Image guided biopsy of her2 positive lesions

Abstract

The current invention relates to a method for determining HER2 expression in one or more lesions of a subject, said method comprises: a. Administering a tracer to said subject, wherein said tracer comprises a radiolabel coupled to a HER2 binding biomolecule; b. Performing a total body Positron emission tomography / computed tomography (PET / CT) scan of said subject; c. Determining the uptake of said tracer in said lesions; d. Performing an initial lesion classification based on said tracer uptake, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative. Aforementioned method can be used for assessing interlesional, intralesional and / or temporal heterogeneity of HER2 expression in a subject. Aforementioned method can also be used for evaluating a cancer therapy.

Description

[0001] IMAGE GUIDED BIOPSY OF HER2 POSITIVE LESIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for determining HER.2 expression in one or more lesions of a subject by administering a tracer to said subject, performing a total body PET / CT scan of said subject and performing an initial lesion classification, wherein each of said one or more lesions is classified as either HER.2 positive or HER2 negative. By determining HER2 expression in one or more lesions of said subject, a targeted biopsy can be for instance performed.

[0004] In a second aspect, the present invention also relates to use of said aforementioned method for assessing heterogeneity of HER2 expression in a subject and for evaluating a cancer therapy.

[0005] BACKGROUND

[0006] The classification of patients according to HER2 status is based upon IHC and ISH. The problem of accurate HER2-status assessment, however, is compounded by the dynamics of receptor expression during the disease course (also known as temporal heterogeneity) and by expression heterogeneity (interlesional and intralesional). This may lead to sampling errors. Furthermore, with the current standard method for HER2-status assessment (ERBB2 IHC assay and the ASCO-CAP score) the scoring accuracy in the low range is poor. These inaccuracies might entail giving a treatment with possible adverse effects to patients who will not benefit, or denying patients a potentially efficacious treatment.

[0007] The present invention aims to resolve at least some of the problems and disadvantages mentioned above.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a method for determining HER2 expression in one or more lesions of a subject according to claim 1. More particular, said method comprises: a. Administering a tracer to said subject, wherein said tracer comprises a radiolabel coupled to a HER.2 binding biomolecule; b. Performing a total body Positron emission tomography / computed tomography (PET / CT) scan of said subject; c. Determining the uptake of said tracer in said lesions; d. Performing an initial lesion classification based on said tracer uptake, wherein each of said one or more lesions is classified as either HER.2 positive or HER2 negative.

[0010] Preferred embodiments of the method are shown in any of the claims 2 to 13.

[0011] A specific preferred embodiment relates to an invention according to claim 2, wherein said method further comprises selection of one or more lesions classified as HER2 positive and taking one or more biopsy samples from said one or more positively classified lesions.

[0012] In a second aspect, the present invention relates to use of aforementioned method according to claims 14-15. More particular, aforementioned method can be used for assessing interlesional, intralesional and / or temporal heterogeneity of HER2 expression in a subject. Aforementioned method can also be used for evaluating a cancer therapy.

[0013] By performing a total body PET / CT scan of the subject, an overview of HER2 expression in the whole body can be obtained. This is in contrast with taking a biopsy, where only a small portion of the body is sampled and analyzed for HER2 expression. As such, the method of the current invention can assist in the decision to avoid unnecessary invasive tissue biopsy as well as guide such a procedure to a tissue location where a valid diagnostic biopsy sample can be obtained. Furthermore, as the HER2 binding biomolecule is very sensitive, the method of the invention allows to detect HER2-expressing lesions, even in patients previously determined to be HER2-low or negative and in small lesions which might have gone unnoticed using different imaging techniques such as [18F]FDG-PET.

[0014] DESCRIPTION OF FIGURES

[0015] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses. Throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.

[0016] Figure 1 shows a schematic representation of a method for determining HER.2 expression and selecting one or more HER.2 positive lesions for taking one or more biopsy samples according to an embodiment of the present invention. The method of the invention allows to select the right drug for the right patient.

[0017] Figure 2 shows a schematic representation of traditional, current and possible future classification, wherein the method according to an embodiment of the present invention allows to discriminate between HER2 0 and HER2 Ultra Low patients.

[0018] Figure 3 shows an algorithm for defining HER2 spectrum of expression according to ASCO / CAP guidelines. The method according to an embodiment of the present invention allows to discriminate between HER2 0 and HER2 Ultra Low patients.

[0019] Figure 4 shows an image obtained by a PET scan, annotated with the maximum standardized uptake value (SUV) value, defined here as SUVmax, the Region Of Interest (ROI)peak and the highest-uptake region. Peak SUV (SUVpeak) is defined as the average SUV within a small, fixed-size region of interest (ROIpeak) centered on a high-uptake part of the tumor. Said SUVpeak can be used in a method according to an embodiment of the present invention for performing an initial lesion classification, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative.

[0020] Figures 5-7 show RP-HPLC chromatograms (RP-HPLC-UV 280 nm profile, full view in A, zoomed view in B) of protein batches comprising either an immunoglobulin single variable domain according to SEQ ID NO: 1 (Figure 5, GMP batch) or SEQ ID NO: 2 (Figure 6, development batch and Figure 7, GMP batch).

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention concerns a method for determining HER2 expression in one or more lesions of a subject and performing an initial lesion classification, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative. By determining HER2 expression in one or more lesions of said subject, a targeted biopsy can be performed. Furthermore, the present invention concerns use of said aforementioned method for assessing heterogeneity of HER.2 expression in a subject and for evaluating a cancer therapy.

[0023] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0024] As used herein, the following terms have the following meanings:

[0025] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0026] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0027] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0028] "Biomolecule" as used herein can refer to peptides, small molecules, scaffold proteins, antibodies, antibody fragments or antibody mimetics. "HER.2 binding biomolecules" are of particular interest in the current invention and include biomolecules that are directed against or specifically bind to HER2.

[0029] "Scaffold proteins" as used herein refer to proteins that simultaneously bind two or more other proteins, and organize binding partners into a functional unit. Radiolabeled scaffold Proteins, DARPin G3 and ADAPT6 ([99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6), can for instance be used for visualization of HER2 expression. Albumin-binding domain-derived affinity proteins (ADAPTS), such as ADAPT6, are a new type of small (46-59 amino acids) protein useful as probes for molecular imaging.

[0030] "Antibody mimetics" are defined as molecules that can bind to antigens similar to antibodies; however, they are not generated by the immune system and have no structural relation to the antibodies. They are mostly unrelated protein scaffolds consisting of o-helices, p-sheets, or random coils that can bind to specific targets and could be designed to incorporate novel binding sites through common protein engineering strategies. Antibody mimetics include for instance affibody molecules. "Affibody molecules" or "affibodies" are small, robust proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies. Affibody molecules are based on the immunoglobulin G domain of staphylococcal protein A. In the current invention, Affibody molecules that selectively bind to HER2 receptors are of specific interest. Such anti-HER2 affibody molecules include for instance ABY-025 and ABY-027. The second generation of Affibody molecule, "ABY-025", binds selectively to HER2 receptors with picomolar affinity. These Affibody molecules are in clinical development for tumor diagnosis. Anti-HER2 Affibody molecule, fused with albumin binding domain (ABD), denoted as ABY-027, labeled with Lutetium-177 provided reduction of renal and hepatic uptake of radioactivity in mice xenografts.

[0031] As used herein, the terms "polypeptide", "protein", "peptide" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0032] "Antibody" as used herein refers to antibodies which comprise two heavy chains, each comprising a constant region and a variable region. These heavy chains are linked by disulfide bridges at the so-called hinge region. In addition, each heavy chain is linked to a light chain (also comprising a constant region and variable region) through further disulfide bridges in an arrangement that is often referred to as forming an overall "Y” shape. Each variable region has three complementary determining regions (CDRs). Together the variable region of a light chain and heavy chain define the binding specificity of the antibody for the target. "Antibody fragment" as used herein refers to an entity which is less than a full antibody, for example a variable region from a heavy and / or light chain, a single chain variable region, a Fab fragment, a variable region and a portion of a constant region, a heavy chain, a light chain, a single chain or the like and including conjugates of each of the same. A variable region from a heavy chain or a light chain can be considered as a basic functional binding unit of antibody and is sometimes referred to as a domain antibody. Alternatively, a variable region from a heavy chain and light chain can be associated together, for example by covalent bonds to provide what is referred to as a "single chain variable fragment (scFv)", and comprises three CDRs from the heavy and three CDRs from the light chain (nominally referred to as Hl, H2, H3 for the heavy chain and LI, L2 and L3 for the light chain), in the same way as a complete antibody.

[0033] "Immunoglobulin single variable domain" as used herein defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain (which is different from conventional immunoglobulins or their fragments, wherein typically two immunoglobulin variable domains interact to form an antigen binding site). It should however be clear that the term "immunoglobulin single variable domain" does comprise fragments of conventional immunoglobulins wherein the antigen binding site is formed by a single variable domain.

[0034] Generally, an immunoglobulin single variable domain will have an amino acid sequence comprising 4 framework regions (FR1 to FR4) and 3 complementarity determining regions (CDR1 to CDR3), preferably according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), or any suitable fragment thereof (which will then usually contain at least some of the amino acid residues that form at least one of the complementarity determining regions). Immunoglobulin single variable domains comprising 4 FRs and 3 CDRs are known to the person skilled in the art and have been described.

[0035] Typical, but non-limiting, examples of immunoglobulin single variable domains include light chain variable domain sequences (e.g. a VL domain sequence) or a suitable fragment thereof, or heavy chain variable domain sequences (e.g. a VH domain sequence or VHH domain sequence) or a suitable fragment thereof, as long as it is capable of forming a single antigen binding unit. Thus, according to a preferred embodiment, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g. a VL domain sequence) or a heavy chain variable domain sequence (e.g. a VH domain sequence); more specifically, the immunoglobulin single variable domain is a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or a heavy chain variable domain sequence that is derived from a heavy chain antibody. The immunoglobulin single variable domain may be a domain antibody ("dAB" or "dAb"), or a single domain antibody ("sdAB" or "sdAb"), or a VHH domain sequence or another immunoglobulin single variable domain, or any suitable fragment of any one thereof. The immunoglobulin single variable domains, generally comprise a single amino acid chain that can be considered to comprise 4 "framework sequences" or FR's and 3 "complementary determining regions" or CDR's (as defined herein). It should be clear that framework regions of immunoglobulin single variable domains may also contribute to the binding of their antigens. The delineation of the CDR sequences (and thus also the FR sequences) can be based on the IMGT unique numbering system for V-domains and V-like domains. Alternatively, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from Camelids.

[0036] It should be noted that the immunoglobulin single variable domains as binding domain moiety in their broadest sense are not limited to a specific biological source or to a specific method of preparation. The term "immunoglobulin single variable domain" encompasses variable domains of different origin, comprising mouse, rat, rabbit, donkey, human, shark or camelid variable domains. According to specific embodiments, the immunoglobulin single variable domains are derived from shark antibodies (the so-called immunoglobulin new antigen receptors or IgNARs), more specifically from naturally occurring heavy chain shark antibodies, devoid of light chains, and are known as VNAR domain sequences. Preferably, the immunoglobulin single variable domains are derived from camelid antibodies. More preferably, the immunoglobulin single variable domains are derived from naturally occurring heavy chain camelid antibodies, devoid of light chains, and are known as VHH domain sequences.

[0037] The term "VHH domain sequence" is, as used herein, is interchangeably with the term "single domain antibody fragment (sdAb)" and refers to a single domain antigen binding fragment. It particularly refers to a single variable domain derived from naturally occurring heavy chain antibodies and is known to the person skilled in the art. VHH domain sequences are usually derived from heavy chain only antibodies (devoid of light chains) seen in camelids and consequently are often referred to as VHH antibody or VHH sequence. Camelids comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example Lama pacos, Lama glama, Lama guanicoe, Vicugna pacos and Lama vicugna). The small size and unique biophysical properties of VHH domain sequences excel conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. VHH domain sequences are stable, survive the gastro-intestinal system and can easily be manufactured. Therefore, VHH domain sequences can be used in many applications including drug discovery and therapy, but also as a versatile and valuable tool for purification, functional study and crystallization of proteins.

[0038] The VHH domain sequences of the invention generally comprise a single amino acid chain that can be considered to comprise 4 "framework regions" or FR's and 3 "complementarity determining regions" or CDR's, according to formula (1) (as defined above). The term "complementarity determining region" or "CDR" refers to variable regions in VHH domain sequences and contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the VHH domain sequences for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions." The VHH domain sequences have 3 CDR regions, each non-contiguous with the others (termed CDR1, CDR2, CDR3). The delineation of the FR and CDR sequences is often based on the IMGT unique numbering system for V-domains and V-like domains. Alternatively, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from Camelids. As will be known by the person skilled in the art, the VHH domain sequences can in particular be characterized by the presence of one or more Camelidae hallmark residues in one or more of the framework sequences (according to Kabat numbering).

[0039] One preferred class of immunoglobulin single variable domains of the current invention corresponds to the VHH domains of naturally occurring heavy chain antibodies, also called "VHH domain sequences" or "single domain antibody fragment (sdAb)".

[0040] Such VHH domain sequences can generally be generated or obtained by suitably immunizing a species of Camelid with a desired target, (i.e. so as to raise an immune response and / or heavy chain antibodies directed against a desired target), by obtaining a suitable biological sample from said Camelid (such as a blood sample, or any sample of B-cells), and by generating VHH sequences directed against the desired target, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person. Alternatively, such naturally occurring VHH domains against the desired target can be obtained from naive libraries of Camelid VHH sequences, for example by screening such a library using the desired target or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in W09937681, W00190190, W003025020 and WO03035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries may be used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in W00043507. Yet another technique for obtaining VHH domain sequences directed against a desired target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and / or heavy chain antibodies directed against a desired target), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, or any sample of B-cells), and then generating VHH domain sequences directed against the desired target starting from said sample, using any suitable technique known per se. For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO02085945 and in WO04049794 can be used.

[0041] A particularly preferred class of immunoglobulin single variable domains of the invention comprises VHH domain sequences with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized" , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person and on the basis of the prior art on humanization. Again, it should be noted that such humanized VHH domain sequences of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. Humanized VHH domain sequences may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. Such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring VHH with the amino acid residues that occur at the same position in a human VH domain, such as a human VH3 domain. The humanizing substitutions should be chosen such that the resulting humanized VHH domain sequences still retain the favourable properties of VHH domain sequences as defined herein. The skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand.

[0042] Also within the scope of the invention are natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "variants") of the immunoglobulin single variable domain of the invention as defined herein.

[0043] Thus, according to one embodiment of the invention, the term "immunoglobulin single variable domain of the invention" in its broadest sense also covers such variants, in particular variants of the sdAb of SEQ ID NO: 1 or of SEQ ID NO: 2. Generally, in such variants, one or more amino acid residues may have been replaced, deleted and / or added, compared to the sdAbs of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and / or in one or more of the CDR's. Variants, as used herein, are sequences wherein each or any framework region and each or any complementarity determining region shows at least 80% identity, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity or, still even more preferably 99% identity with the corresponding region in the reference sequence, as can be measured electronically by making use of algorithms such as PILEUP and BLAST (Altschul et al. 1990, J Mol Biol 215: 403; Higgins & Sharp 1989, CABIOS 5: 151). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www / ncbi. nlm.nih.gov / ). Such variants of immunoglobulin single variable domains may be of particular advantage since they may have improved potency or other desired properties.

[0044] By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and / or an amino acid residue may be replaced by another amino acid residue. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the immunoglobulin single variable domain of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the immunoglobulin single variable domain of the invention (i.e. to the extent that the immunoglobulin single variable domain is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the immunoglobulin single variable domain thus obtained.

[0045] According to embodiments, variants of the immunoglobulin single variable domains, in particular the sdAb of the present invention may have a substitution, deletion or insertion, of 1, 2 or 3 amino acids in one, two or three of the CDRs, more specifically (i) in CDR1 or CDR2 or CDR3; (ii) in CDR1 and CDR2, or, in CDR1 and CDR3, or, in CDR2 and CDR3; (iii) in CDR1 and CDR2 and CDR3. More preferably, variants of the immunoglobulin single variable domains, in particular the sdAbs, of the present invention may have a conservative substitution (as defined herein) of 1, 2 or 3 amino acids in one, two or three of the CDRs, more specifically (i) in CDR1 or CDR2 or CDR3; (ii) in CDR1 and CDR2, or, in CDR1 and CDR3, or, in CDR2 and CDR3; (iii) in CDR1 and CDR2 and CDR3.

[0046] Further, depending on the host organism used to express the immunoglobulin single variable domain of the invention, such deletions and / or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups.

[0047] Examples of modifications, as well as examples of amino acid residues within the immunoglobulin single variable domain sequence, that can be modified, methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g. by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the immunoglobulin single variable domain, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the immunoglobulin single variable domain of the invention. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including single domain antibody fragments). Such functional groups may for example be linked directly (for example covalently) to an immunoglobulin single variable domain of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and / or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments. In an embodiment, site-directed pegylation is used, in particular via a cysteine-residue. For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in an immunoglobulin single variable domain of the invention, an immunoglobulin single variable domain of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and / or C-terminus of an immunoglobulin single variable domain of the invention, all using techniques of protein engineering known per se to the skilled person. Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and / or post-translational modification, depending on the host cell used for expressing immunoglobulin single variable domain of the invention.

[0048] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5: 151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). A "deletion" is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid. Within the context of a protein, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or a fragment thereof may contain more than one deletion.

[0049] An "insertion" or "addition" is that change in an amino acid or nucleotide sequences which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein. "Insertion" generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while "addition" can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. Within the context of a protein or a fragment thereof, an insertion or addition is usually of about 1, about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion. A "substitution", as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

[0050] As used herein, the term "cancer" or "tumor" refers to any neoplastic disorder, for instance breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer, cancer in the esophagus and gastroesophageal cancer. Neoplastic disorders related to HER.2 overexpression are of particular relevance in the current invention, for instance HER2-related breast and gastroesophageal cancer.

[0051] "Lesion" as used herein refers to any type of unidentified mass in the body and includes for instance a neoplastic lesion or an inflammatory lesion. Infective and inflammatory diseases can for instance mimic tumor malignancy. Infective etiologies, especially tuberculosis and fungal infection such as histoplasmosis and aspergillosis are common cause for a pulmonary nodule, mass, or nodal disease that may mimic malignancy. The method of the current invention helps in the determination of the ideal site for biopsy and identification of other disease foci.

[0052] "Radionuclide" as used herein refers to an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus.

[0053] As used herein, the term "diagnosing" or grammatically equivalent wordings, means determining whether or not a subject suffers from a particular disease or disorder.

[0054] As used herein, "prognosing" or grammatically equivalent wordings, means estimating the likely course and outcome of a disease. The prognosis of a patient diagnosed with cancer is often viewed as the chance that the disease will be treated successfully and that the patient will recover.

[0055] As used herein, "treating cancer" or "treating a subject or individual having cancer" or "cancer treatment" includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease. In particular, it includes inhibition of the replication of cancer cells, inhibition of the spread of cancer, reduction in tumor size, lessening or reducing the number of cancerous cells in the body, and / or amelioration or alleviation of the symptoms of cancer. A treatment is considered therapeutic if there is a decrease in mortality and / or morbidity, and may be performed prophylactically, or therapeutically. Generally the "subjects" are mammals or mammalian, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the subjects will be humans.

[0056] "Targeting moiety" or "targeting agent" as used herein refers to an agent possessing binding affinity for a specific molecular target or biomarker, HER.2 in the current invention. In the current invention such "targeting moiety" or "targeting agent" is a HER2 binding biomolecule coupled to a detectable label (a radionuclide) to form a targeted tracer that can for instance be used for in vivo medical imaging.

[0057] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0058] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0059] The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0060] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0061] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0062] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0063] Detailed description

[0064] Worldwide, breast carcinoma has the highest cancer incidence rate in females. Both the expression of hormone receptors and of the human epidermal growth factor 2 (HER2) receptor are important for prognosis and therapy choices. Therapies targeting the HER2 receptor have been developed over the last two decades, including kinase inhibitors, trastuzumab, pertuzumab and trastuzumab-based antibody-drug conjugates (ADC). Receptor expression and genome status are predictive biomarkers for these therapies; in HER2-positive breast carcinoma patients, these therapies improve the overall survival. Current diagnosis of HER2 expression or gene amplification relies on the immunohistochemistry (IHC) and / or in situ hybridization (ISH) tests on pathological specimens. IHC measures the expression of HER2 receptors on the cellular surface of a biopsied tissue sample, which can be graded as 0, 1+, 2+, or 3+. Following the American Society of Clinical Oncology and College of American Pathologists (ASCO-CAP), HER2-positive patients are defined as those with an immunohistochemistry (IHC) score of 3+, or those with IHC 2+ but a positive scoring on in situ hybridization (ISH). In recent years, a new category emerged, termed "HER2-low" (1+ and 2+ with negative ISH), as these patients also benefit from the second-generation ADC trastuzumab-deruxtecan (see figures 2 and 3 for an overview of HER2 classification). This makes the correct assessment of HER2 expression levels and the identification of such HER2-low disease even more important, as the detection of low levels of HER2-expression could be key for selecting the right drug for the right patient. As described above, HER.2 status is determined by IHC and / or ISH on biopsy specimens. Biopsies are invasive but allow the assessment of many required molecular parameters, albeit in only a small fragment of the disease present in the patient. However, increasing data confirms the heterogeneous expression of HER.2 within and between cancer lesions, increasing the risk for sampling errors and false negative results and necessitating the need for multiple biopsies.

[0065] In a first aspect, the invention provides a method for determining HER2 expression in one or more lesions of a subject, said method comprises: a. Administering a tracer to said subject, wherein said tracer comprises a radiolabel coupled to a HER2 binding biomolecule; b. Performing a total body Positron emission tomography / computed tomography (PET / CT) scan of said subject; c. Determining the uptake of said tracer in said lesions; d. Performing an initial lesion classification based on said tracer uptake, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative.

[0066] As described above, current diagnosis of HER2 expression or gene amplification relies on the immunohistochemistry (IHC) and / or in situ hybridization (ISH) tests on pathological specimens (biopsies). Inter- and intra-lesion and temporal heterogeneity introduces a challenge when performing tissue sampling. Depending on the selected lesion or biopsy location within the targeted lesion, the HER2 expression might differ, influencing the IHC HER2 status diagnosis and the subsequent treatment path. HER2 status has been observed to be different between the primary and recurrent breast cancer in 3%-10% of patients, and even turns out to be inconsistent between and within one single lesion. Within the HER2 0 and HER2 negative tumors, 37.3% of temporal heterogeneity is even reported, mainly driven by HER2 status changes within HER2-nul and HER2-low tumors. This heterogeneity drove recurrent malignant breast cancer guideline changes, recommending a new biopsy and accompanied reassessment of the biological marker status.

[0067] The method according to the current invention allows a superior diagnostic performance because all tumor lesions can be evaluated for HER2 expression with a single total body scanning procedure (PET-CT scan). By performing a total body PET / CT scan of the subject, an overview of HER.2 expression in the whole body can be obtained. This is in contrast with taking a biopsy, where only a small portion of the body is sampled and analyzed for HER.2 expression. As such, the method of the current invention can assist in the decision to avoid unnecessary invasive tissue biopsy as well as guide such a procedure to a tissue location where a valid diagnostic biopsy sample can be obtained.

[0068] Whole body assessment of HER2 expression allows to reveal said heterogeneous expression of HER2 and enables identification of HER2 positive tumor lesions. Several targeting molecules or HER2 binding biomolecules can be used as imaging tracers: monoclonal antibodies and their fragments, peptides, protein scaffolds, antibody mimetics and others.

[0069] Each has its own specific pharmacokinetics, which influences the choice of radionuclide.

[0070] In an embodiment, said HER2 binding biomolecule is an affibody. In another preferred embodiment, said HER2 binding biomolecule comprises an immunoglobulin single variable domain directed against or specifically binding to HER2.

[0071] Immunoglobulin single variable domains (also known as "single domain antibodies", abbreviated as "SdAbs") are small (12-15kDa), have a high stability, a nanomolar affinity and low immunogenicity. Radiolabeled immunoglobulin single variable domains target their antigen rapidly and are eliminated by the kidneys. This implies they can be labeled with short-lived radionuclides such as gallium-68 or fluorine-18, reducing the radioactive dose to the patient in comparison with the longer-lived radionuclides needed for radiolabeling of monoclonal antibodies (Abs). Therefore, these immunoglobulin single variable domains allow for same-day imaging, making them ideal probes for molecular imaging.

[0072] As the immunoglobulin single variable domain directed against or specifically binding to HER2 is very sensitive, the method of the invention allows to detect HER2- expressing lesions, even in patients previously determined to be HER2-low or negative and in small lesions which might have gone unnoticed using different imaging techniques such as [18F]FDG-PET. Immunoglobulin single variable domains are ideally suited as targeting moiety for detecting HER2 positive lesions. Immunoglobulin single variable domains have a low molecular weight and a high solubility. Immunoglobulin single variable domains are antibody derived molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain (which is different from conventional immunoglobulins or their fragments, wherein typically two immunoglobulin variable domains interact to form an antigen binding site). Immunoglobulin single variable domains exhibit faster pharmacokinetics as compared to monoclonal antibodies or larger antibodyfragments. This results in higher contrast and shorter waiting times after intravenous injection. As such, the current invention further allows to acquire images quickly post-administration of said tracer. In a preferred embodiment, said PET / CT scan is performed between 30 and 300 minutes, more preferably between 40-240 minutes, more preferably between 60-120 minutes, such as 90 minutes after administrating said tracer.

[0073] The HER2 proto-oncogene encodes the production of a 185 kDa cell surface receptor protein known as the HER.2 protein or receptor. This gene is also sometimes referred to as neu, HER2 / neu or c-erbB-2.

[0074] 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.

[0075] 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 overexpression 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.

[0076] 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. 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. Thus, the signal transduction mechanisms mediated by growth factor receptors are important targets for inhibiting cell replication and tumor growth.

[0077] In approximately 25 % of all breast cancer patients, there is an overexpression of the HER.2 gene due to amplification thereof. This overexpression of HER.2 protein correlates with several negative prognostic variables, including estrogen receptornegative status, high S-phase fraction, positive nodal status, mutated p53, and high nuclear grade. Over-expression of HER.2 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 and cancer in the esophagus.

[0078] A single domain antibody (sdAb) targeting the HER2 receptor (2Rsl5D) has been developed. In an embodiment, said immunoglobulin single variable domain directed against or specifically binding to HER2 comprises an amino acid sequence having at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 100% amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO:2, wherein SEQ ID NO: 1 has the following sequence:

[0079] "QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWH KESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSS".

[0080] And wherein SEQ ID NO:2 has the following sequence:

[0081] "DVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWH KESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSS". In an embodiment, the immunoglobulin single variable domain comprises an amino acid sequence chosen from the group consisting of: a. SEQ ID NO: 1 or SEQ ID NO: 2 b. Polypeptides that have at least 80% amino acid identity with SEQ ID NO: 1 or SEQ ID NO: 2, c. Polypeptides that have 1, 2 or 3 amino acid difference with SEQ ID NO: 1 or SEQ ID NO: 2.

[0082] Said immunoglobulin single variable domain directed against or specifically binding to HER.2 comprising an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO:2 shows a high specificity, a strong antigen-binding affinity, a low immunogenicity and favorable pharmacokinetics.

[0083] The N-terminal glutamine (Q) in SEQ ID NO: 1 is switched to an aspartic acid (D) in SEQ ID NO: 2. This allows to avoid the formation of N-pyroglutamic acid, which creates two different protein species. When using an immunoglobulin single variable domain comprising an N-terminal glutamine (Q) a risk of deamidation exists, which results in a random and uncontrollable process resulting in a more undefined product and the need for extra purification. Extra purification steps result in substantial product loss and lower yields. Figures 5-7 show reversed-phase high-performance liquid chromatography (RP-HPLC) chromatograms of protein batches comprising either an immunoglobulin single variable domain according to SEQ ID NO: 1 (Figure 5, GMP batch) or SEQ ID NO: 2 (Figure 6, development batch and Figure 7, GMP batch). The integration results are compared in Table 1 below. A significant postpeak is observed for the protein batches comprising an immunoglobulin single variable domain according to SEQ ID NO: 1 (Post-peak relative retention time (RRT) 1.05 = 38%) compared to the protein batches comprising an immunoglobulin single variable domain according to SEQ ID NO: 2. This is explained by the N-terminal glutamine present in the immunoglobulin single variable domain according to SEQ ID NO: 1 resulting in N-pyroglutamic acid after production and purification. This variant is more hydrophobic and is eluting as a post-peak on RP-HPLC. The N- terminal amino acid is switched to an aspartic acid in SEQ ID NO: 2, preventing N- terminal cyclization from occurring.

[0084] This was also confirmed by RP-HPLC-UV-MS data. The UV 280 nm profile of protein batches comprising an immunoglobulin single variable domain according to SEQ ID NO: 1 showed a significant post-peak. This peak was identified as a variant with N- terminal cyclization of the glutamine to pyroglutamic acid. This variant is absent in protein batches comprising an immunoglobulin single variable domain according to SEQ ID NO: 2 (no cyclization possible on the N-terminal D) (data not shown).

[0085] Besides this, only slight differences in other product related variants are observed between the protein batches comprising either an immunoglobulin single variable domain according to SEQ ID NO: 1 (Figure 5) or SEQ ID NO: 2 (Figure 6-7).

[0086] Table 1: RP-HPLC results for protein batches comprising either an immunoglobulin single variable domain according to SEQ ID NO: 1 or SEQ ID NO: 2 In an embodiment, said immunoglobulin single variable domain is a recombinant protein produced by an expression host. In an embodiment, said expression host is a bacterial host such as Escherichia coli E. coli) or Bacillus subtilis. In an alternative embodiment, said expression host is a yeast such as Saccharomyces cerevisiae or P. pastoris Komagataella phaffii). In alternative embodiments, said expression host is an insect cell line (e.g. Sf9 or Sf21), a mammalian cell line (e.g. Chinese Hamster Ovary (CHO) Cells or Human Embryonic Kidney (HEK 293) Cells), a plant host (e.g. Nicotiana benthamiana or Arabidopsis thaliana), or an algal host (e.g. Chlamydomonas reinhardtii') . In an alternative embodiment, said recombinant protein is produced by a cell-free expression system.

[0087] As used herein, the term "specifically recognizing" or "specifically binding to" or simply "specific for" refers to the ability of an immunoglobulin single variable domain to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens and does not necessarily imply high affinity (as defined further herein). In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). The terms "specifically bind", "selectively bind", "preferentially bind", and grammatical equivalents thereof, are used interchangeably herein. The term "affinity", as used herein, refers to the degree to which an immunoglobulin single variable domain binds to an antigen so as to shift the equilibrium of antigen and immunoglobulin single variable domain toward the presence of a complex formed by their binding. Thus, for example, where an antigen and antibody (fragment) are combined in relatively equal concentration, an antibody (fragment) of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant is commonly used to describe the affinity between the antibody (fragment) and the antigenic target. Typically, the dissociation constant is lower than 10’5M. Preferably, the dissociation constant is lower than 10’6M, more preferably, lower than 10’7M. Most preferably, the dissociation constant is lower than 10’8M.

[0088] An immunoglobulin single variable domain that can specifically bind to and / or that has affinity for a specific antigen or antigenic determinant (e.g. epitope) is said to be "against" or "directed against" said antigen or antigenic determinant. In an embodiment, said radiolabel comprises a radionuclide selected from the group consisting of fluor 18 (18F), indium 111 (mln), copper 61 (61Cu), copper 64 (64Cu), gallium 67 (67Ga), gallium 68 (68Ga), technetium 99m (99mTc).

[0089] As described above, immunoglobulin single variable domains can be labeled with short-lived radionuclides such as gallium-68 or fluorine-18, reducing the radioactive dose to the patient in comparison with the longer-lived radionuclides needed for radiolabeling of monoclonal antibodies (Abs). In a preferred embodiment, said immunoglobulin single variable domain is labeled with gallium 68 (68Ga). Such a immunoglobulin single variable domain labeled with gallium 68 (68Ga) provides rapid, homogeneous and deep tissue penetration (few minutes), rapid clearance of unbound fraction (few hours) and has a low blood circulation time, a high signal-to- noise ratio and a high stability even under chemical and physicochemical stringent conditions. Furthermore, Gallium 68 is optimal for PET imaging as it confers a low irradiation burden for the subject (decay tl / 2 = 68 min) and a high spatial resolution of the imaging analysis. Furthermore, multiple scans are possible in a short time frame and68Ga generators provide flexibility, convenience, and broad access.

[0090] As will be clear to the skilled person, a specific modification of the tracer may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, DTPA (Diethylentriaminepentaacetic acid) and derivatives (including 1B4M-DTPA derivatives and CHX-A"-DTPA derivatives), DOTA (1,4,7,10- Tetraazacyclododecane-l,4,7,10-tetraacetic acid) and derivatives (including DOTA- GA derivatives, DOTAM derivatives, DO3A and derivatives, DO2A and derivatives, CB-DO2A derivatives and DO3AM derivatives), NOTA (1,4,7-Triazacyclononane- 1,4,7-triacetic acid) and derivatives (including NODA derivatives, NODA-GA derivatives, NO2A derivatives, NOTAM derivatives, NOPO derivatives and TRAP derivatives), HBED (N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid) and derivatives (including HBED-CC derivatives, HBED-CI derivatives, HBED-CA derivatives, HBED-AA derivatives and SHBED derivatives), DEPA (7-[2-(bis- carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl- 1,4,7, 10-tetraaza- cyclododec-l-yl-acetic acid) and derivatives, picolinic acid (PA) based chelators and derivatives (including H2dedpa, H4octapa, H2azapa and H5decapa and derivatives), HEHA (l,2,7,10,13-hexaazacyclooctadecane-l,4,7,10,13,16-hexaacetic acid) and derivatives, TETA (l,4,8,ll-tetraazacyclotetradecane-l,4,8,ll-tetraacetic acid) and derivatives (including TE2A derivatives, CB-TE2A derivatives, CB-TE1A1P derivatives, CB-TE2P derivatives, MM-TE2A derivatives and DM-TE2A derivatives), NETA ([2-(4,7-bis-carboxymethyl-[l,4,7]triazacyclononan-l-yl-ethyl]-2- carbonylmethyl-amino]-tetraacetic acid) and derivatives (including C-NETA derivatives and NE3TA derivatives), AAZTA (l,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-methylperhydro-l,4-diazepine) and derivatives, DATA (6-amino-l,4-diazepine-triacetic acid) and derivatives, TCMC (1,4,7,10- tetraaza-l,4,7,10-tetra(2-carbamoylmethyl)cyclododecane) and derivatives, PCTA (3,6,9, 15-tetraazabicyclo [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetic acid) and derivatives, Macropa (6-[[16-[(6-carboxypyridin-2-yl)methyl]-l,4,10,13- tetraoxa-7,16 diazacyclooctadec-7-yl]methyl]-4-isothiocyanatopyridine-2- carboxylic acid) and derivatives, THP (tris(hydroxypyridinone)) and derivates, DFO (deferoxamine) and derivatives, BCPA (N, N'-l,4-Butanediylbis[3-(2- chlorophenyl)acrylamide]) and derivatives, MAG-2 (2-Mercaptoacetyldiglycyl) and derivatives, MAG-3 (2-Mercaptoacetyltriglycyl) and derivatives, MAS-3 (mercaptoacetyltriserine) and derivatives, HYNIC (Hydrazinonicotinic acid) and derivatives and RESCA (Restrained Complexing Agent). In an embodiment, functional groups, such as maleimide, NCS and NHS, are added to the chelating agent in order to allow various conjugation methods. For instance, the isothiocyanate function (R-NCS) allows formation of stable thiourea bonds at alkaline pH with free amines. NHS is another example of an amine-reactive linker.

[0091] In a preferred embodiment, said tracer is a [68Ga]Ga-NOTA-anti-HER2- immunoglobulin single variable domain, wherein said immunoglobulin single variable domain has a sequence according to SEQ ID NO: 1 or according to SEQ ID NO: 2. A phase I study confirmed that use of said [68Ga]Ga-NOTA-anti-HER2-immunoglobulin single variable domain PET / CT is a safe procedure with a radiation dose similar to other short-lived PET tracers. The tracer accumulates in HER2-positive metastases, with optimal image quality at 90 minutes after injection.

[0092] PET / CT imaging of cancer with combined positron emission tomography (PET) and x-ray computerized tomography (CT) scanners has become a standard component of diagnosis and staging in oncology. In addition to cancer detection and staging, PET / CT imaging is becoming more important as a quantitative monitor of individual response to therapy and an evaluation tool for new drug therapies. There are several methods for measuring the rate and / or total amount of tracer accumulation in tumors. PET scanners are designed to measure the in vivo radioactivity concentration [kBq / ml], which is directly linked to the tracer concentration. Typically, however, it is the relative tissue uptake of the tracer that is of interest. The two most significant sources of variation that occur in practice are the amount of injected radionuclide and the patient size. To compensate for these variations, at least to first order, the standardized uptake value (SUV) is commonly used as a relative measure of tracer uptake. In an embodiment of the invention, the uptake of said tracer is quantified by means of the SUV. The basic expression for SUV is described in formula 1 below: (formula 1) where "r" is the radioactivity activity concentration [kBq / ml] measured by the PET scanner within a region of interest (ROI), "a"' is the decay-corrected amount of injected radionuclide [kBq], and "w" is the weight of the patient [g], which is used a surrogate for a distribution volume of tracer. If all the injected tracer is retained and uniformly distributed throughout the body, the SUV everywhere will be 1 g / ml regardless of the amount of tracer injected or patient size. SUVs are dimensionless under the assumption that 1 ml of tissue weights 1 gm. Both approaches are used in practice. The use of lean body mass for w has also been suggested to account for the lower uptake of tracer by adipose tissue. The use of SUVs as a measurement of relative tissue / organ uptake facilitates comparisons between patients, and has been suggested as a basis for diagnosis.

[0093] As described above, in the method according to the current invention, a total body Positron emission tomography / computed tomography (PET / CT) scan of said subject is performed and the uptake of said tracer is determined. In an embodiment, the standardized uptake values (SUVs) of said tracer in said lesions is determined.

[0094] In a further embodiment, the SUV comprises the "SUVpeak" value. Peak SUV (SUVpeak) is defined as the average SUV within a small, fixed-size region of interest (ROIpeak) centered on a high-uptake part of the tumor. In another embodiment, the SUV comprises the "SUVmean" value. The mean SUV is defined by computing the average SUV within a region of interest (ROI). In another embodiment, the SUV comprises the maximum standardized uptake value, defined here as "SUVmax". SUVmax and ROIpeak are illustrated in Figure 4.

[0095] In a preferred embodiment, said initial lesion classification wherein each of said one or more lesions is classified as either positive or negative, is based upon a predetermined cut-off standardized uptake value (SUV), such as a SUVpeak value. In an embodiment, said cut-off SUVpeak value lies between 1.5 and 5. In a preferred embodiment, said cut-off SUVpeak value is 2, wherein lesions having SUVpeak values below 2 are classified as negative and lesions having SUVpeak values above 2 are classified as positive. In an embodiment, other or additional parameters are used to quantify the tracer uptake and classify the lesions as either HER.2 positive or HER.2 negative. Other or additional parameters might include the number and / or size of the lesions.

[0096] Preliminary data (not shown) demonstrates that [68Ga]Ga-NOTA-anti-HER2- immunoglobulin single variable domain PET / CT showed more sensitive and more specific determination of disease extent than [18F]FDG PET / CT.

[0097] In an embodiment, said initial lesion classification shows a specificity of at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%. In an embodiment, said initial lesion classification shows a sensitivity of at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%.

[0098] In a preferred embodiment, said tracer is administered intravenously.

[0099] In an embodiment, a dose of between 50 and 500 MBq, more preferably between 50-250 MBq, more preferably between 80-220 MBq, more preferably between 100- 200 MBq of radiolabeled biomolecule is administered.

[0100] In an embodiment, a dose of between 25 ug - 1 mg, more preferably between 50- 500 ug, more preferably between 100 - 300 ug of radiolabeled biomolecule is administered.

[0101] In an embodiment, said method further comprises selection of one or more positively classified lesions for taking one or more biopsy samples. As the lesions can be classified as either HER.2 negative or HER2 positive according to the method of the current invention, one or more lesions that are classified as positive can be selected for taking a biopsy sample. As such, the method of the current invention can assist in the decision to avoid unnecessary invasive tissue biopsy as well as guide such a procedure to a tissue location where a valid diagnostic biopsy sample can be obtained. The method of the current invention thus allows to reduce the sampling error, thereby decreasing the amount of false negative results.

[0102] In an embodiment, the method further comprises taking said one or more biopsy samples and analyzing said biopsy samples for HER.2 expression. In an embodiment, said biopsy samples are analyzed for HER.2 expression by means of immunohistochemistry (IHC) and / or in situ hybridization (ISH). IHC measures the expression of HER2 receptors on the cellular surface of a biopsied tissue sample, which can be graded as 0, 1+, 2+, or 3+. In situ hybridization (ISH) measures gene amplification.

[0103] In particular, IHC is an essay that identifies and describes the HER2 protein expression pattern and intensity on the cell membrane of breast cancer cells, while ISH detects the presence of gene amplification using HER2 and CEP17 probes. Based on the completeness, intensity, and percentage of cells in which the staining is identified, HER2 IHC is scored using a three-tiered system, from 0 to 3+. In the case of an equivocal result (score 2+), ISH is used as a reflex test. Specifically, when ISH shows an average HER2 copy number >6.0 signals / cell, the case is HER2+. Taken together, HER2+ breast cancer is defined by a 3+ IHC score or 2+ IHC and positivity of ISH, while tumors with HER2 IHC scores of 1+ or 2+ without ISH amplification are defined as HER2-low. More recently, beyond the striking and established efficacy of HER2-directed therapies in HER2+ breast cancer, the possibility of targeting HER2 has been explored also in HER2-low breast cancer. A subset of breast cancer patients that resulted negative for HER2 biomarker assessment by ISH / IHC, benefited from anti-HER2 therapy. A subset of HER2-negative tumors tested in core biopsy samples might respond to targeted therapy due to intra-tumor heterogeneity phenomena.

[0104] As described above, the method of the current invention allows to determine HER2 expression in the whole body, thereby enabling targeted biopsies of HER2 positive lesions. Furthermore, in a preferred embodiment, the method of the current invention allows to detect HER2 Ultra Low lesions. Said category often remained undetected using previous screening techniques.

[0105] In a preferred embodiment of the method of the current invention, said subject is classified based on HER2 status, wherein said classification is based on said initial lesion classification and said biopsy sample analysis. In an preferred embodiment, the method of the current invention is used to reduce false negative results. False negative results occur for instance often in metastatic breast cancer patients with progressive disease. Hence, in an embodiment, the method of the current invention is used for determining HER.2 expression in one or more lesions of a subject, wherein said subject suffers from metastatic breast cancer with progressive disease.

[0106] In an embodiment, the method of the current invention allows to classify said subject as HER.2 Ultra low.

[0107] In an embodiment, the subject can be classified either as HER2 null (or "HER2- negative"), HER2 Ultra low, HER2 Low or HER2 High (or"HER2 positive"), based on said initial lesion classification by means of a PET / CT scan and said targeted biopsy sample analysis.

[0108] Presence of HER2 over-expression indicates susceptibility towards HER2-specific treatment options, such as administration of HER2-specific antibodies or other binding molecules with affinity for the HER2 receptor. 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.

[0109] 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, cancer in the esophagus and gastro-esophageal cancer.

[0110] 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.

[0111] HER2 status in breast cancer is assessed to select patients eligible for targeted therapy with anti-HER2 therapies. According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), the HER2 test positivity is defined by protein overexpression (score 3+) at immunohistochemistry (IHC) and / or gene amplification at in situ hybridization (ISH). As described above, the introduction of novel anti-HER2 compounds, however, is changing this paradigm because some breast cancers with lower levels of protein expression (i.e. score 1+ / 2+ with no gene amplification) benefited from HER.2 antibody-drug conjugates (ADC). Recently, a potential for HER2 targeting in HER2 "ultra-low" (i.e. score 0 with incomplete and faint staining in <10% of tumor cells) has been highlighted. All these novel findings are transforming the traditional dichotomy of HER2 status and have dramatically raised the expectations in this field. Lately, tumors with low levels of HER2 expression (i.e. IHC 1+ or 2+ with negative ISH), also referred to as HER2 "low" breast cancers, have been shown impressive response rates and progression- free survival (PFS) after ADC-based treatments. Another rising layer of complexity is represented by the possible clinical relevance of the incomplete and faint staining in <10% of tumor cells displayed by a subset of IHC score 0 breast cancers. This HER2 "ultra-low" phenotype might explain some promising evidence on treatment response in HER2-negative breast cancer.

[0112] In an embodiment, said subject receives a different treatment based on said classification.

[0113] Targeted therapies for HER2 over-expressing tumors are currently available, such as monoclonal antibodies (trastuzumab and pertuzumab) that specifically bind the extracellular domain of HER2, and specific tyrosine kinase inhibitors (lapatinib) that interact with the intracellular domain of HER2.

[0114] More recently, trastuzumab-emtansine and trastuzumab-deruxtecan (T-DXd), have appeared as new therapeutic options for HER2-overexpressing cancers. These antibody-drug conjugates (ADC) showed efficacy in cancers with a lower level of expression (sometimes referred to as "HER2 Low cancer"). Of note, even if the HER2 pathway activation is lower in HER2-low than in HER2+ breast cancer, the new anti- HER2 ADCs allow its targeting. New ADCs contain a strong chemotherapeutic payload that is guided against tumor cells thanks to their, even low, HER2 positivity.

[0115] As described above, "HER2-low" patients show 1+ and 2+ scoring on IHC with negative ISH. Trastuzumab-deruxtecan (T-DXd), an HER2-directed ADC containing a topoisomerase I inhibitor, demonstrated an impressive progression-free survival benefit in HER2-positive pretreated breast cancer patients also in earlier lines, as emerged by the data of DESTINY-Breast 03 trial presented at ESMO 2021 congress. Progression-free survival (PFS) is defined as the time from random assignment in a clinical trial to disease progression or death from any cause.

[0116] T-DXd showed promising activity in a phase lb trial including HER2-low patients (Overall response rate (ORR) 37%; PFS 11.1 months; median overall survival (mOS) 29.4 months). Similarly, trastuzumab-duocarmazine demonstrated interesting clinical activity in HER2-positive metastatic breast cancer patients in the phase I study (ORR 28% in HR-positive and 40% in HR-negative HER2-low metastatic breast cancer) (Banerji et al., 2019). Overall response rate (ORR) is defined as the proportion of patients who have a partial or complete response to therapy; it does not include stable disease and is a direct measure of drug tumoricidal activity. Median overall survival (mOS) is the amount of time after which 50% of the patients have died and 50% have survived.

[0117] ADCs can overcome some of the limitations encountered by monoclonal antibodies vehiculating and releasing a cytotoxic payload that can be internalized also by the surrounding cells that do not express HER2 (bystander effect). In this perspective, the characteristic of the ADC, mainly the cytotoxic activity of the payload, the drugantibody ratio (DAR), and the cleavability of the linker through which the payload is charged on the antibody, make the difference. T-DM1 is composed of trastuzumab and DM1 (emtansine), a tubulin polymerization inhibitor, linked through an uncleavable linker with a DAR of 3.5. T-DM1 needs to be internalized and the antibody degraded before executing its cytotoxicity. The mandatory internalization prevents the possibility of targeting HER2 non-expressing surrounding cells. In T- Dxd, on the contrary, trastuzumab is conjugated with deruxtecan, a topoisomerase I inhibitor with a DAR of eight via a cleavable but stable linker.

[0118] In an embodiment, a lesion-specific treatment or a combination treatment is performed based on said initial lesion classification. For instance, ablation or surgical resection of non-HER2 lesions can be performed, while the HER2 positive lesions can be treated with HER2-targeted therapy.

[0119] HER2-expression in the disease course is dynamic, showing temporal heterogeneity. HER2 status can for instance change after neoadjuvant chemotherapy or during metastatic progression. As such, in an embodiment, said HER.2 expression in said lesions of said subject are re-evaluated after a period of time, said re-evaluation comprises repeating steps a- c of the aforementioned method and performing a follow-up lesion classification based on said uptake values of said tracer, wherein each of said one or more lesions is reclassified as either positive or negative.

[0120] For instance, tumor response could be evaluated after application of HER2 targeted therapy and would potentially allow the treating physicians to adapt the therapy more accurately (eg. introductions of treatment holidays, recession or changes). As such, the method of the current invention allows to assess the treatment effect. For instance an early confirmation of the treatment response can be obtained by the method of the current invention, allowing for instance to introduce treatment recession or holidays (shorten treatment duration and accompanied comorbidities). Or early identification of HER.2 treatment resistance can be identified, allowing to refine therapy options faster.

[0121] Furthermore, HER2 changes following neoadjuvant treatment or throughout tumor progression is a potential indicator of poor prognosis. As such, by detecting temporal heterogeneity, a more accurate prognosis can be provided. In the context of the present invention, prognosing an individual suffering from or suspected to suffer from cancer refers to a prediction of the survival probability of an individual having cancer or prediction of the relapse risk which is related to the invasive or metastatic behavior (i.e. malignant progression) of tumor tissue or cells. In an embodiment, said prognosis comprises cancer staging. Cancer staging is determined based on the size of the cancer and if or to what extent the cancer has spread to other parts of the patient's body. The tracer of the current invention allows for an improved pharmacokinetic profile compared to other tracers currently known in the field of medical imaging. As such, a more accurate prognosis (including a more accurate cancer stage) can be determined. This allows to identify the most optimal treatment (for instance certain ongoing clinical trials) and improves the disease outcome (e.g. death, chance of recovery, recurrence).

[0122] As described above, HER2 status has been observed to be different between the primary and recurrent breast cancer in 3%-10% of patients, and even turns out to be inconsistent between and within one single lesion. Within the HER2 0 and HER2 negative tumors, 37.3% of temporal heterogeneity is even reported, mainly driven by HER2 status changes within HER2-nul and HER2-low tumors. As such, in a further aspect, the method of the current invention is used for assessing heterogeneity of HER.2 expression in a subject. In an embodiment, the method of the current invention is used for assessing interlesional heterogeneity of HER.2 expression in a subject. In an embodiment, the method of the current invention is used for assessing intralesional heterogeneity of HER2 expression in a subject. In an embodiment, the method of the current invention is used for assessing temporal heterogeneity of HER2 expression in a subject, wherein said HER2 expression in said lesions of said subject are re-evaluated after a period of time, said re-evaluation comprises repeating steps a-c of aforementioned method and performing a followup lesion classification based on said uptake of said tracer, wherein each of said one or more lesions is reclassified as either positive or negative.

[0123] In addition to cancer detection and staging, PET / CT imaging is becoming more important as a quantitative monitor of individual response to therapy. As described above, said HER2 expression in said lesions of said subject can be re-evaluated after a period of time, for instance after cancer therapy, for evaluating a certain cancer therapy. Said re-evaluation comprises repeating steps a-c of aforementioned method and performing a follow-up lesion classification based on said uptake of said tracer, wherein each of said one or more lesions is reclassified as either positive or negative.

[0124] In an embodiment, the method of the current invention can thus be used for evaluating a cancer therapy. In an embodiment, aforementioned method is used in pre- and posttherapy assessment. As such, the method of the current invention can enable monitoring of the response to therapy, thereby making treatment personalized and more efficient. As described above, tumor response could be for instance evaluated after application of HER2 targeted therapy and would potentially allow the treating physicians to adapt the therapy more accurately (eg. introductions of treatment holidays, recession or changes). An early confirmation of the treatment response can be obtained by the method of the current invention, allowing for instance to introduce treatment recession or holidays (shorten treatment duration and accompanied comorbidities) or early identification of HER2 treatment resistance can be identified, allowing to refine therapy options faster.

[0125] In an embodiment, the tracer is comprised in a pharmaceutical composition, wherein said pharmaceutical composition comprises further at least one of a pharmaceutically acceptable carrier, adjuvant or diluent. The tracer or pharmaceutical composition can be administered by any appropriate route. Suitable routes of administration include, but are not limited to, orally, parenterally, intravenously, intradermally, intramuscularly or subcutaneously, rectally, intraperitoneally, via inhalation or via buccal administration, via sublingual route, or topically e.g. transdermally.

[0126] In some embodiments, the tracer or pharmaceutical composition can be administered through urogenital routes to reach to targets, such as internal organs, topical lesions, or organs can be accessed by instillation (such as urinary bladder, vaginal cannel) more effectively and efficiently.

[0127] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient suitable for rendering the composition administrable orally, parenterally, intravenously, intradermally, intramuscularly or subcutaneously, rectally, intraperitoneally, via inhalation or via buccal administration, via sublingual route, or topically e.g. transdermally.

[0128] The tracer can be admixed or compounded with a conventional, pharmaceutically acceptable excipient. It will be understood by those skilled in the art that a mode of administration, vehicle, excipient or carrier conventionally employed and which is inert with respect to the tracer can be utilized for preparing and administering the pharmaceutical compositions of the present invention. The formulations for use in a subject comprise the tracer, together with one or more acceptable excipients, and optionally therapeutic agents. The excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0129] The pharmaceutical composition may be conveniently made available in a unit dosage form, whereby such formulations may be prepared by any of the methods generally known in the pharmaceutical arts. The combination of the tracer with the one or more adjuvants is then physically treated to present the formulation in a suitable form for delivery (e.g., forming an aqueous suspension, by entrapping the drug in liposomes or microemulsions which are compatible with body tissues).

[0130] Adjuvants or accessory ingredients for use in the formulations of the present invention can include any pharmaceutical ingredient commonly deemed acceptable in the art, such as mixtures, buffers, solubility enhancers, and the like. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms can be sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0131] Suitable vehicles that can be used to provide parenteral dosage forms of the compounds of the invention include, without limitation: sterile water; water for injection USP; saline solution; phosphate buffered saline; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a compound of the invention as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0132] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may further contain additional agents, such as antioxidants, buffers, bacteriostats, and solutes, which render the formulations isotonic with the blood of the intended recipient. In an embodiment, the composition comprises additives that reduce unwanted retention of the immunoglobulin single variable domain of the invention (or conjugate of the present invention) in the body, such as positively charged amino acids that have been shown to reduce kidney retention. The formulations may include aqueous and non-aqueous sterile suspensions, which contain suspending agents and thickening agents.

[0133] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0134] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0135] The present invention also provides convenient pharmaceutical kits. Such kits can comprise the tracer and, typically, a pharmaceutically acceptable carrier. The kit can also further comprise conventional kit components, such as needles for use in injecting the compositions, one or more vials for mixing the composition components, and the like, as are apparent to those skilled in the art. In addition, instructions, either as inserts or as labels, indicating quantities of the components, guidelines for mixing the components, and protocols for administration, can be included in the kits.

[0136] Preferably, the pharmaceutical composition comprises a therapeutically effective amount (or an amount effective for diagnosis / prognosis) of a tracer, and at least one of a pharmaceutically acceptable carrier, adjuvant or diluent.

[0137] A 'carrier', or 'adjuvant', in particular a 'pharmaceutically acceptable carrier' or 'pharmaceutically acceptable adjuvant' is any suitable excipient, diluent, carrier and / or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. So, pharmaceutically acceptable carriers are inherently non-toxic and nontherapeutic, and they are known to the person skilled in the art. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Carriers or adjuvants may be, as a non limiting example, Ringer's solution, dextrose solution or Hank's solution. Non aqueous solutions such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" mean the amount needed to achieve the desired result or results. As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0138] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0139] The present invention will be now described in more details, referring to examples that are not limitative.

[0140] EXAMPLES AND / OR DESCRIPTION OF FIGURES

[0141] Figure 1 shows a schematic representation of a method for determining HER.2 expression and selecting one or more HER.2 positive lesions for taking one or more biopsy samples according to an embodiment of the present invention. The method of the invention allows to select the right drug for the right patient.

[0142] Figure 2 shows a schematic representation of traditional, current and possible future HER 2 classification. Traditional and current classification solely rely on IHC / ISH, whereas the method according to an embodiment of the present invention includes a total body PET / CT scan of the subject using a tracer comprising a radiolabel coupled to a HER2 binding biomolecule. The method according to an embodiment of the present invention classifies each of the lesions as either HER2 positive or HER2 negative and allows to perform targeted biopsies. In contrast to traditional and current classification, the method according to an embodiment of the invention allows to discriminate between HER2 0 and HER2 Ultra Low patients.

[0143] Figure 3 shows an algorithm for defining HER2 spectrum of expression according to ASCO / CAP guidelines. HER2-low diseases are identified by IHC score 2+ with negative ISH or IHC score 1 + . Breast cancer is scored 2+ in case of weak-moderate complete membrane staining in >10% of tumor cells or if the membrane staining is intense but in <10% of tumor cells. Score 1+ is defined by faint or barely perceptible incomplete membrane staining in >10% of tumor cells. Gene amplification by ISH is assessed as detailed in the text boxes, according to the types of probes employed. Among the IHC score 0 category, two different types of expression are present, namely a complete lack of expression and the faint or barely perceptible incomplete membrane staining in <10% of tumor cells. Albeit HER2-negative, this latter type of tumor indeed shows the expression of the protein and can be described as HER.2 "ultra-low". The continuous versus the discontinued line depicts the different levels of evidence in the current clinical practice. Abbreviations, IHC, immunohistochemistry; ISH, in situ hybridization; HER.2, human epidermal growth factor receptor 2. *, dual-probe ISH should be performed for final result.

[0144] Source: Breast Biomarker Reporting, CAP Cancer Protocol Templates, vl.4.1.1, November 2021 update.

[0145] Figure 4 shows an image obtained by a PET scan, annotated with the maximum standardized uptake value (SUV) value, defined here as SUVmax, the Region Of Interest (ROI)peak and the highest-uptake region. Peak SUV (SUVpeak) is defined as the average SUV within a small, fixed-size region of interest (ROIpeak) centered on a high-uptake part of the tumor. Said SUVpeak or the SUV comprises the maximum standardized uptake value, defined here as "SUVmax" can be used in a method according to an embodiment of the present invention for performing an initial lesion classification, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative.

[0146] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. Method for determining HER.2 expression in one or more lesions of a subject, said method comprises: a. Administering a tracer to said subject, wherein said tracer comprises a radiolabel coupled to a HER.2 binding biomolecule; b. Performing a total body Positron emission tomography / computed tomography (PET / CT) scan of said subject; c. Determining the uptake of said tracer in said lesions; d. Performing an initial lesion classification based on said tracer uptake, wherein each of said one or more lesions is classified as either HER2 positive or HER2 negative.

2. Method according to claim 1, wherein said method further comprises selection of one or more lesions classified as HER2 positive and taking one or more biopsy samples from said one or more positively classified lesions.

3. Method according to claim 2, further comprising analyzing said one or more biopsy samples for HER2 expression by means of immunohistochemistry (IHC) and / or in situ hybridization (ISH).

4. Method according to any of the previous claims, wherein said HER2 binding biomolecule comprises an immunoglobulin single variable domain directed against or specifically binding to HER2.

5. Method according to claim 4, wherein said immunoglobulin single variable domain directed against or specifically binding to HER2 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO:2.

6. Method according to any of the previous claims, wherein said radiolabel comprises a radionuclide selected from the group consisting of fluor 18 (18F), indium 111 (mln), copper 61 (61Cu), copper 64 (64Cu), gallium 67 (67Ga), gallium 68 (68Ga), technetium 99m (99mTc).

7. Method according to any of the previous claims, wherein said HER2 binding biomolecule is coupled to a chelating agent selected from the group consisting of DTPA and derivatives, DOTA and derivatives, NOTA and derivatives, HBED and derivatives, DEPA and derivatives, picolinic acid based chelators and derivatives, HEHA and derivatives, TETA and derivatives, NETA and derivatives, AAZTA and derivatives, DATA and derivatives, TCMC and derivatives, PCTA and derivatives, Macropa and derivatives, THP and derivates, DFO and derivatives, BCPA and derivatives, MAG-2 andderivatives, MAG-3 and derivatives, MAS-3 and derivatives, HYNIC and derivatives and RESCA.

8. Method according to any of the previous claims 3-7, wherein based on said initial lesion classification and said biopsy sample analysis said subject is classified based on HER2 status.

9. Method according to claim 8, wherein said subject can be classified as HER2 Ultra low.

10. Method according to any of the previous claims 8-9, wherein based on said classification of said subject said subject receives a different treatment.

11. Method according to any of the previous claims, wherein a lesion-specific treatment or a combination treatment is performed based on said initial lesion classification.

12. Method according to any of the previous claims, wherein said PET / CT scan is performed between 30 and 300 minutes after administrating said tracer.

13. Method according to any of the previous claims, wherein said HER2 expression in said lesions of said subject are re-evaluated after a period of time, said re-evaluation comprises repeating steps a-c of the method according to claim 1 and performing a follow-up lesion classification based on said uptake of said tracer, wherein each of said one or more lesions is reclassified as either HER2 positive or HER2 negative.

14. Use of the method according to any of the previous claims 1-13, for assessing interlesional, intralesional and / or temporal heterogeneity of HER2 expression in a subject.

15. Use of the method of any of the claims 1 to 13, for evaluating a cancer therapy.

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