WO2014013016A1

WO2014013016A1 - Method for determining the her2 status of a malignancy

Info

Publication number: WO2014013016A1
Application number: PCT/EP2013/065184
Authority: WO
Inventors: Jörgen Carlsson; Mattias SANDSTRÖM; Dan Sandberg; Jens SÖRENSEN
Original assignee: Affibody Ab
Priority date: 2012-07-20
Filing date: 2013-07-18
Publication date: 2014-01-23

Abstract

A method for determining the HER2 status of a malignancy is provided. The method comprises administering, to a mammalian subject having a malignancy suspected of being HER2 positive, a tracer molecule comprising a HER2 binding polypeptide; measuring the amount of said tracer molecule present in said malignancy using medical imaging equipment at two time points t₁and t₂ to yield a first and second value; comparing said first and second values, and if said second value is greater than said first value, concluding that the malignancy of said subject is HER2 positive.

Description

METHOD FOR DETERMINING THE HER2 STATUS OF A MALIGNANCY

Field of the invention

The present invention is related to the diagnosis and characterization of malignancies suspected of being characterized as HER2 positive.

Background

HER2 and its role in cancer diseases

The HER2 proto-oncogene encodes the production of a 185 kDa cell surface receptor protein known as the HER2 protein or receptor (Hynes NE et al (1994) Biochim Biophys Acta 1 198:165-184). This gene is also sometimes referred to as neu, HER2/neu or c-erbB-2. Neu was first discovered in rats that had been treated with ethyl nitrosourea, and exhibited mutation of this gene (Shih C et al (1981 ) Nature 290:261 -264). The mutated version of neu results in the production of a constitutively active form of the receptor, and constitutes a potent oncogene that can transform cells at low copy number (Hynes NE et al, supra).

Normal cells express a small amount of HER2 protein on their plasma membranes in a tissue-specific pattern. No known ligand to HER2 has been elucidated; however, HER2 has been shown to form heterodimers with HER1 (the epidermal growth factor receptor, EGFR), HER3 and HER4 in complex with the ligands for these receptors. Such heterodimer formation leads to the activated HER2 receptor transmitting growth signals from outside the cell to the nucleus, thus controlling aspects of normal cell growth and division (Sundaresan S et al (1999) Curr Oncol Rep 1 :16-22).

In tumor cells, errors in the DNA replication system may result in the existence of multiple copies of a gene on a single chromosome, which is a phenomenon known as gene amplification. Amplification of the HER2 gene leads to an increased transcription of this gene. This elevates HER2 mRNA levels and increases the concomitant synthesis of HER2 protein, which results in HER2 protein over-expression on the surface of these tumor cells. This over-expression can result in HER2 protein levels that are 10- to 100-fold greater than those found in the adjacent normal cells. This, in turn, results in increased cell division and a concomitantly higher rate of cell growth.

Amplification of the HER2 gene is implicated in transformation of normal cells to the cancer phenotype (Hynes NE et al, supra; Sundaresan S et al, supra).

Over-expression of HER2 protein is thought to result in the formation of homodimers of HER2, which in turn results in a constitutively active receptor (Sliwkowski MX ef a/ (1999) Semin Oncol 26(4 Suppl 12):60-70). Under these conditions, growth-promoting signals may be continuously transmitted into the cells in the absence of ligands. Consequently, multiple intracellular signal transduction pathways become activated, resulting in unregulated cell growth and, in some instances, oncogenic transformation (Hynes NE et al, supra). Thus, the signal transduction mechanisms mediated by growth factor receptors are important targets for inhibiting cell replication and tumor growth.

Breast cancer is the most common malignancy among women in the United States, with 226870 new cases projected to occur in 2012 (Siegel R et al (2012) CA Cancer J Clin 62:10-29). In approximately 25 % of all breast cancer patients, there is an over-expression of the HER2 gene due to amplification thereof (Slamon DJ ef a/ (1989) Science 244:707-712). This over-expression of HER2 protein correlates with several negative prognostic variables, including estrogen receptor-negative status, high S-phase fraction, positive nodal status, mutated p53, and high nuclear grade (Sjogren S et al (1998) J Clin Oncol 16(2):462-469). According to Slamon et al {supra), the amplification of the HER2 gene was found to correlate strongly with shortened disease-free survival and shortened overall survival of node-positive patients.

For these reasons, it has been, and is still, an important goal to further pursue investigations into the role of HER2 in the pathogenesis and treatment of breast cancer. The identification of molecules that interact with HER2 forms one part of this effort.

Preclinical in vitro studies have examined whether inhibition of HER2 activity could affect tumor cell growth. Treatment of SK-BR-3 breast cancer cells over-expressing HER2 protein with 4D5, one of several murine anti- HER2 monoclonal antibodies, did indeed inhibit tumor cell proliferation, compared with treatment with a control monoclonal antibody. Administration of 4D5 to mice bearing human breast and ovarian cancers (xenografts) that over-express HER2 prolonged their tumor-free survival time. Similar studies demonstrated the growth inhibition by anti-HER2 monoclonal antibodies in human gastric cancer xenografts in mice (Pietras RJ et al (1994) Oncogene 9:1829-1838).

Among the approaches to inhibiting the HER2 protein abundantly present on tumor cell surfaces with an antibody, one therapy has become commercially available during recent years. Thus, the humanized variant of monoclonal antibody 4D5, or trastuzumab, is marketed for this purpose by F Hoffman-La Roche and Genentech under the trade name of Herceptin^®.

Over-expression of HER2 has thus been described for breast cancer. It has also been connected to i.a. ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer (Holbro et al, Annu. Rev. Pharmacol. Toxicol. 2004. 44:195-217) and cancer in the esophagus (Ekman et al, Oncologist 2007; 12;1 165-1 177, see in particular pages 1 170-1 171 ).

Notwithstanding the obvious advantages shown by antibody therapy against cancers characterized by over-expression of HER2 protein, the fact remains that a variety of factors have the potential of reducing antibody efficacy (see e.g. Reilly RM ef a/ (1995) Clin Pharmacokinet 28:126-142). These include the following: (1 ) limited penetration of the antibody into a large solid tumor or into vital regions such as the brain; (2) reduced extravasation of antibodies into target sites owing to decreased vascular permeability; (3) cross-reactivity and nonspecific binding of antibody to normal tissues, reducing the targeting effect; (4) heterogeneous tumor uptake resulting in untreated zones; (5) increased metabolism of injected antibodies, reducing therapeutic effects; and (6) rapid formation of HAMA and human antihuman antibodies, inactivating the therapeutic antibody.

In addition, toxic effects have been a major obstacle in the

development of therapeutic antibodies for cancer (Carter P (2001 ) Nat Rev Cancer 1 :1 18-129; Goldenberg DM (2002) J NucI Med 43:693-713; Reichert JM (2002) Curr Opin Mol Ther 4:1 10-1 18). Cross-reactivity with healthy tissues can cause substantial side effects for unconjugated (naked) antibodies, which side effects may be enhanced upon conjugation of the antibodies with toxins or radioisotopes. Immune-mediated complications include dyspnoea from pulmonary toxic effects, occasional central and peripheral nervous system complications, and decreased liver and renal function. On occasion, unexpected toxic complications can be seen, such as the cardiotoxic effects associated with the HER2 targeting antibody trastuzumab (Schneider JW et al (2002) Semin Oncol 29(3 suppl 1 1 ):22-28). Radioimmunotherapy with isotope-conjugated antibodies also can cause bone marrow suppression.

Despite the recent clinical and commercial success of the currently used anticancer antibodies, a substantial number of important questions thus remain concerning the future of the use of antibodies. As a consequence, the continued provision of agents with a comparable affinity for HER2 remains a matter of substantial interest within the field, as well as the provision of uses of such molecules in the diagnosis and treatment of disease.

HER2 binding Z variant molecules and diagnostic use thereof

Molecules related to protein Z, derived from domain B of

staphylococcal protein A (SPA) (Nilsson B ef a/ (1987) Protein Engineering 1 , 107-133), have been selected from a library of randomized such molecules using different interaction targets (see e.g. WO95/19374; Nord K et al (1997) Nature Biotechnology 15, 772-777; WO2005/000883; WO2005/075507;

WO2006/092338; WO2007/065635). In WO2005/003156, a substantial number of Z variants with an ability to interact with HER2 is disclosed. Baum et al (2010), J Nucl Med 51 (6):892-897, describes the use of one of these variants, denoted Z_HER2:342 or ABY-002, for molecular imaging in human patients.

WO2009/080810 discloses HER2-binding polypeptides with a re- engineered scaffold compared to the Z variants of WO2005/003156, as well as use of such re-engineered polypeptides for the diagnosis in general of cancer diseases in mammalian subjects characterized by the over-expression of HER2. In the experiments of WO2009/080810, the new polypeptides are used for molecular imaging studies in mice with a view to visualize HER2- bearing tumors. These experiments are also disclosed in Ahlgren et al (2010), J Nucl Med 51 (7):1 131 -1 138.

On a poster presentation (P2-09-01 ) at the San Antonio Breast Cancer Symposium held 6-10 December 201 1 in San Antonio, TX, USA, authors Sandberg et al disclosed the use of one of the polypeptides disclosed in WO2009/080810 for medical imaging in human patients. The results were also summarized in the conference abstracts published in Cancer Res 71 (24 Suppl):273s (201 1 ). Disclosure of the invention

It is an object of the present invention to provide additional insights into the diagnosis and prognosis of malignancy disease suspected to feature HER2 over-expression.

Another object is to provide a new method for discriminating between HER2-positive and HER2-negative manifestations of cancer.

These and other objects are met by the different aspects of the invention as claimed in the appended claims.

Thus, the invention provides a method for determining the HER2 status of a malignancy, comprising

• administering, to a mammalian subject having a malignancy suspected of being HER2 positive, a tracer molecule comprising

- a HER2 binding polypeptide which comprises the amino acid sequence

EXiRNAYWEIA LLPNLTNQQK RAFIRKLYDD PSQSSELLX₂E AKKLNDSQ

wherein Xi in position 2 is M, I or L, and X2 in position 39 is S or C

(SEQ ID NO:1 ); and

- a radionuclide suitable for medical imaging;

• measuring the amount of said tracer molecule present in said malignancy using medical imaging equipment

- at a first time point ti within the range from 15 minutes to 6 hours from administration, to yield a first value; and - at a second time point t₂ within the range from 75 minutes to 42 hours from administration and at least 60 minutes after ti, to yield a second value;

• comparing said first and second values, and

- if said second value is greater than said first value, concluding that the malignancy of said subject is HER2 positive; and

- if said second value is equal to or less than said first value, concluding that the malignancy of said subject is HER2 negative. The purpose of the method according to the invention is to determine the status of a malignancy with regard to HER2. The HER2 status of a malignancy may for example consist in an over-expression of the HER2 receptor on the surfaces of tumor cells, in which case the malignancy is considered to be "HER2 positive". Presence of HER2 over-expression, in turn, indicates susceptibility towards HER2-specific treatment options, such as administration of HER2-specific antibodies or other binding molecules with affinity for the HER2 receptor. As explained in the background section, several such HER2-specific treatment options are available on the market (e.g. Herceptin®) and in development. Thus, in a related aspect, the invention also provides a method of treatment of a HER2 positive malignancy, comprising i) concluding that a malignancy in a subject is HER2 positive using the determining method according to the invention, and ii) treating the malignancy using HER2-specific treatment.

Thus, the inventive method may for example be useful in diagnosis and/or molecular characterization of a cancer selected from the non-limiting group of breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer, prostate cancer and cancer in the esophagus. In this context, molecular characterization relates to the characterization of HER2 expression, i.e. for example to determine whether or not there is any expression of HER2, to determine the amount of HER2 expression, e.g.

before and after treatment which can be accomplished by obtaining images before and after treatment, and to determine the extent or anatomical content of HER2 expression, e.g. for surgical purposes. Administration of tracer molecule to the mammalian subject to be investigated may be done in any known way, including by intravenous injection.

Measuring the amount of tracer molecule present in the malignancy is performed using medical imaging equipment in known fashion, such as through acquiring radioactivity counts or images of radiation density, or derivatives thereof such as radiation concentration calculated from

attenuation-corrected radioactivity count images, for example from SPECT or PET.

In one embodiment of the determining method according to the invention, the medical imaging equipment used is positron emission tomography (PET) equipment and said radionuclide is suitable for medical imaging using PET. The skilled person is aware of what radionuclides are available for use with PET. For example, the radionuclide may be selected from the group consisting of ⁶⁸Ga, ^110mln, ¹⁸F, ⁴⁵Ti, ⁴⁴Sc, ⁶¹Cu, ⁶⁶Ga, ⁶⁴Cu, ⁵⁵Co, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ¹²⁴l and ⁷⁶Br.

Due to the logistics involved in a PET examination of a subject, combined with the typical half-lifes of the radionuclides used and the desire to obtain a good discriminatory power between HER2 positive and HER2 negative malignancies, time points ti and t₂ may be selected according to the following general, non-limiting, guidelines when the determination method according to the invention uses PET.

In one embodiment, when PET scanning is used, time point ti is within the range from 30 minutes to 6 hours from administration.

In one embodiment, when PET scanning is used, time point t₂ is within the range from 150 minutes to 36 hours from administration.

In a more specific embodiment, time point ti is within the range from 30 to 90 minutes from administration. In such cases, the radionuclide may in particular be selected from the group consisting of ⁶⁸Ga, ^110mln, ¹⁸F, ⁴⁵Ti, ⁴⁴Sc and ⁶¹Cu. In such embodiments, time point t₂ may for example be within the range from 150 minutes to 8 hours from administration. In particular embodiments using ⁶⁸Ga and ^110mln, time point t₂ may for example be within the range from 150 minutes to 5 hours from administration.

In an alternative specific embodiment, time point ti is within the range from 2 to 6 hours from administration. In such cases, the radionuclide may in particular be selected from the group consisting of ⁶⁶Ga, ⁶⁴Cu, ⁵⁵Co, ⁷² As, ⁸⁶Y, ⁸⁹Zr, ¹²⁴l and ⁷⁶Br. In such embodiments, time point t₂ may for example be within the range from 20 to 36 hours from administration.

In one embodiment of the determining method according to the invention, the medical imaging equipment used is single-photon emission computed tomography (SPECT) equipment and said radionuclide is suitable for medical imaging using SPECT. The skilled person is aware of what radionuclides are available for use with SPECT. For example, the

radionuclide may be selected from the group consisting of ¹¹¹ In, ^99mTc, ¹²³l, ¹³¹ l and ⁶⁷Ga.

Due to the logistics involved in a SPECT examination of a subject, combined with the typical half-lifes of the radionuclides used and the desire to obtain a good discriminatory power between HER2 positive and HER2 negative malignancies, time points ti and t₂ may be selected according to the following, non-limiting, general guidelines when the determination method according to the invention uses SPECT.

In one embodiment, when SPECT scanning is used, time point ti is within the range from 30 minutes to 6 hours from administration, such as from 30 to 90 minutes or from 2 to 6 hours.

In one embodiment, when SPECT scanning is used, time point t₂ is within the range from 16 to 36 hours from administration. In more specific embodiments, time point t₂ may be within the range from 150 minutes to 24 hours from administration, such as from 16 to 24 hours.

In one embodiment, when SPECT scanning is used, time point ti is within the range from 2 to 6 hours from administration, and t₂ is within the range from 20 to 36 hours from administration. In such cases, the

radionuclide may in particular be selected from the group consisting of ¹¹¹ In, In another embodiment, time point ti is within the range from 30 to 90 minutes from administration, and t₂ is within the range from 150 minutes to 24 hours from administration, such as from 16 to 24 hours. In such cases, the radionuclide may in particular be ^99mTc, but may in certain cases also be any one of the other indicated SPECT radionuclides.

Even more specific embodiments of the determining method according to the invention are provided in the following Table 1 , in which specific ranges for ti and t₂ are provided for each one of a list of radionuclides contemplated for use with the invention:

Table 1 : Selection of ti and t₂ ran es for certain radionuclides

Regardless of the general guiding time points given in the table above, it may also frequently be possible to use any of the listed SPECT

radionuclides within the time frames typical for PET examination, such as listed for ^99mTc in the table. Depending on the selection of radionuclide and imaging equipment, and taking the above information into account, the skilled person is able to find measuring time points ti and t₂ that afford the necessary discriminatory power in order to distinguish HER2 positive from HER2 negative

malignancies. For further guidance, and regardless of radionuclide and medical imaging equipment used, the following relationship may be useful as a non-limiting approximation in order to guide selection of the time points for measuring the amount of tracer: 5^*ti < t₂ < 7* . The determining method according to the invention also comprises a step of comparing the measured values and making a conclusion concerning the malignancy's HER2 status. As stated above, if the second value is greater than the first value, i.e. if the amount of measured tracer has increased from time point ti to time point t₂, this warrants a conclusion that the malignancy is HER2 positive. In a more specific embodiment, the criterion for HER2 positivity is that the second value is more than 20 % greater than the first value, for example that the second value is more than 25 %, more than 30 %, more than 35 %, more than 40 %, more than 45 %, more than 50 %, more than 55 %, more than 60 %, more than 65 %, more than 75 %, more than 100 %, more than 125 %, more than 150 %, more than 200 %, more than 300 %, more than 400 %, more than 500 %, more than 750 %, or more than 1000 % greater than the first value.

With regard to the tracer molecule for use in the determining method according to the invention, it comprises a HER2 binding polypeptide, which has an amino acid sequence that comprises

EXiRNAYWEIA LLPNLTNQQK RAFIRKLYDD PSQSSELLX₂E AKKLNDSQ wherein Xi in position 2 is M, I or L, and X₂ in position 39 is S or C

(SEQ ID NO:1 ), or in some cases more preferably

YAKEX1 RNAYW EIALLPNLTN QQKRAFIRKL YDDPSQSSEL

LX₂EAKKLNDS Q

wherein Xi in position 5 is M, I or L, and X₂ in position 42 is S or C (SEQ ID NO:2), or in some cases more preferably

AEAKYAKEXiR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ

SSELLX₂EAKK LNDSQ

wherein Xi in position 9 is M, I or L, and X₂ in position 46 is S or C

(SEQ ID NO:3), or in some cases more preferably

ESEKYAKEX₁R NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ

SSELLX₂EAKK LNDSQ,

wherein Xi in position 9 is M, I or L, and X₂ in position 46 is S or C

(SEQ ID NO:4).

A polypeptide comprising any of the sequences SEQ ID NO: 1 -4 exhibits advantages in connection with the inventive method, for example in comparison with the HER2 binding polypeptides disclosed in

WO2005/003156, while retaining the capacity of those previously described polypeptides to bind the target HER2. Non-limiting examples of such advantages, exhibited by one or more embodiments of the polypeptide according to the invention, are listed on pages 6-7 of WO2009/080810.

For the purpose of carrying out the inventive method, different modifications of, and/or additions to, the polypeptide may be performed in order to tailor the polypeptide to the specific use intended. Such modifications and additions are described in more detail below, and may comprise additional amino acids comprised in the same polypeptide chain, or labels that are chemically conjugated or otherwise bound to the polypeptide.

Expressions like "binding affinity for HER2", "HER2 binding" and the like refer to a property of a polypeptide which may be tested e.g. by the use of surface plasmon resonance technology, such as in a Biacore^® instrument (GE Healthcare). HER2 binding affinity may be tested in an experiment wherein HER2, or a fragment thereof, e.g. the extracellular domain, or a fusion protein thereof, is immobilized on a sensor chip of the instrument, and a sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing HER2, or a fragment thereof, e.g. the extracellular domain, is passed over the chip. The skilled person may then interpret the sensorgrams obtained to establish at least a qualitative measure of the polypeptide's binding affinity for HER2. If a quantitative measure is sought, e.g. with the purpose to establish a certain K_D value for the interaction, it is again possible to use surface plasmon resonance methods. Binding values may e.g. be defined in a Biacore^® 2000 instrument (GE Healthcare). HER2, or a fragment thereof, e.g. the extracellular domain, is immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. K_D values may then be calculated from the results, using e.g. the 1 :1 Langmuir binding model of the BIAevaluation 3.2 software provided by the instrument manufacturer.

The invention also encompasses using polypeptides in which the HER2 binding polypeptide described above is present as a HER2 binding domain, to which additional amino acid residues have been added at either terminal. These additional amino acid residues may play a role in the binding of HER2 by the polypeptide, but may equally well serve other purposes, related for example to one or more of the production, purification,

stabilization, coupling or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for purposes of chemical coupling. An example of this is the addition of a cysteine residue, for example at the very first or very last position in the polypeptide chain, i.e. at the N or C terminus. A cysteine residue to be used for chemical coupling may also be introduced by replacement of another amino acid on the surface of the protein domain, preferably on a portion of the surface that is not involved in target binding. Such additional amino acid residues may also comprise a "tag" for purification or detection of the polypeptide, such as a hexahistidyl (His6) tag, or a "myc" tag or a "FLAG" tag for interaction with antibodies specific to the tag. The skilled person is aware of other alternatives.

In a specific embodiment of the inventive methods, using a polypeptide which has additional amino acid residues, the HER2 binding polypeptide comprises the amino acid sequence AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5).

In another specific embodiment, the HER2 binding polypeptide comprises the amino acid sequence

ESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK (SEQ ID NO:6).

The "additional amino acid residues" discussed above may also constitute one or more polypeptide domain(s) with any desired function, such as the same binding function as the first, HER2-binding domain, or another binding function, or an enzymatic function, or a fluorescent function, or mixtures thereof.

Thus, the inventive methods encompass using multimers of a polypeptide comprising any of the sequences SEQ ID NO: 1 -6. It may be of interest in the present methods to obtain even stronger binding of HER2 than is possible with one domain of a polypeptide. In this case, the provision of a multimer, such as a dimer, trimer or tetramer, of the polypeptide may provide the necessary avidity effects. The multimer may consist of a suitable number of HER2 binding polypeptide domains as disclosed herein, having the same or different amino acid sequences. The linked polypeptide "units" in such a multimer may be connected by covalent coupling using known organic chemistry methods, or expressed as one or more fusion polypeptides in a system for recombinant expression of polypeptides, or joined in any other fashion, either directly or via a linker, for example an amino acid linker. The invention encompasses using polypeptides in which the HER2 binding polypeptide described above has been provided with a label group, such as at least one radioactive isotope, for example for purposes of detection of the polypeptide. In particular, the invention encompasses using a radiolabeled polypeptide consisting of a radiochelate of a HER2 binding polypeptide as described above and a radionuclide, such as a radioactive metal.

A majority of radionuclides have a metallic nature and metals are typically incapable to form stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of targeting proteins with radioactive metals is performed with the use of chelators, multidentate ligands, which form non-covalent compounds, called chelates, with the metal. In an embodiment of the invention, the coupling of a radionuclide to the HER2 binding polypeptide is enabled through the provision of a chelating

environment, through which the radionuclide may be coordinated, chelated or complexed to the polypeptide.

In some specific embodiments of the methods according to the invention, a polyaminopolycarboxylate chelator is used to couple a

radionuclide to the HER2 binding polypeptide. Preferably, the polypeptide then comprises at least one cysteine, and most preferably it compises only one cysteine. The cysteine(s) may be present in the polypeptide either initially or added at a later stage as one or more additional amino acids.

A skilled person could also foresee a number of other chelators, capable to chelate such cores as "naked " Me, Me=O, O=Me=O, Me≡N, Me(CO)3, or HYNIC-Me-co-ligand(s) core (wherein Me is a radioactive isotope of Tc or Re), which can be attached to a polypeptide site-specifically during peptide synthesis or conjugated to a recombinantly produced polypeptide using known conjugation chemistry for radiolabeling. Preferably, such chelators have a hydrophilic character. A good overview of such chelators is provided in Liu S and Edwards DS (1999) Chem Rev. 99(9):2235- 68.

One can distinguish two classes of polyaminopolycarboxylate chelators: macrocyclic and acyclic.

The most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetraacetic acid) (see below). Several different DOTA-based compounds suitable for use as chelators with the HER2 binding polypeptides in the methods according to the invention are commercially available, for example from Macrocyclics Inc., USA, and examples are shown below:

A B

benzyl-DOTA, C is DOTA-TFP ester, and D is thiol-reactive maleimido-mono- amide DOTA.

The high kinetic inertness, i.e. the slow rate of dissociation of metal from DOTA, favors stable attachment of a radionuclide. However, elevated temperatures are required for labeling due to a slow association rate. For this reason, DOTA derivatives are widely used for labeling of short peptides, which are relatively insensitive to heating to 60-90 °C.

One preferred derivative for use as chelator in the present invention is

1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tris-acetic acid-10- maleimidoethylacetamide.

As disclosed above, the HER2 binding polypeptide may for example comprise the amino acid sequence SEQ ID NO:5. In a more specific embodiment, the polypeptide comprises SEQ ID NO:5 and has a

tetraazacycio compound coupled to amino acid residue C61 . In a particularly preferred embodiment, the tetraazacycio compound is 1 ,4,7,10- tetraazacyclododecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide, which has been coupled to the polypeptide through reaction of the SH group of residue C61 with the maleimido moiety of the tetraazacycio compound. One particularly preferred embodiment of the invention is thus a HER2 binding polypeptide comprising the amino acid sequence

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide.

The most commonly used acyclic polyaminopolycarboxylate chelators are different derivatives of DTPA (diethylenetriamine-pentaacetic acid), such as those shown in the schematic below, where A is DTPA, B is the amino- reactive derivative isothiocyanatobenzyl-DTPA, C is the amino-reactive derivative semirigid 2-(para-isothiocyanatobenzyl)-6-methyl-DTPS (IB4M), and D is the amino-reactive derivative CHX-A"-DTPA.

It has been found that backbone-modified semi-rigid variants of DTPA provide adequate stability for labeling with ⁹⁰Y of e.g. Zevalin^®. Though acyclic chelators are less inert, and consequently, less stable than

macrocyclic ones, their labeling is rapid enough even at ambient temperature. For this reason, they might be preferred for labeling of monoclonal antibodies, which cannot tolerate heating. Detailed protocols for coupling of polyaminopolycarboxylate chelators to targeting proteins and peptides have been published by Cooper and co-workers (Nat. Protoc. 1 : 314-7. 2006) and Sosabowski and Mather (Nat. Protoc. 1 : 972-6, 2006).

A HER2 binding polypeptide for use in the methods according to the invention coupled to a polyaminopolycarboxylate chelator is useful to provide a radiolabeled polypeptide consisting of a radiochelate of the HER2 binding polypeptide coupled to the chelator and a radionuclide suitable for medical imaging, said radionuclide for example being selected from the group consisting of ¹¹¹ ln, ⁶⁸Ga, ⁶⁴Cu, ⁸⁹Zr, ⁴⁵Ti, ⁵⁵Co, ⁸⁶Y, ⁶¹Cu, ⁶⁶Ga, ⁶⁷Ga, ^{1 10m}ln and ⁴⁴Sc, wherein the radionuclide is complexed with the HER2 binding polypeptide via the chelating environment.

One particularly preferred radiolabeled polypeptide for use in the methods according to the invention is a radiochelate of a HER2 binding polypeptide having the amino acid sequence

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide and ¹¹¹ ln.

One particularly preferred radiolabeled polypeptide for use in the methods according to the invention is radiochelate of a HER2 binding polypeptide having the amino acid sequence

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide and ⁶⁶Ga, ⁶⁷Ga or ⁶⁸Ga, in particular ⁶⁸Ga.

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide and ⁶¹Cu or ⁶⁴Cu, in particular ⁶⁴Cu.

One particularly preferred embodiment of a radiolabeled polypeptide according to the invention is radiochelate of a HER2 binding polypeptide having the amino acid sequence

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-ths-acetic acid-10-maleimidoethylacetamide and ⁸⁹Zr.

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-ths-acetic acid-10-maleimidoethylacetamide and ⁵⁵Co.

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C (SEQ ID NO:5) coupled to 1 ,4,7,10-tetraazacyclo- dodecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide and ⁸⁶Y.

In an alternative embodiment of the invention, the polypeptide comprises a tetradentate chelating environment characterized as an N₃S chelator. As the term N₃S indicates, the four attaching 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 attachment of the radioactive metal.

An N₃S chelator may be provided through a suitable choice of amino acid residues in the amino acid sequence of the polypeptide.

For example, an N₃S chelating environment may be provided through the coupling of mercaptoacetyl to the N-terminus of the polypeptide. In this embodiment, the mercaptoacetyl provides the necessary S atom, whereas the first three N atoms in the peptide backbone constitute the three N atoms of the tetradentate chelator. Preferably, mercaptoacetyl is coupled to a polypeptide comprising the amino acid sequence SEQ ID NO:4 as the most N-terminal amino acid sequence, resulting in a polypeptide comprising the amino acid sequence maESEKYAKEXiR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ

SSELLX₂EAKK LNDSQ,

wherein ma is mercaptoacetyl, Xi in position 9 is M, I or L, and X₂ in position 46 is S or C. In a particularly preferred embodiment, the polypeptide has the sequence:

maESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ

SSELLSEAKK LNDSQAPK,

wherein ma is mercaptoacetyl (SEQ ID NO:7).

In an alternative embodiment, the three N groups may be provided by the N atoms of three consecutive peptide bonds in the polypeptide chain, of which the last is a cysteine residue, which comprises an SH group on its side- chain. The S atom of the cysteine side-chain constitutes the S atom in the N₃S chelator. Alternatively speaking, the N₃S chelator is provided in the form of a tripeptide sequence XXC, wherein X is any amino acid residue. Such a tripeptide sequence may be comprised in the polypeptide according to the invention either initially or added as one or more additional amino acids. It is preferred that the cysteine residue is the one situated at the C-terminal end of SEQ ID NO:5 or that an additional cysteine is situated at the C-terminal of any of SEQ ID NO:1 -4 or 6-7, optionally with additional amino acid residues between the last amino acid residue of SEQ ID NO:1 -4 or 6-7 and the C- terminal cysteine. The cysteine residue may also be followed by one or more other amino acid residue(s).

A polypeptide which comprises a tetradentate chelating environment characterized as an N₃S chelator, provided either by mercaptoacetyl coupled to the N-terminal and the nitrogen atoms of three consecutive peptide bonds, or by the nitrogen atoms of three consecutive peptide bonds and a cysteine residue at the C-terminal of the polypeptide, may be used to provide a radiolabeled polypeptide consisting of a radiochelate of the HER2 binding polypeptide and a radionuclide suitable for medical imaging, said radionuclide in particular embodiments being ^99mTc (see for example Engfeldt et al (2007) Eur. J. Nucl. Med. Mol. Imaging 34(5):722-22; Engfeldt ef a/ (2007) Eur. J. Nucl. Med. Mol. Imaging 34(1 1 ):1843-53). In some embodiments, the radionuclide is complexed with the HER2 binding polypeptide via the chelating environment.

When the polypeptide comprises two, three or more XXC tripeptides, it is preferably the terminal one that is used to complex the radionuclide. When the polypeptide is radiolabeled in this way, the other XXC tripeptide(s) is/are preferably protected.

One particularly preferred embodiment of a radiolabeled polypeptide for use in a method according to the invention is a radiochelate of a HER2 binding polypeptide having the amino acid sequence maESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK, wherein ma is mercaptoacetyl (SEQ ID NO:7) and ^99mTc.

A radiolabeled polypeptide for use in the methods according to the invention may also be obtained by indirect labeling of a HER2 binding polypeptide as described above. Preferably, the polypeptide comprises at least one cysteine, and most preferably it comprises only one cysteine. The cysteine(s) may be present in the polypeptide either initially or added at a later stage as one or more additional amino acids. For labeling with various radionuclides, for example selected from the group consisting of ¹⁸F, ⁷⁶Br, ⁷²As, ¹²³l, ¹²⁴l and ¹³¹1, intermediate "linker molecules" are used for labeling. Such a linker should contain two functional moieties, one providing rapid and efficient radiolabeling, and another enabling rapid and efficient coupling to the proteins, e.g. to amine groups, or preferably to the thiol group of a unique cysteine. For example a malemide group reacts with thiol groups to form a stable thioether bond. It is possible to first react the "linker molecule" with the radiolabel and subsequently with the thiol group of the protein, as described for example in Kramer-Marek G et al (2008), EJNMMI 35:1008-1018 and Kiesewetter et al (2008), J Fluor Chem 129:799-805, using ¹⁸F as the radiolabel. Alternatively, the linker molecule is first reacted with the thiol group of the protein, and then conjugated to the radiolabel. This has for example been described by Cheng et al (2008), J Nucl Med 49:804-13, again using ¹⁸F as the radioactive isotope. Several alternatives have been thoroughly investigated for

radioiodination, where radiolabeling of the linker molecule has been done e.g. on an activated phenolic ring or an aromatic ring with a suitable leaving group. A detailed protocol for preparation of non-labeled linker and indirect radioiodination using N-succinimidyl 3-[^*l]iodobenzoate is provided in

Vaidyanathan G and Zalutsky MR (2006), Nat Protoc 1 (2):707-13. An example of indirect radioiodination using N-succinimidyl trimethylstannyl- benzoate is illustrated in the schematic below.

In this schematic, the linker molecule is first radioiod inated in acidic conditions and then coupled to free amine (N-terminal of the ε-amino group of lysine) in alkaline conditions. Both meta- and para-iododerivatives of benzoate have been described in the literature.

The linker molecule may comprise an aryl group linking the radiolabel

(e.g. at the 2-, 3- or 4- position), provided that when the radiolabel is ⁷⁶Br, then the linker molecule does not include a phenolic OH-group. Non-limiting examples of linker molecules comprises a heterocyclic imide that can be used to link the inventive polypeptide and radiolabel include N-[2- benzamidoethyl]malemide, 4-maleimidobenzophenone (BPMal), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride (MPBH), and maleimidobenzoyl-N- hydroxysuccinimide ester (MBS).

N-[2-benzamidoethyl]malemide may be reacted with ¹⁸F to form N-[2- (4-(¹⁸F-fluorobenzamido)ethyl]malemide. Likewise it may be reacted with ⁷⁶Br as described by Cai et al (J. Nucl. Med. 47:1 172-80, 2006). To summarize, the method of the present invention may, in their various embodiments, use as tracer molecule the variants described above. In other words, when referring to the "HER2 binding polypeptide" in this description, this term is intended to encompass the HER2 binding polypeptide alone, but also all those molecules based on this polypeptide described above that e.g. incorporate the HER2 binding polypeptide as a moiety in a fusion protein and/or are conjugated to a label, a chelator, a therapeutic and/or diagnostic agent and/or are provided with additional amino acid residues as a tag or for other purposes.

In one embodiment of the method according to the invention, the method comprises, before the administration step, a preparatory step of preparing a tracer molecule, which step comprises mixing of a HER2 binding polypeptide as defined herein with a radionuclide suitable for medical imaging.

In a more specific embodiment, the method comprises, before the administration step, a preparatory step of preparing a radiolabeled

polypeptide of i) a polypeptide whose amino acid sequence consists of SEQ ID NO:5 conjugated to 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tris-acetic acid- 10-maleimidoethylacetamide with ii) ¹¹¹ In, which step comprises mixing the conjugate with ¹¹¹ In in an appropriate buffer preventing formation of non- soluble colloids, for example (but not limited to) acetate or citrate buffer.

In another specific embodiment of the imaging method according to the invention, the method comprises, before the administration step, a

preparatory step of preparing a radiolabeled polypeptide of i) a polypeptide whose amino acid sequence consists of SEQ ID NO:5 conjugated to 1 ,4,7,10- tetraazacyclododecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide with ii) ⁶⁶Ga, ⁶⁷Ga or ⁶⁸Ga, in particular ⁶⁸Ga, which step comprises mixing the conjugate with ⁶⁶Ga, ⁶⁷Ga or ⁶⁸Ga in an appropriate buffer preventing formation of non-soluble colloids, for example (but not limited to) acetate or citrate buffer. In another specific embodiment of the imaging method according to the invention, the method comprises, before the administration step, a

preparatory step of preparing a radiolabeled polypeptide of i) a polypeptide whose amino acid sequence consists of SEQ ID NO:7 with ii) ^99mTc, which step comprises mixing the polypeptide with ^99mTc-pertechnetate in the presence of appropriate reducing agent, e.g. stannous chloride or fluoride, in an appropriate buffer. This might be performed also in the presence of an intermediate weak chelator, e.g. tartrate, or citrate or gluconate.

preparatory step of preparing a radiolabeled polypeptide of i) a polypeptide whose amino acid sequence consists of SEQ ID NO:5 conjugated to 1 ,4,7,10- tetraazacyclododecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide with ii) ⁶¹Cu or ⁶⁴Cu, in particular ⁶⁴Cu, which step comprises mixing the conjugate with ⁶¹Cu or ⁶⁴Cu in an appropriate buffer preventing formation of non-soluble colloids, for example (but not limiting to) acetate or citrate buffer.

In some embodiments of the method of the invention, said mammalian subject is a human being.

In some embodiments of the method of the invention, said malignancy originates from a cancer disease selected from breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer, prostate cancer and cancer in the esophagus.

Also provided, in yet another aspect of the invention, is a radiolabeled polypeptide as described above for use as the tracer molecule in any one of the inventive methods described herein. The invention will now be illustrated in detail through the description of experiments conducted in accordance therewith. The examples to follow are not to be interpreted as limiting. In the examples, reference is made to the appended figures. Brief description of the figures

Figure 1 shows a plot of average [¹¹¹ ln]ABY-025 tumor kinetics in breast cancer lesions that are HER2 positive (a HercepTest score on staining of 3+, n = 4, one lesion per subject) or HER2 negative (a HercepTest score on staining of 1 + ,n = 2, or 0 ,n = 1 , in a total of two subjects).

Figure 2 shows a sub-analysis of average tumor uptake of [¹¹¹ ln]ABY- 025 of all relevant lesions detected by FDG-PET (from 1 to 15 lesions per subject ) in HER2 positive subjects (n = 4). When normalized to the amount of radioactivity measured at 4 h after injection, the uptake increased at the 24 h time point (non-parametric Kruskal-Wallis ANOVA, p = 0.01 ).

Figure 3 shows a sub-analysis of average tumor uptake of [¹¹¹ ln]ABY- 025 of all relevant lesions detected by FDG-PET (from 5 to 35 lesions per subject) in HER2 negative subjects (n = 3). When normalized to the amount of radioactivity measured at 4 h after injection, the uptake decreased at the 24 h time point and tended to decrease further at 48 h (non-parametric Kruskal-Wallis ANOVA, p = 0.04).

Figure 4 shows a box plot of average [¹¹¹ ln]ABY-025 uptake in all relevant lesions detected by FDG-PET (from 1 to 35 lesions per subject). When normalized to the 4 h time point, there is a significant difference

(p < 0.05) on the group level between HER2 positive (n = 4) and HER2 negative (n = 3) subjects.

Example 1

Summary

A clinical study was conducted at Uppsala University Hospital, in which 7 subjects with metastatic breast cancer were investigated with gamma camera imaging of [¹¹¹ ln]ABY-025 uptake in tumors, to evaluate HER2 expression.

The data analysis shows that HER2 positive (n = 4 at subject level) and HER2 negative (n = 3 at subject level) breast cancer metastases can be differentiated with 100 % specificity and sensitivity using [¹¹¹ ln]ABY-025 SPECT imaging and studying the change in uptake between two different time points.

Materials and methods

Test product and dose: The tracer molecule denoted ABY-025 in the following is the HER2 binding polypeptide with SEQ ID NO:5 coupled to DOTA as described above. Following the procedure described in Example 3a of WO2009/080810, ABY-025 was labeled with ¹¹¹ In and used in the following experiments. For each subject, approximately 100 g ABY-025 was labeled with approximately 150 MBq ¹¹¹ In and administered in a single dose as an intravenous injection.

Subject disposition: The subjects were enrolled into the following groups, based on the HER2 expression in their original primary tumors as determined by the standard commercial test HercepTest™:

Study procedure: All subjects were first evaluated with [¹⁸F]FDG PET imaging before administration of [¹¹¹ ln]ABY-025. After administration, subjects were evaluated with gamma camera for Whole Body Scans (WBS) and Single Photon Emission Computed Tomography (SPECT). For WBS, the scan time points were 0.5, 4, 24 and 48 hours post injection. For SPECT, two scans were made at each time point and the intended scan time points were 4, 24 and 48 hours.

Biopsies were obtained from current metastases in all subjects except 006, and tested for HER2 status using HercepTest™. Evaluation of diagnostic efficacy

(A) Tumor-to-normal uptake ratio at 24 hours post administration

Column 3 in the table below was derived by taking the [¹¹¹ ln]ABY-025 uptake value (measurement of radioactivity counts concentration from SPECT imaging data at the 24 hr imaging time point) in a representative metastasis ("lesion") and dividing that uptake value with a background value from a region in the lung.

5 A "high" tumor-to-lung ratio (>5) for the ABY-025 value was obtained in all those metastases that exhibited a high HER2 expression (= 3+) in the biopsy IHC test.

Subject 004 was originally diagnosed as having a HER2 positive primary tumor, but the current metastases were HER2 negative. Re- 10 evaluation of the primary tumor showed that it was heterogeneous with both HER2 positive and HER2 negative cell clones. Apparently, the HER2 negative clones had caused the current metastatic disease. This illustrates the potential clinical utility of re-assessing the HER2 status during clinical progression of disease, using imaging with [¹¹¹ ln]ABY-025.

15

(B) Change in tumor uptake from 4 to 24 hrs

A different approach is to compare the uptake kinetics within each tumor by defining "baseline" as the radioactivity concentration at a first time point, here at 4 h post administration, and then normalize the uptake at a 20 second time point by dividing it with the uptake measured at the first time point. The result is expressed below as "percent of uptake at 4 h", i.e. the baseline is set to 100 %.

As shown below in different types of figures, all HER2 positive tumors increase the uptake from 4 to 24 h, while all HER2 negative tumors decrease 25 their uptake. Example 2 Summary

A clinical study is conducted at Uppsala University Hospital, in which subjects with metastatic breast cancer are investigated with PET imaging of [⁶⁸Ga]ABY-025 uptake in tumors, to evaluate HER2 expression.

The data analysis is expected to show that HER2 positive and HER2 negative breast cancer metastases can be differentiated with high specificity and sensitivity using [⁶⁸Ga]ABY-025 PET imaging and studying the change in uptake between two different time points.

Materials and methods

Test product and dose: The tracer molecule denoted ABY-025 in the following is the HER2 binding polypeptide with SEQ ID NO:5 coupled to DOTA as described above. ABY-025 is labeled with ⁶⁸Ga essentially as described in Tolmachev ef a/ (2010; Eur J Nucl Med Mol Imaging 37:1356-67) and used in the following experiments. For each subject, approximately 100 pg or 500 pg ABY-025 is labeled with approximately 500 MBq ⁶⁸Ga and administered in a single dose as an intravenous injection of approximately 8 ml.

Subject disposition: The subjects are enrolled into groups based on the HER2 expression in their original primary tumors as determined by the standard commercial test HercepTest™ or fluorescent in situ hybridization (FISH).

Study procedure: All subjects are first evaluated with [¹⁸F]FDG PET imaging before administration of [⁶⁸Ga] ABY-025. After administration, subjects are evaluated with PET camera imaging directly post-injection (dynamic acquisition during 45 minutes) and static images at 60, 120 and 240 minute time points. Low-dose CT scans are performed during each

[⁶⁸Ga]ABY-025 PET acquisition at -5, 60, 120 and 240 minutes. The procedures for image acquisition follow established routines for the PET camera system used. Data analysis: For example, the amount of tracer uptake in a tumor measured at the 240 minute time point (considered as "t₂") is compared to the amount measured at the 60 minute time point (considered as "ti"), in the same way as described in Example 1 for time points 4 and 24 hours after administration of [¹¹¹ ln]ABY-025. The results are expected to show an increase of uptake between these two time points for HER2 positive tumors.

Claims

1 . Method for determining the HER2 status of a malignancy, comprising

· administering, to a mammalian subject having a malignancy suspected of being HER2 positive, a tracer molecule comprising

- a HER2 binding polypeptide which comprises the amino acid sequence

EXiRNAYWEIA LLPNLTNQQK RAFIRKLYDD PSQSSELLX₂E AKKLNDSQ

wherein Xi in position 2 is M, I or L, and X₂ in position 39 is S or C (SEQ ID NO:1 ); and

- a radionuclide suitable for medical imaging;

- at a first time point ti within the range from 15 minutes to 6 hours from administration, to yield a first value; and

- at a second time point t₂ within the range from 75 minutes to 42 hours from administration and at least 60 minutes after ti, to yield a second value;

• comparing said first and second values, and

- if said second value is equal to or less than said first value, concluding that the malignancy of said subject is HER2 negative.

2. Method according to claim 1 , in which said medical imaging equipment is positron emission tomography equipment and said radionuclide is suitable for medical imaging using positron emission tomography.

3. Method according to claim 2, in which said radionuclide is selected from the group consisting of ⁶⁸Ga, ^110mln, ¹⁸F, ⁴⁵Ti, ⁴⁴Sc, ⁶¹Cu, ⁶⁶Ga, ⁶⁴Cu, ⁵⁵Co, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ¹²⁴l and ⁷⁶Br.

4. Method according to any one of claims 2-3, in which said time point ti is within the range from 30 minutes to 6 hours from administration.

5. Method according to claim 4, in which said radionuclide is selected from the group consisting of ⁶⁸Ga, ^110mln, ¹⁸F, ⁴⁵Ti, ⁴⁴Sc and ⁶¹Cu, and said time point ti is within the range from 30 to 90 minutes from administration.

6. Method according to claim 4, in which said radionuclide is selected from the group consisting of ⁶⁶Ga, ⁶⁴Cu, ⁵⁵Co, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ¹²⁴l and ⁷⁶Br, and said time point ti is within the range from 2 to 6 hours from

administration.

7. Method according to any one of claims 2-6, in which said time point t₂ is within the range from 150 minutes to 36 hours from administration.

8. Method according to claim 5, in which said time point t₂ is within the range from 150 minutes to 8 hours from administration.

9. Method according to claim 8, in which said radionuclide is selected from the group consisting of ⁶⁸Ga and ^110mln, and said time point t₂ is within the range from 150 minutes to 5 hours from administration.

10. Method according to claim 6, in which said time point t₂ is within the range from 20 to 36 hours from administration.

1 1 . Method according to claim 1 , in which said medical imaging equipment is single-photon emission computed tomography equipment and said radionuclide is suitable for medical imaging using single-photon emission computed tomography.

12. Method according to claim 1 1 , in which said radionuclide is selected from the group consisting of ¹¹¹ ln, ^99mTc, ¹²³l, ¹³¹ l and ⁶⁷Ga.

13. Method according to any one of claims 1 1 -12, in which said time point ti is within the range from 30 minutes to 6 hours from administration, such as from 30 to 90 minutes or from 2 to 6 hours.

14. Method according to any one of claims 1 1 -13, in which said time point t₂ is within the range from 150 minutes to 36 hours from administration, such as from 150 minutes to 24 hours or from 20 to 36 hours.

15. Method according to any one of claims 1 1 -14, in which said radionuclide is ^99mTc and said time point ti is within the range from 30 to 90 minutes from administration.

16. Method according to any one of claims 1 1 -15, in which said radion uucclliiddee iiss ^99mTTcc aanndd ssaaiidd ttiimmee ppooiinntt t₂ is within the range from 150 minutes to 24 hours from administration.

17. Method according to any one of claims 1 1 -14, in which said radionuclide is selected from the group consisting of ¹¹¹ In, ¹²³l, ¹³¹1 and ⁶⁷Ga, and said time point t₂ is within the range from 20 to 36 hours from

administration.

18. Method according to any preceding claim, in which the relationship between time points ti and t₂ is defined by 5^*ti < t₂≤7^*ti .

19. Method according to any preceding claim, in which said HER2 binding polypeptide comprises the amino acid sequence

YAKEXiRNAYW EIALLPNLTN QQKRAFIRKL YDDPSQSSEL LX₂EAKKLNDS Q wherein Xi in position 5 is M, I or L, and X₂ in position 42 is S or C (SEQ ID NO:2).

20. Method according to claim 19, in which said HER2 binding polypeptide comprises the amino acid sequence

AEAKYAKEXiR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLX₂EAKK LNDSQ

wherein Xi in position 9 is M, I or L, and X₂ in position 46 is S or C (SEQ ID NO:3).

21 . Method according to claim 19, in which said HER2 binding polypeptide comprises the amino acid sequence

ESEKYAKEXiR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLX₂EAKK LNDSQ

wherein Xi in position 9 is M, I or L, and X₂ in position 46 is S or C (SEQ ID NO:4).

22. Method according to claim 20, in which said HER2 binding polypeptide comprises the amino acid sequence

AEAKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPKVD C

(SEQ ID NO:5).

23. Method according to claim 21 , in which said HER2 binding polypeptide comprises the amino acid sequence

ESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK (SEQ ID NO:6).

24. Method according to any preceding claim, in which said HER2 binding polypeptide comprises a cysteine residue either already present in the sequence or as an additional amino acid residue or through replacement of a surface exposed amino acid residue that is not involved in HER2 binding.

25. Method according to claim 24, in which said HER2 binding polypeptide comprises a chelating environment provided by a

polyaminopolycarboxylate chelator coupled to the polypeptide via a thiol group.

26. Method according to claim 25, wherein the

polyaminopolycarboxylate chelator is 1 ,4,7,10-tetraazacyclododecane- 1 ,4,7,10-tetraacetic acid or a derivative thereof.

27. Method according to claim 26, wherein the 1 ,4,7,10- tetraazacyclododecane-1 , 4, 7, 10-tetraacetic acid derivative is 1 ,4,7,10- tetraazacyclododecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide.

28. Method according to any preceding claim, in which said HER2 binding polypeptide consists of SEQ ID NO:5 coupled to 1 ,4,7,10- tetraazacyclododecane-1 ,4,7-tris-acetic acid-10-maleimidoethylacetamide.

29. Method according to claim 25, wherein the

polyaminopolycarboxylate chelator is diethylenetnaminepentaacetic acid or derivatives thereof.

30. Method according to any one of claims 25-29, in which said tracer molecule comprises a radiochelate of said HER2 binding polypeptide and a radionuclide selected from the group consisting of ¹¹¹ In, ⁶⁸Ga, ⁶⁴Cu, ⁸⁹Zr, ⁴⁵Ti,

31 . Method according to claim 30, in which said HER2 binding polypeptide is as specified in claim 28 and said radionuclide is ⁶⁸Ga.

32. Method according to claim 30, in which said HER2 binding polypeptide is as specified in claim 28 and said radionuclide is ¹¹¹ ln.

33. Method according to claim 24, wherein the cysteine residue is the one situated at the C-terminal end of SEQ ID NO:5 or the additional cysteine is situated at the C-terminal of any of SEQ ID NO:1 -4 or 6-7, optionally with additional amino acid residues between the last amino acid residue of SEQ ID NO:1 -4 or 6-7 and the C-terminal cysteine, wherein the cysteine residue optionally is followed by one or more other amino acid residue(s), and wherein a chelating environment constituted by a N₃S chelator motif is provided by the nitrogen atoms of three consecutive peptide bonds in the stretch of amino acid resides constituted by the cysteine and the two preceding amino acid residues, together with the SH group of the cysteine residue.

34. Method according to any one of claims 1 -23, in which said HER2 binding polypeptide further comprises a mercaptoacetyl coupled to the N- terminal thereof, whereby a chelating environment constituted by a N₃S chelator motif is provided by the nitrogen atoms of the first three consecutive peptide bonds from the N-terminal, together with the SH group of the mercaptoacetyl.

35. Method according to claim 34, wherein the first three consecutive peptide bonds from the N-terminal are provided by the amino groups of residues ESE.

36. Method according to any one of claims 34-35, in which said HER2 binding polypeptide has the sequence:

maESEKYAKEMR NAYWEIALLP NLTNQQKRAF IRKLYDDPSQ SSELLSEAKK LNDSQAPK

wherein ma is mercaptoacetyl (SEQ ID NO:7).

37. Method according to any one of claims 33-36, in which said tracer molecule comprises a radiochelate of said HER2 binding polypeptide and

38. Method according to claim 37, in which said HER2 binding polypeptide is as specified in claim 36 and said radionuclide is ^99mTc.

39. Method according claim 24, in which said tracer molecule

comprises said HER2 binding polypeptide linked via a linker molecule to a radionuclide suitable for medical imaging selected from the group consisting of ¹⁸F, ⁷⁶Br, ⁷²As ¹²³l, ¹²⁴l and ¹³¹ l.

40. Method according to any preceding claim, comprising, before the administration step, a step of preparing said tracer molecule, which step comprises mixing said HER2 binding polypeptide with said radionuclide.

41 . Method according to claim 40, wherein said HER2 binding polypeptide and radionuclide are as specified in claim 31 .

42. Method according to claim 40, wherein said HER2 binding polypeptide and radionuclide are as specified in claim 32.

43. Method according to claim 40, wherein said HER2 binding polypeptide and radionuclide are as specified in claim 38.

44. Method according to any preceding claim, wherein said mammalian subject is a human.

45. Method according to any preceding claim, wherein said malignancy originates from a cancer disease selected from breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer, prostate cancer and cancer in the esophagus.