Novel HER2-binding polypeptide

Abstract

Provided is a HER2 binding polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Also provided is a radiolabeled polypeptide comprising a radioactive chelate of a HER2 binding polypeptide and a radionuclide. Also provided are methods and uses of the polypeptide in therapeutic, diagnostic and prognostic applications, for example relating to cancers characterized by overexpression of HER2.

Description

[Technical Field]

[0001] This disclosure relates to a novel polypeptide that binds to human epidermal growth factor receptor 2 (HER2). This disclosure also relates to the use of such HER2-binding polypeptides as diagnostic agents, prognostic agents and / or pharmaceuticals, and more particularly to the use of such polypeptides in the diagnosis, prognosis and / or treatment of cancer forms characterized by HER2 overexpression. [Background technology]

[0002] HER2 is a transmembrane receptor belonging to the receptor tyrosine kinase superfamily. HER2 is overexpressed in a significant proportion of breast, gastroesophageal, and ovarian cancers, and elevated HER2 expression is associated with poor prognosis. Since tumors with high HER2 expression respond to specific targeted therapies, information on a patient's HER2 expression level is crucial for disease management. Currently, the only method used to determine HER2 expression status is analysis of biopsy material; however, this is invasive and complex, requiring multiple sample collections, and makes it difficult to elucidate heterogeneous HER2 expression or changes in HER2 expression in response to treatment.

[0003] On the other hand, radionuclide molecular imaging is expected to non-invasively detect HER2 in both primary tumors and metastases without producing false-negative results due to sampling errors. The first clinical trial demonstrating this approach was conducted using trastuzumab, a monoclonal antibody labeled with the single-photon emitter indium-111. Further advances in the use of antibody-based probes have been achieved with the use of zirconium-89, a long-lived positron emitter, and positron emission tomography (PET), resulting in improved sensitivity and resolution. Nevertheless, imaging probes based on therapeutic antibodies have a slow elimination rate from the blood. As a result, image contrast is low and the dose burden is high (even 4-5 days after injection). By using smaller imaging probes, such as those based on design scaffold proteins, it is possible to shorten the optimal imaging time to a few hours after injection, increasing image contrast and consequently improving sensitivity.

[0004] For example, one useful scaffold protein for generating HER2-binding molecules for radionuclide imaging is the protein Z derivative of domain B of Staphylococcus protein A. The Z variant is based on a 58-amino acid cysteine-free scaffold and may be chemically synthesized or produced in bacteria by recombinant DNA technology. The Z variant is further characterized by its small size, high thermal stability, site-specific radiolabeling capability, and potentially very high target affinity. Different generations of HER2-binding Z variants are disclosed in International Publication Nos. 2005 / 003156, 2009 / 080810, and 2015 / 028550.

[0005] The first-generation HER2-binding Z variant (see International Publication No. 2005 / 003156) demonstrated excellent imaging capabilities for HER2-expressing tumors in preclinical studies (Orlova et al (2006), Cancer Res 66:4339-4348; Ahlagren et al (2009) J Nucl Med 50:781-789) and clinical studies (Baum et al (2010), J Nucl Med 51(6):892-897). In the second generation, the Z variant scaffold was extensively redesigned, with 11 of the 45 amino acids in the unbinding region substituted (International Publication No. 2009 / 080810, Feldwisch et al (2010) J Mol Biol 398:232-247). These substitutions improved the peptide synthesis yield and storage stability of the Z variant molecule. Residual binding to IgG and IgM was essentially eliminated to facilitate blood clearance, and the substitution of alanine with serine increased the hydrophilicity of the surface exposed to water.

[0006] This second-generation HER2-bound Z variant is DOTA conjugate 68 It is used in PET imaging in the form of a Ga-labeled radiotrace molecule. This tracer has shown excellent sensitivity and specificity in detecting HER2-expressing metastases in breast cancer patients (Sorensen et al (2016), Theranostics 6(2):262-271). In typical clinical trials using this tracer, the effective dose is in the range of 5–6 mSv, which is considerably lower than the typical dose for PET imaging with antibody reagents (14–22 mSv). PET imaging is relatively readily available in North America and Western Europe, but less so in South America, Asia, Africa, and some European countries. SPECT is more common in these regions. 111In has been used as a label and the same imaging molecule has also been tested for SPECT (Sorensen et al (2014), J Nucl Med 55(5):730 - 735). However, this radionuclide is expensive and requires a medium - energy collimator, resulting in reduced resolution and sensitivity.

[0007] From a clinical perspective, 99m the use of 99mTc (T1 / 2 = 6 hours) is much more attractive. Since this radionuclide is produced by a generator, it is inexpensive and easily accessible. 99m The decay scheme of 99mTc is almost ideal for radiolabeling of pharmaceuticals because of its optimal half - life, gentle dose, ability to use a low - energy high - resolution collimator, and improved imaging sensitivity. 99m The labeling of the first - generation Z - variant with 99mTc has been well - investigated, especially using peptide - based chelators. Depending on the composition of such chelators and the position of the chelator in the Z - variant molecule, 99m it has been shown that the biodistribution and excretion pathway of the 99mTc - labeled molecule can be significantly altered (Hofstrom et al (2013), J Med Chem 56:4966 - 4974). Furthermore, by placing a cysteine residue at the C - terminus of the Z - variant sequence (thus generating the C - terminal sequence - KVDC, SEQ ID NO: 4), 99m it has been shown that an N3S chelator useful for stable coupling of 99mTc is formed (Ahlgren et al (2009), J Nucl Med 50:781 - 789). The 99mTc - labeled HER2 - binding molecule Z 99m showed improved target specificity and high contrast in imaging HER2 expression in a mouse model (tumor - to - blood ratio 121 ± 24, 4 hours after injection). However, Z HER2:2395 had high renal uptake (140 - 190%ID / g, 4 hours after injection), which is not desirable for imaging. HER2:2395

[0008] 188Re is also being tested in relation to this. 188 Re is a high-energy, beta-emitting radioactive isotope with a short half-life (16.98 hours), making it suitable for the production of radiopharmaceuticals for the diagnosis and treatment of malignant tumors. Its short half-life makes it safer for patients, staff, and the environment. 188 Radiation from Re contains both beta and gamma rays. High beta emission (784 keV) ensures high therapeutic efficacy, while low gamma emission (155 keV) allows operators to obtain visual information using conventional gamma cameras. Furthermore, rhenium generators can be easily provided to clinical settings.

[0009] It has been found that both the amino acid composition and the order of amino acid residues in peptide-based chelators affect their in vivo distribution and targeting characteristics (Wallberg et al (2011), J Nucl Med 52:461-469; Altai et al (2012), Amino Acids 5:1975-1985). First-generation HER2-binding Z variant containing a C-terminal GGGC chelate sequence (SEQ ID NO: 5). 99m Tc-Z HER2:V2 This showed the best image contrast. 99m The Tc](Tc=O)-GGGC complex has been shown to act as a non-residual label. The radioactivity (activity) that was rapidly reabsorbed and internalized in the kidney was rapidly excreted. In tumors where the internalization of the Z variant molecule was slow, the radioactivity was retained for a long time due to the high affinity of the Z variant for HER2 on the cell membrane (Wallberg et al (2011), supra). This resulted in good image construction, 99m Tc-Z HER2:V2 This combination was achieved with low renal uptake.

[0010] Based on these results, the -GGGC chelator is considered a candidate label for second-generation HER2-binding Z variants. However, rearranging amino acids in the scaffold sequence carries the risk of generating additional chelating pockets due to clustering of amino acids with electron-donating side chains. It has been suggested that such side chains may be involved in technetium complexation (Cyr et al (2007), J Labelled Comp Radiopharm 50(suppl 1):S9). Complexation leads to loss of label site specificity and alters the stability and pharmacokinetics of the conjugate. For example, Z HER2:V2 Introducing the HEHEHE tag (SEQ ID NO: 6) to the N-terminus of the construct significantly altered its in vivo distribution, particularly increasing renal uptake (Lindberg et al (2012), Tumour Biol 33(3):641-651). Furthermore, 99m Tc-Z HER2:V2 Even a slight modification, such as introducing an N-terminal purification tag, has been shown to have adverse effects on labeling in this case, in a medical setting (Cai et al (2020), Iran J Radiol e96419, published online on 20 January 2020). Furthermore, another Z variant, the EGFR-binding molecule Z EGFR:2377 The binding site composition contains a chelate pocket that competes with the -GGGC chelator, rendering its use impossible (Oroujeni et al (2018), Amino Acids 50:981-994).

[0011] In conclusion, the redesigned HER2-specific Z variant uses the -GGGC chelate sequence as its scaffold. 99m It can hardly be said to be suitable for stable marking by Tc. Disclosure of the present invention

[0012] The purpose of this disclosure is to satisfy the requirement of providing drugs with affinity for HER2 by providing polypeptides characterized by specific binding to HER2.

[0013] A related objective of this disclosure is a HER2-binding polypeptide that exhibits little to no nonspecific binding.

[0014] Another object of this disclosure is to provide a HER2-binding polypeptide that can be readily used as a portion in a fusion polypeptide.

[0015] Another objective is to provide a HER2-binding polypeptide that solves one or more of the known problems experienced with existing antibody reagents.

[0016] A further objective of this disclosure is to provide HER2-binding polypeptides that can be used for therapeutic purposes.

[0017] A further object of this disclosure is to provide HER2-binding polypeptides that can be used for diagnostic purposes.

[0018] A further object of this disclosure is to provide HER2-binding polypeptides that can be used for prognostic applications.

[0019] A related objective is to identify new forms of treatment, inhibition, and / or targeting in clinical settings for cancers characterized by HER2 protein overexpression.

[0020] Another object of this disclosure is to provide HER2-binding polypeptides that can be produced using recombinant protein expression or synthesized by chemical peptide synthesis.

[0021] A related objective is to provide HER2-binding polypeptides that can be labeled with the resulting radionuclide without adding any additional steps to conjugate the polypeptide with a chelate group.

[0022] Another objective is to find HER2-binding polypeptides that exhibit improved stability compared to known HER2-binding agents and, optionally, extend the shelf life during storage.

[0023] Another object of this disclosure is to obtain HER2-binding polypeptides that exhibit low antigenicity and / or immunogenicity when used in vivo in mammals.

[0024] Another object of this disclosure is to provide a HER2-binding polypeptide that exhibits beneficial in vivo distribution when administered to mammals.

[0025] These and other objectives are achieved by various aspects of the present disclosure. Accordingly, in the first aspect, the present disclosure relates to a HER2-binding polypeptide having an amino acid sequence, This provides a HER2-binding polypeptide containing AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LSESQGGGC (SEQ ID NO: 1).

[0026] Accordingly, the present inventors have identified a HER2-binding polypeptide containing SEQ ID NO: 1, for example, SEQ ID NO: 2, which is previously disclosed in Wallberg et al (2011, supra), and is referred to below as H2BPP2. HER2:V2 , and the Z of Ahlagren et al (2009, supra) having sequence number 3, which is shown below as H2BPP3. HER2:2395 We have found that it exhibits remarkable advantages compared to [the other]. Non-limiting examples of such advantages that may be individually demonstrated by one or more embodiments of the polypeptide according to the present aspects of this disclosure are as follows: The disclosed polypeptide contains fewer amino acid residues, such as asparagine and aspartic acid, which can cause problems such as low yields and success rates in the chemical synthesis of polypeptide sequences. The disclosed polypeptide has fewer amino acid residues that confer surface hydrophobicity and therefore has a more hydrophilic profile than the related HER2-binding polypeptides previously described. This means fewer problems with low solubility and aggregation. While we do not wish to be bound by theory, it is now thought that the more hydrophilic properties may also have the effect of shifting the in vivo distribution of the polypeptide, upon administration to the host, from the hepatobiliary pathway (excretion via the liver) to the more desirable renal pathway (excretion via the kidneys). The disclosed polypeptide contains few amino acid residues associated with polypeptide stability issues, such as asparagine, aspartic acid, and the dipeptide asparagine-proline. Asparagine is easily deamidated, aspartic acid is easily isomerized, and the asparagine-proline bond is easily cleaved, all of which contribute to heterogeneity of the final product. The disclosed polypeptide lacks the amino acid residues (Silverman GJ, Int. Rev. Immunol. 1992; 9(1): 57-78) that have been found to increase interaction with immunoglobulins containing VH3-derived heavy chain variable domains in similar sequence configurations. While we do not wish to be bound by theory, it is currently believed that such amino acid residue substitutions in the polypeptide according to the present invention would reduce the antigenicity of the polypeptide upon administration to the host. The disclosed polypeptides contain fewer potential T cell epitopes than previously known HER2-binding Z variants, as shown in Example 4 below, and are therefore considered to have lower immunogenicity.

[0027] Polypeptides according to this embodiment of the Disclosure can be used as substitutes for HER2-binding antibodies and known HER2-binding Z variants in a variety of applications. In non-limiting examples, the polypeptides are useful in the treatment of cancers characterized by HER2 overexpression, in inhibiting cell signaling by binding to HER2 on the cell surface, in both in vivo and in vitro diagnosis and / or prognosis of cancers characterized by HER2 overexpression, in targeting other therapeutic, diagnostic, or prognostic agents to cells overexpressing HER2, in histochemical methods for detecting HER2, in separation methods, and in other applications. Polypeptides according to this embodiment can be demonstrated to be useful in any method that depends on the affinity of the reagent for HER2. Therefore, the polypeptides can be used as detection reagents, capture reagents, or separation reagents in such methods, as diagnostic and / or prognostic agents for in vivo or in vitro diagnosis and / or prognosis, as therapeutic agents themselves, or as a means for targeting other therapeutic or diagnostic agents to the HER2 protein. The methods for using polypeptides in vitro according to the embodiments of this disclosure can be carried out in various forms, such as on microtiter plates, protein arrays, biosensor surfaces, and tissue sections.

[0028] Without departing from the scope of this instruction, various modifications and / or additions to the polypeptide according to the embodiments of this disclosure can be made to adapt the polypeptide to a particular intended use. Such modifications and additions are described in more detail below and may include further amino acids contained in the same polypeptide chain, or labels and / or therapeutic agents that are chemically conjugated or otherwise attached to the polypeptide according to the embodiments of this disclosure.

[0029] As used herein, the terms “HER2 binding” and “binding affinity to HER2” refer to the properties of a polypeptide that can be tested, for example, by the use of ELISA or surface plasmon resonance (SPR) technology.

[0030] For example, as described in the following examples, HER2 binding affinity can be tested by immobilizing HER2 or its fragments on a sensor tip of a surface plasmon resonance (SPR) apparatus and passing a sample containing the polypeptide to be tested through the tip. Alternatively, the polypeptide to be tested is immobilized on a sensor tip of the apparatus and passed through a sample containing HER2 or its fragments through the tip. A person skilled in the art can then interpret the results obtained by such an experiment to establish at least a qualitative measurement of the binding affinity of the polypeptide to HER2. For example, the K of the interaction D Surface plasmon resonance (SPL) can also be used when quantitative measurement is desired to determine the value. The binding value can be defined, for example, using a Biacore T200 instrument (Cytiva) or a ProteOn XPR 36 (Bio-Rad) instrument. HER2 is preferably immobilized on the instrument's sensor chip, and polypeptide samples for which affinity is to be sought are prepared by serial dilution and injected in a random order. The results are then analyzed using, for example, the 1:1 Langmuir binding model in the BIAevaluation 4.1 software, or other suitable software provided by the instrument manufacturer. D The value can be calculated.

[0031] These aspects of the disclosure also include polypeptides in which the above-mentioned HER2-binding polypeptide exists as a HER2-binding domain with additional amino acid residues added to either end, particularly the N-terminus. These additional amino acid residues may play a role in the binding of the polypeptide to HER2, but may also serve other purposes related to one or more of the following: polypeptide production, purification, stabilization, coupling, or detection. Such additional amino acid residues may include one or more amino acid residues added for the purpose of chemical coupling. Such additional amino acid residues may also include “tags” for the purification or detection of the polypeptide, such as a hexahistidyl (H6) tag, or a “myc” tag or a “FLAG” tag. Such tags may enable, for example, interaction with a tag-specific antibody, or possibly immobilized metal affinity chromatography (IMAC). Those skilled in the art are aware of other alternatives.

[0032] The "further amino acid residues" described above may also constitute one or more polypeptide domains having any desired function, such as the same binding function as the first HER2-binding domain, or a different binding function, or an enzymatic function, or a fluorescent function, or a combination thereof.

[0033] Accordingly, this disclosure encompasses a polypeptide polymer comprising SEQ ID NO: 1. For example, when using the polypeptide according to the present invention in the diagnosis or treatment of cancer, or in a method for purifying HER2, it may be important to obtain a stronger HER2 binding than is possible with a single polypeptide according to the present invention. In this case, the provision of a polypeptide polymer, e.g., a dimer, trimer, or tetramer, can provide the necessary avidity effect. The polymer may consist of a suitable number of polypeptides according to the present invention. The linked polypeptide "units" in the polymer according to the present invention may be covalently linked using known organic chemical methods, or expressed as one or more fusion polypeptides in a system for recombinant polypeptide expression, and may be linked directly or via a linker, e.g., an amino acid linker, or in any other manner.

[0034] Furthermore, "heterogeneous" fusion polypeptides in which the polypeptide containing SEQ ID NO: 1 constitutes a first domain or first part, and the second and further parts have functions other than binding to HER2, are also envisioned and within the scope of this disclosure. The second and further parts (or more) of such fusion polypeptides may include binding domains having affinity for targets other than HER2. Non-limiting examples of targets for such second and further parts (or more) may be selected from the group consisting of CD3, CD137(4-1BB), CTLA-4, EGFR, HER3, VEGF, PD1, PD-L1, and cMet. As a result, a fusion polypeptide having at least one HER2-binding domain and at least one domain having affinity for the other target molecule is obtained. This makes it possible to create multispecific reagents that can be used in several applications, such as therapeutic and / or diagnostic and / or prognostic agents, or reagents for capture, detection, or separation. The preparation of such multispecific polymers of polypeptides in which at least one polypeptide domain contains SEQ ID NO: 1 can be carried out as described above for several HER2-binding "unit" polymers.

[0035] A second or further part(s) may include a variant of the domain derived from Staphylococcus aureus protein A, for example, a Z variant, or an unrelated native or recombinant protein (or a fragment thereof that retains the binding ability of a native or recombinant protein) having binding affinity to the target.

[0036] Another example of such a binding protein that has affinity for human serum albumin and can be used as a fusion partner with the polypeptide according to the present invention is one of the albumin-binding domains of protein G of Streptococcus strain G148 (Nygren P-Å et al (1988) Mol Recogn 1:69-74), e.g., the GA1, GA2, or GA3 domain. The GA3 domain of protein G is also called ABD, i.e., the albumin-binding domain. Derivatives of ABD having improved properties compared to the wild-type GA3 domain of protein G are disclosed, for example, in International Publication Nos. 2009 / 016043, 2012 / 004384, and 2014 / 048977. Accordingly, a fusion polypeptide of the HER2-binding polypeptide containing SEQ ID NO. 1 and the albumin-binding domain of Streptococcus protein G is also within the scope of this disclosure. When the disclosed polypeptide is administered to human subjects as a diagnostic, prognostic, therapeutic, or targeted agent, the fusion of the polypeptide with a serum albumin-binding portion can be demonstrated to be beneficial in that the in vivo half-life of such fusion protein is likely to be longer than that of the HER2-binding portion alone (this principle is described, for example, in International Publication No. 91 / 01743).

[0037] Other possibilities for the preparation of fusion polypeptides are also considered. Accordingly, polypeptides according to the first aspect of this disclosure may be covalently bonded with a second or further part(s) that exhibit other functions in addition to, or instead of, target binding. One example is the fusion of one or more polypeptides comprising SEQ ID NO: 1 with an enzymatically active polypeptide that functions as a reporter or effector part. Examples of reporter enzymes that can be bound to the polypeptide comprising SEQ ID NO: 1 to form a fusion protein are known to those skilled in the art and include enzymes such as β-galactosidase, alkaline phosphatase, horseradish peroxidase, and carboxypeptidase. Other options for the second and further parts(s) of the fusion polypeptide according to the present invention include fluorescent polypeptides, such as green fluorescent protein, red fluorescent protein, luciferase, and variants thereof.

[0038] Other options for second and further portions(s) of the fusion polypeptide according to the present invention include portions(s) for therapeutic uses. In therapeutic uses, other molecules may also be covalently or noncovalently bonded to the polypeptide of the present invention by other means. Non-limiting examples include enzymes (e.g., carboxypeptidases) for antibody-directed enzyme prodrug therapy (ADEPT) applications using the polypeptide according to the present invention for effector enzyme targeting; proteins for mobilizing effector cells and other components of the immune system; cytokines, e.g., IL-2, IL-12, IFNγ, TNF, IP-10, GM-CSF; procoagulation factors, e.g., tissue factor, von Willebrand factor; toxins, e.g., lysine A, pseudomonas exotoxin, calicheamicin, maytansinoids; and toxic small molecules, e.g., auristatin analogs, doxorubicin.

[0039] With regard to the above description of the fusion protein incorporating the HER2-binding polypeptide according to the present invention, it should be noted that the designations "first," "second," and "further parts" are made for clarity to distinguish one HER2-binding part from the other functional part. These designations are not intended to refer to the actual order of different domains in the polypeptide chain of the fusion protein. Therefore, for example, the first part may appear at the N-terminus, middle, or C-terminus of the fusion protein without limitation.

[0040] In one embodiment of the first aspect of this disclosure, the HER2-binding polypeptide is SEQ ID NO: 1.

[0041] The disclosure further includes polypeptides in which the above-mentioned HER2-binding polypeptide comprises at least one labeling group, such as a fluorophore, biotin, or radioisotope, for the purpose of polypeptide detection.

[0042] In particular, a second aspect of this disclosure relates to a radiolabeled polypeptide comprising a radiochelate of a HER2-binding polypeptide and a radionuclide according to the first aspect.

[0043] Most radionuclides have metallic properties. Since metals typically cannot form stable covalent bonds with elements contained in proteins and peptides, labeling target proteins with radioactive metals is often performed using chelators, i.e., polydentate ligands that form non-covalent compounds called chelates with the metal. In the polypeptide according to the present invention, the incorporation of radionuclides is made possible by providing a chelating environment through which the radionuclides can coordinate, chelate, or complex with the polypeptide.

[0044] A polypeptide according to the first embodiment comprises a tetradentate chelate environment characterized as an N3S chelator. As the term N3S indicates, the four binding groups of such a chelator are formed from three nitrogen atoms and one sulfur atom. In an N3S chelator, the N and S atoms are spatially arranged to provide a suitable “pocket” for complexing or binding of a radioactive metal. In the polypeptide according to this disclosure, the N3S chelator is obtained by selecting -GGGC as the C-terminal amino acid residue of SEQ ID NO: 1.

[0045] Therefore, a polypeptide according to the first embodiment, comprising a tetradentate chelate environment characterized as an N3S chelator, provided by the nitrogen atoms of three consecutive peptide bonds and the cysteine ​​residue at the C-terminus of the polypeptide, can be used to provide a radiolabeled polypeptide according to the second embodiment, comprising a radiochelate of a HER2-binding polypeptide with a radionuclide.

[0046] In one embodiment, the radionuclide is suitable for medical imaging. In a more detailed embodiment, the radionuclide is 99m Tc, 51 Mn, 52m Mn, 52 Mn, 186 Re and 188 Selected from the group consisting of Re. In a particular embodiment, a radionuclide suitable for medical imaging is 99m It is Tc.

[0047] In another embodiment, the radionuclide is suitable for treatment. In a more detailed embodiment, the radionuclide is 186 Re and 188 Selected from the group consisting of Re. In a particular embodiment, the radionuclide suitable for treatment is 188 It is Re.

[0048] In these embodiments of the radiolabeled polypeptide according to the second aspect, the radionuclide is complexed with the HER2-binding polypeptide via a chelation environment provided by the C-terminal GGGC sequence.

[0049] This disclosure also includes compositions comprising a polypeptide according to the first embodiment or a radiolabeled polypeptide according to the second embodiment.

[0050] A third aspect of the present disclosure provides a composition comprising the HER2-binding polypeptide of the first aspect and at least one pharmaceutically acceptable excipient or carrier. The composition may preferably be formulated for purposes selected from storage, transport, lyophilization, concentration, or any combination thereof. In certain embodiments, the composition according to this aspect is suitable for use as a pharmaceutical, in vivo diagnostic, or in vivo prognostic agent without further processing. In another embodiment, the composition is suitable for lyophilization. Lyophilization of the composition according to this embodiment yields a lyophilized powder of the polypeptide. The lyophilized powder of the polypeptide constitutes a further fourth aspect of the present disclosure, which may offer advantages in terms of transport, storage, handling, and radiolabeling of the polypeptide when the polypeptide is intended to be used as a pharmaceutical or in vivo diagnostic or prognostic agent.

[0051] A fifth aspect of this disclosure provides a composition comprising a radiolabeled polypeptide of the third aspect and at least one pharmaceutically acceptable excipient or carrier. Preferably, the at least one pharmaceutically acceptable excipient or carrier is selected to enable or enhance the administration of the radiolabeled polypeptide to a subject to be treated or imaged.

[0052] A person skilled in the art of pharmaceutical compositions and formulations can select an appropriate excipient or carrier for a given use in a HER2-binding polypeptide or a radiolabeled polypeptide and prepare a composition according to the third or fifth embodiment without excessive experimentation.

[0053] This disclosure also encompasses various embodiments of using the above-described HER2-binding polypeptides and compositions, whether radiolabeled or not. This disclosure also encompasses various methods for therapeutic, diagnostic and prognostic purposes in which the polypeptide is useful due to its binding properties. When the term “HER2-binding polypeptide” is referred to in the following description of these uses and methods, this term is intended to encompass not only the HER2-binding polypeptide alone, but also all molecules based on the above polypeptide that incorporate the HER2-binding polypeptide, for example, as a portion in a fusion protein, and / or conjugate it to a label, chelator, therapeutic and / or diagnostic agent, and / or have further amino acid residues as a tag or for other purposes. As described above, such fusion proteins, derivatives, etc., form part of this disclosure. “HER2-binding polypeptide” is also intended to encompass compositions and powders of polypeptides or radiolabeled polypeptides according to the third, fourth and fifth embodiments described above.

[0054] Accordingly, in a sixth aspect, the present invention relates to an in vivo imaging method in the body of a mammalian subject, including a human, having or suspected of having cancer characterized by HER2 overexpression, wherein the method is -Administering a radiolabeled polypeptide according to a second embodiment, containing a radionuclide suitable for medical imaging, into the body of a mammalian subject, and The present invention provides a method comprising the step of obtaining an image of at least a portion of a body of a subject using a medical imaging device, wherein the image indicates the presence of a radionuclide in the body. In one embodiment, the image is obtained within 1 to 72 hours, for example, 1 to 24 hours, after administration of a radiolabeled polypeptide into the body. The time from administration to image acquisition depends on the half-life of the radionuclide used. The image acquisition step can be repeated two, three or more times, thereby obtaining a series of images. This is useful when attempting to track the in vivo distribution of a targeted drug over time, enabling pharmacokinetic studies. Those skilled in the art will understand that any number of images can be obtained to achieve the required degree of temporal resolution in such studies.

[0055] In one embodiment of the imaging method according to this aspect, the administration is intravenous.

[0056] In one embodiment of the imaging method according to this embodiment, the method includes a preparation step of preparing a radiolabeled polypeptide according to the second embodiment before the administration step, the preparation step of mixing the polypeptide according to the first embodiment with a radionuclide suitable for medical imaging.

[0057] In a more detailed embodiment of the imaging method according to this embodiment, the method includes, before the administration step, i) a polypeptide whose amino acid sequence contains or consists of SEQ ID NO: 1, and ii) 99m The preparation step includes preparing a radiolabeled polypeptide with Tc, wherein the polypeptide is dissolved in a suitable buffer in the presence of a suitable reducing agent, such as stannous chloride, ascorbic acid, or gentisic acid. 99m The preparation step may also be carried out in the presence of a weak chelator of the intermediate (e.g., tartrate, citrate, or gluconate).

[0058] If the polypeptide is present in the form of a lyophilized powder according to the fourth embodiment, the preparation step preferably also includes a first step of reconstituting the powder in a buffer suitable for radiolabeling and administration.

[0059] In a related seventh aspect, the present disclosure relates to a method for diagnosing cancer characterized by HER2 overexpression, - To perform an in vivo imaging method according to the sixth aspect, - A method is provided which includes establishing the diagnosis based on the obtained images.

[0060] In another related eighth aspect, the present disclosure relates to a method for establishing the prognosis of cancer characterized by HER2 overexpression, - To perform an in vivo imaging method according to the sixth aspect, - A method is provided which includes establishing the prognosis based on the obtained images.

[0061] Furthermore, in a ninth aspect, the present disclosure provides a method for treating a mammalian subject, including a human, having cancer characterized by HER2 overexpression, the method comprising administering to the subject a therapeutically effective amount of a radiolabeled polypeptide according to a second aspect, comprising a radionuclide suitable for treatment.

[0062] In one embodiment of the treatment method according to this aspect, the administration is intravenous.

[0063] In one embodiment of the therapeutic method according to this embodiment, the method includes a preparation step of preparing a radiolabeled polypeptide according to the second embodiment before the administration step, the preparation step of mixing the polypeptide according to the first embodiment with a radionuclide suitable for therapeutic use.

[0064] In a more detailed embodiment of the treatment method according to this embodiment, the preparation step is: - Mix the polypeptide according to the first embodiment with a perrhenium salt in a suitable buffer in the presence of a suitable reducing agent (this mixing may be carried out in the presence of a weak intermediate chelator, such as tartrate, citrate, or gluconate), or -The polypeptide according to the first embodiment is mixed with a rhenium-reactive intermediate, such as a tricarbonyl complex.

[0065] In a more detailed embodiment of the treatment method according to this embodiment, the preparation step of this method is performed before the administration step by: i) a polypeptide whose amino acid sequence includes or consists of SEQ ID NO: 1, and ii) 188 The preparation step includes preparing a radiolabeled polypeptide with Re, wherein the polypeptide is converted to perrhenium acid in a suitable buffer in the presence of a suitable reducing agent, such as stannous chloride, ascorbic acid, or gentisic acid. 188 This includes mixing with Re. The preparation step may also be carried out in the presence of a weak chelator of the intermediate (e.g., tartrate, citrate, or gluconate).

[0066] If the polypeptide is present in the form of a lyophilized powder according to the fourth embodiment, the preparation step preferably also includes a first step of dissolving the powder in a buffer suitable for radiolabeling and administration.

[0067] In some embodiments of these diagnostic, prognostic, and therapeutic embodiments of the present invention, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, colorectal cancer, prostate cancer, bladder cancer, salivary gland cancer, lung cancer (particularly non-small cell lung cancer), and esophageal cancer (particularly gastric and gastroesophageal junction cancer). In a particular embodiment, the cancer is breast cancer.

[0068] This disclosure, while originating from these cancer types, also encompasses binding and targeting of HER2-positive metastatic lesions localized in other parts of the body. In particular, HER2-positive metastatic lesions in the brain are considered.

[0069] The disclosure further provides a polypeptide according to the first embodiment, a radiolabeled polypeptide according to the second embodiment, a composition according to the third embodiment, a lyophilized powder according to the fourth embodiment, or a composition according to the fifth embodiment for use as an in vivo diagnostic agent, an in vivo prognostic agent, or a pharmaceutical agent in any of the methods according to the sixth, seventh, eighth, and ninth embodiments.

[0070] Further process-related aspects of this disclosure are based on the remarkable discovery that it is possible to radiolabel the polypeptides disclosed herein at temperatures lower than those previously considered practical or efficient. In connection with this, it has been observed that radiolabeling at such low temperatures results in less generation of undesirable byproducts of the reaction. This disclosure encompasses a method for radiolabeling a polypeptide according to a first aspect, comprising the step of radiolabeling at a temperature of 60–85°C, e.g., 65–80°C, e.g., 65–75°C, and particularly about 70°C, e.g., 70°C. Those skilled in the art will understand that the radiolabeling method according to this aspect may be incorporated into the preparation of radiolabels included in some embodiments of the imaging and therapeutic methods described above.

[0071] Another aspect of this disclosure relates to a nucleic acid molecule comprising a sequence encoding a polypeptide according to the first aspect.

[0072] Further aspects of this disclosure relate to nucleic acid molecules of the above aspects, and expression vectors comprising other nucleic acid elements that enable the production of polypeptides by expression of nucleic acid molecules.

[0073] Another aspect of this disclosure relates to a host cell comprising an expression vector of the said aspect.

[0074] The latter three aspects of the present invention are tools for producing polypeptides according to the present invention, and those skilled in the art will be able to obtain and put into practical use such tools without undue burden, given the information herein relating to the polypeptide to be expressed and the current level of the art in the field of recombinant protein expression.

[0075] The polypeptides relating to this disclosure may also be produced by other known means, including chemosynthesis or expression in various prokaryotic or eukaryotic hosts, including plants and transgenic animals.

[0076] Herein, various aspects of this disclosure will be described in detail through a description of experiments conducted in accordance with each aspect. The following examples are not to be constrained. Please refer to the attached figures in the examples. [Brief explanation of the drawing]

[0077] [Figure 1] This is a list of amino acid sequences for a novel HER2-binding polypeptide containing a C-terminal GGGC chelator (H2BPP1, SEQ ID NO: 1), a HER2-binding reference polypeptide containing a C-terminal GGGC chelator (H2BPP2, SEQ ID NO: 2), and a HER2-binding reference polypeptide containing a C-terminal KVDC chelator (H2BPP3, SEQ ID NO: 3).

[0078] [Figure 2] This figure shows the circular dichroism spectra of (A) H2BPP1 and (B) H2BPP2 collected at 20°C before (solid line) and after (dashed line) variable temperature measurement (VTM).

[0079] [Figure 3A] This figure (black line) shows the RP-UPLC-MS total ion chromatogram (TIC) of H2BPP1 after heat treatment at 90°C for 60 minutes. The chromatogram of the untreated reference sample (gray line) is included for comparison. [Figure 3B] This is an enlarged chromatogram of Figure 3A. The chromatogram of the untreated reference sample (gray line) is included for comparison. [Figure 3C] This figure (black line) shows the RP-UPLC-MS total ion chromatogram (TIC) of H2BPP2 after heat treatment at 90°C for 60 minutes. The chromatogram of the untreated reference sample (gray line) is included for comparison. [Figure 3D] Figure 3C is an enlarged chromatogram. The chromatogram of the untreated reference sample (gray line) is included for comparison.

[0080] [Figure 4] This figure shows a single-cycle kinetics (SCK) SPR sensorgram (black line) indicating the binding of H2BPP1 to (A) human HER2 and (B) cynomolgus monkey HER2, which were continuously injected at concentrations of 0.3 and 3 nM, respectively, at the time points indicated by the gray vertical lines.

[0081] [Figure 5] This figure shows an SCK SPR sensorgram (black line) indicating no binding of H2BPP1 to human (A)HER1, (B)HER3, and (C)HER4, as continuously injected and analyzed at specified concentrations at the time points indicated by the gray vertical lines.

[0082] [Figure 2] This figure shows the SDS-PAGE analysis of the 99mTc-labeled polypeptides (1) 99mTc-H2BPP1, (2) 99mTc-H2BPP2, and (3) 99mTc-H2BPP3, respectively. Signal (4) corresponds to 99mTcO4-. The signal is measured in digital light units (DLU) and is proportional to the radioactivity at a given point in the lane in the SDS-PAGE gel.

[0083] [Figure 7] This figure shows the in vitro binding specificity of (A, D)99mTc-H2BPP1, (B, E)99mTc-H2BPP2, and (C, F)99mTc-H2BPP3 to HER2-expressing SKOV-3 (A, B, C) and BT-474 (D, E, F) cell lines. A 500-fold molar excess of non-radioactive labeled polypeptide was added to pre-saturate HER2. Data are shown as mean (n=3) ± SD.

[0084] [Figure 8]This figure shows the normalized cell retention rates of (A, D)99mTc-H2BPP1, (B, E)99mTc-H2BPP2, and (C, F)99mTc-H2BPP3 in HER2-expressing SKOV-3 (A, B, C) and BT-474 (D, E, F) cell lines. Data are shown as mean (n=3) ± SD. If no error bars are visible, the error bars are smaller than the dot symbols.

[0085] [Figure 9A] This figure shows the incorporation of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, into the blood of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9B] This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the lungs of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9C] This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the liver of female NMRI mice 4 hours after injection. [Figure 9D] This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the spleen of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9E] This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the stomach of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9F]This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the kidneys of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9G] This figure shows the uptake of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, in the muscle of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein. [Figure 9H] This figure shows the incorporation of the novel 99mTc-labeled polypeptide H2BPP1, as well as the reference polypeptides H2BPP2 and H2BPP3, into the bone of female NMRI mice 4 hours after injection. 1 μg (60 kBq, 100 μl in PBS) of the labeled polypeptide was injected into the tail vein.

[0086] [Figure 10] This figure shows the in vivo specificity of 99mTc-H2BPP1 in HER2-negative Ramos xenografts and HER2-positive SKOV-3 xenografts 4 hours after injection. Data are shown as mean (n=4) ± SD.

[0087] [Figure 11] This figure shows (A) the in vivo distribution and (B) the tumor-to-organ ratio of indicated 99mTc-H2BPP1 and 99mTc-H2BPP2 in BALB / C nu / nu mice carrying HER2-expressing SKOV-3 xenografts, 4 hours after injection. Data are expressed as mean (n=4) ± SD.

[0088] [Figure 12] This figure shows imaging of HER2-positive SKOV-3 xenografts (right mouse) and HER2-negative Ramos xenografts (left mouse) in BALB / C nu / nu mice using 99mTc-H2BPP1.

[0089] [Figure 13] This figure shows the chromatogram obtained by RP-HPLC analysis of 188Re-H2BPP1 after labeling at 70°C.

[0090] [Figure 14] This figure shows the in vitro binding specificity of 188Re-H2BPP1 to HER2-expressing (A)SKOV-3 and (B)SK-BR-3 cell lines. A 400-fold molar excess of non-radioactive labeled polypeptide was added to the control dish for HER2 pre-saturation ("blockage"). Data are shown as mean (n=3) ± SD.

[0091] [Figure 15A] This figure shows the in vivo distribution of imaging ligands in non-tumor-bearing mice. (A) Results of Na / I cotransporter saturation in the salivary glands of NMRI mice. Addition of NaI to the drinking water of one group of mice ("blocked") significantly reduced radioactivity uptake in the salivary glands (p<0.0013). Data are shown as mean (n=4) ± SD. An asterisk indicates a statistically significant difference (p<0.05 in an unpaired t-test). [Figure 15B] This figure shows the in vivo distribution of imaging ligands in non-tumor-bearing mice. (B) Comparison of the in vivo distribution of 188Re-H2BPP1 and 99mTc-H2BPP1 in NMRI mice 4 hours after injection. Data are shown as mean (n=4) ± SD. An asterisk indicates a statistically significant difference (p<0.05 in an unpaired t-test).

[0092] [Figure 16] This figure shows imaging of HER2-positive SKOV-3 xenografts in BALB / C nu / nu mice one and four hours after injection of 188Re-H2BPP1. The arrows point to tumors (T) and kidneys (K).

[0093] [Figure 17A](A) This figure shows a comparison of 188Re-H2BPP1 in BALB / C nu / nu mice with HER2-expressing SKOV-3 and HER2-negative Ramos xenografts (data are shown as mean (n=4) ± SD). [Figure 17B] (B) This figure shows imaging of HER2-positive SKOV-3 xenografts (left mouse) and HER2-negative Ramos xenografts (right mouse) in BALB / C nu / nu mice injected with 188Re-H2BPP1 (arrows point to tumors (T)).

[0094] [Figure 18A] (A) This figure shows the growth of individual tumors in mice treated with 188Re-H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg, 16 MBq, 3 times). [Figure 18B] (B) This figure shows the growth of individual tumors in mice treated with a vehicle (3 times with a 10% ethanol aqueous solution). [Figure 18C] (C) This figure shows the growth of individual tumors in mice treated with unlabeled H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg three times).

[0095] [Figure 19A] This figure shows the (A) survival rate of mice treated with 188Re-H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg, 16 MBq, 3 times), vehicle (3 times in 10% ethanol aqueous solution), or unlabeled H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg, 3 times). [Figure 19B] This figure shows (B) the average body weight of mice treated with 188Re-H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg, 16 MBq, 3 times), vehicle (3 times in 10% ethanol aqueous solution), or unlabeled H2BPP1 (dissolved in 10% ethanol aqueous solution, 5 μg, 3 times). [Example 1]

[0096] The following examples disclose the generation and characterization of H2BPP1 (SEQ ID NO: 1), a novel HER2-targeted polypeptide designed to have a cysteine-containing peptide-based chelator (GGGC, SEQ ID NO: 5) at its C-terminus. The in vitro and in vivo properties of H2BPP1 were compared with those of two previously tested HER2-binding polypeptides: H2BPP2 (SEQ ID NO: 2), which similarly contains a C-terminal GGGC chelator, and H2BPP3 (SEQ ID NO: 3), which contains a C-terminal KVDC chelator (SEQ ID NO: 4) (Wallberg et al (2011), J Nucl Med 52:461-469; Ahlagren et al (2010), J Nucl Med 50:781-789). The amino acid residues that interact with HER2 are identical in all three polypeptides, but the novel polypeptide H2BPP1 differs from H2BPP2 and H2BPP3 in terms of its scaffold residues. Here, the latter two are identical except for the C-terminal chelate residue (Figure 1). Because changes in the scaffold can lead to undesirable changes in properties, such as loss of site specificity of radiolabeling and changes in in vivo distribution and imaging properties, new preclinical evaluations are necessary for the clinical application of novel radiotracers. For this purpose, radiolabeling was performed using Tc-99m. The tests described in the following examples surprisingly showed that, despite some amino acid changes in the H2BPP1 scaffold compared to H2BPP2 and H2BPP3, a beneficial reduction in labeling efficiency and renal uptake was retained. In conclusion, favorable in vitro and in vivo properties, such as HER2 binding, immunogenicity profile, in vivo distribution, and tumor targeting, which make this novel polypeptide suitable for use in diagnostic and therapeutic applications, were demonstrated for H2BPP1.

[0097] Example 1 Preparation and purity analysis of HER2-binding polypeptides method Polypeptide chemical synthesis: Novel HER2-conjugated polypeptide H2BPP1 (SEQ ID NO: 1) and reference polypeptide H2BPP2 (SEQ ID NO: 2), both containing GGGC chelate sequences at the C-terminus, were custom-ordered from Almac (Edinburgh, UK). The polypeptides were prepared by chemical synthesis, purified by RP-HPLC, converted to acetic acid-containing buffer, and delivered as lyophilized products. Prior to characterization and radiolabeling, the lyophilized polypeptides were stored at -20°C.

[0098] Recombinant production: The reference polypeptide H2BPP3 (SEQ ID NO: 3) was produced as described above (Ahlgren et al (2008), Bioconjugate Chem 19:235-243).

[0099] SE-HPLC and RP-UPLC-MS Analysis: The purity of H2BPP1 and H2BPP2 was analyzed by size exclusion high-performance liquid chromatography (SE-HPLC) and reverse-phase ultrafast chromatography-mass spectrometry (RP-UPLC-MS). Lyophilized polypeptides were dissolved in PBS (KCl 2.68 mM, KH2PO4 1.47 mM, NaCl 136.9 mM, Na2HPO4 8.1 mM, pH 7.4), EDTA 2 mM was added, and the mixture was incubated for 2 hours with occasional stirring. SE-HPLC analysis was performed on an Agilent 1100 HPLC system (Agilent Technologies) connected to a Superdex peptide GL 10 / 300 column (Cytiva) at a flow rate of 0.3 mL / min using PBS as the running buffer. RP-UPLC-MS analysis was performed using an Acquity UPLC CSH-C18, 1.7 μm, 2.1 × 150 mm column (Waters) connected to an Agilent 1290 UPLC system (Agilent Technologies) equipped with API-ES and a 6130 single quadrupole MSD. Elution was performed by a linear gradient of 10–60% acetonitrile in 0.1% TFA at a flow rate of 0.2 mL / min for 24 minutes.

[0100] result Peptide synthesis, purification, and lyophilization were successfully performed for both H2BPP1 and H2BPP2. The purity of both polypeptides was determined to be over 99% by SE-HPLC and over 92% by RP-UPLC. RP-HPLC-MS analysis, based on the integration of the 220 nm signal, identified the predicted masses corresponding to the monomeric polypeptide and Cys-Cys-mediated dimeric polypeptide, respectively, in the fractions shown in Table 1. In conclusion, the purity of the two polypeptides was comparable and considered sufficient for further in vitro and in vivo analysis.

[0101] [Table 1]

[0102] Example 2 Stability testing of unlabeled HER2-binding polypeptides method Circular Dichroism Spectroscopy: The thermal stability and refolding after thermal denaturation of the novel polypeptide H2BPP1 (SEQ ID NO: 1) and the reference polypeptide H2BPP2 (SEQ ID NO: 2) were measured using circular dichroism spectroscopy. Lyophilized polypeptides were dissolved in PBS, incubated for 2 hours, and then sterile filtered through a 0.22 μm Millex-GV syringe filter (Millipore). EDTA was added until the final concentration was 2 mM. The polypeptide samples were diluted to 0.5 mg / mL in PBS. Measurements were performed using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) with a cell having a 1 mm path length. For each polypeptide, the first CD spectrum measuring the ellipticity from 250 to 195 nm was recorded at 20°C. Thermal stability was determined by recording the ellipticity at 221 nm during variable temperature measurement (VTM, 5°C / min from 20°C to 90°C). After cooling to 20°C, a second CD spectrum in the 250nm to 195nm range was recorded.

[0103] Long-term stability evaluation: The stability of H2BPP1 (SEQ ID NO: 1) and H2BPP2 (SEQ ID NO: 2), which were freeze-dried and stored at -20°C, or dissolved in PBS + 2 mM EDTA and stored at -80°C and 5°C, was monitored for up to 6 months. RP-UPLC and SE-HPLC analyses performed as described in Example 1 were used for evaluation.

[0104] High-temperature stability evaluation: H2BPP1 (SEQ ID NO: 1) and H2BPP2 (SEQ ID NO: 2) were dissolved in PBS. To 100 μg of each polypeptide, 56 μg of SnCl, 75 μg of Na4EDTA, and 2.8 mg of sodium gluconate were added. The stability of the polypeptides after treatment under radiolabeling conditions at 90°C for 60 minutes was evaluated using an RP-UPLC connected in series with a mass spectrometer (Vion-Q-Tof, Waters). Analysis was performed using an Acquity UPLC CSH-C18, 1.7 μm, 2.1 × 100 mm column (Waters) at a flow rate of 0.2 mL / min, with elution by a linear gradient of acetonitrile 10-50% in 0.1% formic acid for 24 minutes.

[0105] result Circular Dichroism Spectroscopy: The melting temperatures Tm, determined from variable temperature measurements using circular dichroism spectroscopy, were 62°C for H2BPP1 and 68°C for H2BPP2. These were considered high for Z variant polypeptides. Furthermore, CD spectra measured before and after thermal induction denaturation showed complete refolding and a typical α-helix structure in both polypeptides (Figure 2).

[0106] Long-term stability evaluation: When stored lyophilized at -20°C, or dissolved in PBS + 2mM EDTA and stored at -80°C or 5°C for up to 6 months, no difference in stability was observed between H2BPP1 and H2BPP2. Table 2 summarizes the RP-UPLC results.

[0107] High-temperature stability assessment: RP-UPLC-MS total ion chromatograms (TICs) indicate that the novel polypeptide H2BPP1 is more stable than the reference polypeptide H2BPP2 after heat treatment under preferred labeling conditions (90°C, 60 minutes), as can be concluded from the fact that more post-peak variants were generated for H2BPP2 (comparing Figure 3B and Figure 3D). These variants are most likely deamidated polypeptide variants.

[0108] [Table 2] * Dissolve in PBS (pH 7.4) + 2 mM EDTA.

[0109] Example 3 Binding analysis of unlabeled HER2-binding polypeptides This example presents an SPR binding test performed using the novel polypeptide H2BPP1 (SEQ ID NO: 1) to evaluate its binding affinity and specificity to HER2. Interspecies interactions were investigated by analyzing binding to human, cynomolgus monkey, rat, and mouse HER2, respectively. To confirm the HER2 binding specificity of H2BPP1, binding to closely related receptor tyrosine kinases HER1 (epidermal growth factor receptor, also known as EGFR), HER3, and HER4 was evaluated.

[0110] method SPR interspecies binding analysis: In the first experiment, the binding affinity of H2BPP1 to human, cynomolgus monkey, rat, and mouse HER2-containing Fc chimeric proteins was measured using a Biacore 8K instrument (Cytiva). Recombinant human ErbB2 / HER2-Fc (Sino Biological, catalog number 10004-H02H), cynomolgus monkey ErbB2 / HER2-Fc (Sino Biological, catalog number 90295-C02H), rat ErbB2 / HER2-Fc (Sino Biological, catalog number 80079-R02H), and mouse ErbB2 / HER2-Fc (Sino Biological, catalog number 50714-M02H) were each diluted to 10 μg / mL in immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized on CM5 sensor chips (Cytiva) according to the manufacturer's instructions by EDC / NHS coupling chemistry using an amine coupling kit type 2 (Cytiva). The results of sensor chip immobilization were as follows: human HER2 Fc chimeric protein 2342 RU, cynomolgus monkey HER2 Fc chimeric protein 2066 RU, rat HER2 Fc chimeric protein 2385 RU, and mouse HER2 Fc chimeric protein 1674 RU. Single-cycle kinetics (SCK) was used to inject H2BPP1 onto the chip surface, allowing for a series of analytes to be injected in one cycle without regeneration. For background subtraction, a blank cycle using HBS-EP+ (HEPES 10 mM, NaCl 150 mM, EDTA 3 mM, surfactant P-20 0.05%, pH 7.4) was performed before each analyte cycle. H2BPP1 was continuously injected at concentrations of 0.3 and 3 nM for 240 seconds, followed by a dissociation time of 900 seconds. The flow rate was 50 μl / min, and HBS-EP+ was used as the running buffer. The surface was regenerated by injecting a regeneration buffer (25 mM HCl) once for 30 seconds. The binding rate constant (k) was calculated using the 1:1 binding model of Biacore 8K Insight Evaluation software. a ), dissociation rate constant (k d ) and the dissociation equilibrium constant (K D ) was calculated.

[0111] SPR specificity analysis: In the second experiment, the specificity of H2BPP1 was investigated by analyzing its binding to other members of the ErbB receptor family. Immobilization and sample analysis of recombinant human EGFR / Her1-Fc (R&D Systems, catalog no. 344-ER), ErbB2 / HER2-Fc (R&D Systems, catalog no. 1129-ER), ErbB3 / Her3-Fc (R&D Systems, catalog no. 348-RB), and ErbB4 / Her4-Fc (R&D Systems, catalog no. 1131-ER) were performed in the same manner as described for interspecies binding analysis, except that 1) H2BPP1 was injected sequentially at concentrations of 0.19, 0.38, and 0.75 nM, respectively, and 2) the dissociation time was 1200 seconds. Immobilization of human HER Fc chimeric proteins onto sensor chips resulted in 1592 RU for HER1, 1704 RU for HER2, 2942 RU for HER3, and 2056 RU for HER4.

[0112] result The binding affinity and specificity of the novel H2BPP1 to HER2 were evaluated by surface plasmon resonance (SPL) measurements.

[0113] SPR heterologous binding analysis: In the first analysis, H2BPP1 was injected into four Biacore sensor chip channels containing immobilized human, cynomolgus monkey, rat, and mouse HER2 Fc chimeric proteins, respectively, at SCK concentration series (0.3 and 3 nM). H2BPP1 showed binding to human and cynomolgus monkey HER2 (Figure 4), but not to rat or mouse HER2 (not shown). The binding rate constant (k) for H2BPP1 binding to human and cynomolgus monkey HER2-Fc was calculated. a ), dissociation rate constant (k d ) and the dissociation equilibrium constant (K D The results are summarized in Table 3.

[0114] [Table 3]

[0115] SPR specificity analysis: In the second SPR analysis, the HER2 binding specificity of H2BPP1 was investigated by analyzing its binding to other members of the ErbB receptor family, HER1, HER3, and HER4, respectively. The binding kinetics for human HER2 were consistent with the data shown in Table 3, and K D The concentration was determined to be 100 pM, but no binding to HER1, HER3, or HER4 was detected (Figure 5).

[0116] Example 4 In silico immunogenicity assessment This example describes the in silico immunogenicity evaluation of the amino acid sequences of a novel HER2-binding polypeptide, H2BPP1, and a reference polypeptide, H2BPP2, using a web-based tool for HLA binding prediction. For use in human trials, the least immunogenic polypeptide is highly preferred.

[0117] method We performed in silico predictive analysis of a novel polypeptide, H2BPP1 (SEQ ID NO: 1), and a reference polypeptide, H2BPP2 (SEQ ID NO: 2), using a web-based MHC II binding prediction tool from the Immune Epitope Database (IEDB, National Institute of Allergy and Infectious Disease, NIH: http: / / tools.iedb.org / mhcii / ). This tool predicts the binding affinity of the peptide (15-mer by default) to selected HLA alleles, and the affinity is ranked in relation to the peptide's reference set. Percentile ranks can be used to identify high-affinity binding epitopes that are likely to be immunogenic. For background information and further details on the IEDB tool, see Vita et al (2019), Nucleic Acids Res 47:339-343. The query parameters used were the IEDB default parameters (Methods: "IEDB Recommendation 2.22", HLA set: complete HLA reference set (27 alleles), peptide length: 15-mer). The data were grouped based on percentile rank (≤1%, 1-2%, 2-3%, and 3-10%), and the number of unique core epitopes (9 amino acids) and alleles per threshold group and query sequence was tallied. Peptides with scores above 10% were classified as unimportant.

[0118] result The potential presence of immunogenic epitopes in two peptides was evaluated in silico using an IEDB web-based MHC II binding prediction tool. Table 4 shows the number of distinct core epitopes predicted for the novel polypeptide H2BPP1 and the reference polypeptide H2BPP2, respectively. Any single epitope whose affinity for different alleles fell within different thresholds was counted multiple times (once for each threshold). Furthermore, the number of distinct HLA alleles was counted and aggregated for each sequence and threshold (Table 5). A total of 27 alleles were used for evaluation, and therefore the maximum score was 27.

[0119] [Table 4] [Table 5]

[0120] Comparing the predicted epitopes in H2BPP1 with those in H2BPP2, the main difference is a single 1-2% threshold epitope that is present at amino acid residues 40-48 in the reference polypeptide H2BPP2 but is not predicted to be present in the novel polypeptide H2BPP1. Furthermore, the difference from the 3-10% threshold epitope is a further epitope at positions 5-13 and another at positions 30-52, which are predicted in the reference polypeptide H2BPP2 but not in the novel polypeptide H2BPP1 (the scaffold residues are the most different between these two polypeptides). In conclusion, the novel polypeptide H2BPP1 has fewer potentially immunogenic epitopes compared to the reference polypeptide H2BPP2, making H2BPP1 preferable for development for human use.

[0121] Example 5 Radiolabeling of HER2-binding polypeptides In this example, the novel polypeptide H2BPP1 (SEQ ID NO: 1) and two reference polypeptides H2BPP2 (SEQ ID NO: 2) and H2BPP3 (SEQ ID NO: 3) were used to produce technetium-99 ( 99m Radiolabeling of Tc) with metastable nuclear isomers is described. Analysis performed to evaluate radiochemical yield, purity, and stability is also shown.

[0122] method Disulfide bond reduction: Prior to labeling, each polypeptide was treated with dithiothreitol (DTT, Merck) to reduce the disulfide bonds formed between cysteine ​​residues. For this purpose, a DTT solution (15 μl, 1 M degassed Milli-Q water) was mixed with the polypeptide (500 μl, 2 mg / mL in degassed PBS). The mixture was incubated at 37°C for 2 hours. The reduced polypeptides were equilibrated with PBS and purified using an eluting NAP-5 column (Cytiva). Each eluted polypeptide was aliquoted (50 μg in 50 μl) and then stored at -80°C until use.

[0123] 99m Labeling with Tc: A lyophilized labeling kit containing 75 μg of tin(II) chloride dihydrate (Fluka Chemika), 5 mg of sodium gluconate (Celsus Laboratories), and 100 μg of tetrasodium ethylenediaminetetraacetate (EDTANa4) (Sigma-Aldrich) was prepared as described above (Ahlgren et al (2010), Nucl Med Biol 37:539-546). The Ultra TechneKow generator (Mallinckrodt) was eluted with sterile 0.9% sodium chloride (Mallinckrodt). 99m Tc was obtained as pertechnetium salt. Each polypeptide was radiolabeled by adding the contents of the lyophilized kit, dissolved in 100 μl of degassed PBS, to 50 μg of polypeptide. Generator elution was performed on the reaction mixture. 99m 100 μl (150-250 MBq) of Tc-pertechnetiumate was added, and the vial was degassed to protect the mixture from oxidation. The reaction vial was thoroughly mixed by vortexing and incubated at 90°C for 1 hour.

[0124] Evaluation of radioactive labeling: The radiochemical yield of each polypeptide was analyzed using ITLC-SG strips (Agilent Technologies) and instant thin-layer chromatography unfolded in PBS (polypeptide: retention factor (Rf) = 0.0, 99mOther forms of Tc (Rf=1.0). The level of reductive hydrolysis technetium colloid (RHT) in the product was measured using pyridine:acetic acid:water (5:3:1.5) as the mobile phase. 99m Tc colloid: Rf = 0.0 99m Other forms of Tc and radiolabeled polypeptides (Rf=1.0) were also examined. Since the radiochemical yield was over 95% for all conjugates, further purification was not performed. The ITLC results were validated by SDS-PAGE using NuPAGE 4-12% Bis-Tris gels (both Invitrogen) in MES buffer. A Cyclone Storage Phosphor system (Perkin-Elmer) was used for quantitative measurement of radioactivity distribution in both ITLC strips and electrophoretic gels.

[0125] To further cross-validate the radioactive ITLC data, RP-HPLC was performed using a LaChrom Elite® system (VWR-Hitachi) equipped with a series-connected L-2130 pump, UV detector (L-2400), and radiation flow detector (Bioscan). 99m Purity analysis of the Tc-labeled compound was performed using an analytical column (Luna® 5μm C18, 100Å, 150×4.6mm column, Phenomenex). The RP-HPLC conditions were as follows: Solvent A: 10mM TFA in Milli-Q water, Solvent B: 10mM TFA in acetonitrile, UV detection at 220nm, gradient elution: B5-70% for 0-15 minutes, B70-95% for 15-18 minutes, B5% for 19-20 minutes, flow rate 1.0 mL / min.

[0126] To evaluate the in vitro stability of the radiolabeled conjugate, each fraction (10 μl, 0.4 μg) of the newly labeled polypeptide was incubated with an excess of PBS (40 μl) at 37°C for 1 hour and 4 hours. The test was performed using triplicates. 99m Tc release was monitored by ITLC as described above.

[0127] result All three polypeptide variants are, 99m The conjugates were successfully labeled with Tc, and both the radiolabeling yield and radiochemical purity exceeded 95%. All conjugates were stable during incubation at 37°C for 4 hours in the presence of excess PBS. 99 Less than 3% release of mTc was observed. The results of radiolabeling and in vitro stability tests are summarized in Table 6.

[0128] [Table 6]

[0129] To cross-validate the ITLC data, SDS-PAGE, RP-HPLC, and analysis were performed. SDS-PAGE analysis (Figure 6) did not reveal any high or low molecular weight radioactivity bands, indicating no signs of aggregation, degradation, or release of radionuclides. Radioactive RP-HPLC analysis showed that the retention time for all polypeptides was approximately 8.6–9.2 minutes, and the retention time of the labeled polypeptide (measured by a radioactivity detector) was the same as that of the unlabeled polypeptide (measured by a UV detector). This confirmed the authenticity of the labeled peptide.

[0130] Example 6 In vitro characterization of radiolabeled HER2-binding polypeptides In this embodiment, three prepared as described in Example 5 99m This paper describes the characterization of Tc-labeled polypeptides using in vitro cell-based assays.

[0131] material Cell Culture: HER2-expressing ovarian cancer SKOV-3 and breast cancer BT-474 cell lines obtained from the American Type Culture Collection (ATCC) were used. Cells were cultured in RPMI medium (Flow Irvine) (hereinafter referred to as "complete medium") supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and PEST consisting of 100 IU / mL penicillin and 100 mg / mL streptomycin. 6 Cells were seeded into cell culture dishes at a density of cells / dish. A set of three dishes was used for each data point in in vitro studies of binding specificity, cell retention, and cell processing.

[0132] In vitro binding specificity: In the control dish, SKOV-3 and BT-474 cells were pre-saturated with 500-fold excess unlabeled H2BPP3 for 15 minutes. 0.5 nM of each labeled polypeptide was then added. 99m Tc-H2BPP1 (new), 99m Tc-H2BPP2 (see reference) and 99m Tc-H2BPP3 (reference) was added to the test dish and control dish, respectively, and the cells were incubated in a humidified incubator (5% CO2, 37°C) for 1 hour. The medium was discarded, the cells were washed with cold serum-free medium, and then trypsin-EDTA solution (0.5 mL per dish) was added, and the cells were incubated for a further 10 minutes. The detached cells were diluted in 0.5 mL of complete medium, resuspended, and transferred to fraction tubes. The radioactivity of the cells was measured using an automated gamma counter (1480 Wizard, Wallac) equipped with a NaI(TI) detector, and cell-bound radioactivity was calculated.

[0133] In vitro binding affinity: 99mThe binding kinetics of Tc-labeled polypeptides H2BPP1 (novel), H2BPP2 (reference), and H2BPP3 (reference) to the HER2 receptor were measured using a LigandTracer Yellow instrument (Ridgeview Instruments). SKOV-3 cells were seeded in localized areas of cell culture dishes (Nunclon®, size 100620, NUNC A / S). Measurements were performed at room temperature (RT) to prevent internalization. 99m The uptake curves were recorded for Tc-labeled polypeptides at concentrations of 0.33, 1, and 3 nM. Afterward, the radioactive medium was removed, and a fresh non-radioactive medium was added to record the dissociation curve. The data were analyzed using Interaction Map software (Ridgeview Diagnostics) to determine the binding rate, dissociation rate, and equilibrium dissociation constant (K). D The calculation was performed using duplicate.

[0134] Cell processing studies: Cell processing was investigated using previously described and validated methods (Wallberg and Orlova (2008), Cancer Biother Radiopharm 23:435-442). Briefly, SKOV-3 and BT-474 cells (1 × 10⁶) were used. 6 Cells (dish) were incubated with 0.5 nM each of labeled H2BPP1 (novel), H2BPP2 (reference), and H2BPP3 (reference) at room temperature for 1 hour. The medium was then removed, the cells washed, fresh medium was added, and the cells were placed in a humidified incubator at 37°C. Internalized fractions were determined by acid washing at 0, 1, 2, 4, and 6 hours of incubation. Membrane-bound polypeptides were removed from the cells by treatment with 4 M urea solution in 0.1 M glycine buffer (pH 2.5) for 5 minutes on ice. Cellular debris containing internalized conjugates was detached by treatment with 1 M NaOH. Cellular radioactivity was measured using an automated gamma counter (1480 Wizard, Wallac) equipped with a NaI(TI) detector, and the proportions of membrane-bound and internalized radioactivity were calculated.

[0135] result In vitro binding specificity: The HER2 binding specificity of H2BPP1 (novel), H2BPP2 (reference), and H2BPP3 (reference) polypeptides was evaluated using saturation experiments. When cells were pre-saturated with unlabeled H2BPP3, binding was significantly reduced (p<5×10⁻¹⁰). -6 (Figure 7) It was confirmed that binding to both the novel and reference polypeptides was HER2-mediated.

[0136] In vitro binding affinity: Measured using LigandTracer. 99m Table 7 summarizes the binding dynamics of Tc-labeled polypeptides H2BPP1 (novel), H2BPP2 (reference), and H2BPP3 (reference) to the HER2 receptor in SKOV-3 cells. Best-fit binding of all conjugates to the SKOV-3 cell line was achieved using a 1:1 model. Interaction map calculations showed rapid binding followed by very slow dissociation for all conjugates, resulting in a picomolar equilibrium dissociation constant (K). D ) was obtained.

[0137] [Table 7]

[0138] Cell processing tests: Results from cell retention and internalization experiments showed that internalization of all polypeptides was slow (Figure 8). Reference polypeptide 99m Tc-H2BPP3, after 6 hours of incubation, produces a polypeptide containing a GGGC chelator, i.e., a novel polypeptide. 99m Tc-H2BPP1 (65.8±2.1% and 81.5±2.3% for SKOV-3 and BT-474, respectively) and reference polypeptide 99m A slight tendency was observed for higher retention rates (87.6±3.5% and 87.3±3.5% for SKOV-3 and BT-474 cells, respectively) compared to Tc-H2BPP2 (76.8±4.0% and 86.9±1.2% for SKOV-3 and BT-474 cells, respectively).

[0139] Conclusion: Novel polypeptide 99m Tc-H2BPP1 has an affinity of 58±2 pM (K D It was shown to specifically bind to HER2-expressing cells. This is favorable for maintaining radioactivity in tumors, despite the slow rate of internalization.

[0140] Example 7 In vivo biodistribution study method Animal handling: Animal experiments were conducted in accordance with national laws regarding work with laboratory animals. Approval was granted by Uppsala's Ethical Committee for Animal Research. These experiments used groups of four mice per data point.

[0141] In vivo distribution in non-tumor-bearing mice: Novel polypeptides 99m To confirm that the redesign of the Tc-H2BPP1 scaffold does not have a strong adverse effect on the in vivo behavior of the polypeptide, the in vivo distribution in female NMRI mice 4 hours after injection was compared with that of a reference polypeptide. 99m Tc-H2BPP2 and 99m The in vivo distribution of Tc-H2BPP3 was compared. Non-tumor-bearing mice (body weight: 25.5 ± 2.4 g) were subjected to each 99m One μg (60 kBq, 100 μl in PBS) of Tc-labeled polypeptide was injected via tail vein. Four hours later, the mice were euthanized by an overdose of anesthesia (Rompun / Ketalar), followed by cardiac puncture to collect blood samples. Organ and tissue samples were collected and weighed. Organ radioactivity was measured using a gamma spectrometer (1480 Wizard, Wallac) equipped with a NaI(TI) detector, and organ uptake was calculated as a percentage of the injected dose per gram of tissue (%ID / g).

[0142] In vivo distribution in tumor-bearing mice: In BALB / C nu / nu mice carrying HER2-positive SKOV-3 xenografts, the in vivo distribution and targeting characteristics were evaluated. To construct the xenografts, 107 Individual SKOV-3 cells were subcutaneously injected into the right hind limb of female BALB / c nu / nu mice. As a specific control, HER2-negative Ramos xenografts were used. 5×10 6 Individual Ramos cells (ATCC) were subcutaneously transplanted into the left hind limb of 5 female BALB / c nu / nu mice. The experiment was conducted 2 weeks after cell transplantation. The average body weight of the animals was 18.2±0.7 g. The average tumor weights were 0.06±0.02 and 0.03±0.01 g for SKOV-3 and Ramos xenografts, respectively. At 1, 4, 8, and 24 hours after injection in mice with SKOV-3 xenografts 、99m The biodistribution of 99m Tc-H2BPP1 was measured. Three groups of tumor-bearing mice were 99m injected with 4 μg (80 kBq, 100 μl in PBS) of 99m Tc-H2BPP1 via the tail vein. To measure the biodistribution 24 hours after injection, one group of mice was injected with 640 kBq, but the injected protein mass was the same 4 μg. The 99m HER2 specificity of 99m Tc-H2BPP1 accumulation in tumors was evaluated by injecting 4 μg (80 kBq, 100 μl in PBS) of

[0143] Tc-H2BPP1 into one group of animals with HER2-negative Ramos xenografts (the average body weight of the mice was 17.6±1.2 g) and measuring the biodistribution 4 hours after injection. For comparison, one group of mice was injected with the reference polypeptide 99mTc-H2BPP1 was intravenously injected. Four hours after injection, the mice were imaged using a nanoScan SPECT / CT scanner (Mediso Medical Imaging Systems). The mice were euthanized by CO2 asphyxiation immediately before being placed in the camera. Computed tomography (CT) acquisition was performed with the following parameters: energy peak 50 kV, 670 μA, 480 projections, acquisition time 2.29 minutes. SPECT acquisition was performed with the following parameters: 99m Tc energy peak 140 keV, window width 20%, matrix 256×256, acquisition time 1 hour. CT images were reconstructed in real time using Nucline 2.03 software (Mediso Medical Imaging Systems). The raw data of SPECT were reconstructed using Tera-Tomo (trademark) 3D SPECT reconstruction technology.

[0144] Results In vivo distribution in non-tumor-bearing mice: In NMRI mice 4 hours after injection, the novel polypeptide 99m Tc-H2BPP1 as well as two reference polypeptides 99m Tc-H2BPP2 and 99m The comparison of the in vivo distributions of Tc-H2BPP3 is shown in Fig. 9. 99m Tc-H2BPP2 and 99m The in vivo distribution data of Tc-H2BPP3 were in very good agreement with the data previously obtained for these reference polypeptides (Wallberg et al (2011), supra; Ahlgren et al (2010), supra). The GGGC-containing polypeptide 99m Tc-H2BPP1 (5.9±2.1% ID / g) and 99m The renal uptake of Tc-H2BPP2 (7.9±2.1% ID / g) was 99m significantly lower than that of Tc-H2BPP3 (183.8±27.3% ID / g) (p<0.0005). In other words, 99m Tc-H2BPP1 and 99m the renal uptake of Tc-H2BPP2 was 99mThe renal uptake of Tc-H2BPP3 was 1 / 25 to 1 / 30 of that of other Tc-H2BPP3 molecules. Furthermore, 99m Tc-H2BPP1 and 99m Tc-H2BPP2 is taken up in the lungs, liver, spleen, stomach, muscles, and bones. 99m It was significantly lower (p<0.05) than Tc-H2BPP3 in the liver. 99m Tc-H2BPP1 and 99m To import data from Tc-H2BPP2, 99m The uptake was approximately 1 / 4 compared to that of Tc-H2BPP3. This low uptake is a desirable characteristic because it reduces the background radioactivity required for imaging liver metastases, which are common in breast cancer. (Novel polypeptide) 99m Tc-H2BPP1 also, 99m It also showed significantly lower blood concentrations than Tc-H2BPP3.

[0145] In vivo distribution in tumor-bearing mice: Experiments conducted in nude mice carrying human cancer xenografts showed that HER2-expressing SKOV-3 xenografts were distributed 4 hours after injection. 99m We demonstrated that Tc-H2BPP1 uptake was 175 times higher than in HER2-negative Ramos xenografts (p<0.0005) (Figure 10). This confirmed that tumor accumulation was HER2-specific. Novel polypeptides were observed 1, 4, 8, and 24 hours after injection. 99m Table 8 shows the in vivo distribution data for Tc-H2BPP1. 99m Tc-H2BPP1 demonstrated efficient targeting, as tumor uptake was already 24±7% ID / g one hour after injection. 99mDespite the fact that the Tc](Tc=O)-GGGC complex has been shown to act as a non-residualizing label, the retention of radioactivity over time in tumors was good. While we do not wish to be bound by theory, the very high affinity of the binding polypeptide to HER2 is the basis for such beneficial properties. There was no significant difference in tumor uptake at 1 hour and 8 hours after injection, and tumor uptake decreased by only half by 24 hours after injection. In contrast, clearance from normal tissue was rapid. Radioactivity in the kidneys decreased very rapidly over time (40±1% ID / g at 1 hour after injection compared to 5.3±0.3% ID / g at 24 hours after injection). Due to this in vivo distribution pattern, by 4 hours after injection, 99m The tumor-to-organ ratio for Tc-H2BPP1 is favorably high (Table 9).

[0146] [Table 8]

[0147] [Table 9]

[0148] In the same batch of nude mice with human SKOV-3 xenografts 4 hours after injection 99m Tc-H2BPP1 and 99m A direct comparison of the in vivo distribution of Tc-H2BPP2 (Figure 11) demonstrated that the in vivo distributions of these polypeptides are very similar, meaning that changes in the scaffold of the novel polypeptides do not have any negative effect on the in vivo distribution. Furthermore, there were no significant differences in the tumor-to-organ ratio between the two conjugates, except for the tumor-to-bone ratio. The tumor-to-kidney ratio was at approximately the same level for both conjugates. 99m Tc-H2BPP1 and 99m For Tc-H2BPP2, the values ​​were 2.2±0.5 and 1.9±0.2, respectively.

[0149] In vivo imaging: The results of microSPECT / CT imaging (Figure 12) are as follows: 99m We demonstrated that high-contrast visualization of HER2 expression in HER2-positive tumors is possible using Tc-H2BPP1. A SKOV-3 tumor in the right hindlimb was clearly and with high contrast visualization 4 hours after injection. Consistent with in vivo distribution data, radioactivity uptake in the tumor was considerably higher than in the kidney. As expected, radioactivity uptake in HER2-negative Ramos xenografts was much lower than in HER2-positive SKOV-3 xenografts.

[0150] conclusion Even with extensive redesign of the scaffold for the novel polypeptide H2BPP1, using the GGGC chelator designed within the C-terminus of the polypeptide... 99 The imaging properties of the polypeptide labeled with mTc were not impaired. This novel probe, 99m Tc-H2BPP1 delivered a superior tumor-to-blood ratio compared to HER2 imaging probes for single-photon emission computed tomography (SPECT) described in previous literature. The inventors stated that, 99m We conclude that Tc-H2BPP1 is a promising candidate for further clinical application trials due to its high tumor-to-organ ratio and low renal uptake.

[0151] Example 8 Dose assessment for human use method Novel polypeptide 99m To assess the dose of Tc-H2BPP1 in humans, the uptake values ​​in mice were obtained using the established "percent kg / g method" (Stabin (2008), Fundamentals of Nuclear Medicine Dosimetry, New York, NY: Springer; pp. 83-86), as follows: (%IA / organ) human =[(%IA / g) animal × (kg TBweight ) animal × (gorgan / (kg TBweight ) human ] The calculation was performed according to the following criteria (where "%IA" represents the percentage of administered radioactivity and "TBweight" represents total body weight).

[0152] Human organ uptake was calculated using the organ weights of a standard adult female (Report of the Task Group on Reference Man, ICRP Publication 23 (1975). Pergamon Press, Oxford). Residence time was calculated as the area under the curve of an exponential fit to the human time-radioactivity curve. Remaining internal residence time is based on radioactivity in the carcass. Absorbed dose was estimated using OLINDA / EXM 1.0 software.

[0153] result Novel Polypeptides in Humans 99m Table 10 shows the estimated absorbed dose of Tc-H2BPP1. According to calculations using OLINDA / EXM 1.0, the effective dose should be 0.00066 mSv / MBq (effective dose equivalent: 0.00116 mSv / MBq).

[0154] [Table 10]

[0155] Example 9 188 Radioactive labeling of H2BPP1 by Re method 188 Labeling polypeptides with Re: 188 Re was measured using 4 mL of sterile 0.9% sodium chloride. 188 W / 188 The perrhenium salt was obtained by elution using a Re-generator (both manufactured by OncoBeta GmbH, Germany). Radioactive labeling was performed using two methods: 1) low eluate volume (less than 1500 μl) or 2) high eluate volume (4000 μl).

[0156] Labeling with low eluate volume: The contents of one lyophilized kit containing 1 mg of tin(II) chloride dihydrate, 5 mg of sodium gluconate, and 4100 μg of EDTANa, dissolved in 1.25 M sodium acetate at pH 4.2, were added to 200 μg of H2BPP1 to make a total volume of 100 μl. 188 Up to 1500 μl (up to 300 MBq) of Re-containing generator eluate was added. The mixture was incubated at 70°C for 60 minutes, then cooled at room temperature for 5 minutes. Labeling was performed using a size-exclusion NAP-5 column as described in Example 5. 188 Re-H2BPP1 was purified.

[0157] Labeling with high eluate volume: The contents of one lyophilized kit containing 2 mg of tin(II) chloride dihydrate, 50 mg of sodium gluconate, and 4200 μg of EDTANa, dissolved in 1.25 M sodium acetate, pH 4.2, were added to 200 μg of H2BPP1 to make a total volume of 100 μl. 188 4000 μl of Re-containing generator eluate was added. 440 μg of ascorbic acid (2 mg / mL in 1.25 M sodium acetate buffer (pH 4.2)) was added to the reaction vial, and the mixture was incubated at 70°C for 60 minutes, then cooled to room temperature for 5 minutes. Subsequently, the total amount of ascorbic acid in the reaction vial was adjusted to 1 mg using a 5 mg / mL ascorbic acid PBS solution. Purification was performed using an Oasis HLB 6cc Vac Cartridge (Waters). The cartridge was activated by passing 5 mL of 50% ethanol aqueous solution through it, and then deactivated by passing 5 mL of water through it. The reaction mixture was passed through the cartridge, followed by 20 mL of water. The cartridge was further washed with 500 μl of 25% ethanol aqueous solution and purified with 1 mL of 50% ethanol aqueous solution. 188 Re-H2BPP1 was eluted. For subsequent use, 188 Re-H2BPP1 was diluted with water to a final ethanol concentration of 5-10%. The purified samples were analyzed by ITLC and RP-HPLC as described in Example 5.

[0158] result Labeling with a low eluate volume yielded a radiochemical yield of 98.6 ± 0.5%. The radiochemical purity of the product was over 98%. 188 Re-H2BPP1 was used in the characterization, in vivo distribution, and imaging tests described in Examples 10-11.

[0159] Labeling with a high eluate volume yielded a radiochemical yield of 85 ± 10%. The radiochemical purity of the product was over 97%. 188 Re-H2BPP1 was used in the experimental therapeutic trial described in Example 12.

[0160] Example 10 188 In vitro characterization of Re-H2BPP1 HER2-binding polypeptide In this example, the preparation was made as described in Example 9. 188 This report describes the characterization of Re-H2BPP1 using in vitro cell-based assays.

[0161] method Cell culture: HER2-expressing ovarian cancer SKOV-3 and breast cancer SK-BR-3 cell lines (ATCC) were used. Cells were cultured in complete medium (see Example 6) and placed in cell culture dishes for 10 minutes. 6 Seeds were seeded at a density of cells / dish. A set of three dishes was used for each data point in the in vitro binding specificity test.

[0162] In vitro binding specificity: In the control dish, SKOV-3 and SK-BR-3 cells were pre-saturated by incubation with 400-fold molar excess unlabeled H2BPP1 (200 nM) for 15 minutes. Subsequently, 0.5 nM labeled polypeptide was added. 188Re-H2BPP1 was added to both the test and control dishes, and the cells were incubated in a humidified incubator (5% CO2, 37°C) for 1 hour. The medium was discarded, the cells were washed with cold serum-free medium, and then trypsin-EDTA solution (0.5 mL per dish) was added, and the cells were incubated for a further 10 minutes. The detached cells were diluted in 0.5 mL of complete medium, resuspended, and transferred to fraction tubes. The radioactivity of the cells was measured using an automated gamma counter (1480 Wizard, Wallac) equipped with a NaI(TI) detector, and cell-bound radioactivity was calculated.

[0163] In vitro binding affinity: 188 The binding kinetics of Re-H2BPP1 to the HER2 receptor were measured using a LigandTracer Yellow instrument (Ridgeview Instruments) as described in Example 6.

[0164] result In vitro binding specificity: Using saturation experiments, 188 The HER2 binding specificity of Re-H2BPP1 was evaluated. When cells were pre-saturated with unlabeled H2BPP1, binding was significantly reduced (p<5×10⁻⁶). -5 (Figure 14) 188 It was confirmed that Re-H2BPP1 binding is HER2-mediated.

[0165] In vitro binding affinity: Measured using LigandTracer 188 Table 11 summarizes the binding dynamics of Re-H2BPP1 to the HER2 receptor in SKOV-3 cells. For all conjugates, the best match for binding to SKOV-3 cells was achieved using a 1:2 model. In the interaction map calculation, 188 The dominant interaction of Re-H2BPP1 shows rapid binding followed by very slow dissociation, resulting in an equilibrium dissociation constant (K D A low value for ) was obtained, meaning a very high affinity was achieved.

[0166] [Table 11]

[0167] Conclusion Novel polypeptide 188 Re-H2BPP1 was shown to specifically bind to HER2-expressing cells with a predominant affinity (K D ) of 5 ± 3 pM. This is favorable for the retention of radiation energy in tumors.

[0168] Example 11 In Vivo Biodistribution and Imaging Study Method Animal handling: Animal experiments were conducted in accordance with national laws regarding work with experimental animals. Approval was given by the Ethical Committee for Animal Research in Uppsala.

[0169] Biodistribution in non-tumor-bearing mice: 188 To determine whether radioactive pertechnetate is released during the renal catabolism of Re-H2BPP1 and to obtain data for dose estimation in humans, a biodistribution study was performed (Example 13). NMRI mice were randomly divided into groups of four animals each. The average body weight of the animals at the time of the experiment was 29.6 ± 3.4 g. Each mouse was intravenously injected with 5 μg (210 kBq) of Re-H2BPP1 in 100 μl of 0.9% sterile saline 188 In the first experiment, one group of mice was administered 5% sodium iodide (NaI) in drinking water 24 hours before the experiment to saturate the salivary gland Na / I cotransporter, and another group was administered drinking water without addition. Biodistribution was measured 4 hours after injection. In further experiments, NaI in drinking water was administered to all groups 24 hours before the experiment. At 0.5, 1, 4, 24, and 48 hours after injection, the biodistribution of Re-H2BPP1 was measured as described in Example 7 188 The results obtained 4 hours after injection were compared with the results at the same time points obtained in Example 7 for Tc-H2BPP1 to evaluate any effect of the label on biodistribution. 99m

[0170] In vivo distribution in tumor-bearing mice: In BALB / C nu / nu mice carrying HER2-positive SKOV-3 xenografts, the in vivo distribution and targeting characteristics were evaluated. To construct the xenografts, 10 7 SKOV-3 cells were subcutaneously injected into the right hind limb of female BALB / c nu / nu mice. HER2-negative Ramos xenografts were used as a specificity control. 5 × 10 6 100 Ramos cells (ATCC) were subcutaneously transplanted into the left hind limbs of 5 female BALB / c nu / nu mice. The experiment was performed 2 weeks after cell transplantation. The mean body weight of the animals was 18.0 ± 1.0 g. The mean tumor weights were 0.160 ± 0.120 g and 0.7 ± 0.3 g for SKOV-3 and Ramos xenografts, respectively. 1, 4, 8, and 24 hours after injection in mice with SKOV-3 xenografts 188 The in vivo distribution of Re-H2BPP1 was measured in three groups of tumor-bearing mice. 188 Re-H2BPP1 10 μg (263 kBq, 100 μl in PBS) was injected via tail vein. To measure the in vivo distribution 24 and 48 hours after injection, 470 kBq was injected into two groups of mice, but the amount of protein injected was the same at 10 μg. 188 To evaluate the HER2 specificity of Re-H2BPP1 accumulation, a group of animals (with a mean body weight of 19.8 ± 0.6 g) carrying HER2-negative Ramos xenografts were subjected to the study. 188 Re-H2BPP1 10 μg (265 kBq, 100 μl in PBS) was injected, and its in vivo distribution was measured 4 hours after injection. The in vivo distribution in tumor-bearing mice was measured in the same manner as described above for NMRI mice.

[0171] In vivo imaging: SPECT / CT imaging was performed on small animals to confirm the in vivo distribution results. One SKOV-3 xenograft mouse and one Ramos xenograft mouse were given 2.4 MBq / 10 μg. 188Re-H2BPP1 was intravenously injected. Mice with SKOV-3 xenografts were imaged 1 hour and 4 hours after injection under sevoflurane anesthesia using a nanoScan SPECT / CT scanner (Mediso Medical Imaging Systems). Subsequently, mice with SKOV-3 and Ramos xenografts were euthanized by CO2 asphyxiation immediately before being placed in the camera and imaged side by side simultaneously. Computed tomography (CT) acquisition was performed with the following parameters: energy peak 50 kV, 670 μA, 480 projections, acquisition time 2.29 minutes. SPECT acquisition was performed with the following parameters: 188 Re energy peak 150 keV, window width 20%, matrix 256×256, acquisition time 20 minutes. CT images were reconstructed in real time using Nucline 2.03 software (Mediso Medical Imaging Systems). The raw data of SPECT were reconstructed using Tera-Tomo (trademark) 3D SPECT reconstruction technology.

[0172] Results In vivo distribution in non-tumor-bearing mice: The results of the first experiment to evaluate the blockade of the Na / I cotransporter in NMRI mice are shown in Figure 15A. As a result of the blockade, radioactive uptake in the salivary gland significantly decreased (p<0.0013). Free radioactive perrhenate ( 188 ReO4 - ) is taken up by the salivary gland by the Na / I cotransporter, indicating that blockade of the cotransporter by cold iodide results in the release of radioactive perrhenate during the catabolism of Re-H2BPP1. Free radioactive perrhenate may increase the dose absorbed in normal tissues. To avoid this, clinically, the Na / I cotransporter is blocked by administering iodine to patients in the form of sodium iodide, iodine in milk, or Lugol's iodine during radionuclide therapy. This approach can also be used for treatment with Re-H2BPP1. Therefore, further experiments were performed in mice after adding NaI to the drinking water.

[0173] ​​​​ Technetium and rhenium are chemical analogs, but they differ in their chemical properties, and as a result, 99m Tc and 188 Radiopharmaceuticals labeled with Re exhibit different biological behaviors. Such differences 188 Re-H2BPP1 and 99m To confirm that it does not cause undesirable effects on the in vivo distribution of Tc-H2BPP1, the results obtained in NMRI mice 4 hours after injection were compared (Figure 15B). 188 Re-H2BPP1 uptake was found to be high in the blood, lungs, liver, and bones. Unexpectedly, it was also high in the kidneys. 188 Re retention rate 99m The retention rate of Tc is lower, which should lead to a lower absorbed dose in the kidneys. Since the kidneys are an important organ in radionuclide therapy, 188 This characteristic of Re-H2BPP1 is favorable for its application in the treatment of H2BPP1. 188 Table 12 shows the in vivo distribution data for Re-H2BPP1. The characteristics of its in vivo distribution include its presence in the blood and from other organs and tissues. 188 The clearance of Re-H2BPP1 was rapid. The small amount accumulated in the intestinal contents suggests that the hepatobiliary excretion pathway was used to remove it from the body. 188 This suggests that it played no substantial role in the emission of Re-H2BPP1. The very important point is that, 188 The renal uptake of Re-H2BPP1 decreased rapidly.

[0174] [Table 12] * The data is presented as the %ID for the entire sample containing the contents. ** The data is presented for the entire sample.

[0175] In vivo distribution in tumor-bearing mice: In BALB / C nu / nu mice carrying HER2-expressing SKOV-3 xenografts. 188The in vivo distribution data for Re-H2BPP1 are shown in Figures 16-17 and Table 13. The data is consistent with the in vivo distribution data in normal NMRI mice. 188 Re-H2BPP1 rapidly disappeared from normal tissues, including blood and kidneys. Tumor uptake was high, already at 31±4% ID / g one hour after injection (Table 13). By this point, tumor uptake was higher than that in most organs and comparable to renal uptake. However, while tumor uptake remained high over time, renal radioactivity decreased rapidly. Four hours after injection, tumor uptake was already more than eight times that of renal uptake. This created favorable conditions for radionuclide therapy. In vivo distribution data was confirmed by experimental imaging using microSPECT / CT (Figure 16).

[0176] 188 To confirm the HER2 specificity of tumor targeting using Re-H2BPP1, uptake in HER2-positive SKOV-3 and HER2-negative Ramos lymphoma xenografts was compared 4 hours after injection (Figure 17). Uptake in HER2-positive xenografts was 245 times higher than in HER2-negative xenografts. This indicates that 188 This clearly demonstrates that tumor uptake of Re-H2BPP1 is HER2-mediated.

[0177] [Table 13] * The data is presented as the %ID for the entire sample containing the contents. ** The data is presented for the entire sample.

[0178] Example 12 Radiation therapy for HER2-positive xenografts method Animal handling: Animal experiments were conducted in accordance with national laws regarding work using laboratory animals. Approval was granted by Uppsala's Ethical Committee for Animal Research.

[0179] Experimental radionuclide therapy of xenografts: The effect of H2BPP1 on xenografts with high levels of HER2 expression was investigated using SKOV-3 cells. 10 BALB / c nu / nu mice were given 10 7 One SKOV-3 cell was subcutaneously transplanted. Seven days later, three groups of mice (n=10 per group) were treated as follows: Group A: 188 Re-labeled H2BPP1 (5 μg, 16 MBq, dissolved in 150 μl of 10% ethanol aqueous solution), Group B: Vehicle (150 μl of 10% ethanol aqueous solution, control group), Group C: Unlabeled H2BPP1 (5 μg, dissolved in 150 μl of 10% ethanol aqueous solution, control group to evaluate whether treatment with the protein itself has any effect on tumor growth). Each group received three doses at 48-hour intervals. In accordance with ethical approvals, the study was conducted for 90 days from the first drug administration. The presence and size of tumors were assessed twice a week using slide calipers. Mice were euthanized if weight loss exceeded 10% in one week or 15% after the start of treatment, or if the tumor grew larger than 1 mL or became ulcerated. At the start of treatment, the tumor volume was as follows: Group A: 101 ± 28 mm 3 , Group B: 85±43mm 3 , Group C: 86±25mm 3 The difference in tumor size between groups was not significant (p>0.05, one-way ANOVA with Bonferroni correction for multiple comparisons).

[0180] result Figures 18-19 show the results of experimental radionuclide therapy of xenografts. Figure 18 shows the growth of individual tumors in each group. Tumor growth was rapid in the control group. The tumor volume doubling time was 12 ± 7 days in group B (treated with vehicle) and 9 ± 2 days in group C (treated with unlabeled H2BPP1). There was no significant difference in tumor doubling time between these two groups (p>0.05, unpaired t-test), which suggests that treatment with unlabeled H2BPP1 has no antitumor effect. All animals in group B were sacrificed by day 51 due to reaching the tumor volume limit or due to tumor ulceration. In group C, the last animal was sacrificed by day 37. The tumor growth pattern differed in group A. A decrease in tumor volume was observed from day 19 after the start of treatment (Figure 18A). At this point, the mean tumor volume was significantly smaller than that in the two control groups (p<0.05, one-way ANOVA with Bonferroni correction for multiple comparisons). Some tumors resumed growth with a slight delay. However, three animals were still alive at the end of the study (day 90).

[0181] The median survival time in the treatment group (A) was 68 days, which was significantly longer than that of the control group (log-rank Mantel-Cox test <0.0001) (Group B: 29 days, Group C: 27.5 days, Figure 19A). There was no difference in survival time between mice treated with the vehicle (Group B) and those treated with unlabeled H2BPP1 (Group C) (log-rank Mantel-Cox test p>0.05).

[0182] The treatment was well-tolerated. The behavior and appearance of mice in the treatment group were no different from those of animals in the control group. There was no significant difference in the mean body weight of animals between the treatment group and the control group (Figure 19B).

[0183] conclusion Experimental preclinical treatments are, 188 Treatment with Re-H2BPP1 slowed the growth of HER2-expressing tumor xenografts, significantly extended the survival time of mice carrying such xenografts, and demonstrated no adverse effects on the animals.

[0184] Example 13 Dose evaluation for use in humans Method Based on the biodistribution data in non-tumor-bearing mice (Example 11), the upscaling of the dose evaluation of Re-H2BPP1 was performed as described in Example 8. 188 The upscaling of the dose evaluation of Re-H2BPP1 was carried out as described in Example 8.

[0185] Results Novel polypeptide in humans 188 The estimated absorbed dose of Re-H2BPP1 is shown in Table 14. According to the calculation using OLINDA / EXM 1.0, the effective dose should be 0.0683 mSv / MBq (effective dose equivalent: 0.0683 mSv / MBq).

[0186] [Table 14]

[0187] List of items of the embodiment 1. A HER2-binding polypeptide having an amino acid sequence comprising AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LSESQGGGC (SEQ ID NO: 1).

[0188] 2. The HER2-binding polypeptide according to item 1, having an amino acid sequence consisting of SEQ ID NO: 1.

[0189] 3. A radiolabeled polypeptide comprising a radioactive chelate of the HER2-binding polypeptide according to item 1 or 2 and a radionuclide.

[0190] 4. The radiolabeled polypeptide according to item 3, wherein the radionuclide is suitable for medical imaging.

[0191] 5. The radionuclide is 99m Tc, 51 Mn, 52m Mn, 52 Mn, 186 Re and 188A radiolabeled polypeptide selected from the group consisting of Re, as described in item 4.

[0192] 6. The radioactive nuclide 99m Tc, a radiolabeled polypeptide as described in item 5.

[0193] 7. A radiolabeled polypeptide as described in item 3, wherein the radionuclide is suitable for treatment.

[0194] 8. The radioactive nuclide 186 Re and 188 A radiolabeled polypeptide selected from the group consisting of Re, as described in item 7.

[0195] 9. The radioactive nuclide 188 Re, a radiolabeled polypeptide as described in item 8.

[0196] 10. A composition comprising a HER2-binding polypeptide as described in item 1 or 2, or a radiolabeled polypeptide as described in any one of items 3 to 9, and at least one pharmaceutically acceptable excipient or carrier.

[0197] 11. An in vivo imaging method in the body of a mammal, including a human, that has or is suspected of having cancer characterized by HER2 overexpression, wherein the method is -Administering into a mammalian subject a radiolabeled polypeptide, optionally included in the composition described in item 10, as described in any one of items 4 to 6, and A method comprising the step of obtaining one or more images of at least a part of a body of a subject using a medical imaging device, wherein the images indicate the presence of a radionuclide in the body.

[0198] 12. A method according to item 11, comprising, prior to the administration step, a step of preparing a radiolabeled polypeptide according to any one of items 4 to 6, wherein the step comprises mixing the polypeptide according to item 1 or 2 with a radionuclide suitable for medical imaging.

[0199] 13. A method for diagnosing cancer characterized by HER2 overexpression, - Perform the in vivo imaging method described in item 11 or 12. - A method comprising establishing the diagnosis based on the obtained images.

[0200] 14. A method for establishing the prognosis of cancer characterized by HER2 overexpression, - Perform the in vivo imaging method described in item 11 or 12. - A method comprising establishing the prognosis based on the obtained images.

[0201] 15. The method according to any one of items 11 to 14, wherein the cancer is selected from breast cancer, ovarian cancer, gastric cancer, colorectal cancer, prostate cancer, bladder cancer, salivary gland cancer, lung cancer, and esophageal cancer.

[0202] 16. The method according to item 15, wherein the cancer is breast cancer.

[0203] 17. A method for treating a mammalian subject, including a human, having cancer characterized by HER2 overexpression, comprising the step of administering to the subject an effective amount for treatment of a radiolabeled polypeptide according to any one of items 7 to 9, optionally included in the composition according to item 10.

[0204] 18. A method according to item 17, comprising, prior to the administration step, a step of preparing a radiolabeled polypeptide according to any one of items 7 to 9, wherein the step comprises mixing the polypeptide according to item 1 or 2 with a radionuclide suitable for treatment.

[0205] 19. The method according to item 17 or 18, wherein the cancer is selected from breast cancer, ovarian cancer, gastric cancer, colorectal cancer, prostate cancer, bladder cancer, salivary gland cancer, lung cancer, and esophageal cancer.

[0206] 20. The method according to item 19, wherein the cancer is breast cancer.

[0207] 21. HER2-binding polypeptides as described in item 1 or 2, for use as pharmaceuticals.

[0208] 22. A HER2-binding polypeptide for use as described in item 21, wherein the use described herein is a use described in any one of items 17 to 20.

[0209] 23. A HER2-binding polypeptide as described in item 1 or 2, for use as an in vivo diagnostic agent.

[0210] 24. A HER2-binding polypeptide for use as described in item 23, wherein the use described herein is a use described in any one of items 13 and 15-16.

[0211] 25. A HER2-binding polypeptide as described in item 1 or 2, for use as an in vivo prognostic agent.

[0212] 26. A HER2-binding polypeptide for use as described in item 23, wherein the use described herein is a use described in any one of items 14 to 16.

[0213] 27. A radiolabeled polypeptide as described in any one of items 3-6, for use as an in vivo diagnostic agent.

[0214] 28. A radiolabeled polypeptide for use as described in item 25, wherein the use is a use described in any one of items 13 and 15-16.

[0215] 29. A radiolabeled polypeptide described in any one of items 3-6, for use as an in vivo prognostic drug.

[0216] 30. A radiolabeled polypeptide for use as described in item 29, wherein the use is a use described in any one of items 14 to 16.

[0217] 31. Radiolabeled polypeptides as described in items 3 and 7-9, for use as pharmaceuticals.

[0218] 32. A radiolabeled polypeptide for use as described in item 31, wherein the use is a use described in any one of items 17 to 20.

[0219] 33. The composition described in item 10 for use as an in vivo diagnostic agent.

[0220] 34. The composition for use according to item 33, wherein the use is the use described in any one of items 13 and 15-16.

[0221] 35. The composition described in item 10 for use as an in vivo prognostic agent.

[0222] 36. The composition for use according to item 35, wherein the use is the use described in any one of items 14 to 16.

[0223] 37. The composition described in item 10 for use as a pharmaceutical.

[0224] 38. The composition for use according to item 37, wherein the use is the use described in any one of items 17 to 20.

[0225] 39. A nucleic acid encoding a polypeptide as described in item 1 or 2.

[0226] 40. An expression vector containing the nucleic acid described in item 39.

[0227] 41. Host cells containing the expression vector described in item 40.

Claims

1. A HER2-conjugated polypeptide containing the amino acid sequence AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LSESQGGGC (SEQ ID NO: 1).

2. The HER2-binding polypeptide according to claim 1, wherein the amino acid sequence consists of SEQ ID NO:

1.

3. A radiolabeled polypeptide comprising a radioactive chelate of a HER2-binding polypeptide and a radionuclide as described in claim 1 or 2.

4. The radiolabeled polypeptide according to claim 3, wherein the radionuclide is suitable for medical imaging.

5. The polypeptide according to claim 4, wherein the radioactive nuclide is selected from the group consisting of 99mTc, 51Mn, 52mMn, 52Mn, 186Re, and 188Re.

6. The polypeptide according to claim 4, wherein the radioactive nuclide is 99mTc.

7. The radiolabeled polypeptide according to claim 3, wherein the radionuclide is suitable for therapeutic use.

8. The radiolabeled polypeptide according to claim 6, wherein the radioactive nuclide is selected from the group consisting of 186 Re and 188 Re.

9. The radiolabeled polypeptide according to claim 6, wherein the radioactive nuclide is 186Re.

10. A composition comprising a HER2-binding polypeptide according to claim 1 or 2 or a radiolabeled polypeptide according to any one of claims 3 to 9, and at least one pharmaceutically acceptable excipient or carrier.

11. A HER2-binding polypeptide according to claim 1 or 2, for use as a pharmaceutical, as an in vivo diagnostic agent, or as an in vivo prognostic agent.

12. A radiolabeled polypeptide according to any one of claims 3 to 6, for use as an in vivo diagnostic agent.

13. The aforementioned use is a method for diagnosing cancer characterized by HER2 overexpression, i) An in vivo imaging method in mammals, including humans, that have or are suspected of having cancer characterized by HER2 overexpression, - Administering into the body of a mammalian subject a radiolabeled polypeptide according to any one of claims 3 to 6, which is optionally included in the composition according to claim 10, and - A method comprising the step of obtaining one or more images of at least a part of the body of a subject using a medical imaging device, wherein the images indicate the presence of radionuclides in the body, and ii) Use in a method comprising establishing the diagnosis based on the obtained image, the radiolabeled polypeptide for use according to claim 12.

14. A radiolabeled polypeptide according to any one of claims 3 to 6, for use as an in vivo prognostic agent.

15. The aforementioned use is a method for establishing the prognosis of cancer characterized by HER2 overexpression, i) An in vivo imaging method in mammals, including humans, that have or are suspected of having cancer characterized by HER2 overexpression, - Administering into the body of a mammalian subject a radiolabeled polypeptide according to any one of claims 3 to 6, which is optionally included in the composition according to claim 10, and - A method comprising the step of obtaining one or more images of at least a part of the body of a subject using a medical imaging device, wherein the images indicate the presence of radionuclides in the body, and ii) Use in a method comprising establishing the prognosis based on the obtained image, the radiolabeled polypeptide for use according to claim 14.

16. A radiolabeled polypeptide according to any one of claims 3 to 9, for use as a pharmaceutical.

17. The radiolabeled polypeptide for use according to claim 16, wherein the use is in a method of treating a mammalian subject, including a human, having cancer characterized by overexpression of HER2, the method comprising administering to the subject an effective amount for treatment of a radiolabeled polypeptide according to any one of claims 3 to 9, which is optionally included in the composition according to claim 10.

18. A nucleic acid encoding the polypeptide described in claim 1 or 2.

Images