WO2008049866A1 - New chelating compound - Google Patents

Abstract

A chelating compound of the formula ma-Xaa1-Xaa2-Xaa3-R is provided, wherein - ma is mercaptoacetyl; - Xaa1, Xaa2 and Xaa3 are amino acid residues defined as follows: o any one or two of Xaa1, Xaa2 and Xaa3 is/are the amino acid residue Ser; o in case two of Xaa1, Xaa2 and Xaa3 are Ser, the remaining amino acid residue is selected from the group consisting of Glu, Arg and Lys; o in case one of Xaa1, Xaa2 and Xaa3 is Ser, the remaining amino acid residues are both Glu; and - R has a biological function and is selected from the group consisting of polypeptides, PNA, DNA, RNA, and mixtures thereof. Also provided are chelates of this compound with radioactive metal atoms, as well as methods and uses employing the chelating compound.

Description

NEW CHELATING COMPOUND

Field of the invention

The present invention relates to compounds useful for in vivo imaging. More particularly, the invention relates to a chelating compound and a chelate thereof with a radioactive metal atom. The present invention furthermore relates to methods of diagnosis, wherein the chelating compound is used.

Background of the invention

Molecular targeting of chemical structures within the body of a subject has been evaluated, for example for purposes of in vivo diagnostics of cancer disease. The targeting of tumor specific chemical structures is called tumor targeting. Molecules used for detection in the various applications of molecular targeting are called targeting agents. Monoclonal antibodies, fragments of monoclonal antibodies and peptides have been evaluated as targeting agents. The large size of antibodies, however, causes slow blood clearance, slow extravasation and slow tumor penetration. Therefore, the use of peptide receptor ligands for targeting of receptors that are overexpressed in malignant tumors is today a common approach in radionuclide tumor targeting. The small size of such peptides improves extravasation and tumor penetration. At the same time, a large proportion of short peptides (smaller than 60 kD) is rapidly cleared via the kidneys. This reduces radioactivity concentration in blood and healthy tissues. In this way, a high contrast (tumor-to-non-tumor ratio) is obtained when the peptides are used in the context of in vivo imaging. However, peptides are often re-absorbed in proximal tubulae after glomerular filtration, which can cause the accumulation of excessive radioactivity in the kidneys. Such high radioactivity accumulation in kidneys complicates the imaging of targets located close to the kidneys.

US 4 861 869 discloses bifunctional coupling agents suitable for forming conjugates with biomolecules and for complexing with radionuclide atoms. Examples of such coupling agents are various salts of modified mercaptoacetyl-glycyl-glycyl-glycinate, which are coupled to different antibody variants.

EP 284 071 A2 discloses similar labeling of biomolecules with a mercaptoacetyl-glycyl-glycyl-glycyl moiety for complexing with a radionuclide. US 5 980 861 is directed to the labeling of polynucleotides via a chelator compound, which chelator compound is either mercaptoacetyl-glycyl- glycyl-glycyl or mercaptoacetyl-seryl-seryl-seryl.

The above references are examples of using a tetradentate chelating group, which comprises three amide nitrogens from a peptide backbone and a sulfur from the terminal mercapto group. Formula (II) on page 6 of

EP 284 071 A2 is a schematic illustration of the geometry of such a complex.

Chelating groups such as those described in the above references are collectively denoted N3S or N₃S chelators, due to the three N's and one S involved. The work to date relating to such N3S chelators has been centered on mercaptoacetyl-glycyl-glycyl-glycyl, which in the present text is denoted ma-Gly-Gly-Gly. It is also referred to in the literature as MAG3 or MAG3. ma-Gly-Gly-Gly with different protective groups has been utilized for labeling of antibodies (Lei et al, Nucl Med Biol 23(7):917-922 (1996)) and their

Fab (Kim et al, Nucl Med Biol 29(2):139-142 (2002)) and dsFv fragments (Kobayashi et al, Cancer Res 56:3788-3795 (1996)), peptides (Qu et al, Nucl

Med Commun 22(2):203-215 (2001 ); Dechstoforo and Mather, Bioconjugate

Chem 10:431 -438 (1999)), and antisense DNA (Zhang et al, Eur J Nucl Med

27(11 ):1700-1707 (2000)), demonstrating good in vivo stability and, often, good pharmacokinetics. Some work has also been done on alternatives to ma-Gly-Gly-Gly.

Thus, in Appl Radiat lsot 50(4):723-732 (1999), Chang et al report on studies performed on mercaptoacetyl-seryl-seryl-seryl, i.e. ma-Ser-Ser-Ser, also denoted MAS3 or MAS₃.

Other alternative N3S chelators were studied by Zhu et al in Nucl Med Biol 28:703-708 (2001 ). In order to develop and test an alternative synthesis route, the authors synthesized ma-Gly-Gly-Gly as well as glutamic acid or phenylalanine containing variants ma-Glu-Gly-Gly and ma-Phe-Gly-Gly. The effect on biodistribution in mice was studied via coupling of these chelators to the 7 kD HNE2 protein.

Another biodistribution study was carried out by Verbeke et al and reported in Nucl Med Biol 27:769-779 (2000). However, this group used another type of chelator with only two amino acid residues coupled to the mercaptoacetyl group.

Previous experiments with mercaptoacetyl-thpeptide chelators, N3S chelators, have not yielded optimal results with regard to excretion pathways of the radiolabeled targeting agent. In particular, excretion via the hepatobiliary pathway leads to significant accumulation of activity in the liver and the intestines. This may necessitate imaging the day after injection, instead of imaging at the day of injection, in order to detect expression in extrahepatic metastases in the abdomen area and ovarian carcinoma. Therefore, it is desirable to effect a shift from the hepatobiliary pathway to the renal pathway. At the same time, when the desired properties of reduced hepatobiliary excretion are balanced by a high rate of clearance by the kidneys, it is desirable to minimize re-absorption in the kidneys. No chelator moiety for conjugation with a targeting agent has hitherto been presented which offers the requisite balance in this regard. Therefore, improved targeting agents are called for, which reduce the radioactivity concentration in healthy organs and at the same time enable high sensitivity in for example in vivo imaging.

Description of the invention One object of the present invention is to meet this demand, by developing new and improved chelating compounds that can function as targeting agents when complexed to a suitable radioactive metal atom.

Another object of the present invention is to provide chelates of such compounds with radioactive metal atoms, which chelates show high sensitivity for target structures.

A related object is to provide chelates of such compounds with radioactive metal atoms, which chelates reduce the radioactivity accumulation in healthy tissue. Yet another object of the present invention is to provide methods for preparing said chelates of compounds with radioactive metal atoms.

Still another object of the present invention is to provide means and methods that enable targeted in vivo imaging for diagnostic and/or pharmacokinetic studies.

Thus, in a first aspect of the invention, there is provided a chelating compound of the formula: ma-Xaa1 -Xaa2-Xaa3-R wherein - ma is mercaptoacetyl;

- Xaa1 , Xaa2 and Xaa3 are amino acid residues defined as follows: o any one or two of Xaa1 , Xaa2 and Xaa3 is/are the amino acid residue Ser; o in case two of Xaa1 , Xaa2 and Xaa3 are Ser, the remaining amino acid residue is selected from the group of amino acid residues consisting of GIu, Arg and Lys; o in case one of Xaa1 , Xaa2 and Xaa3 is Ser, the remaining amino acid residues are both GIu residues.

- R has a biological function and is selected from the group consisting of polypeptides, PNA, DNA, RNA, and mixtures thereof.

Thus, the chelating compound according to the invention has a chelating moiety, ma-Xaa1 -Xaa2-Xaa3, and a moiety conferring a biological function, R. In the present specification, variants of the chelating moiety of the inventive chelating compounds are denoted ma-Xaa1 -Xaa2-Xaa3, each of said Xaa:s being an amino acid represented in the standard three-letter code. A chelating moiety in a chelating compound according to the invention, in which moiety Xaa1 is GIu, Xaa2 is Ser and Xaa3 is GIu, is thus denoted ma- Glu-Ser-Glu, GIu and Ser being the three letter representations of glutamic acid and serine, respectively. In one embodiment of the present invention, the ma-Xaa1 -Xaa2-Xaa3 chelating moiety comprises one or two Ser, the remaining two or one amino acid residues being GIu. For example, Xaa3 may be GIu, and the inventive compound be selected from ma-Ser-Glu-Glu-R, ma-Glu-Ser-Glu-R and ma- Ser-Ser-Glu-R. Alternatively, Xaa3 may be Ser, and the inventive compound be selected from ma-Ser-Glu-Ser-R, ma-Glu-Glu-Ser-R and ma-Glu-Ser-Ser- R.

In the experiments described below, a combination of one serine and two glutamic acid residues (ma-Glu-Ser-Glu, ma-Ser-Glu-Glu and ma-Glu- Glu-Ser) was found to significantly reduce renal uptake in comparison with a chelating moiety studied for comparative purposes, ma-Glu-Glu-Glu, in an unexpected fashion. In addition, it was found that accumulated radioactivity in the gastrointestinal tract for the inventive compounds was less than 5 % of the total radioactivity in Balb/C mice, meaning that radiolabeled compounds of the above general formula are useful for imaging of tumors in the abdominal area. Thus, the inventive compounds have good tumor absorption as well as rapid clearance from normal organs and tissue. In imaging applications, radiolabeled targeting agents must have high tumor-to-background ratios, in order to obtain images with a high sensitivity.

In another embodiment, ma-Xaa1 -Xaa2-Xaa3 comprises two Ser, the remaining amino acid residue being selected from Arg and Lys. For example, the remaining amino acid residue may be Arg, and the inventive compound be selected from ma-Ser-Ser-Arg-R, ma-Ser-Arg-Ser-R and ma-Arg-Ser-Ser- R. Alternatively, the remaining amino acid residue may be Lys, and the inventive compound be selected from ma-Ser-Ser-Lys-R, ma-Ser-Lys-Ser-R and ma-Lys-Ser-Ser-R. In a preferred embodiment, the inventive compound is selected from ma-Ser-Arg-Ser-R and ma-Ser-Lys-Ser-R. As demonstrated in the experiments recounted below, the incorporation of one positively charged amino acid residue in the chelating moiety of the inventive compound unexpectedly provides the desired shift in excretion from the hepatobiliary pathway to the renal pathway. At the same time, renal uptake increased only marginally. In a comparative experiment also reported below, a chelating moiety having three positively charged amino acids, e.g. ma-Lys-Lys-Lys, instead caused an unwanted increase in liver uptake as well as an accumulation of radioactivity in the kidneys.

The inventive compound thus provides the structural modification ma- Xaa1 -Xaa2-Xaa3- on a functional or "effector" biomolecule R, which may be a polypeptide, a DNA molecule, an RNA molecule, a PNA molecule or a mixture of any two or more of these. The structural modification provides a chelating moiety available for binding of a radioactive metal atom M.

The biomolecular moiety R provides at least one specific biological function, for example selected from the group consisting of selective interaction with a target substance, in which case R may comprise a targeting moiety for directing the inventive compound towards a specific molecular structure; enzymatic action; therapeutic action; and combinations thereof. In one embodiment, the R moiety is capable of selective interaction with a target substance. This may interchangeably be expressed as the R moiety "being capable of binding to" or "having (a binding) affinity for" the target substance, etc.

Target substances are preferably structures found inside, on the surface of, or outside cells in the body of a mammalian subject, and are preferably relevant target substances for a medical application, such as cancer diagnostics and/or therapy. Target substances may be molecules bound to a cell surface; molecules bound to intracellular structures; intracellular molecules; components of extracellular matrix; or extracellular molecules. In the case of R being capable of selective interaction with a molecule bound to a cell surface, such a molecule may be selected from cell surface antigens, cell surface receptors, cell adhesion molecules and cell surface bound products of a reporter gene within the cell nucleus. Non-limiting examples of molecules bound to cell surfaces are PSMA, EpCAM, CEA, GRPR, NMBR, BRS3, CCK1 R, GASR, NTR-1 , NPY-Y2R, GLP1 R, NK1 R, VPAC1 R, VPAC2R, PACR, EDNR, SSR1 -5, TRLR1 -13, uPAR, PPARs, GLUTR, CD antigens including CD4, CD11 a, CD25, CD44, CD52, CD54, CD56, CD64, CD133, CD135, CD213a, CD213b; tyrosine kinase receptors including EGFR, HER2, ERBB3, ERBB4, cMET, RON, Tie1 , Tie2, IGF1 R, IR, antigens specific for neovasculature including VEGFR1 -3, PDGF receptors α and β; G-protein coupled receptors (GPCR) including chemokine receptors; apoptosis related receptors such as TRAILR 1-4, FASR, receptors within the TNFR family; cell adhesion molecules including integrins, cadherins, selectins, desmosomal cadherins, membrane proteoglycans, desmin, syndecan and ion channels.

In the case of R being capable of selective interaction with a molecule bound to an intracellular structure, non-limiting examples of such a molecule are cytoskeletal proteins, keratin family, lamins, vimentin, desmin, actin, tubulin, GFAP and melanin.

In the case of R being capable of selective interaction with an intracellular molecule, non-limiting examples of such a molecule are kinases, phosphatases and transcription factors, including members of the MAPK kinase, JAK-STAT, EGFR, VEGFR, insulin, integrin, mTOR, NF-κB, Notch, P53, Wnt, TGFb, TNFRs, ToIIR, FAK and phosphatidylinositol signalling pathways, molecules involved in apoptosis including members of the caspase cascade and those involved in the mitochondrial pathway for apoptosis and cell cycle progression including cyclins and cyclin dependent kinases, as well as protein regulatory molecules including the SOCS box containing proteins, ubiquitinating enzymes, sumoylating proteins, the proteasome complex, heat shock proteins and melanin.

In the case of R being capable of selective interaction with an extracellular molecule, non-limiting examples of such a molecule are cytokines, such as members of the TNF and IFN families, interleukins and colony stimulating factors, chemokines including the C, CC, CXC and CX3C families, hormones, such as oestrogen, GH, PRL, erythropoietin, activin, TSH, LSH, ACTH, melatonin, NPY, gastrin, and growth factors, such as FGFs, PDGFs, VEGFs, IGFI s, TGFb, EGF, Heregulin and Neuregulins, plasma proteins, including enzymatically active proteins or inhibitors such as the MMP, ADAM and serpin families, high abundant serum proteins, complement factors, hormone and cytokine binding factors and proteins involved in lipid transportation.

In the case of R being capable of selective interaction with components of extracellular matrix, non-limiting examples of such molecules are collagen and laminin families, fibronectin, elastin, endostatins, thrombospondins, tenascin and fibulin. As stated above, the moiety R, having a biological function, may be a polypeptide. In one embodiment of the invention, R is a polypeptide with from 3 to 200 amino acid residues. In a preferred embodiment, R may have from 6 to 120 amino acid residues, for example from 30 to 70 amino acid residues. In another preferred embodiment, R may be a polypeptide with from 3 to 30 amino acid residues, for example from 3 to 10 amino acid residues, such as 8 amino acid residues.

As described briefly in the Background section, much work has been done developing peptides as targeting agents, targeted towards their peptide receptors (reviewed in Reubi, Endocr Rev 24(4):389-427 (2003)). Such peptides may be comprised in the biological function moiety R in a chelating compound according to the invention, and be labeled with a radionuclide via the ma-Xaa1 -Xaa2-Xaa3 chelating moiety. As a non-limiting list of examples, R may comprise a polypeptide which is selected from peptide targeting agents based on somatostatin, bombesin, CCK, neurotensin, NPY, GLP1 and NK1.

In one embodiment, R may comprise an antibody molecule, or an antibody fragment or an antibody derivative, which is capable of selective interaction with a target substance (i.e. its antigen). Non-limiting examples of antibodies, antibody fragments and antibody derivatives are polyclonal antibodies, monospecific antibodies, monoclonal antibodies, Fab' fragments, scFv:s and domain antibodies (dAb:s).

In another embodiment of the present invention, R comprises a binding polypeptide, i.e. a polypeptide capable of selective interaction with a target substance, which is derived from a non-antibody scaffold. Such a binding polypeptide may be engineered using a naturally occurring protein, or domain(s) thereof, as a scaffold (i.e. a starting-point structure). Protein engineering may for example be used to obtain libraries of polypeptide variants, from which libraries suitable binding polypeptides are subsequently selected. Such libraries may be constructed by combinatorial protein engineering, making possible the evolution of highly specific binding polypeptides through randomization of a given number of amino acid residues in the scaffold polypeptide. Suitable procedures for isolation of binding polypeptides from combinatorial libraries are e.g. phage display, ribosome display, yeast two-hybrid system, mRNA display, SELEX (System Evolution of Ligands by Exponential Enrichment) and protein fragment complementation assays (PCA). Non-limiting examples of non-antibody scaffolds for use as starting point for the development of binding polypeptides are three-helix domains, lipocalins, ankyrin repeat domains, cellulose binding domains, Y crystallins, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain, zinc fingers, avimers, microproteins and conotoxins.

In a preferred embodiment, R comprises a binding polypeptide which has been engineered using as scaffold a three-helix polypeptide domain. Non-limiting examples of such three-helix polypeptide domains are any three- helix domain of Protein G from Streptococcus spp., such as the albumin binding domain (ABD) of Protein G from Streptococcus pyogenes strain G418; any domain of Protein A from Staphylococcus aureus, such as the protein Z variant of the B domain of Protein A from Staphylococcus aureus. In some embodiments of the invention, R comprises a binding polypeptide which has been engineered using as scaffold a three-helix polypeptide domain with structural properties derived from any protein A domain, including variants displaying alternative surfaces both on the target interaction face and on other sides of the molecule. In specific embodiments of the present invention, R comprises a polypeptide with an amino acid sequence selected from those listed below in Table 1. In Table 1 , amino acid sequences are given in the standard one-letter code. Table 1 Non-limiting examples of amino acid sequences that may be included in R

As an alternative to, or in combination with, the polypeptides discussed above, R in the inventive compound may also comprise a polynucleotide molecule, such as DNA, RNA, PNA or mixtures thereof. Such a polynucleotide molecule may also be capable of selective interaction with a target substance. For example, single stranded nucleic acids, called aptamers or decoys, fold into well-defined three-dimensional structures and may bind to a target substance with high affinity and specificity (see for review Bunka et al, Nat Rev Microbiol 4(8):588-596 (2006)). For example, Hicke et a/ (J Nucl Med 47(4):668-678 (2006)) reported the use of aptamers that bind to the extracellular matrix protein tenascin-C for imaging of brain and breast tumor xenografts. Another type of polynucleotide targeting agents are polynucleotides that are complementary to the DNA or mRNA of genes amplified or over-expressed in tumors (i.e. antisense polynucleotides). Any polynucleotide ligand for use as the R moiety in the present invention may be of RNA, DNA or PNA origin, or be a mixture of nucleotides with different backbones. An example of labeling of a PNA molecule with a known N3S chelator was presented by Chang et al (1999, supra), whereas Zhang et al (J Nucl Med 46(6):1052-1058 (2005)) used a known N3S chelator to label antisense DNA. Analogous procedures may be followed using the novel chelator moiety described herein.

The chelating compound according to the invention is suitably produced by known processes. In the case of R being a polypeptide, the compound may be prepared using expression of a gene encoding the amino acid sequence of interest, optionally including the three amino acid residues of the chelating moiety ma-Xaa1 -Xaa2-Xaa3-. The mercaptoacetyl group may then be conjugated to the purified polypeptide via known coupling chemistry. Alternatively, the compound can be produced by chemical synthesis. By choosing a synthetic production route rather than recombinant expression, the chelating moiety can be introduced site-specifically and the modified peptide can be purified to homogeneity.

As explained in detail above, and in keeping with the various objects of the invention, a chelating compound according to the first aspect of the invention may be complexed with a radionuclide for the purposes of various medical applications. Thus, in a second aspect thereof, the present invention provides a chelate of a chelating compound as described above with a radioactive moiety selected from M, M=O and O=M=O, wherein M is a radioactive metal atom.

In one embodiment of this aspect of the invention, the radioactive metal atom is suitable for in vivo imaging applications, i.e. applications wherein it is of interest to obtain images of the distribution of the radioactive metal atom within a body of a subject. A radionuclide for use in such applications is suitably one that emits gamma radiation with an energy of approximately 100-300 keV or positrons. As is known to the person of skill in the field, various isotopes of technetium are preferred metal atoms for use in imaging applications. Thus, in one embodiment of the present invention, M is selected from the group consisting of ^99mTc, ⁹⁴Tc and ⁹⁶Tc, preferably ^99mTc. SPECT, Single Photon Emission Computer Tomography, and PET, Positron Emission Tomography are the two techniques that are most frequently used for in vivo detection and visualization of radionuclide distribution in mammalian subjects. SPECT utilizes gamma-quanta, which are emitted from the nuclei of radioactive nuclides. ^99mTc (with a half-life of 6 h) has preferably been used in SPECT, due to the energy of gamma-quanta emitted (140 keV; nearly ideal for gamma-camera/SPECT imaging) and to its short half-life, which considerably reduces the radiation burden on patients. Furthermore, ^99mTc half-life is compatible with imaging using polypeptides.

A chelate according to the invention can be used for in vivo imaging. Chelates of the inventive compound with a radionuclide possess physio- chemical properties which in vivo results in improved biodistribution, compared with previously known compounds, such as the previously described ^99mTc:ma-Gly-Gly-Gly based chelates. As will be explained in detail in the Examples, the present inventors have carried out biodistribution studies in mice (normal NMRI mice or Balb/C nu/nu mice bearing SKOV-3 xenografts) and gamma-camera imaging, which visualized the distribution of chelates. The inventive chelate was found to give a lowered hepatobiliary excretion and a shift in excretion to the renal pathway. Chelates of the inventive compound thus reduce the accumulation of radioactivity in the liver and the intestines and makes possible for example tumor imaging with high contrast in the abdomen area at the day of injection. Unexpectedly and importantly, the renal uptake of a chelate of the inventive compound was only slightly increased in comparison with known chelates. Instead, renal retention was low, which is considered to be an effect of prevented re-absorption of the compound from primary urine in proximal tubulae of the kidneys. Reduction of renal retention for such a chelate thus decreases the radioactivity uptake in the kidney. This significant combination, displayed by the present invention, of reduction of hepatobiliary excretion on the one hand and reduction of renal retention on the other, provides the excellent biodisthbution properties needed in many medical applications. In a further, third, aspect of the invention, there is provided a method of preparing the chelate according to the second aspect. The method comprises mixing, in the presence of a reducing agent, of a compound according to the first aspect of the invention with a radioactive moiety MO₄ ^", wherein M is a radioactive metal atom. In order to prepare a chelate suitable for in vivo imaging applications, the initial chemical and isotopic form of the radioactive moiety may be selected from [""¹Tc]TcO₄ ^", [⁹⁴Tc]TcO₄ ^" and [⁹⁶Tc]TcO₄ ^", preferably [^99mTc]TcO₄\

In the method according to this aspect of the invention, mixing is carried out in the presence of a reducing agent. As non-limiting examples, said reducing agent may be SnCI₂ or SnF₂.

Optionally, the mixing is carried out in the additional presence of a weak chelator, which acts as an intermediate in the transfer of M to the inventive chelating compound. Non-limiting examples of such a weak intermediate chelator are tartrate, glucoheptonate and citrate. According to a fourth aspect thereof, the present invention provides a method of in vivo imaging of the body of a mammalian, including human, subject, which method comprises the steps of:

- administering a chelate according to the invention into the body of a mammalian subject; and - obtaining an image of at least a part of the subject's body using a medical imaging instrument, said image indicating the presence of M inside said body. In one embodiment of this inventive method, the step of obtaining an image is repeated at least three times, whereby a series of images is obtained. This embodiment is useful when one seeks to follow the biodisthbution over time of a targeting agent, and allows for pharmacokinetic studies. The skilled person appreciates that any number of images could be obtained, in order to achieve the requisite degree of time resolution in such a study.

In one embodiment of the imaging method according to the invention, the method comprises, before the administration step, a preparatory step of preparing a chelate according to the second aspect of the invention, which step comprises mixing, in the presence of a reducing agent, of a compound according to the first aspect with a compound selected from [""¹Tc]TcO₄ ^", [⁹⁴Tc]TcO₄ ^" and [⁹⁶Tc]TcO₄ ^", preferably [^99mTc]TcO₄\ Optionally, a weak intermediate chelator as discussed above may also be used. In related aspects, the present invention provides the chelate according to the second aspect of the invention for use in diagnostics. Also provided is use of said chelate in the preparation of a diagnostic agent for imaging in vivo of the body of a mammalian, including human, subject.

The invention will now be illustrated in a non-limiting manner by the following Example and Figures.

Brief description of the figures

Figure 1 illustrates a gamma-camera image of tumor-bearing mice using ^99mTc:ma-Gly-Gly-Gly-Z_HER2342 as targeting agent. Arrows point to caecum (C), kidneys (K) and tumors (T).

Figure 2 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Gly-Gly-Gly-Z_HER2342 (^99mTc-maGGG-Z_HER2₃₄₂) and ^99mTc:ma-Ser- Ser-Ser-Z_HER2342 (^99mTc-maSSS-Z_HER2342) in normal NMRI mice 4 h post- injection (p.i.).

Figure 3 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Ser-Ser-Ser-Z_HER2342 (^99mTc-maSSS-Z_HER2342) in Balb/C nu/nu mice bearing HER2-expressing SKOV-3 xenografts 4 h p.i. Figure 4 illustrates a gamma-camera image of a mouse bearing HER2- expressing SKOV-3 xenograft using ^99mTc:ma-Ser-Ser-Ser-Z_HER2342- Arrows point to caecum (C), kidneys (K) and tumors (T).

Figure 5 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Ser-Ser-Ser-Z_HER2₃₄₂ (^99mTc-maSSS-Z_HER2342), ^99mTc:ma-Glu-Glu- Glu-Z_HER2342 (^99mTc-maEEE-Z_HER2342) and ^99mTc:ma-Lys-Lys-Lys-Z_HER2342 (^99mTc-maKKK-Z_HER2342) in normal NMRI mice 4 h p.i.

Figure 6 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Ser-Ser-Ser-Z_HER2342 (^99mTc-maSSS-Z_HER2342), ^99mTc:ma-Glu-Glu- Glu-Z_HER2342 (^99mTc-maEEE-Z_HER2342), ^99mTc:ma-Glu-Glu-Ser-Z_HER₂342 (^99mTc- maEES-Z_HER2342), ^99mTc:ma-Ser-Glu-Glu-Z_HER2342 (^99mTc-maSEE-Z_HER2342) and ^99mTc:ma-Glu-Ser-Glu-Z_HER2342 (^99mTc-maESE-Z_HER2342) in normal NMRI mice 4 h p.i.

Figure 7 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Ser-Ser-Ser-Z_HER2342 (^99mTc-maSSS-Z_HER₂342), ^99mTc:ma-Ser-l_ys- Ser-Z_HER2342 (^99mTc-maSKS-Z_HER2342) and ^99mTc:ma-Lys-Lys-Lys-Z_HER2342 (^99mTc-maKKK-Z_HER2342) in normal NMRI mice 4 h p.i.

Figure 8 is a diagram showing the biodistribution of targeting agents ^99mTc:ma-Ser-Ser-Ser-Z_HER2342 (^99mTc-maSSS-Z_HER₂342), ^99mTc:ma-Glu-Glu- Glu-Z_HER2342 (^99mTc-maEEE-Z_HER2342), ^99mTc:ma-Ser-Glu-Glu-Z_HER₂342 (^99mTc- maSEE-Z_HER₂342), ^99mTc:ma-Glu-Glu-Ser-Z_HER₂342 (^99mTc-maEES-Z_HER₂342), and ^99mTc:ma-Glu-Ser-Glu-Z_HER2342 (^99mTc-maESE-Z_HER2342) in Balb/C nu/nu mice bearing SKOV-3 xenografts 4 h p.i.

Figure 9 is a diagram showing the biodistribution of targeting agent ^99mTc:ma-Ser-Lys-Ser-Z_HER2342 (^99mTc-maSKS-Z_HER2342) in Balb/C nu/nu mice bearing SKOV-3 xenografts 4 h p.i.

Figure 10 illustrates a gamma-camera image of a mouse bearing HER2-expressing SKOV-3 xenograft using ^99mTc:ma-Glu-Ser-Glu-Z_HER2342- Arrows point to kidneys (K) and tumors (T). Figure 1 1 illustrates the specificity of uptake of ^99mTc:ma-Ser-l_ys-Ser-

ZH_ER2 342 and ^99mTc:ma-Glu-Ser-Glu-Z_HER2342 in HER2-expressing tumor xenografts. For blocking, a large molar excess of non-labeled Z_HER2 343 was pre-injected in the animals. Data are presented as average percent of injected activity per gram (% IA/g) in four animals ± SEM.

In Figures 1 , 4 and 10, contours of the animals were derived from the digital photographs and superimposed over the gamma-camera images to facilitate interpretation thereof.

In Figures 2-3 and 5-9, data are presented as an average percent of injected activity per gram (% IA/g) of four animals ± SEM. The data for intestines are presented as radioactivity per whole intestines with content, and expressed as % IA per whole sample (typical weight approximately 3 grams).

Example

Outline

In certain forms of breast and ovarian cancer, tumor cells overexpress the protein kinase HER2 on their surfaces. Polypeptides capable of selective interaction with HER2 have been described, such as a monoclonal antibody to HER2 (trastuzumab, marketed as Herceptin® by Genentech and Roche) and engineered combinatorial proteins, for example those designated

ZHER2342 and Z_HER2477 (Orlova et al, Cancer Research 66:4339-48 (2006)). In the studies reported below, the HER2 binding polypeptide Z_{HER2 342} was provided with chelating moieties as described herein, thus forming chelating compounds according to the invention, and with other N3S chelating moieties for comparative purposes.

Peptide synthesis

The HER2 binding molecule Z_{HER2 342} was synthesized on a 433A Peptide Synthesizer (Applied Biosystems) using standard Fmoc chemistry, essentially as described in Engfeldt et al, Chembiochem 6:1043-50 (2005).

The peptide Z_HER2342

(VEN KFN KEM RNAYWEIALLPNLNNQQKRAFI RSLYDDPSQSAN LLAEA-

KKLNDAQAPK; SEQ ID NO:2) was assembled on an Fmoc amide resin with a substitution level of 0.67 mmol/g. The N-terminal Fmoc group was removed by 20 % piperidine-NMP. Acylation reactions were performed in NMP with 10 molar equivalents of amino acid, activated with 2-(1 H-benzothazol-1-yl)- 1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1 - hydroxybenzothazole (HOBt) (Advanced ChemTech, Louisville, USA). The 17 underlined residues in the sequence above were double coupled. Unreacted amino groups were capped with acetic anhydride (Applied Biosystems, UK).

The chelating functions ma-Gly-Gly-Gly, ma-Ser-Ser-Ser, ma-Glu-Glu- GIu and ma-Lys-Lys-Lys (all for comparison) and ma-Ser-Glu-Glu, ma-Glu- Ser-Glu, ma-Glu-Glu-Ser and ma-Ser-Lys-Ser (illustrating the invention) were introduced by manual synthesis. The N-terminal Fmoc protecting group was removed by incubation with 20 % piperidine-NMP for 20 min. All manual acylation reactions were performed with 5 molar equivalents of amino acid, HBTU and HOBt and 10 equivalents of N-ethyldiisopropylamine (DIEA, Lancaster Synthesis, UK) in NMP for 20 min. All reactions were monitored by the Kaiser test, and repeated when the yield was found unsatisfactory.

The final deprotection and release of peptides from the resin support was performed as a one-step procedure by treatment with TFA/ethanedithiol(EDT)/thisopropylsilane (TIS)/H₂O (94:2.5:2.5:1 ) for 2 h. This was followed by three rounds of extraction in te/t-butyl methyl ether-H₂O (50:50) (Merck), filtration and lyophilization. The product was analyzed by analytical RP-HPLC using a 4.6 x 150 mm polystyrene/divenylbenzene matrix column with a particle size of 5 μm (GE Healthcare, Sweden), a 20 min gradient of 20-60 % B (A: 0.1 % TFA-H₂O, B: 0.1 % TFA-CH₃CN) and a flow rate of 1 ml/min. The peptides were purified using a 20 min gradient of 25-45 % B. The pure product was lyophilized prior to dissolution in 10 mM NH₄Ac pH 5,5, and the final peptide concentration was determined by amino acid analysis.

To verify the identity of the peptides, part of the fractions from the HPLC analysis were lyophilized, dissolved in 5 M urea, 0.2 M NH₄HCOs, and 30 mM DTT and incubated for 1 h to reduce disulfides before capping free thiol groups with iodoacetamide (0.1 M, 1 h, Sigma, Germany), and finally the peptide mass was analyzed on a Q-Tof™ Il mass spectrometer fitted with an ESI source (Waters Corporation, Micromass MS Technologies, UK). The Maximum Entropy™ 1 (MaxEnti ) algorithm was used to deconvolute isotopic and charge state information.

ZHER2342 was successfully synthesized using Fmoc solid phase peptide synthesis. The synthetic yield was determined to 21 % by analytical RP- HPLC. The total yield of chelating polypeptides was 13-17 %. The peptides were purified to more than 90 % purity. The mass of each peptide was determined by ESI-MS, and the experimentally determined molecular weights correlated well with the theoretically calculated values (Table 2).

Melting point analysis

Variable temperature measurements were performed using a Jasco J- 810 spectropolahmeter (JASCO, Tokyo, Japan). Samples were diluted to a concentration of 50 μM in PBS, pH 7.4. A cell with an optical path length of 1 mm was used. CD spectra from 250 to 195 nm were obtained at 20 ⁰C before and after melting tests. For melting point measurements, the absorbance was measured at 221 nm and the temperature was increased by 5 °C/min from 20 to 90 ⁰C.

All conjugates generated very similar characteristic α-helical spectra. Identical CD spectra were taken before and after a melting point test was performed, indicating that the peptides regained their original fold after melting. The conjugates all had melting points between 64-68 ⁰C, as indicated in Table 2.

Biosensor analysis

Real-time biospecific interaction analysis was used to study the binding kinetics of Z_HER2 342 per se and the chelating compounds based on Z_HER2342 with chelating moieties on a Biacore 2000 instrument (Biacore, Sweden). Recombinant extracellular domain of HER2 (generous gift from G Adams, Fox Chase Cancer Center, Philadelphia) and the control protein human serum albumin, (HSA, KabiVitrum, Sweden) were diluted to 20 μg/ml in 10 mM NaAc pH 4.5 and immobilized onto a CM5 sensor chip (Biacore). The immobilization level was 500 RU (Response Units) for the HER2 surface and 900 RU for HSA. Samples were diluted in HBS (10 mM HEPES, 150 mM NaCI, 3.4 mM EDTA, 0.005 % Surfactant P20, pH 7.4) to concentrations ranging from 0.3 to 10 nM. The flow rate was set to 50 μl/min with HBS as running buffer, and the samples were injected for 5 min followed by 10 min dissociation and regeneration with 20 μl HCI (20 mM). The BiaEval software (Biacore) and the Langmuir 1 :1 binding model were used for the kinetic analysis.

Z_HER2342 per se had an apparent dissociation constant of 76 pM. The corresponding values for the different chelator containing polypeptides proved to be somewhat higher, as shown in Table 2.

Labeling with "¹⁷¹Tc and stability tests

^99mTc was obtained as pertechnate by elution of Ultra-TechneKow generator (Tyco, Holland) with sterile 0.9 % NaCI (Mallinckrodt Medical BV, Holland). Before labeling, the freeze-dhed chelating derivatives of Z_{HER2 342} were dissolved in degassed MiIIi-Q water and stored at -20 ⁰C. Analysis of the labeled derivatives of Z_HER2342 was performed using glass fiber sheets impregnated with silica gel for instant thin layer chromatography (ITLC™ SG, Gelman Sciences Inc, USA). To determine labeling yield and stability, ITLS™ SG strips were eluted with PBS. Validation experiments demonstrated that pertechnetate, as well as tartrate, glucoheptonate and cysteine complexes of ^99mTc migrate with the eluent front, while derivatives of Z_HER2342 do not move under these conditions. To determine the presence of reduced hydrolyzed technetium, ITLC was eluted with pyridine:acetic acid:water (5:3:1.5). When this eluent was used, the technetium colloids stayed, while radiolabeled derivatives of Z_HER2342, as well as pertechnetate and tartrate complexes of ^99mTc, migrated with the solvent front.

Thawed conjugate solution in ultra-pure water (20 μl, 1 mg/ml) was mixed with 20 μl 0.15 M NaOH, and 10 μl of freshly prepared SnCI₂'2H₂O solution in 0.01 M HCI (1 mg/ml) was added followed by 200 μl of ^99mTc- eluate. The mixture was carefully mixed and incubated at room temperature for 60 min. Use of alkaline conditions gave a labeling yield of approximately 90 % for all conjugates synthesized. The presence of reduced, hydrolyzed technetium was less than 2 % in all experiments.

After labeling, the ^99mTc-labeled Z_{HER2 342} variants were isolated from unreacted technetium and other low-molecular weight components using size-exclusion chromatography with a disposable NAP-5 column (GE Healthcare, Sweden) pre-equilibrated with PBS. Separation was performed according to the manufacturer's instruction. The use of PBS as an eluent made it possible to obtain ^99mTc-labeled Z_{HER2 342} directly in an injectable form after purification. Losses of labeled conjugate during purification were on the order of 13-17 %. According to ITLC analysis of the purified product, its purity was always higher than 95 %. Labels were stable in PBS. Stability tests were performed, in which labeled Z_{HER2 342} molecules were incubated in PBS or in PBS with 300 molar equivalents of cysteine. Stability was analyzed by ITLC. After a one hour incubation with a 300-fold excess of cysteine at 37 ⁰C, more than 90 % of the radioactivity was associated with the Z_{HER2 342} molecule, which indicates strong binding of ^99mTc. Binding specificity of "^mTc-labeled Z_HER2342 variants to HER2-expressing SKOV-3 cells

SKOV-3 cells bearing 1.2 x 10⁶ HER2 receptors per cell were cultivated on Petri dishes with a diameter of 3.5 cm to a cell density of 2-5 x 10⁵ CeIIs per dish.

Labeled conjugates, 0.8 ng/10⁵ cells, were added to two groups of Petri dishes containing SKOV-3 cells. One group of dishes was pre-saturated with a 1000-fold excess of non-labeled recombinant Z_{HER2 342} molecules 10 min before labeled molecules were added. The cells were incubated for 1 h at 37 ⁰C and incubation media was collected. The cell dishes were washed with cold serum-free medium and treated with 0.5 ml trypsin-EDTA solution (0.05 % trypsin, 0.02 % EDTA in PBS buffer, Flow Irvine, UK) for 10 min at 37 ⁰C. When cells were detached, 0.5 ml complete medium was added to every dish and cells were re-suspended. The cell suspension was collected for radioactivity measurements. Cell-associated radioactivity (C) was measured on an automated gamma-counter with a 3-inch NaI(TI) detector (1480 WIZARD, Wallac OY, Finland) in parallel with 1 ml corresponding incubation medium (M). The fraction of added radioactivity bound to cells was calculated as % of bound radioactivity = C x 100 % / (C + M). The results of this binding specificity experiment showed that binding of all ^99mTc-labeled Z_{HER2 342} chelators could be prevented by receptor saturation (data not shown).

Animal studies The animal study was approved by the local Ethics Committee for

Animal Research at Uppsala University, Sweden. In order to create an in vivo tumor model, female outbred Balb/c nu/nu mice (10-12 weeks old at arrival) were used in the in vivo experiments. SKOV-3 tumors were grafted by subcutaneous injection of approximately 7 x 10⁶ cells in the right hind leg. Xenografts were allowed to develop during eight weeks. Biodistribution in normal NMRI mice

To quantify hepatobiliary excretion, groups of four NMRI mice were injected with approximately 1 μg ^99mTc-labeled Z_HER2342 (approximately 100 kBq). The biodistribution measurement was performed 4 h after injection (pi). Mice were intraperitoneal^ injected with a lethal dose of ketamine HCI

(Ketalar®, Pfizer) and xylazine HCI (Rompun®, Bayer). The mice were killed with heart puncture with a 1 ml syringe rinsed with diluted heparin (Leo Pharma). Organs and tissue samples of blood, lung, liver, spleen, kidney, stomach, salivary glands, thyroid and muscle were excised and collected in weighed plastic bottles. Intestines were collected together with their content. Organs and tissue samples were weighed and their radioactivity was recorded using an automatic gamma-spectrometer with standard ^99mTc protocol. The background was measured for each sample holder in the same way. Tissue uptake values were calculated as percent of injected activity per gram tissue (% IA/g).

Radioactivity in kidneys and intestine with contents were selected as a measure of renal and hepatobiliary excretion. Other organs were selected to evaluate in vivo stability of the technetium label. An elevated level of blood radioactivity would be an indication of transchelation of ^99mTc to blood plasma proteins, while formation of radio colloids or macroaggregates should be reflected in an elevated uptake in liver, spleen or lungs. Re-oxidation and release of pertechnetate during catabolism of the label should cause increased uptake of radioactivity in stomach, salivary gland and thyroid.

Biodistribution in mice with SKOV-3 xenografts

To study biodistribution in Balb/c nu/nu mice bearing SKOV-3 xenografts, four animals per conjugate were used. For ^99mTc:ma-Glu-Ser-Glu- ZHER2342 and ^99mTc:ma- Ser-Lys-Ser-Z_HER2342, an extra group of four mice was subcutaneously injected with 750 μg of non-labeled recombinant Z_{HER2 342} in 250 μl PBS 1 h before the radioactive injection (blocking experiment). Mice were subcutaneously injected with the various ^99mTc:ma- Xaa1 -Xaa2-Xaa3-Z_HER2342 variants in 100 μl PBS (approximately 100 kBq). At 4 h pi, mice were injected intraperitoneal^ with a lethal dose of ketamine HCI (Ketalar®, Pfizer) and xylazine HCI (Rompun®, Bayer). The mice were killed with heart puncture with a 1 ml syringe rinsed with diluted heparin (Leo Pharma). The mice in the blocking group were killed 4 h pi. The samples were treated as in the previously described experiments on biodistribution in normal NMRI mice (see above).

Statistical analysis

An unpaired f-test of the biodistribution data was performed using the GraphPad Prism software (Graph-Pad Software Inc, San Diego, USA). The differences were considered significant if the p values were less than 0.05.

Gamma-camera imaging

Experimental tumor imaging was performed five hours after injection in mice bearing SKOV-3 xenografts. All animals were intravenously injected (tail) with 3 MBq (3 μg) ^99mTc:ma-Gly-Gly-Gly-Z_HER2342 (comparison), 9^9mTc:ma-Ser-Ser-Ser-Z_HER2342 (comparison) or ^99mTc:ma-Glu-Ser-Glu- Z_{HER2 342} (illustrating the invention). Five hours after injection of the "Relabelled conjugates, all animals were killed by overdosing of ketamine/xylazine. Imaging was performed using a Siemens e.cam gamma- camera equipped with an LEHR collimator. Static images were collected during 10 minutes.

Results pertaining to comparative chelators

Gamma-camera images of mice with HER2-expressing xenografts using ^99mTc:ma-Gly-Gly-Gly-Z_HER2342 demonstrated capability of this substance to image tumors (Figure 1 ). However, a majority of the radioactivity accumulation was found in the abdomen area. Dissection of imaged animals and subsequent radioactivity measurements showed that this radioactivity was contained in the caecum content. This was considered to be evidence of hepatobiliary excretion of the conjugate. As discussed in the above description of the invention, the hepatobiliary excretion pathway is undesirable, since it generates a high and long-lasting background in the abdomen. The biodistribution experiment in normal mice demonstrated that the use of the ma-Ser-Ser-Ser chelator enabled a reduction of the level of hepatobiliary excretion three-fold, at a cost of moderate increase of radioactivity uptake in the kidneys (Figure 2). The biodistribution experiment in tumor-bearing mice confirmed the capacity of ^99mTc:ma-Ser-Ser-Ser-Z_HER2342 to target HER2-expressing tumor xenografts. Radioactivity accumulation in the tumors exceeded the accumulation in all organs and tissues, except the kidneys (Figure 3). The tumor accumulation was even higher than the average accumulation in the gastrointestinal tract, with account taken of the fact that the typical sample weight of intestines was 3 gram.

A gamma camera image (Figure 4) demonstrated improvement in contrast in comparison with ^99mTc:ma-Gly-Gly-Gly-Z_HER2342- Still, a radioactivity accumulation in caecum was clearly seen, which caused a modest contrast between tumor and abdominal area.

When all three amino acids in a chelating ma-Xaa1 -Xaa2-Xaa3 moiety of the Z_HER2342 derivatives were lysine or glutamic acid, hepatobiliary excretion was to a high extent suppressed in normal mice (Figure 5). At the same time, a shift to a renal excretion pathway caused an appreciable increase in renal uptake. Kidney accumulation was 95 ± 23 % IA/g (percent injected activity per gram) for ^99mTc:ma-Glu-Glu-Glu-Z_HER2342 and 127 ± 9 % IA/g for ^99mTc:ma-Lys-Lys-Lys-Z_HER2342 (compared with 18 ± 4 % IA/g for ^99mTc:ma-Ser-Ser-Ser-Z_HER2342) Besides, use of ma-Lys-Lys-Lys as a chelator caused high radioactivity uptake in liver, 6.8 ± 2.5 % IA/g (compared with 0.48 ± 0.28 % IA/g for ^99mTc:ma-Ser-Ser-Ser-Z_HER2₃₄₂ and 0.21 ± 0.01 % IA/g for ^99mTc:ma-Glu-Glu-Glu-Z_HER2342).

Results pertaining to chelators of the invention

Unexpectedly, the shift to renal excretion was accompanied by much smaller increase of radioactivity accumulation in kidneys, when only part of serines was substituted by glutamic acids or lysines (Figure 6). Thus, the radioactivity concentration in kidneys for ma-Glu-Glu-Ser and ma-Ser-Glu-Glu was less than half of that of ma-Glu-Glu-Glu, and in the case of ma-Glu-Ser- GIu, the difference was more than four-fold. Apparently, the reduction in renal uptake was not caused by an impaired clearance, since the radioactivity concentration in all other organs and in the rest of the body was at approximately the same level as in the case of ma-Glu-Glu-Glu. Most likely, but without wishing to be bound by this theory, the conjugates were excreted by glomerular filtration, while their re-absorption from primary urine was diminished in comparison with ma-Glu-Glu-Glu.

A similar effect was observed for the ma-Ser-Lys-Ser chelator (Figure 7). This chelator moiety provided as low radioactivity concentration in intestine as ma-Lys-Lys-Lys, but the renal uptake was reduced nearly fourfold (from 127 ± 9 %IA/g to 33 ± 2 %IA/g). Additionally, the use of ma-Ser- Lys-Ser reduced liver uptake twelve-fold in comparison with ma-Lys-Lys-Lys.

The tumor-targeting properties of ^99mTc:ma-Glu-Glu-Glu-Z_HER2342, ^99mTc:ma-Ser-Glu-Glu-Z_HER2342, ^99mTc:ma-Glu-Glu-Ser-Z_HER2342, ^99mTc:ma- Glu-Ser-Glu -Z_HER2342, and ^99mTc:ma-Ser-Lys-Ser-Z_HER2342 were verified in mice bearing HER2-expressing SKOV-3 xenografts (Figures 8 and 9). In general, the biodistribution of the inventive chelating compounds was characterized by quick clearance of radioactivity from blood and all organs and tissues. The radioactivity concentration in tumors was higher than in other organs or tissues except for the kidneys. The combination of a reasonably good retention of the radioactive metal atom in tumors on the one hand, and quick blood and whole-body clearance on the other, generated a good contrast (i.e. tumor-to-non-tumor ratio) with respect to a majority of the organs and tissues. As a confirmation of the biodistribution studies, gamma-camera imaging of ^99mTc:ma-Glu-Ser-Glu-Z_HER2342 (Figure 10) showed that high contrast images of HER2-expressing xenografts in mice can be produced using a conjugate according to the invention. The only organ with apparent accumulation of radioactivity apart from the tumor was the kidney. Though it might create certain problems with detection of HER2-expression in metastases in close proximity to kidneys, the position of kidneys in the body is well-defined, which would help in the interpretation of the gamma-camera images. The reduction in kidney uptake further reduces this problem. Importantly, radioactivity in the abdominal area was very low, and a high contrast of tumor imaging in this area was possible.

To control specificity of tumor uptake, additional groups of animals were pre-injected with a large excess of non-labeled, recombinant Z_{HER2 342} in order to saturate HER2 receptors in tumor xenografts. Then, ^99mTc:ma-Ser- Lys-Ser-Z_HER2342 and ^99mTc:ma-Glu-Ser-Glu-Z_HER2342 were injected. As shown in Figure 11 , injection of a blocking amount of "cold" Z_HER2342 reduced significantly the uptake of radioactivity in tumors, thus demonstrating the specificity of the Z_HER2342 targeting agent.

Claims

1. Chelating compound of the formula:

ma-Xaa1 -Xaa2-Xaa3-R wherein

- ma is mercaptoacetyl;

- Xaa1 , Xaa2 and Xaa3 are amino acid residues defined as follows: o any one or two of Xaa1 , Xaa2 and Xaa3 is/are the amino acid residue Ser; o in case two of Xaa1 , Xaa2 and Xaa3 are Ser, the remaining amino acid residue is selected from the group consisting of GIu, Arg and Lys; o in case one of Xaa1 , Xaa2 and Xaa3 is Ser, the remaining amino acid residues are both GIu; and

- R has a biological function and is selected from the group consisting of polypeptides, PNA, DNA, RNA, and mixtures thereof.

2. Compound according to claim 1 , in which one or two of Xaa1 , Xaa2 and Xaa3 is/are Ser, and the remaining two or one of Xaa1 , Xaa2 and Xaa3 is/are GIu.

3. Compound according to claim 2, wherein o Xaa3 is GIu; and o the dipeptide Xaa1 -Xaa2 is selected from Ser-Glu, Glu-Ser and Ser-Ser.

4. Compound according to claim 3, wherein o Xaa3 is GIu; and o the dipeptide Xaa1 -Xaa2 is selected from Ser-Glu and Glu-

Ser.

5. Compound according to claim 4, wherein o Xaa1 is Ser; o Xaa2 is GIu; and o Xaa3 is GIu.

6. Compound according to claim 4, wherein o Xaa1 is GIu; o Xaa2 is Ser; and o Xaa3 is GIu.

7. Compound according to claim 3, wherein o Xaa1 is Ser; o Xaa2 is Ser; and o Xaa3 is GIu.

8. Compound according to claim 2, wherein o Xaa3 is Ser; and o the dipeptide Xaa1 -Xaa2 is selected from Ser-Glu, Glu-Ser and GIu-GIu.

9. Compound according to claim 8, wherein o Xaa1 is Ser; o Xaa2 is GIu; and o Xaa3 is Ser.

10. Compound according to claim 8, wherein o Xaa1 is GIu; o Xaa2 is GIu; and o Xaa3 is Ser.

11. Compound according to claim 8, wherein o Xaa1 is GIu; o Xaa2 is Ser; and o Xaa3 is Ser.

12. Compound according to claim 1 , in which two of Xaa1 , Xaa2 and Xaa3 are Ser, and the remaining one of Xaa1 , Xaa2 and Xaa3 is selected from Arg and Lys.

13. Compound according to claim 12, in which two of Xaa1 , Xaa2 and Xaa3 are Ser, and the remaining one of Xaa1 , Xaa2 and Xaa3 is Arg.

14. Compound according to claim 13, wherein o Xaa1 and Xaa2 are Ser; o Xaa3 is Arg.

15. Compound according to claim 13, wherein o Xaa2 and Xaa3 are Ser; o Xaa1 is Arg.

16. Compound according to claim 13, wherein o Xaa1 and Xaa3 are Ser; o Xaa2 is Arg.

17. Compound according to claim 12, in which two of Xaa1 , Xaa2 and Xaa3 are Ser, and the remaining one of Xaa1 , Xaa2 and Xaa3 is Lys.

18. Compound according to claim 17, wherein o Xaa1 and Xaa2 are Ser; o Xaa3 is Lys.

19. Compound according to claim 17, wherein o Xaa2 and Xaa3 are Ser; o Xaa1 is Lys.

20. Compound according to claim 17, wherein o Xaa1 and Xaa3 are Ser; o Xaa2 is Lys.

21. Compound according to claim 12, wherein o Xaa1 and Xaa3 are Ser; o Xaa2 is selected from the group consisting of Arg and Lys.

22. Compound according to any one of claims 1 -21 , wherein said biological function is selected from the group consisting of selective interaction with a target substance; enzymatic action; therapeutic action; and combinations thereof.

23. Compound according to claim 22, wherein said biological function comprises selective interaction with a target substance.

24. Compound according to claim 23, wherein said target substance is selected from the group consisting of molecules bound to a cell surface; molecules bound to intracellular structures; intracellular molecules; components of extracellular matrix; or extracellular molecules.

25. Compound according to claim 24, wherein said target substance is a molecule bound to a cell surface selected from the group consisting of cell surface antigens, cell surface receptors, cell adhesion molecules, and cell surface bound products of a reporter gene.

26. Compound according to claim 25, wherein said molecule bound to a cell surface is selected from the group consisting of PSMA, EpCAM, CEA, GRPR, NMBR, BRS3, CCK1 R, GASR, NTR-1 , NPY-Y2R, GLP1 R, NK1 R, VPAC1 R, VPAC2R, PACR, EDNR, SSR1 -5, TRLR1 -13, uPAR, PPARs, GLUTR, CD antigens including CD4, CD11 a, CD25, CD44, CD52, CD54, CD56, CD64, CD133, CD135, CD213a, CD213b; tyrosine kinase receptors including EGFR, HER2, ERBB3, ERBB4, cMET, RON, Tie1 , Tie2, IGF1 R, IR, antigens specific for neovasculature including VEGFR1 -3, PDGF receptors α and β; G-protein coupled receptors (GPCR) including chemokine receptors; apoptosis related receptors such as TRAILR 1-4, FASR, receptors within the TNFR family; cell adhesion molecules including integrins, cadherins, selectins, desmosomal cadherins, membrane proteoglycans, desmin, syndecan and ion channels.

27. Compound according to claim 24, wherein said target substance is a molecule bound to an intracellular structure, which molecule is selected from the group consisting of cytoskeletal proteins, keratin family, lamins, vimentin, desmin, actin, tubulin, GFAP and melanin.

28. Compound according to claim 24, wherein said target substance is an intracellular molecule selected from the group consisting of kinases, phosphatases and transcription factors, including members of the MAPK kinase, JAK-STAT, EGFR, VEGFR, insulin, integrin, mTOR, NF-κB, Notch, P53, Wnt, TGFb, TNFRs, ToIIR, FAK and phosphatidylinositol signalling pathways, molecules involved in apoptosis including members of the caspase cascade and those involved in the mitochondrial pathway for apoptosis and cell cycle progression including cyclins and cyclin dependent kinases, as well as protein regulatory molecules including the SOCS box containing proteins, ubiquitinating enzymes, sumoylating proteins, the proteasome complex, heat shock proteins and melanin.

29. Compound according to claim 24, wherein said target substance is an extracellular molecule selected from the group consisting of cytokines, such as members of the TNF and IFN families, interleukins and colony stimulating factors, chemokines including the C, CC, CXC and CX3C families, hormones, such as oestrogen, GH, PRL, erythropoietin, activin, TSH, LSH, ACTH, melatonin, NPY, gastrin, and growth factors, such as FGFs, PDGFs, VEGFs, IGFI s, TGFb, EGF, Heregulin and Neuregulins, plasma proteins, including enzymatically active proteins or inhibitors such as the MMP, ADAM and serpin families, high abundant serum proteins, complement factors, hormone and cytokine binding factors and proteins involved in lipid transportation.

30. Compound according to claim 24, wherein said target substance is a component of extracellular matrix selected from the group consisting of collagen and laminin families, fibronectin, elastin, endostatins, thrombospondins, tenascin and fibulin.

31. Compound according to any preceding claim, wherein R is a polypeptide.

32. Compound according to claim 31 , wherein said polypeptide has from 3 to 200 amino acid residues.

33. Compound according to claim 32, wherein said polypeptide has from 6 to 120 amino acid residues.

34. Compound according to claim 33, wherein said polypeptide has from 30 to 70 amino acid residues.

35. Compound according to claim 32, wherein said polypeptide has from 3 to 30 amino acid residues.

36. Compound according to claim 35, wherein said polypeptide has from 3 to 10 amino acid residues.

37. Compound according to claim 36, wherein said polypeptide has 8 amino acid residues.

38. Compound according to any one of claims 35-37, R comprises a polypeptide which is selected from peptide targeting agents based on somatostatin, bombesin, CCK, neurotensin, NPY, GLP1 and NK1.

39. Compound according to any one of claims 23-38, wherein R comprises a binding polypeptide selected from the group consisting of an antibody, an antibody fragment and an antibody derivative.

40. Compound according to any one of claims 23-38, wherein R comprises a binding polypeptide derived from a non-antibody scaffold.

41. Compound according to claim 40, wherein said scaffold is selected from the group consisting of three-helix domains, lipocalins, ankyrin repeat domains, cellulose binding domains, Y crystallins, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain, zinc fingers, avimers, microproteins and conotoxins.

42. Compound according to claim 41 , wherein said scaffold is a three- helix polypeptide domain.

43. Compound according to claim 42, wherein said scaffold is based on any three-helix domain of Protein G from Streptococcus spp.

44. Compound according to claim 43, wherein said scaffold is the albumin binding domain of Protein G from Streptococcus pyogenes strain G418.

45. Compound according to claim 42, wherein said scaffold is based on any domain of Protein A from Staphylococcus aureus.

46. Compound according to claim 45, wherein said scaffold is the protein Z variant of the B domain of Protein A from Staphylococcus aureus.

47. Compound according to any one of claims 1 -32, wherein R comprises a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:1-12 in Table 1.

48. Compound according to any one of claims 1 -32, wherein R comprises a polynucleotide molecule selected from DNA, RNA, PNA and mixtures thereof.

49. Compound according to claim 48, wherein said polynucleotide molecule comprises a DNA molecule.

50. Compound according to claim 48, wherein said polynucleotide molecule comprises an RNA molecule.

51. Compound according to claim 48, wherein said polynucleotide molecule comprises a PNA molecule.

52. Compound according to any one of claims 48-51 , wherein said polynucleotide molecule comprises an aptamer.

53. Compound according to any one of claims 48-51 , wherein said polynucleotide molecule comprises an antisense polynucleotide.

54. Chelate of a compound according to any one of claims 1 -53 with a radioactive moiety selected from M, M=O and O=M=O, wherein M is a radioactive metal atom.

55. Chelate according to claim 54, wherein M is suitable for in vivo imaging applications.

56. Chelate according to claim 55, wherein M is selected from the group consisting of ^99mTc, ⁹⁴Tc and ⁹⁶Tc.

57. Chelate according to claim 56, wherein M is ^99mTc.

58. Method of preparing a chelate according to any one of claims 54- 57, comprising mixing, in the presence of a reducing agent, of a compound according to any one of claims 1 -53 with a radioactive moiety MO₄ ^", wherein M is a radioactive metal atom.

59. Method of preparing a chelate according to any one of claims 55- 56, comprising mixing, in the presence of a reducing agent, of a compound according to any one of claims 1 -53 with a compound [""¹Tc]TcO₄ ^", [⁹⁴Tc]TcO₄ ^" and [⁹⁶Tc]TcO₄ ^".

60. Method of preparing a chelate according to claim 57, comprising mixing, in the presence of a reducing agent, of a compound according to any one of claims 1 -53 with [^99mTc]TcO₄\

61. Method according to any one of claims 58-60, in which said reducing agent is selected from the group consisting of SnCb and SnF₂.

62. Method according to any one of claims 58-61 , in which said mixing is carried out in the presence of a weak chelator, which acts as an intermediate in transfer of M to the compound according to any one of claims 1 -53.

63. Method according to claim 62, in which said weak chelator is selected from the group consisting of tartrate, glucoheptonate and citrate.

64. Method of in vivo imaging of the body of a mammalian, including human, subject, comprising the steps: - administering a chelate according to any one of claims 55-57 into the body of a mammalian subject; and - obtaining an image of at least a part of the subject's body using a medical imaging instrument, said image indicating the presence of M inside said body.

65. Method according to claim 64, in which the step of obtaining an image is repeated at least three times, whereby a series of images is obtained.

66. Method according to any one of claims 64-65, comprising, before the administration step, a step of preparing a chelate according to any one of claims 55-56, comprising mixing, in the presence of a reducing agent, of a compound according to any one of claims 1 -53 with a compound selected from [^99mTc]TcO₄ ^", [⁹⁴Tc]TcO₄ ^" and [⁹⁶Tc]TcO₄ ^".

67. Method according to any one of claims 64-65, comprising, before the administration step, a step of preparing a chelate according to claim 57, comprising mixing, in the presence of a reducing agent, of a compound according to any one of claims 1 -53 with [""¹Tc]TcO₄ ^".

68. Chelate according to any one of claims 55-57 for use in diagnostics.

69. Use of a chelate according to any one of claims 55-57 in the preparation of a diagnostic agent for imaging in vivo of the body of a mammalian, including human, subject.