US20120010153A1 - Novel tumor-targeting compounds - Google Patents

Novel tumor-targeting compounds Download PDF

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US20120010153A1
US20120010153A1 US13/144,416 US201013144416A US2012010153A1 US 20120010153 A1 US20120010153 A1 US 20120010153A1 US 201013144416 A US201013144416 A US 201013144416A US 2012010153 A1 US2012010153 A1 US 2012010153A1
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cancer
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peptide
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Andre Koltermann
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Sku Asset Management GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to novel compounds targeting tumor markers; a method for synthesis of such compound; and use of such compound in treating cancer.
  • immunotoxins i.e. compounds that are conjugates of toxic drugs to antibody molecules or protein ligands to receptors on human cancer cells.
  • antibodies that attach to cancer cells are used as conjugates with toxins in an attempt to minimize any delivery of the toxin to non-pathogenic cells.
  • Immunotoxins have been developed e.g. by linking tumor-specific antibodies to Ricin, Pseudomonas exotoxin, Diptheria toxin, and Tumor necrosis factor alpha (TNF-a).
  • the described approaches have significant disadvantages, e.g. in terms of unspecific toxicity, low efficacy, and high cost of manufacturing. Consequently, there is a need to provide anti-tumor compounds that are specific for tumor cells and that can be manufactured at lower costs. In particular, there is a need to provide compounds with anti-tumor efficacy and specificity that can be manufactured entirely by chemical synthesis from standard chemical building blocks.
  • the invention provides compounds with anti-tumor efficacy that are conjugates comprised of a backbone structure and one or more synthetic binders, whereby the backbone structure is a synthetic chemical structure that comprises one or more effector functions, flexible linker structures, as well as one or more coupling sites for the binders.
  • the effector functions within the backbone structure act as ligands to pathogen pattern recognition receptors (PRRs), in particular comprising pathogen-associated molecular patterns (PAMPS), and/or activates PRRs.
  • PRRs pathogen pattern recognition receptors
  • PAMPS pathogen-associated molecular patterns
  • the backbone structure resembles the Y structure of immunoglobulins with the effector function at the base of the compound and two flexible arms where the binders are located (see FIG. 1 ). Therefore, the compound comprises one branching point between flexible linker structures (the arms) as in immunoglobulins or in further embodiments additional branching points. The coupling sites for the binders are located at the end of such flexible linker structures.
  • the invention provides compounds that specifically target tumor cell markers.
  • tumor cell markers include human Epidermal Growth Factor Receptor (EGFR); human Vascular Endothelia Growth Factor Receptor (VEGFR); ErbB-2 receptor (Her-2); Fibroblast Growth Factor Receptor (FGFR); integrins, including alpha-3-beta-1 integrin and alpha-4-beta-1 integrin; lectins, including human beta-galactoside-binding lectin 3 (Galectin-3, or Gal-3); c-Met (hepatocyte growth factor receptor, HGFR or scatter factor, SF).
  • EGFR Epidermal Growth Factor Receptor
  • VEGFR Vascular Endothelia Growth Factor Receptor
  • FGFR Fibroblast Growth Factor Receptor
  • integrins including alpha-3-beta-1 integrin and alpha-4-beta-1 integrin
  • lectins including human beta-galactoside-binding lectin 3 (Galect
  • the invention provides a compound that comprises, two or more binders (resembling the head portion of the immunoglobulin structure);
  • a backbone structure comprising at least one coupling site for the binder, at least one linker structure, and at least one effector (to resemble the base of the immunoglobulin structure), whereby the effector comprises one of the following sequences: SEQ ID NO 24; fML (N-formyl-Met-Leu); SEQ ID NO 25 fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26 fMLF (N-formyl-Met-Leu-Phe), and whereby the linker structure is composed of a polypeptide, of polyethylene glycol, or a combination thereof.
  • binding preferably refers to any molecular structure, which interacts/binds with a second molecular structure, in particular with a tumor marker on a target tumor cell.
  • the binder of the invention can be of natural or synthetic origin and be modified or unmodified. Binders of the present invention include small molecules, peptides, peptide mimetics, modified or unmodified amino acids, oligonucleotides, as well as fragments, derivatives, analogs, chimerics or polymers of the aforementioned. Other examples of binders include synthetically produced small molecules.
  • small molecule preferably refers to a bioactive molecule with preferably a molecular weight of below 500 Da. Small molecules are molecules other than peptides, proteins, DNA or RNA.
  • tumor marker refers to any structure which is sufficiently specific for cancer cells, therewith enabling their discrimination from other host structures. Any structure which is capable of distinguishing host structures from cancer cells either quantatively or qualitatively can serve as a tumor marker, as e.g. in the case of overexpressed cell surface proteins that are also present on other host cells, but in lower quantity.
  • the interactions between binders and tumor markers can comprise any kind of chemical or physical interaction, e.g. hydrogen bonds, van-der-Waals forces or hydrophobic interactions.
  • the specificity of the marker can be due either to its selective expression or its enhanced expression in or on or at the proximity of cancer cells. Examples for tumor markers include receptors, ion channels and other cell-surface proteins.
  • effector preferably refers to any molecular structure which modulates signal transduction via a PRR.
  • the effectors of the present invention include small molecules, saccharides, peptides, peptide mimetics, natural or other amino acids, oligonucleotides as well as fragments, derivatives, analogs, chimerics or polymers thereof. Effectors include lipopolysaccharides, teichoic acids, nucleotides with unmethylated CpG motifs, double-stranded RNA, mannans, and formyl peptides. One important function of the effectors can be the immune cell activation. Effectors are preferably angonist of PRR, i.e.
  • Effectors especially include molecular structures that have direct or indirect stimulatory, modifying or activating effects on the innate immune system.
  • Cells of the innate immune system are effective in destroying and removing pathogens as well as diseased tissues or cells.
  • Cells of the innate immune system that are activated by effectors include macrophages, neutrophils including polymorphonuclear neutrophils, lymphocytes and NK cells.
  • linker structure preferably refers to any structure which is able to physically link two molecular entities whereby these entities can exhibit their intended function.
  • Linkers can be composed of a variety of chemical structures, for example, polyethylene glycols, peptides, ethers, esters.
  • activation when used in combination with cells or receptors (and quantitative measurement or qualitative assessment thereof) preferably refers to any direct or indirect immune response, including, for example, infiltration, degranulation, rolling, chemotaxis, phagocytosis, endocytosis, increased expression or activity of various catabolic or degradative enzymes (e.g., elastases), oxidative burst, production or release of hydrogen peroxide and other highly reactive oxygen species, intracellular calcium flux, cell polarization, and changes in inositol metabolism and signaling.
  • catabolic or degradative enzymes e.g., elastases
  • oxidative burst oxidative burst
  • production or release of hydrogen peroxide and other highly reactive oxygen species e.g., intracellular calcium flux, cell polarization, and changes in inositol metabolism and signaling.
  • determinants of activation include increased expression and production of leukotrienes, complement, chemokines, cytokines, chemoattractant factors, interleukins, or interferons. Methods for measuring these activities are well known to those skilled in the art. (See e.g., William E. Paul, Fundamental Immunology, Lippincott Williams and Wilking Publishers. 1999; John E. Coligen et al., Current Protocols in Immunology, John Wiley & Sons, New York, N.Y., 1999.)
  • substantially homologous when used in connection with amino acid sequences, preferably refers to sequences which are substantially identical to or similar in sequence, giving rise to a homology in conformation and thus to similar biological activity. The term is not intended to imply a common evolution of the sequences.
  • substantially homologous sequences are at least 50%, more preferably at least 80%, identical in sequence, at least over any regions known to be involved in the desired activity. Most preferably, no more than five residues, other than at the termini, are different.
  • the divergence in sequence, at least in the aforementioned regions is in the form of “conservative modifications”.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the first sequence which has for example 100 residues, at least 30, preferably at least 40, more preferably at least 50, even more preferably at least 60, and even more preferably at least 70, 80 or 90 residues are aligned).
  • non-immunoglobulin refers to a synthetic molecule that is not a natural immunoglobulin or a natural immunoglobulin with a synthetic portion. However, according to the invention the molecule more or less resembles the general structure of an immunoglobulin.
  • FIG. 1 Comparison of functionalities, size and intramolecular distances of the inventive compound with the structure of an immunoglobulin
  • FIG. 2 Structure 1 according to a preferred embodiment of the invention, with two binder peptides comprising the peptide sequence, SEQ ID NO 1 YHWYGYTPQNVI, one effector comprising the peptide sequence, SEQ ID NO 25 fMLP, and PEG as linker.
  • FIG. 3 Structure 2 according to a preferred embodiment of the invention, with two binder peptides comprising the peptide sequence, SEQ ID NO 1 YHWYGYTPQNVI, one effector comprising the peptide sequence, SEQ ID NO 26 fMLF, and PEG as linker.
  • FIG. 4 Variations of structure 2 with different chemical structures for coupling the binders to the flexible linker structures.
  • the binder peptide sequence, SEQ ID NO 1 YHWYGYTPQNVI could be replaced by a variation thereof (truncated or single amino acid residues replaced) as well as by other peptide sequences providing binding capability to tumor markers.
  • the binder can be attached via its C terminus (as in structures B and E) or via its N terminus (as in structures A, C and D).
  • FIG. 5 Binding studies with EGFR and the compound according to structure 1.
  • FIG. 6 Chemotaxis assays with the compound according to structure 2.
  • SAB refers to the structure 2
  • fMLF refers to a fMLF peptide only used as control.
  • the present invention provides novel compounds which enable an effective treatment of tumor cells.
  • anti-tumor compounds that are conjugates comprised of a backbone structure and one or more synthetic binders, whereby the backbone structure is a synthetic chemical structure that comprises one or more effectors, one or more flexible linker structures, as well as one or more coupling sites for the binders.
  • the backbone structure of the inventive compound provides the basic functions of the compound, i.e. (i) the effector function, (ii) sites to which binders can be coupled, and (iii) a flexible framework to enable interaction of the compound with both, cells of the innate immune system as well as with target cells bearing tumor markers. Besides these functions the backbone structure is preferably selected to have no other side activity.
  • the backbone structure preferably resembles in size and function the structure of an immunoglobulin G, i.e. (i) the effector function is located at the base of an Y-shaped structure like the Fc part of an immunoglobulin G; (ii) Two (or more) flexible linker structures are attached to the base via branching points like the Fab fragments in an immunoglobulin G; (iii) The binders are attached to the end of the flexible linker structures like the CDRs which are located at the tips of the Fab fragments in an immunoglobulin G.
  • the length of the flexible linkers when they are completely extended can be approximately 8 nm. This is in the same range as the length of an Fab fragment thereby providing the ability to bind to two binding sites (e.g. receptors on the surface of a cell) located in the same distance as immunoglobulins (see FIG. 1 ).
  • the at least one effector within the backbone structure is any substance which can act as a ligand to a pathogen pattern recognition receptor (PRR), in particular substances that comprise pathogen-associated molecular patterns (PAMPS), and/or activate PRRs, to exhibit at least one of their biological functions within the innate immune system.
  • PRR pathogen pattern recognition receptor
  • PAMPS pathogen-associated molecular patterns
  • the innate immune system represents a defense mechanism that recognizes a few highly conserved structures present in many different pathogens.
  • Such pathogen-associated molecular patterns include LPS from the gram-negative bacterial cell wall, peptidoglycans, lipotechoic acids from the gram-positive bacterial cell wall, the sugar mannose common in many microbial glycolipids and glycoproteins, fucose, N-acetyl glucosamine, bacterial DNA, N-formylmethionine found in bacterial proteins, double-stranded RNA from viruses, and glucans from fungal cell walls.
  • the targeted PRRs of the invention include inter glia mannose receptors (MR), formyl peptide receptors (FPRs), toll-like receptors (TLRs), CD14, and nucleotide-binding oligomerization domain proteins (NOD). Binding of ligands to these receptors also promotes the synthesis and secretion of intracellular regulatory molecules (immune modulating signals) such as cytokines that are crucial to initiating innate immunity and adaptive immunity.
  • intracellular regulatory molecules such as cytokines that are crucial to initiating innate immunity and adaptive immunity.
  • effectors of the present invention are peptides, formylated peptides, in particular N-formyl methionine peptides, peptidoglycans, acylated lipopeptides, e.g.
  • diacylated mycoplasmal lipopeptides or triacylated bacterial lipopeptide and tryacyl lipopeptide Pam3CSSNA
  • diaminopimelic acid containing desmuramyi peptide gamma-D-glutamyl-meso-DAP; iE-DAP
  • muramyldipeptide MDP
  • the at least one effector activates a formyl peptide receptor (FPR).
  • FPR formyl peptide receptor
  • Activation of FPRs leads to several phases of neutrophil response and activation, including chemoattraction, stimulation of production and release of immunosignaling molecules (e.g., interleukins, cytokines, etc.), as well as degranulation, a cellular process that includes the production and release of both chemical (e.g., hydrogen peroxide and other reactive oxygen radical species) and enzymatic agents (e.g., elastase and other digestive enzymes) capable of mediating destruction of the foreign agent or pathogen.
  • chemical e.g., hydrogen peroxide and other reactive oxygen radical species
  • enzymatic agents e.g., elastase and other digestive enzymes
  • FPR eponymous formyl peptide receptor
  • FPRL1 and FPRL2 are related to FPR by sequence homology but appear to be functionally distinct.
  • the cellular response mediated by the formyl peptide receptor (FPR) includes cellular polarization and transmigration, generation of superoxide O2 radicals through respiratory burst oxidase, degranulation and release of a variety of various degradative enzymes, as well as phagocytosis.
  • the effector interacts with the FPR and therewith induces at least one of the above mentioned cellular responses.
  • the effector contains N-formyl methionine residues. More preferably, the effector comprises one of the following N-terminal amino acid sequences: N-formyl-methionine-leucine (SEQ ID NO 24, fML); N-formyl-methionine-leucine-phenylalanine (SEQ ID NO 26, fMLF); N-formyl-methionine-leucine-proline (SEQ ID NO 25, fMLP).
  • Particularly preferred effectors are the peptides formyl-methionylleucine (SEQ ID NO 24, N-formyl-Met-Leu; fML), formyl-methionylleucylphenylalanine (SEQ ID NO 26, N-formyl-Met-Leu-Phe; fMLF), and formyl-methionylleucylproline (SEQ ID NO 25, N-formyl-Met-Leu-Pro; fMLP).
  • Formylmethionyl peptides are proinflammatory peptides which are able to stimulate many leukocyte functions.
  • the cellular response mediated by binding of formylated peptide antagonists to the formyl peptide receptor (FPR) includes cellular polarization and transmigration, generation of superoxide O2 radicals through respiratory burst oxidase, degranulation and release of a variety of various degradative enzymes, as well as phagocytosis.
  • the flexible linkers within the backbone structure of the inventive compound are preferably selected to enable the compound to interact with different cell surface proteins.
  • the chemistry of the backbone structure is preferably selected to have no or very low immunogenicity in the human body.
  • the size of the backbone structure is preferably adjusted through the length of the linkers.
  • the size of the inventive compound is selected in a range to facilitate tissue penetration of the compound and/or to adjust the clearance of the compound from the human body to its therapeutic efficacy.
  • linkers include oligomers and polymers of ethylene glycol (PEG), peptides, dendrimers, e.g. polylysine, oligonucleotides comprised of ribonucleic acid (RNA), desoxynucleic acid (DNA), peptide nucleic acids (PNA), polysaccharides, e.g. hydroyethylcellulose or hydroxyethylstarch, dextrans, aminodextrans, polyethers, polyamides, polyesters, polyurethanes.
  • PEG ethylene glycol
  • peptides es
  • dendrimers e.g. polylysine
  • oligonucleotides comprised of ribonucleic acid (RNA), desoxynucleic acid (DNA), peptide nucleic acids (PNA), polysaccharides, e.g. hydroyethylcellulose or hydroxyethylstarch, dextrans, aminodextrans, polyethers, polyamide
  • one or more flexible linkers within the backbone structure are composed of oligomers or polymers of ethylene glycol.
  • Polyethylene glycol is known to have no or very low immunogenicity in the human body.
  • the size of the backbone structure is adjusted by selecting the number of ethylene units used to synthesize the polyethylene glycol linkers.
  • the number of ethylene glycol units within the backbone structure is in the range between 1 and 250, more preferably between 1 and 100. In a particularly preferred aspect of this embodiment the number of ethylene glycol units is in the range between 20 and 80.
  • one or more flexible linkers within the backbone structure are composed of peptide sequences, i.e. oligomers of amino acids.
  • oligomers can be homooligomers (e.g. polylysine, polyglycine, etc.) or can be composed of different amino acids, including standard as well as non-standard amino acids.
  • the number of amino acid residues within the backbone structure that form flexible linker units is in the range between 1 and 100, more preferably between 1 and 50. In a particularly preferred embodiment the number of amino acids residues is in the range between 10 and 25.
  • the peptide oligomers have the sequence (GGGGS) 2-4 , i.e.
  • the sequence GGGGS is repeated 2 to 4 times.
  • Such linkers typically form flexible structures in aqueous solution.
  • the peptide oligomers have the sequence (EAAAK) 2-5 , i.e. the sequence EAAAK is repeated 2 to 5 times.
  • Such linker typically form alpha-helical structures in aqueous solution.
  • the backbone structure of the inventive compound comprises coupling sites that can be used to covalently link binder moieties to the backbone structure.
  • Any chemical residue can be used as coupling site which enables site-specific covalent linking of binder moieties to the backbone structure.
  • Examples of such chemical residues include a carboxylic acid group, an activated carboxyl group (e.g. active ester), an amino group, an aldehyde, an isocyanate, a thiol group, a maleimide, a succinimidyl ester.
  • the coupling site is an amino group and the one or more binder moieties are covalently coupled to the backbone structure via an activated carboxyl group.
  • the coupling sites are amino groups and the one or more binder moieties are peptides which are covalently coupled with the C terminal alpha carboxyl group to the amino group at the coupling site, thereby providing a compound comprising one or more peptide binders with free N termini.
  • the backbone structure preferably further comprises branching points to enable the introduction of either more than one effector, or more than one coupling site, or both.
  • the backbone structure comprises at least one branching point and at least two coupling sites, and flexible linkers are included between the at least one branching point and the at least two coupling sites.
  • branching points include trifunctional monomers such as the amino acids lysine, ornithine and citrulline (one carboxyl group and two amino groups), aspartic acid and glutamic acid (two carboxyl groups and one amino group), cystein (one carboxyl group, one amino group, and one thiol group), or tricarboxylic acids such as citric acid, isocitric acid or aconitic acid (three carboxyl groups), as well as tetrafunctional monomers such as lanthionine (two carboxyl groups and two amino groups).
  • trifunctional monomers such as the amino acids lysine, ornithine and citrulline (one carboxyl group and two amino groups), aspartic acid and glutamic acid (two carboxyl groups and one amino group), cystein (one carboxyl group, one amino group, and one thiol group), or tricarboxylic acids such as citric acid, isocitric acid or aconitic acid (three carboxyl groups), as well as
  • the backbone structure comprises two or more amino acids with an additional amino side group (such as lysine, ornithine or citrulline) as branching points whereby these amino acids are linked by peptide bonds to each other and whereby one of the resulting three or more amino groups (one alpha amino group and two or more other amino groups) serves as coupling site for the effector and the remaining two or more amino groups serve as coupling sites for the binders.
  • the single resulting alpha amino group serves as coupling site for the effector and the two or more other amino groups serve as coupling sites for the binders.
  • a flexible linker is synthesized between one or more of the effectors, branching points and/or binders.
  • the backbone structure has one of the following compositions:
  • EDFECTOR is an effector moiety
  • CO-NH or “NH-CO” symbolizes a peptide bond
  • LM is a linker monomer (e.g. ethylene glycol, or an amino acid)
  • BP is a branching point (e.g. a lysine residue)
  • NH2 is an amino group
  • OH is a hydroxyl group
  • n and “m” are integers.
  • the asterisk “*” marks the coupling sites at which the binders are covalently coupled to the backbone structure.
  • n is between 1 and 100, more preferably between 10 and 50, and even more preferably between 15 and 35. In a particularly preferred embodiment n is 27.
  • m is lower than or equal 10, more preferably lower than or equal 5, and even more preferably m is 2 or 3. In a particularly preferred embodiment m is 2.
  • the backbone structure can be manufactured completely by chemical synthesis.
  • the backbone structure is completely synthesized by solid-phase synthesis. Standard protocols for solid phase synthesis of peptides can be used to synthesize the backbone structure of the inventive compound.
  • the backbone structure is synthesized starting with an amino acid having two amino acid groups (a branching point, e.g. lysine) which is coupled with the carboxyl group to a solid support (e.g. polystyrene).
  • a branching point e.g. lysine
  • the linker structure as well the effector can be coupled to the molecule via activated carboxyl groups.
  • the one or more effectors and/or the one or more linker structures are peptides and are synthesized by coupling the respective amino acid residues via activated carboxyl esters to the molecule.
  • the binders are covalently linked to the synthetic backbone structure via the coupling sites.
  • the inventive compound is manufactured by synthesizing the backbone structure via solid phase synthesis followed by coupling of the binder moieties to the backbone structure.
  • the one or more binders are peptides, and the inventive compound is completely synthesized by solid phase synthesis.
  • binders according to the invention are peptides (linear, cyclic, or branched), peptide mimetics, peptide repeats, oligonucleotides (e.g. DNA, RNA), oligonucleotide analogs (e.g. PNA, DNA/RNA chimera, etc.) or small molecules.
  • the binder is peptide.
  • the length of the peptide is between 1 and 75 amino acid residues, more preferably between 3 and 25 amino acid residues.
  • the binders are peptides comprising specific amino acid sequences. Such sequences can contain the 20 standard amino acids, non-standard amino acids, as well as chemically modified amino acids.
  • non-standard amino adds include carnitine, citrulline, homoarginine, homocitrulline, homocysteine, homophenylalanine, homoproline, hydroxyproline, allo-isoleucine, isoserine, ornithine, phenylglycine, phenylisoserine, allo-threonine, selenocysteine, pyrrolysine, lanthionine, 2-amino isobutyric acid, gamma-amino butyric acid, dehydroalanine (2-amino acrylic acid), beta alanine (3-amino propanoic acid, or a stereo-isomer thereof.
  • the peptide sequences can comprise both, L-amino acids as well as D-amino acids.
  • the one or more binders are cyclic structures.
  • the binders are cyclic peptides.
  • Peptides of 30 or less amino acids are often unstructured in aqueous solutions. These peptides adopt a defined conformation only upon complex formation with partner molecules such as a receptor. Unstructured peptides can be metabolically unstable due to faster clearance from the mammalian body. Stability, bioavailability as well as selectivity for interaction with receptors can be increased by cyclization of such peptides.
  • cyclic peptides are generated by intramolecular disulfide bridging. It has been shown that peptides with intramolecular disulfide bridges are conformationally stable and thereby can be highly selective binders, e.g. for receptors. Examples of such peptides are defensins (pathogen control in vertebrates and invertebrates).
  • cyclic peptides are generated by stably linking the N and the C terminus of the peptide (head-to-tail cyclization) by forming an amide bond between the amino- and the carboxy-terminus.
  • This bond formation leads to homodetic, cyclic peptides.
  • the coupling chemistry used therefore is based on activated esters similar to the chemistry applied during solid phase peptide synthesis.
  • cyclic peptides are generated by side chain-to-side chain cyclization.
  • a thiol side chain e.g. of a cystein residue
  • the C-C double bond e.g. of a 2-amino acrylic acid residue
  • two C-C double bonds e.g. of 2-amino acrylic acid residues
  • Such homo- and heterodetic peptide macrocycles can be equipped with additional functional groups such as aldehydes, ketones and aminooxy groups as handles for covalent attachment to the coupling site in the backbone structure.
  • the stabilities of binder and/or effector peptides are increased and/or the peptides are protected from proteolytic degradation by blocking the amino and/or carboxy termini.
  • the term blocking refers to the introduction of a blocking group to the terminus of the peptide via a covalent modification. Suitable blocking groups do not interfere with the biological activity of the peptides.
  • Acetylation of the amino termini of the peptides is a preferred method of protecting the peptides from proteolytic degradation.
  • Preferred blocking moieties comprise the following acyl moieties (in the order of increasing hydrophobicity): formyl, acetyl, propanoyl, hexanoyl. Amidation of the carboxy termini of the peptides is a further preferred method of protecting the peptides from proteolytic degradation.
  • compounds that comprise one or more binders, whereby the binders bind to one of the following tumor markers: human Epidermal Growth Factor Receptor (EGFR); human Vascular Endothelia Growth Factor Receptor (VEGFR); ErbB-2 receptor (Her-2); Fibroblast Growth Factor Receptor (FGF receptor); Integrins including Alpha-3-beta-1 Integrin and Alpha-4-beta-1 Integrin; human beta-galactoside-binding lectin Galectin-3 (Gal-3); c-Met (Receptor for human Hepatocyte Growth Factor, HGF, or Scatter Factor, SF).
  • EGFR Epidermal Growth Factor Receptor
  • VEGFR Vascular Endothelia Growth Factor Receptor
  • ErbB-2 receptor Her-2
  • Integrins including Alpha-3-beta-1 Integrin and Alpha-4-beta-1 Integrin
  • Such binders preferably comprise one of the following sequences SEQ ID NO 1, YHWYGYTPQNVI; SEQ ID NO 2, CSDSWHYWC; SEQ ID NO 3, CSDHWHYWC; SEQ ID NO 4, CSDYNHHWC; SEQ ID NO 5, CSDWQHPWC; SEQ ID NO 6, KCCYSL; SEQ ID NO 7, MQLPLAT; SEQ ID NO 8, CDGLGDDC; SEQ ID NO 9, CDGWGPNC; SEQ ID NO 10, CLDWDLIC; SEQ ID NO 11, SWKLPPS; SEQ ID NO 12, CPLDIDFYC; SEQ ID NO 13, ANTPCGPYTHDCPVKR; SEQ ID NO 14, YLFSVHWPPLKA; or a fragment thereof with a length of at least four amino acid residues.
  • the peptide binders are covalently linked to the backbone structure via the C terminus in order to provide compounds with binders that have free N termini.
  • the binders comprise one of the following sequences: SEQ ID NO 15, SDSWHYW; SEQ ID NO 16, SDHWHYW; SEQ ID NO 17, SDYNHHW; SEQ ID NO 18, SDWQHPW; SEQ ID NO 19, DGLGDD; SEQ ID NO 20, DGWGPN; SEQ ID NO 21, LDWDLI; SEQ ID NO 22, PLDIDFY; SEQ ID NO 23, GPYTHD in a homo- or heterodetic peptide macrocycle which is formed through a thioether or a C-C double bond.
  • the invention provides compounds of the structure type: (Binder-Linker1)n-Linker2-Effector, wherein the Binder is a peptide and comprises one of the following sequences SEQ ID NO 1, YHWYGYTPQNVI; SEQ ID NO 2, CSDSWHYWC; SEQ ID NO 3, CSDHWHYWC; SEQ ID NO 4, CSDYNHHWC; SEQ ID NO 5, CSDWQHPWC; SEQ ID NO 6, KCCYSL; SEQ ID NO 7, MQLPLAT; SEQ ID NO 8, CDGLGDDC; SEQ ID NO 9, CDGWGPNC; SEQ ID NO 10, CLDWDLIC; SEQ ID NO 11, SWKLPPS; SEQ ID NO 12, CPLDIDFYC; SEQ ID NO 13, ANTPCGPYTHDCPVKR; SEQ ID NO 14, YLFSVHWPPLKA; or a fragment thereof with a length of at least four amino acid residues; the Effector is a peptide and comprises one of the
  • Linker1 and Linker2 are a polypeptide, a polyethylene glycol structure, or combination thereof, or a single covalent bond; n is at least 2.
  • compounds are provided that comprise one or more binders which are operably linked to at least one effector, whereby the binders bind to human Epidermal Growth Factor Receptor (EGFR) and/or related markers on tumor cells, and whereby the at least one effector is any substance which can act as a ligand to a pathogen pattern recognition receptor (PRR), in particular substances that comprise pathogen-associated molecular patterns (PAMPS), and/or activate PRRs.
  • PRR pathogen pattern recognition receptor
  • Such compounds can preferably be used in the treatment of solid tumors, in particular for the treatment of colon, breast, prostate, head & neck, NSCLC, ovarian, bladder, and/or esopheageal cancer.
  • the invention provides a compound that comprises two or more peptides as binders, each comprising one of the following sequences: SEQ ID NO 27, YHWY; SEQ ID NO 28, HWYG; SEQ ID NO 29, WYGY; SEQ ID NO 30, YGYT; SEQ ID NO 31, GYTP; SEQ ID NO 32, YTPQ; SEQ ID NO 33, TPQN; SEQ ID NO 34, PQNV; SEQ ID NO 35, QNVI; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); and
  • linker molecules composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond, whereby the linker molecules covalently link the two or more binders to the at least one effector.
  • the invention provides a compound that comprises two or more peptides as binders comprising the sequence SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile) or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe);
  • an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • binder peptide sequence SEQ ID NO 1, YHWYGYTPQNVI could be replaced by a variation thereof (truncated or single amino acid residues replaced) as well as by other peptide sequences providing binding capability to EGFR.
  • the binder peptide sequence can be attached alternatively via its N terminus or its C terminus.
  • the invention provides compounds of the structure type: (Binder-Linker1)n-Linker2-Effector, wherein the Binder is a peptide and comprises the sequence, SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-lie) or a fragment thereof with a length of at least four amino acid residues; the Effector is a peptide and comprises one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe);
  • Linker1 and Linker2 are a polypeptide, a polyethylene glycol structure, or combination thereof, or a single covalent bond; n is at least 2 and signifies the number of binder molecules bound to the effector.
  • such compounds comprise binders that are peptides with a length between 3 and 75 amino acid residues, more preferably between 4 and 25 amino acid residues, and comprise the sequence, SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile), or a fragment thereof with a length of at least four amino acid residues, preferably SEQ ID NO 36, YGYTPQ; SEQ ID NO 37, WYGYTPQN; or SEQ ID NO 38, HWYGYTPQNV, or a peptide that is substantially homologous to the sequence SEQ ID NO 1, YHWYGYTPQNVI or a fragment thereof.
  • SEQ ID NO 1 YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-I
  • the binder in such compounds is a peptide that comprises at least one of the following sequences: SEQ ID NO 27, YHWY; SEQ ID NO 28, HWYG; SEQ ID NO 29, WYGY; SEQ ID NO 30, YGYT; SEQ ID NO 31, GYTP; SEQ ID NO 32, YTPQ; SEQ ID NO 33, TPQN; SEQ ID NO 34, PQNV; SEQ ID NO 35, QNVI, or a sequence that is substantially homologous to one of these sequences.
  • the peptide has binding affinity to human EGFR (Epidermal Growth Factor Receptor) and/or variants, derivatives or homologues thereof.
  • the oligopeptide has binding affinity to the full-length EGFR or to the truncated receptor EGFRvIII (Epidermal Growth Factor Receptor variant III) or to both.
  • EGFR is overexpressed in a number of pathogenic cell populations such as in colon cancer, head and neck cancer, ovarial cancer, pancreatic cancer, non-small cell lung cancer, breast cancer and glioblastoma.
  • a compound having the formula: (Binder-Linker)n-Effector, wherein the Binder is a peptide comprising the sequence, SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile) or a fragment thereof with a length of at least four amino acid residues;
  • the Effector is a peptide comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe) at the N terminus as well as n lysine residues;
  • the n Linker molecules are polyethylene glycol oligomers;
  • the Effector is covalently attached via the epsilon amino groups of the n lysine residues through an amide bond to terminal carboxy groups of the n Linker molecules;
  • the Binder molecules are covalently attached via the carboxy terminus through an amide bond to a terminal carboxy group of the polyethylene glycol Linker;
  • n is an integer between 2 and 10.
  • the Binder is a peptide with the sequence, SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile);
  • the Effector is a peptide comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe) at the N terminus as well as two lysine residues;
  • the two Linker molecules are polyethylene glycol oligomers;
  • the Effector is covalently attached via the epsilon amino groups of the two lysine residues through an amide bond to terminal carboxy groups of the two Linker molecules;
  • the Binder molecules are covalently attached via the carboxy terminus through an
  • a compound having the formula: (Binder-Linker)2-Effector, wherein the Binder is a peptide with the sequence, SEQ ID NO 1, YHWYGYTPQNVI (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile);
  • the Effector is a peptide comprising the sequence SEQ ID NO 39, fMLPKK (N-formyl-Met-Leu-Pro-Lys-Lys);
  • the two Linker molecules are polyethylene glycol oligomers with a length between 2 and 100 ethylene glycol units;
  • the Effector is covalently attached via the epsilon amino groups of the two lysine residues through an amide bond to terminal carboxy groups of the two Linker molecules;
  • the Binder molecules are covalently attached via the carboxy terminus through an amide bond to a terminal carboxy group of the polyethylene glycol Linker molecules.
  • FIG. 2 Structure 1 according to a preferred embodiment of the invention.
  • FIG. 3 Structure 2 according to a preferred embodiment of the invention.
  • a compound is provided having one of the structures shown in FIG. 4 ( FIG. 4 A-E).
  • compounds are provided that comprise binders to human Vascular Endothelia Growth Factor Receptor (VEGFR) and/or related markers on tumor cells.
  • VEGFR Vascular Endothelia Growth Factor Receptor
  • Such compounds can be used in the treatment of a variety of human cancers that have a VEGFR or related marker.
  • the binders bind to human Vascular Endothelia Growth Factor Receptor (VEGFR) and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 2, CSDHWHYWC; SEQ ID NO 3, CSDHWHYWC; SEQ ID NO 4, CSDYNHHWC; SEQ ID NO 5, CSDWQHPWC; or a fragment thereof with a length of at least four amino acid residues.
  • VEGFR Vascular Endothelia Growth Factor Receptor
  • peptides as binders comprising the sequence, SEQ ID NO 2, CSDHWHYWC; SEQ ID NO 3, CSDHWHYWC; SEQ ID NO 4, CSDYNHHWC; SEQ ID NO 5, CSDWQHPWC; or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • the binder comprises the sequence SEQ ID NO 15, SDSWHYW; SEQ ID NO 16, SDHWHYW; SEQ ID NO 17, SDYNHHW; or SEQ ID NO 18, SDWQHPW; and side chain-to-side chain cyclization is carried out via a thioether, lactam, lactone or carbon-carbon linkage upon reaction between appropriate functional groups.
  • Particularly preferred linkages comprise formation of a thioether bond and formation of a carbon-carbon double bond.
  • Such peptide macrocycles can be equipped with additional functional groups such as aldehydes, ketones and aminooxy groups as handles for covalent attachment to the linker molecule.
  • compounds are provided that comprise binders to human ErbB-2 receptor (Her-2) and/or related markers on tumor cells.
  • Such compounds can preferably be used in the treatment of breast cancer, pancreatic cancer, ovarian cancer, NSCLC and/or lymphoma.
  • the binders bind to human ErbB-2 receptor (Her-2) and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 6, KCCYSL, or a fragment thereof with a length of at least four amino acid residues.
  • two or more peptides as binders comprising the sequence, SEQ ID NO 6, KCCYSL or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • compounds are provided that comprise binders to human Fibroblast Growth Factor Receptor (FGF receptor) and/or related markers on tumor cells.
  • FGF receptor Fibroblast Growth Factor Receptor
  • Such compounds can preferably be used in the treatment of Ovarian cancer.
  • the binders bind to human Fibroblast Growth Factor Receptor (FGF receptor) and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 7, MQLPLAT; or a fragment thereof with a length of at least four amino acid residues.
  • FGF receptor Fibroblast Growth Factor Receptor
  • two or more peptides as binders comprising the sequence, SEQ ID NO 7, MQLPLAT or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • compounds are provided that comprise binders to human Integrins including Alpha-3-beta-1 Integrin and Alpha-4-beta-1 Integrin and/or related markers on tumor cells.
  • Such compounds can preferably be used in the treatment of multiple peritoneal tumors of gastric cancer.
  • the binders bind to human Integrins including Alpha-3-beta-1 Integrin and Alpha-4-beta-1 Integrin and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 8, CDGLGDDC; SEQ ID NO 9, CDGWGPNC; SEQ ID NO 10, CLDWDLIC; SEQ ID NO 11, SWKLPPS; SEQ ID NO 12, CPLDIDFYC; or a fragment thereof with a length of at least four amino acid residues.
  • peptides as binders comprising the sequence, SEQ ID NO 8, CDGLGDDC; SEQ ID NO 9, CDGWGPNC; SEQ ID NO 10, CLDWDLIC; SEQ ID NO 11, SWKLPPS; SEQ ID NO 12, CPLDIDFYC; or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • the binder comprises the sequence SEQ ID NO 19, DGLGDD, SEQ ID NO 20, DGWGPN; SEQ ID NO 21, LDWDLI; SEQ ID NO 11, SWKLPPS; or SEQ ID NO 22, PLDIDFY, and side chain-to-side chain cyclization is carried out via a thioether, lactam, lactone or carbon-carbon linkage upon reaction between appropriate functional groups.
  • Particularly preferred linkages comprise formation of a thioether bond and formation of a carbon-carbon double bond.
  • Such peptide macrocycles can be equipped with additional functional groups such as aldehydes, ketones and aminooxy groups as handles for covalent attachment to the linker molecule.
  • compounds are provided that comprise binders to human beta-galactoside-binding lectin Galectin-3 (Gal-3) and/or related markers on tumor cells.
  • Such compounds can preferably be used in the treatment of breast cancer.
  • the binders bind to human Integrins including Alpha-3-beta-1 Integrin and Alpha-4-beta-1 Integrin and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 13, ANTPCGPYTHDCPVKR or a fragment thereof with a length of at least four amino acid residues.
  • two or more peptides as binders comprising the sequence SEQ ID NO 13, ANTPCGPYTHDCPVKR; or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • the binder comprises the sequence SEQ ID NO 23, GPYTHD and side chain-to-side chain cyclization is carried out via a thioether, lactam, lactone or carbon-carbon linkage upon reaction between appropriate functional groups.
  • Particularly preferred linkages comprise formation of a thioether bond and formation of a carbon-carbon double bond.
  • Such peptide macrocycles can be equipped with additional functional groups such as aldehydes, ketones and aminooxy groups as handles for covalent attachment to the linker molecule.
  • compounds are provided that comprise binders to c-Met (Receptor for human Hepatocyte Growth Factor, HGF, or Scatter Factor, SF) and/or related markers on tumor cells.
  • c-Met Receptor for human Hepatocyte Growth Factor, HGF, or Scatter Factor, SF
  • Such compounds can preferably be used in the treatment of a variety of human tumors.
  • the binders bind to c-Met (Receptor for human Hepatocyte Growth Factor, HGF, or Scatter Factor, SF) and/or related markers on tumor cells, and comprise one of the following sequences: SEQ ID NO 14, YLFSVHWPPLKA or a fragment thereof with a length of at least four amino acid residues.
  • two or more peptides as binders comprising the sequence, SEQ ID NO 14, YLFSVHWPPLKA; or a fragment thereof with a length of at least four amino acid residues; at least one peptide as effector comprising one of the following sequences: SEQ ID NO 24, fML (N-formyl-Met-Leu); SEQ ID NO 25, fMLP (N-formyl-Met-Leu-Pro); SEQ ID NO 26, fMLF (N-formyl-Met-Leu-Phe); an oligomeric linker composed of a polypeptide, of polyethylene glycol, or of a combination thereof, or of a single covalent bond.
  • the peptide SEQ ID NO 1, YHWYGYTPQNVI showing specific binding activity to the tumor marker Erb-B1/EGFR was chosen as binder and the macrophage-activating peptide formyl-MLP as effector to synthesize structure 1.
  • the structure was synthesized chemically by standard methods known in the art and purified by ion exchange chromatography. Chemical identity was verified by mass spectrometry. Unlike immunoglobulins and other known antitumor compounds, structure 1 demonstrated high solubility at pH 7 (up to 10 mg/ml). Size exclusion chromatography as well as dynamic light scattering analysis showed no oligomers or large aggregates and indicated a hydrodynamic radius slightly larger than expected for a molecule of approximately 6 kDa.
  • Binding affinity of structure 1 to human EGFR/Erb-B1 was tested using a fluorescently labelled variant and commercially available EGFR preparations using fluorescence spectroscopy.
  • a fluorescently labelled SECS ID NO 1, YHWYGYTPQNVI 12mer ( FIG. 5 a ) was incubated with EGFR in order to form a complex.
  • a decrease in fluorescence intensity was observed due to displacement of the fluorescent peptide from the complex with EGFR ( FIG. 5 b ).
  • a KD value in the low nM range for the interaction of structure 1 with EGFR was estimated. This finding confirms the notion that multivalency will increase affinity of a binder to the respective receptor. This effect is expected to be relevant when targeting receptors on cell surfaces.
  • FIG. 5 shows HPLC and MS data of an NBD-labelled variant of the 12mer EGFR-binding peptide used in displacement experiments as fluorescent tracer. The observed molar mass of 1703.4 Da is in good agreement with the calculated mass.
  • FIG. 5 b shows binding of the NBD-labeled tracer peptide to EGFR. 50 nM tracer (grey line) were mixed with 50 nM EGFR. Mixing results in an increase of fluorescence intensity (black line).
  • FIG. 5 c shows displacement of the fluorescent tracer peptide from the EGFR complex after adding non-labelled peptide.
  • the EGFR-tracer complex is preformed at 50 nM in PBS buffer at pH 7.5 (black line, excitation at 460 nm). A 10 fold excess of unlabelled peptide is added and the observed decrease in fluorescence intensity (grey line) reflects release of the tracer molecule.
  • FIG. 5 d shows displacement with increasing concentrations of structure 1. A 1 ⁇ M solution of EGFR and NBD-12mer peptide complex is titrated with increasing concentrations of structure 1 leading to displacement of the fluorescent tracer.
  • FIG. 6 The results of the chemotaxis assay with structure 2 are shown in FIG. 6 .
  • Concentration-dependent granulocyte activation of N-formyl peptide conjugates has been compared with activation of the peptide only.
  • Each series of experiments has been repeated 3 times with 200,000 granulocytes each.
  • the structure 2 compound induces a similar (slightly higher) chemotactic behavior (measured by counting migrated cells in response to stimulus) as the reference N-formyl peptide at the same concentration.

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US9994616B2 (en) 2013-10-15 2018-06-12 The University Of Tokyo c-Met protein agonist
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US8470963B2 (en) * 2006-06-30 2013-06-25 Sku Asset Management Gmbh Multifunctional compounds for pharmaceutical purposes
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