paraffin embedded HEK293 that overexpress recombinant HER1, HER2, HER3 or HER4 after transient transfection. The stainings need to be performed with the Ventana BENCHMARK® automated immunohistochemistry platform using the same protocol for the anti-HER2 and the anti-HER3 antibody. For example, the stainings could be performed with the ultraview DAB kit with CC1 Standard, antibody incubation time of 32 min at 37° C., using the ultraWash option and a counter staining with Hematoxylin II for 4 min and Blueing reagent for 8 min. Further, the antibodies are to be validated on xenograft and tumour tissues with known HER2 and HER3 expression levels using the same staining protocol. In addition, the amino acid sequences of heavy and light chains are obtained from the hybridoma clones using standard molecular biology procedures.
Development of a Dual-Binder Recognizing HER2 and HER3
a) Development of Dual-Binder Using Peptide Linkers
We develop dual-binders that consist of two Fab fragments which are connected by a linker of glycine-glycine-glycine-glycine-serine (G4S) units and a hemagglutinin-Tag (YPYDVPDYA) (
HEK293-F cells in suspension are transfected with the plasmids for transient expression of the recombinant dual binder. In total, 50 mL 1×106 cells/ml with a viability of >90% are transfected with the plasmids HC-Dual binder (coding for the protein sequence shown in SEQ ID NO:24), LC_7.2.32 (coding for the protein sequence shown in SEQ ID NO:20) and LC_4.1.43 (coding for the protein sequence shown in SEQ ID NO:22) in a ratio of 1:1:1 using 293-FREE™ Transfection Reagent (Novagen) according to the manufacturer's instructions. After transfection, the HEK293-F cells are incubated for 7 days at 130 rpm, 37° C. and 8% CO2. The cells are then centrifuged at 4° C., 8000 rpm for 20 min. The supernatant containing the dual binder is further filtered using a 0.22 μm STERIFLIP® (Millipore) vacuum filtration system, aliquoted and flash frozen in liquid nitrogen and stored at 20° C.
b) Development of Dual-Binders Using DNA-Based Linkers
To generate dual binders based on hybridization via DNA-linkers, the recombinant Fab-fragments are expressed as fusion proteins encoding a sortase cleavage recognition sequence (SEQ ID NO:23). Next, the recombinant proteins are labeled site-specifically with 17mer (oligo for 4.1.43 labeling shown in SEQ ID NO:26) and 19mer (oligo for 7.2.32 labeling shown in SEQ ID NO:27) oligo molecules which can then hybridize with the available library of linker DNA.
DNA-oligo conjugation using the enzyme Sortase in a transpeptidase reaction
The enzyme sortase is a prokaryotic and proteolytic enzyme that also has a transpeptidase activity (Ton-That et al, PNAS 1999). Here, the enzyme catalyzes a transpeptidase reaction between an LPXTG motive and a glycine residue that is attached to the DNA-oligo.
The recombinant Fab-fragments of 7.2.32 and 4.1.43 are expressed in HEK293 cells. 400 mL 1×106 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 4.1.43 using 293-FREE™ Transfection Reagent (Novagen) according to the manufacturer's instructions.
The proteins are expressed as fusion proteins and carry a C-terminal 6×HIS-Tag at the light chain and a C-terminal Sortase-cleavage sequence at the heavy chain of the Fab fragments. Further, 7.2.32 carries an HA-Tag upstream of the Sortase cleavage-tag.
After transfection, the HEK293-F cells are incubated for 7 days at 130 rpm, 37° C. and 8% CO2. The cells are then centrifuged at 4° C., 8000 rpm for 20 min. The supernatant, containing the recombinant protein, is further filtered using a 0.22 μm STERIFLIP® (Millipore) vacuum filtration system.
The Fab fragments are purified by Nickel affinity-column chromatography and preparative gel filtration using the AKTA EXPLORER FPLC system using standard purification methods. Purity was accessed by SDS-PAGE and analytical gel filtration.
The labeling is performed with 10 μM recombinant Sortase, 50 μM Fab fragment and 200 μM Oligo in a buffer of 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl2, at 37° C. overnight. Next, the labeling reaction is diluted 10 times in 20 mM Tris pH 8.0 and applied to a RESOURCE™ Q ion exchange column (GE Healthcare) which is equilibrated in 20 mM Tris pH 8.0. 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. 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.
The assembly of the dual binder is investigated by analytical gel filtration.
The 7.2.32-Fab-19mer and 4.1.43-Fab-17mer conjugates in an equimolar ratio are mixed in individual experiments with each one of the linker molecules (linker 3-14 that are shown in Example 2.4. The assembly of the dual binder is then investigated using analytical gel filtration.
Analytical size exclusion chromatography is performed using a GE Healthcare SUPERDEX® 200 10/300GL analytical size exclusion chromatography column with a flow rate of 0.5 ml/min 20 mM Tris pH 8.0, 200 mM NaCl is used as a running buffer and 100 μL of sample are injected in each run. The concentration of the Fab-oligo conjugates and the linker is 2.5 μM. The retention time is monitored by a change in absorbance at 280 nm (aromatic absorbance) and 495 nm (fluorescein absorbance). The formation of the dual binder via DNA hybridization is established by differences in retention time between the Fab-oligo conjugate, Fab-Oligo and linker complex and the ternary complex of the two Fab-fragments and the linker.
Generation of Formalin-Fixed and Paraffin Embedded MCF-7 Cells
The MCF-7 cancer cell line is known to express intermediate levels of HER2 and HER3 but no HER1 (DeFazio et al, Int. J. Cancer, 87, 487-498 (2000)) and it is therefore an exemplary study model to detect the induction of HER2/HER3 heterodimerization. Further, HER2/HER3 heterodimers have already been detected in stimulated MCF-7 cells using other methods (Mukherjee et al, PLOS One 6(1), 2011).
The MCF-7 cells are cultured using RPMI 1640 (Gibco) with 2 mM L-Glutamine (Gibco) and 10% FCS and they are grown to optical confluency in a T175 cm2 flask. The confluent cells are starved in RPMI 1640, 2 mM L-Glutamine without FCS for approximately 18 hours. The cells are then stimulated with 20 nM NRG1-β1 (Peprotech) for 15 min at 37° C. As a negative control, cells are left without stimulation for 15 min at 37° C. in fresh cell culture medium without FCS. The cells are fixed with formalin and embedded in paraffin as described (Defazio-Eli et al, Breast Cancer Res 13(2):R44 (2011).
Since phosphorylation of HER3 only occurs after ligand binding, a staining of phosphorylated HER3 should show a difference in activation status of HER3 between treated and non-treated MCF-7 cells that may also indicate different levels of HER2/HER3 heterodimerization. The stimulation of HER3 by NRG1-β1 is validated on the formalin-fixed and paraffin embedded MCF-7 cells with immunohistochemical stainings using the 21D3 (Cell Signaling) antibody that is specific for phosphorylated HER3. The manual immunohistochemical staining is performed using EDTA buffer (Thermo Scientific) for antigen retrieval and the ULTRAVISION™ LP Large Volume Detection System (Thermo Scientific) for antigen detection with the DAB+ chromogen (Dako) according to standard protocols. Only MCF-7 cells that are stimulated with NRG1-β1 stain positively for phosphorylated HER3 and the non-stimulated cells stain negatively.
Detection of HER2/HER3 Heterodimers in MCF-7 Cells
The immunohistochemistry stainings are performed on a Ventana BENCHMARK® XT system. The detection of dual binders with peptide linkers occurrs via the “bridging antibody” anti-HA-Tag antibody C29F4 (Cell Signaling). The Fc-Part of the anti-HA-Tag antibody is detected using the OPTIVIEW DAB detection kit (Ventana). The detection signal may be amplified using the OPTIVIEW Amplification kit (Ventana). Detection of dual binders with DNA-based linkers occurs either via the described “bridging antibody” anti-HA-Tag antibody C29F4 (targeting the HA-Tag at the C-terminus of the 7.2.32 heavy chain), or via the biotin label of the linker molecule that can serve as detection tag for the streptavidin-based iVIEW DAB detection kit (Ventana). The library of dual binders is prepared in individual experiments by mixing the 7.2.32-Fab-19mer and 4.1.43-Fab-17mer in equimolar ratio with each of the linkers (linker 3-14) that are shown in Example 2.4.
3.5 μm cuts of the MCF-7 cell blocks are produced using a microtome (HM355S, Thermo Scientific) and transferred onto a microscope slide. In the automated BENCHMARK® XT staining system, the cells are deparaffinized and treated with CC1 buffer for 32 min for antigen retrieval. For the screening, each dual binder is diluted in Ventana antibody diluent and 100 μL manually applied to the slides in different dilutions between 1:1 and 1:1000, and incubated for 16 min at 37° C. The anti-HA-Tag antibody C29F4 (Cell Signaling) is diluted 1:400 in Ventana antibody diluent and transferred into a dispenser. The anti-HA antibody has the function of a “bridging antibody” as it binds the HA-Tag of the dual binder, and, at the same time, its Fc part can be detected using the anti-rabbit detection system of the Ventana OPTIVIEW DAB detection kit. As a post fixative, 100 μL of the bridging antibody are applied and incubated for 32 min Next, the OPTIVIEW detection and amplification reagents are applied according to manufacturer's recommendations. The staining is analyzed in bright field microscopy.
A positive hit of the screening is a dual-binder that only stains the NRG1131-treated MCF-7 cells with induced HER2/HER3 heterodimerization and but does not stain the negative control of untreated MCF-7 cells (HER2 and HER3 monomers only). Further, the staining is inhibited by the addition of peptides of the epitope, 25 μg/ml HER2(1223-1236), shown in SEQ ID NO:25, or 25 μg/ml HER3(1242-1267), shown in SEQ ID NO:26, to inhibit one of the two binding site of the dual binder since the HER2/HER3 heterodimers should only be detectable if both arms of the dual binder are freely accessible.
While this disclosure has been described as having an exemplary design, the present disclosure may be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within the known or customary practice in the art to which this disclosure pertains.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10196687.7 | Dec 2010 | EP | regional |
| 11173844.9 | Jul 2011 | EP | regional |
This application is a continuation application of U.S. patent application Ser. No. 13/923,646, filed Jun. 21, 2013, which is a continuation of International Application No. PCT/EP2011/073567, filed Dec. 21, 2011, which claims the benefit of European Patent Application No. 10196687.7, filed Dec. 23, 2010, and European Patent Application No. 11173844.9, filed Jul. 13, 2011, the disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13923646 | Jun 2013 | US |
| Child | 15465379 | US | |
| Parent | PCT/EP2011/073567 | Dec 2011 | US |
| Child | 13923646 | US |
