Image-guided biopsy of HER2-positive lesions

Abstract

This invention relates to a method for determining HER2 expression in one or more lesions of a subject, the method comprising: a. administering a tracer to the subject, wherein the tracer comprises a radiolabeled biomolecule conjugated to HER2; b. performing a whole-body positron emission tomography / computed tomography (PET / CT) scan on the subject; c. determining the uptake of the tracer in the lesions; and d. performing an initial lesion classification based on the tracer uptake, wherein the one or more lesions are classified as HER2 positive or HER2 negative. The above method can be used to assess interlesional, intralesional, and / or temporal heterogeneity of HER2 expression in a subject. The above method can also be used to evaluate cancer therapies.

Description

Technical Field

[0001] This invention relates to a method for determining the HER2 expression level in one or more lesions of a subject by administering a tracer to the subject, performing a whole-body PET / CT scan, and initially classifying the lesions; wherein each of the one or more lesions is classified as HER2 positive or HER2 negative. By determining HER2 expression in each of the one or more lesions of the subject, targeted biopsy can be performed, for example.

[0002] In a second aspect, the present invention also relates to the application of the above-described method in assessing the heterogeneity of HER2 expression in subjects and in evaluating cancer therapies. Background Technology

[0003] HER2 status is classified in patients based on IHC and ISH. However, the challenges of accurately assessing HER2 status are compounded by the dynamic changes in receptor expression throughout the disease process (also known as temporal heterogeneity) and expression heterogeneity (inter-lesion and intra-lesion heterogeneity). This can lead to sampling errors. Furthermore, the accuracy of scores in the lower range is poor for current standard methods of HER2 status assessment (ERBB2 IHC testing and ASCO-CAP scoring). These inaccuracies may result in: administering potentially adverse treatments to patients who will not benefit, or depriving patients of the opportunity to receive potentially effective treatments.

[0004] The present invention aims to solve at least some of the above-mentioned problems and defects. Summary of the Invention

[0005] The present invention and its embodiments aim to provide a solution to one or more of the aforementioned deficiencies. To this end, the present invention relates to the method of claim 1 for determining HER2 expression in one or more lesions of a subject. More particularly, the method includes: a. Administering a tracer to the subject, wherein the tracer contains a radiolabeled substance conjugated to a HER2-binding biomolecule; b. Perform whole-body positron emission tomography / computed tomography (PET / CT) on the subject; c. Determine the amount of tracer absorbed in the lesion; d. Initial classification of lesions based on the tracer uptake, wherein each of the one or more lesions is classified as HER2 positive or HER2 negative.

[0006] Preferred embodiments of the method are shown in any one of claims 2 to 13.

[0007] The preferred embodiment relates to the invention of claim 2, wherein the method further includes selecting one or more lesions classified as HER2 positive and obtaining one or more biopsy samples from the one or more positive lesions.

[0008] In a second aspect, the present invention relates to the application of the methods described in claims 14 and 15. More particularly, the aforementioned methods can be used to assess interlesional, intralesional, and / or temporal heterogeneity of HER2 expression in subjects. The methods can also be used to evaluate cancer therapies.

[0009] A whole-body PET / CT scan of the subject provides an overall picture of HER2 expression throughout the body. This contrasts with biopsy, in which only a small portion of the body is sampled and HER2 expression is analyzed. Therefore, the method of this invention can aid in decision-making, avoid unnecessary invasive tissue biopsies, and guide biopsy procedures to locate tissue sites from which effective diagnostic biopsy samples can be obtained. Furthermore, due to the high sensitivity of HER2-binding biomolecules, the method of this invention allows for the detection of HER2-expressing lesions, even in patients previously identified as having low or negative HER2 expression, and when using different imaging techniques (e.g., […]). 18 It can also detect small lesions that may go unnoticed by FDG-PET. Attached Figure Description

[0010] The following description of specific embodiments of the present invention is merely exemplary and is not intended to limit the teachings, their application, or uses. In all the accompanying drawings, corresponding reference numerals denote the same or corresponding parts and features.

[0011] Figure 1 This diagram illustrates an embodiment of the method of the present invention for determining HER2 expression and selecting one or more HER2-positive lesions to obtain one or more biopsy samples. The method of the present invention allows for the selection of the correct medication for the correct patient.

[0012] Figure 2 The diagram illustrates traditional classification, current classification, and possible future classifications, wherein the method of embodiments of the present invention allows for the differentiation between patients with HER2 0 and HER2 ultra-low expression.

[0013] Figure 3 An algorithm for defining HER2 expression profiles according to the ASCO / CAP guidelines is illustrated. The method of embodiments of the present invention allows for the differentiation between patients with HER2 0 and HER2 ultra-low expression.

[0014] Figure 4 Images obtained via PET scans are shown, annotated with the maximum normalized uptake value (SUV), defined here as SUV.max Target Area of ​​Interest (ROI) 峰值 And the area of ​​highest intake. Peak SUV (SUV) 峰值 A region of interest (ROI) is defined as a small, fixed-size region centered on a high-uptake portion of the tumor. 峰值 The average SUV within the range. The SUV 峰值 In a method for initial lesion classification that can be used in embodiments of the present invention, each of the one or more lesions is classified as HER2 positive or HER2 negative.

[0015] Figures 5 to 7 The RP-HPLC chromatogram (RP-HPLC-UV 280 nm chromatogram, A: full view, B: magnified view) of the protein batch is shown, the protein batch containing SEQ ID NO: 1 ( Figure 5 (GMP batch) or SEQ ID NO: 2 ( Figure 6 Research and development batches and Figure 7 Immunoglobulin single variable domain (GMP batch). Detailed Implementation

[0016] This invention relates to a method for determining HER2 expression in one or more lesions of a subject and performing initial lesion classification, wherein the one or more lesions are classified as HER2-positive or HER2-negative. Targeted biopsies can be performed by determining HER2 expression in one or more lesions of the subject. Furthermore, this invention relates to the application of the above method in assessing the heterogeneity of HER2 expression in subjects and in evaluating cancer therapies.

[0017] Unless otherwise defined, all terms disclosed herein, including technical and scientific terms, shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance in incorporating terminology definitions is provided to better understand the teachings of this invention.

[0018] As used herein, the following terms have the following meanings: Unless the context clearly specifies otherwise, as used herein, “a,” “one,” and “the” refer to the singular and plural referents, respectively. For example, “compartment” refers to one or more compartments.

[0019] As used herein, the word "about" when used to describe measurable numerical values, such as parameters, quantities, durations, etc., refers to a variation of less than + / -20%, preferably less than + / -10%, more preferably less than + / -5%, even more preferably less than + / -1%, and still more preferably less than + / -0.1% relative to the specific value, provided that such variation applies to the invention disclosed herein. However, it should be understood that the value referred to by the modifier "about" is itself specifically disclosed.

[0020] As used herein, “comprising,” “including,” and “containing” are synonymous and are inclusive or open-ended terms used to specify the presence of the components listed thereafter, without excluding or precluding the presence of other unlisted components, features, elements, elements, members, or steps known in the art or disclosed in this invention.

[0021] As used herein, "biomolecule" can refer to peptides, small molecules, scaffold proteins, antibodies, antibody fragments, or antibody mimics. "HER2-binding biomolecules" are of particular interest in this invention and include biomolecules that target or specifically bind to HER2.

[0022] As used herein, a "scaffold protein" refers to a protein that simultaneously binds to two or more other proteins and assembles its binding partner into a functional unit. For example, the radiolabeled scaffold protein DARPin G3 binds to ADAPT6 ([[ 99m Tc]Tc-(HE)3-G3 and [ 99m [Tc]Tc-ADAPT6) can be used for the visual detection of HER2 expression levels. Albumin-binding domain-derived affinity proteins (ADAPTs), such as ADAPT6, are a class of novel small proteins (46 to 59 amino acids) suitable for use as molecular imaging probes.

[0023] "Antibody mimics" are defined as molecules that can bind to antigens in a manner similar to antibodies; however, they are not produced by the immune system and are structurally independent of antibodies. They are typically unrelated protein scaffold molecules composed of structures such as α-helices, β-sheets, or random coils, capable of binding to specific targets, and can be modified using conventional protein engineering strategies to introduce new binding sites. Antibody mimics include, for example, affinity molecules. "Affinity molecules" or "affinity molecules" are small, stable proteins that, when engineered, can bind to a large number of target proteins or peptides with high affinity, mimicking the action of monoclonal antibodies. Affinity molecules are based on the immunoglobulin G domain of Staphylococcus aureus protein A. This invention focuses particularly on affinity molecules that selectively bind to the HER2 receptor. Such anti-HER2 affinity molecules include, for example, ABY-025 and ABY-027. The second-generation affinity molecule "ABY-025" can selectively bind to the HER2 receptor with picomolar affinity. These affinity molecules are currently in clinical development for tumor diagnostics. The anti-HER2 affinity molecule ABY-027, fused with the albumin-binding domain (ABD), after being labeled with lutetium-177, can reduce the uptake of radioactivity by the kidneys and liver in mouse xenotransplantation.

[0024] As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to a polymer of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified / derived amino acids, and polypeptides with modified peptide backbones.

[0025] As used herein, an "antibody" refers to an antibody consisting of two heavy chains, each containing a constant region and a variable region. These heavy chains are linked by disulfide bonds in what are called hinge regions. In addition, each heavy chain is connected to a light chain (also containing a constant region and a variable region) via other disulfide bonds; this arrangement is often referred to as forming a unified "Y"-shaped structure. Each variable region contains three complementarity-determining regions (CDRs), and the variable regions of the light and heavy chains together determine the antibody's binding specificity to its target.

[0026] As used herein, "antibody fragment" refers to an entity that does not constitute a complete antibody, such as variable regions of the heavy and / or light chains, single-chain variable regions, Fab fragments, variable regions and partial constant regions, heavy chains, light chains, single chains, etc., and also includes conjugates of the above-mentioned parts. The variable regions of the heavy or light chains can be considered as the basic functional binding units of an antibody, sometimes also referred to as domain antibodies. Alternatively, the variable regions of the heavy and light chains can be linked together, for example, by covalent bonds, to form what is called a "single-chain variable fragment (scFv)," which is identical to a complete antibody and contains the three CDRs of the heavy chain (commonly referred to as H1, H2, H3) and the three CDRs of the light chain (commonly referred to as L1, L2, L3).

[0027] As used herein, an "immunoglobulin single variable domain" is defined as a molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain (this differs from conventional immunoglobulins or fragments thereof, which typically require the interaction of two immunoglobulin variable domains to form an antigen-binding site). However, it should be clarified that the term "immunoglobulin single variable domain" includes conventional immunoglobulin fragments in which the antigen-binding site is composed of a single variable domain.

[0028] Typically, the amino acid sequence of an immunoglobulin single variable domain comprises four framework regions (FR1 to FR4) and three complementation-determining regions (CDR1 to CDR3), preferably conforming to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1) or any suitable fragment thereof (typically containing at least some amino acid residues constituting at least one complementation-determining region). Immunoglobulin single variable domains containing four FR regions and three CDR regions are known to those skilled in the art and have been described.

[0029] Typical non-restricted examples of immunoglobulin single variable domains include: light chain variable region sequences (e.g., VL domain sequences) or suitable fragments thereof, heavy chain variable region sequences (e.g., VH domain sequences or V... HThe immunoglobulin single variable domain (VH domain sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit, is acceptable. Therefore, according to a preferred embodiment, the immunoglobulin single variable domain is a light chain variable region sequence (e.g., VL) or a heavy chain variable region sequence (e.g., VH); more specifically, the immunoglobulin single variable domain is a heavy chain variable region sequence derived from a conventional four-chain antibody, or a heavy chain variable region sequence derived from a heavy chain antibody. The immunoglobulin single variable domain can be a domain antibody (“dAB” or “dAb”), a single-domain antibody (“sdAB” or “sdAb”), or a VH domain. H The H domain sequence or other immunoglobulin monovariable domains, or any suitable fragment of any of the aforementioned domains. An immunoglobulin monovariable domain typically contains a single amino chain, which can be considered to contain four frame sequences (“FRs”) and three complementarity-determining regions (“CDRs”) (as defined herein). Clearly, the frame regions can also participate in antigen binding. The delineation of CDR sequences (and FR sequences) can be based on the unique IMGT numbering system used for V and V-like domains. Alternatively, the delineation of FR and CDR sequences can be based on the V-like domain numbering system applicable to camel-derived V-like domains. H Kabat numbering system for H domain.

[0030] It should be noted that the immunoglobulin single variable domain, as a binding domain portion, is broadly not limited to a specific biological source or a specific preparation method. The term "immunoglobulin single variable domain" encompasses variable regions from various sources, including variable regions from mice, rats, rabbits, donkeys, humans, sharks, or camelids. According to a specific embodiment, the immunoglobulin single variable domain is derived from shark antibodies (referred to as immunoglobulin neoantigen receptors (IgNARs), more specifically, from naturally occurring, light-chain-free heavy-chain shark antibodies, referred to as VNAR domain sequences. Preferably, the immunoglobulin single variable domain is derived from camelid antibodies; more preferably, the immunoglobulin single variable domain is derived from naturally occurring, light-chain-free camelid heavy-chain antibodies, i.e., VNAR domains. H H-domain sequence.

[0031] As used in this article, the term "V" H "H domain sequence" and "single-domain antibody fragment (sdAb)" are used interchangeably, referring to a single-domain antigen-binding fragment. It specifically refers to a single variable domain derived from a natural heavy chain antibody, and is known to those skilled in the art. V H The H domain sequence is usually derived from antibodies in camel-like animals that contain only heavy chains (no light chains), and is therefore often referred to as V. H H antibody or V H H sequence. Camelidae include Old World camels (Bactrian camels, Dromedary camels) and New World camels (e.g., alpacas, llamas, guanacos, llamas). V HThe small size and unique biophysical properties of the H domain sequence make it superior to conventional antibody fragments in recognizing rare or cryptic epitopes and cavities or active sites that bind to protein targets. V H The H domain sequence is stable, tolerant of the gastrointestinal environment, and easy to manufacture. Therefore, V H H-domain sequences have applications in many fields, including drug development and treatment, and are also a versatile and valuable tool for protein purification, functional studies, and crystallization.

[0032] The V of the present invention H The H domain sequence typically contains a single amino acid chain, comprising four "frame regions (FRs)" and three "complementation-determining regions (CDRs)," according to equation (1) (as described above). The term "complementation-determining region (CDR)" refers to the V... H The variable regions in the H domain sequence contain amino acid sequences that can specifically bind to antigen targets; these CDR regions determine the V H H represents the fundamental specificity of a particular antigenic determinant structure. These regions are also known as "hypervariable regions." V H The H domain sequence has three discontinuous CDR regions (referred to as CDR1, CDR2, and CDR3). The delineation of FR and CDR sequences is typically based on the unique IMGT numbering system used for the V and V-like domains. Alternatively, the delineation of FR and CDR sequences can be based on the V-like domain numbering system applicable to camel-derived sources. H The Kabat numbering system for the H field. As is known to those skilled in the art, V H H-domain sequences can be characterized in particular by the presence of one or more camelid-specific residues in one or more frame region sequences (according to Kabat numbering).

[0033] The present invention preferably includes a class of immunoglobulins with a single variable domain corresponding to the V of natural heavy chain antibodies. H H domain, also known as "V" H "H domain sequence" or "single domain antibody fragment (sdAb)".

[0034] This type of V H The H domain sequence can typically be obtained by: appropriately immunizing a camel with the target (to induce an immune response and / or generate a heavy-chain antibody targeting the target), obtaining a suitable biological sample (e.g., a blood sample or any B cell sample) from the camel, and generating a VHH sequence targeting the target sequence from the sample using any known suitable technique. These techniques are readily apparent to those skilled in the art. Alternatively, the H domain sequence can be obtained from the camel's V... H Obtain natural V for the desired target from the original library of the H sequence. HThe H domain, for example, allows for the screening of such libraries using one or more known screening techniques by employing a desired target or at least a portion, fragment, antigenic determinant, or epitope. Such libraries and techniques are described, for example, in WO09937681, WO0190190, WO03025020, and WO03035694. Alternatively, libraries can be used from the original V... H Modified synthetic or semi-synthetic libraries derived from H libraries, such as those derived from the original V library through techniques like random mutagenesis and / or CDR rearrangement. H V obtained from H Library H H-libraries, as described in WO0043507. Another method to obtain V targeting specific points. H The technique involving H-domain sequencing involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., inducing an immune response and / or heavy chain antibodies against the desired target), obtaining a suitable biological sample (e.g., a blood sample or any B cell sample) from the transgenic mammal, and then using any suitable technique known per se to generate V-domain antibodies against the desired target from said sample. H H domain sequence. For example, for this purpose, transgenic mice expressing heavy chain antibodies as described in WO02085945 and WO04049794, as well as further methods and techniques, can be used.

[0035] A particularly preferred class of immunoglobulins of the present invention contains V H The H domain sequence, whose amino acid sequence is similar to that of natural V H The amino acid sequence corresponding to the H domain has been "humanized," i.e., by modifying the natural V... H Humanization is achieved by replacing one or more amino acid residues in the H sequence (particularly the framework region sequence) with amino acid residues from the corresponding positions of the VH domain of a conventional human four-chain antibody. This can be done by methods known in the art, and those skilled in the art will understand how to implement it based on existing techniques related to humanization. Furthermore, it is important to note that the humanization domain V of this invention... H H sequences can be obtained by any known suitable method, and are therefore not strictly limited to those containing natural V. H The H-domain polypeptide is a polypeptide obtained from the starting material. It is related to the corresponding natural V... H Compared to the H domain, humanized V H The H domain sequence may offer several advantages, such as reduced immunogenicity. This humanization typically involves introducing natural V... H One or more amino acid residues in the H sequence are replaced with amino acid residues at the same position in the human VH domain (e.g., the human VH3 domain). The choice of humanization substitution should ensure that the resulting humanized VHH domain sequence still retains the V as defined in this paper. HThe H-domain sequence possesses excellent properties. Those skilled in the art can select appropriate substitutions or combinations to optimize or achieve the excellent properties provided by humanized substitutions and those of natural V. H An ideal balance between the desirable properties of the H domain.

[0036] The scope of this invention also includes natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs of immunoglobulins as defined herein (collectively referred to herein as “variants”).

[0037] Therefore, according to one embodiment of the invention, the term "immunoglobulin single variable domain of the invention" also broadly encompasses such variants, particularly the sdAb variants of SEQ ID NO:1 or SEQ ID NO:2. Typically, such variants may have substitutions, deletions, and / or insertions of one or more amino acid residues relative to the sdAb of the invention as defined herein. Such substitutions, deletions, and / or insertions may occur in one or more frame regions and / or CDRs. As used herein, a variant is a sequence identity of at least 80%, preferably at least 85%, more preferably 90%, further preferably 95%, and most preferably 99%, between each or any frame region and each or any complementarity-determining region and the corresponding region of the reference sequence. Sequence identity can be electronically calculated using algorithms such as PILEUP, BLAST, etc. (Altschul et al. 1990; J Mol Biol 215: 403; Higgins & Sharp 1989, CABIOS 5: 151). The software used for BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI, http: / / www / ncbi.nlm.nih.gov / ). Such immunoglobulin single-variable domain variants may have particular advantages because they may possess higher potency or other desired properties.

[0038] As a non-limiting example, substitutions may be conservative substitutions (as described herein), and / or one amino acid residue may be replaced by another amino acid residue. Therefore, any one or more substitutions, deletions, or insertions, or any combination thereof, made to the immunoglobulin single variable domain of the present invention, as long as they improve the properties of the immunoglobulin single variable domain of the present invention, or at least do not cause undue adverse effects on the desired properties, or the balance or combination of desired properties of the immunoglobulin single variable domain of the present invention (i.e., do not render the immunoglobulin single variable domain unsuitable for its intended use), are included within the scope of the present invention. Those skilled in the art can typically determine and select suitable substitutions, deletions, or insertions, or suitable combinations thereof, based on the disclosure herein, optionally through a limited number of conventional experiments; for example, a limited number of possible sampling points can be introduced and their effects on the performance of the resulting immunoglobulin single variable domain can be determined.

[0039] According to embodiments, the immunoglobulin single-domain variants of the present invention, particularly sdAb, may have one, two, or three amino acids substituted, deleted, or inserted in one, two, or three complementarity-determining regions (CDRs); more specifically: (i) in CDR1, CDR2, or CDR3; (ii) in CDR1 and CDR2, CDR1 and CDR3, or CDR2 and CDR3; (iii) in CDR1, CDR2, and CDR3. More preferably, the immunoglobulin single-domain variants of the present invention, particularly sdAb, may have one, two, or three amino acids conservatively substituted (as defined herein) in one, two, or three CDRs; more specifically: (i) in CDR1, CDR2, or CDR3; (ii) in CDR1 and CDR2, CDR1 and CDR3, or CDR2 and CDR3; (iii) in CDR1, CDR2, and CDR3.

[0040] Furthermore, depending on the host organism used to express the immunoglobulin single variable domain of the present invention, such deletions and / or substitutions can be designed to remove one or more post-translational modification sites (e.g., one or more glycosylation sites), which is entirely feasible to those skilled in the art. Alternatively, substitutions or insertions can be designed to introduce one or more sites for linking functional groups.

[0041] Examples of modifications, examples of amino acid residues that can be modified in immunoglobulin single variable domain sequences, methods and techniques for introducing such modifications, and the potential uses and advantages of such modifications will be apparent to those skilled in the art. For example, such modifications may involve introducing one or more functional groups, residues, or portions (e.g., via covalent linkage or other suitable means) into or on the surface of an immunoglobulin single variable domain, particularly introducing one or more functional groups, residues, or portions that impart one or more desired properties or functions to the immunoglobulin single variable domain of the present invention. Such functional groups and their introduction techniques are clear to those skilled in the art and generally include all the functional groups and techniques mentioned in the background section cited above, as well as known functional groups and techniques for modifying pharmaceutical proteins, particularly antibodies or antibody fragments (including single-domain antibody fragments). For example, such functional groups may be directly (e.g., covalently) linked to the immunoglobulin single variable domain of the present invention, or optionally linked via suitable linkers or spacers, as is also well known to those skilled in the art. One of the most common techniques for prolonging the half-life of pharmaceutical proteins and / or reducing their immunogenicity involves linking suitable pharmaceutically acceptable polymers, such as polyethylene glycol (PEG) or derivatives thereof (e.g., methoxylated polyethylene glycol, mPEG). Generally, any suitable polyethylene glycol glycosylation method applicable to antibodies and antibody fragments in the art can be used. In one embodiment, site-directed polyethylene glycol glycosylation is employed, particularly via cysteine ​​residues. For example, for this purpose, PEG can be linked to naturally occurring cysteine ​​residues in the immunoglobulin monovariable domain of the present invention; the immunoglobulin monovariable domain of the present invention can also be modified to appropriately introduce one or more cysteine ​​residues for linking PEG; or an amino acid sequence containing one or more cysteine ​​residues for linking PEG can be fused to the N-terminus and / or C-terminus of the immunoglobulin monovariable domain of the present invention, all of which employ protein engineering techniques known to those skilled in the art. Another, generally less preferred modification includes N-linked or O-linked glycosylation, which is typically as part of co-translational and / or post-translational modifications, depending on the host cell used to express the immunoglobulin monovariable domain of the present invention.

[0042] As used herein, the term "sequence identity" refers to the degree of similarity between sequences at the nucleotide or amino acid level within an alignment window. Therefore, the "sequence identity percentage" is calculated as follows: Two optimally aligned sequences are compared within the alignment window to determine the number of identical nucleotide bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) in the two sequences, yielding the number of matching sites. This number is then divided by the total number of sites within the alignment window (i.e., the window size), and the result is multiplied by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage can be performed manually or using existing computer programs in the art. Examples of applicable algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol.215: 403 (1990)). Software used for BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). A “deletion” as defined herein is a change in the amino acid or nucleotide sequence relative to the amino acid or nucleotide sequence of the parent polypeptide or nucleic acid, lacking one or more amino acid or nucleotide residues. For proteins, deletions can involve approximately 2, approximately 5, approximately 10, up to approximately 20, up to approximately 30, or up to approximately 50 or more amino acids. Proteins or fragments thereof may contain more than one deletion.

[0043] "Insertion" or "addition" refers to a change in the amino acid or nucleotide sequence compared to the parent protein, by adding one or more amino acid or nucleotide residues. "Insertion" typically refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, while "addition" can be an insertion or the addition of amino acid residues at the N-terminus, C-terminus, or both ends. For proteins or fragments thereof, the number of inserted or added amino acids is typically approximately 1, 3, 5, 10, up to approximately 20, up to approximately 30, or up to more than approximately 50. Proteins or fragments thereof may contain more than one insertion. As used herein, "substitution" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides compared to the parent protein's amino acid or nucleotide sequence. It should be understood that proteins or fragments thereof may have conserved amino acid substitutions that have little or no effect on protein activity. Conserved substitutions include combinations such as: gly and ala; val, ile, leu and met; asp and glu; asn and gin; ser and thr; lys and arg; cys and met; and phe, tyr and trp.

[0044] As used herein, the terms “cancer” or “tumor” refer to any neoplastic condition, such as breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer, esophageal cancer, and gastroesophageal cancer. Neoplastic conditions associated with HER2 overexpression are particularly relevant to this invention, such as HER2-associated breast cancer and gastroesophageal cancer.

[0045] As used herein, a "lesion" refers to any type of mass in the body of undetermined nature, including, for example, neoplastic or inflammatory lesions. Infectious and neoplastic diseases can present with features resembling, for example, malignant tumors. Infectious causes, particularly tuberculosis and fungal infections such as histoplasmosis and aspergillosis, often lead to pulmonary nodules, masses, or lymph node lesions that may resemble malignant tumors. The method of this invention helps determine ideal sites for biopsies and identify lesions of other diseases.

[0046] As used in this article, a “radioactive nuclide” refers to an atom with excess nuclear energy and therefore unstable. Excess nuclear energy can be used in one of three ways: emitted from the nucleus as gamma rays; transferred to an electron in the atom and released as a converted electron; or used to generate and emit new particles (alpha or beta particles) within the nucleus.

[0047] As used in this article, the term “diagnosis” or its grammatically equivalent means determining whether a subject has a specific disease or condition.

[0048] As used in this article, "prognosis," or its grammatically equivalent, refers to the estimated course and outcome of a disease. The prognosis for a patient diagnosed with cancer is typically defined as the likelihood of successful treatment and recovery.

[0049] As used herein, “treating cancer” or “treating a subject or individual with cancer” or “cancer treatment” includes significantly inhibiting disease progression, significantly delaying or reversing disease progression, significantly improving clinical symptoms of the disease, or significantly preventing the onset of clinical symptoms of the disease. Specifically, it includes inhibiting the replication of cancer cells, inhibiting the spread of cancer, reducing tumor volume, reducing or decreasing the number of cancer cells in the body, and / or improving or alleviating cancer symptoms. Treatment is considered effective if mortality and / or morbidity are reduced, and treatment can be administered in a preventative or therapeutic manner.

[0050] Typically, a “subject” is a mammal or mammalian, where these terms are used broadly to refer to an organism belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In many implementations, the subject will be a human.

[0051] As used herein, "targeting moiety" or "targeting agent" refers to an agent having a binding affinity for the specific molecular target or biomarker HER2 in this invention. In this invention, such "targeting moiety" or "targeting agent" is a HER2-binding biomolecule coupled to a detectable label (radionoid) to form a targeting tracer, which may be used, for example, in in vivo medical imaging.

[0052] Furthermore, unless otherwise stated, the terms first, second, third, etc., in the specification and claims are used only to distinguish similar elements and are not necessarily used to describe order or chronological order. It should be understood that the terms can be used interchangeably under appropriate conditions, and the embodiments of the invention described herein can be implemented in an order different from that described or illustrated herein.

[0053] The range of values ​​expressed in terms of endpoints includes all numbers and fractions contained within that range, as well as the endpoints themselves.

[0054] Unless otherwise defined, the expressions “% by weight,” “percentage by weight,” “%wt” or “wt%” in this document and throughout the specification refer to the relative weight of the corresponding component based on the total weight of the formulation.

[0055] Although the terms “one or more” or “at least one”, such as one or more or at least one element in a set of elements, are self-evident, by further example, the term specifically includes references to any one of the elements or any two or more of the elements, for example, any element ≥3, ≥4, ≥5, ≥6 or ≥7, up to all the elements.

[0056] Unless otherwise defined, all terms disclosed herein, including technical and scientific terms, shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes definitions of terms used in the specification to better understand the teachings of this invention. The terms or definitions used herein are provided solely to aid in understanding the invention.

[0057] Throughout this specification, references to "one embodiment" or "a particular embodiment" mean that a specific feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in a particular embodiment" appearing in various places throughout this specification do not necessarily refer to the same embodiment, but may indeed do so. Furthermore, it will be apparent to those skilled in the art, based on this disclosure, that specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Moreover, while some embodiments described herein include certain features but not certain other features included in other embodiments, those skilled in the art will understand that combinations of features from different embodiments are also intended to fall within the scope of this invention. For example, in the following claims, any claimed embodiment can be used in any combination.

[0058] Detailed description

[0059] Breast cancer has the highest incidence rate among women worldwide. The expression of hormone receptors and human epidermal growth factor 2 (HER2) receptors is important for prognosis and treatment selection. Over the past two decades, therapies targeting the HER2 receptor have been developed, including kinase inhibitors, trastuzumab, pertuzumab, and trastuzumab-based antibody-drug conjugates (ADCs). Receptor expression and genomic status are predictive biomarkers for these therapies; in HER2-positive breast cancer patients, these therapies have improved overall survival. Currently, the diagnosis of HER2 expression or gene amplification relies on immunohistochemistry (IHC) and / or in situ hybridization (ISH) of pathological samples. IHC measures the expression of the HER2 receptor on the cell surface of biopsy tissue samples, which can be graded as 0, 1+, 2+, or 3+. According to the American Society of Clinical Oncology and the American Society of Pathologists (ASCO-CAP), HER2-positive patients are defined as those with an immunohistochemistry (IHC) score of 3+, or those with an IHC score of 2+ but a positive in situ hybridization (ISH) score. In recent years, a new category has emerged called "HER2 low expression" (1+ and 2+, with ISH negative), because these patients also benefit from the second-generation ADC trastuzumab-detrastuzumab (see overview of HER2 classification). Figure 2 and Figure 3 This makes accurate assessment of HER2 expression levels and identification of diseases with low HER2 expression even more important, as detecting low levels of HER2 expression may be key to selecting the right treatment for the right patient. As mentioned above, HER2 status is determined by IHC and / or ISH on biopsy samples. Biopsy is invasive, but it can assess many desired molecular parameters, although this assessment is limited to a small subset of the existing disease in the patient. However, increasing data indicate heterogeneous HER2 expression within and around cancer lesions, which can increase the risk of sampling errors and false negatives, thus requiring multiple biopsies.

[0060] In a first aspect, the present invention provides a method for determining HER2 expression in one or more lesions of a subject, the method comprising: a. Administering a tracer to the subject, wherein the tracer contains a radiolabeled substance conjugated to a HER2-binding biomolecule; b. Perform whole-body positron emission tomography / computed tomography (PET / CT) on the subject; c. Determine the amount of tracer absorbed in the lesion; d. Initial classification of lesions based on the tracer uptake, wherein each of the one or more lesions is classified as HER2 positive or HER2 negative.

[0061] As mentioned above, current diagnostic methods for HER2 expression or gene amplification rely on immunochemical (IHC) and / or in situ hybridization (ISH) testing of pathological samples (biopsy). Interlesional, intralesional, and temporal heterogeneity present challenges for tissue sampling. HER2 expression can vary depending on the selected lesion or the biopsy portion within the target lesion, thus affecting the diagnosis of IHC HER2 status and subsequent treatment pathways. Differences in HER2 status have been found between 3% and 10% of patients with primary and recurrent breast cancer, and even between and within individual lesions. Even within HER2 0 and HER2-negative tumors, temporal heterogeneity has been reported in 37.3%, primarily driven by changes in HER2 status between HER2-null and HER2-low expression tumors. This heterogeneity has driven changes in guidelines for recurrent breast cancer, recommending repeat biopsies and reassessment of biomarker status.

[0062] The method of this invention offers superior diagnostic performance because it allows for the assessment of HER2 expression in all tumor lesions using a single whole-body PET-CT scan. By performing a whole-body PET / CT scan on the subject, a comprehensive picture of HER2 expression throughout the body can be obtained. This contrasts with biopsies, in which only a small portion of the body is sampled and analyzed for HER2 expression. Therefore, the method of this invention can aid in decision-making, avoid unnecessary invasive tissue biopsies, and guide biopsies to tissue locations where effective diagnostic biopsy samples can be obtained.

[0063] Systemic assessment of HER2 expression allows for the revelation of heterogeneous HER2 expression and the identification of HER2-positive tumor lesions. Various targeting molecules or HER2-binding biomolecules can be used as imaging tracers: monoclonal antibodies and their fragments, peptides, protein scaffolds, antibody mimics, etc.

[0064] Each molecule has its own specific pharmacokinetics, which affects the selection of radionuclides.

[0065] In one embodiment, the HER2-binding biomolecule is an affinity compound. In another preferred embodiment, the HER2-binding biomolecule comprises an immunoglobulin monovariable domain that targets or specifically binds to HER2.

[0066] Immunoglobulin single variable domains (also known as "single-domain antibodies," abbreviated as "SdAbs") are small (12 kDa to 15 kDa), highly stable, possess nanomolar affinity, and have low immunogenicity. Radiolabeled immunoglobulin single variable domains rapidly target their antigens and are cleared by the kidneys. This means they can be labeled with short-lived radionuclides (such as gallium-68 or fluorine-18), reducing the radiation dose to patients compared to the longer-lived radionuclides required for radiolabeling of monoclonal antibodies (Abs). Therefore, these immunoglobulin single variable domains allow for same-day imaging, making them ideal probes for molecular imaging.

[0067] Because immunoglobulin single variable domains that target or specifically bind to HER2 have extremely high sensitivity, the method of this invention can detect lesions expressing HER2, even in patients previously identified as having low or negative HER2 expression, and when using different imaging techniques (such as […]). 18 Detection is possible even in small lesions that might otherwise go unnoticed by FDG-PET. Immunoglobulin single variable domains (IVVs) are ideal targeting fractions for detecting HER2-positive lesions. IVVs have low molecular weight and high solubility. IVVs are antibody-derived molecules in which the antigen-binding site is located on and formed by a single immunoglobulin domain (unlike conventional immunoglobulins or fragments thereof, which typically require two IVVs to interact to form the antigen-binding site). IVVs exhibit faster pharmacokinetics compared to monoclonal antibodies or larger antibody fragments. This results in higher contrast and shorter waiting times after intravenous injection. Therefore, this invention also allows for rapid image acquisition after administration of the tracer. In a preferred embodiment, the PET / CT scan is performed 30 to 300 minutes, more preferably 40 to 240 minutes, and even more preferably 60 to 120 minutes, for example, 90 minutes, after administration of the tracer.

[0068] The HER2 proto-oncogene encodes the production of a 185 kDa cell surface receptor protein, namely the HER2 protein or receptor. This gene is sometimes also called neu, HER2 / neu, or c-erbB-2.

[0069] Normal cells express small amounts of HER2 protein on their plasma membrane in a tissue-specific manner. No specific HER2 ligand has yet been identified; however, studies have shown that HER2 forms heterodimers with HER1 (epidermal growth factor receptor, EGFR), HER3, and HER4. These heterodimers form when these receptors bind to their respective ligands to form complexes. Once this heterodimer forms, the activated HER2 receptor transmits growth signals from the extracellular space to the nucleus, thereby controlling various aspects of normal cell growth and division.

[0070] In tumor cells, errors in the DNA replication system can lead to multiple copies of a gene on a single chromosome, a phenomenon known as gene amplification. Amplification of the HER2 gene increases its transcriptional level. This results in elevated HER2 mRNA levels and, consequently, increased HER2 protein synthesis, leading to HER2 overexpression on the surface of these tumor cells. This overexpression can result in HER2 protein levels that are 10 to 100 times higher than in adjacent normal cells. This, in turn, leads to increased cell division and, consequently, a faster cell growth rate. HER2 gene amplification is associated with the transformation of normal cells into a cancer phenotype.

[0071] Overexpression of the HER2 protein is thought to promote the formation of HER2 homodimers, leading to sustained receptor activation. Under these conditions, growth-promoting signals can be continuously transmitted into the cell even in the absence of ligands. Therefore, multiple intracellular signaling pathways are activated, resulting in uncontrolled cell growth and, in some cases, carcinogenesis. Thus, growth factor receptor-mediated signal transduction mechanisms are important targets for inhibiting cell replication and tumor growth.

[0072] In 25% of all breast cancer patients, the HER2 gene is overexpressed due to amplification. This overexpression of the HER2 protein is associated with several adverse prognostic variables, including estrogen receptor negativity, high S-phase fraction, lymph node positivity, p53 mutation, and high nuclear grade. Therefore, HER2 overexpression has been reported in breast cancer. It is also associated with ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, lung cancer, and esophageal cancer.

[0073] A single-domain antibody (sdAb) targeting the HER2 receptor (2Rs15D) has been developed. In one embodiment, the immunoglobulin single variable domain targeting 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, wherein SEQ ID NO:1 has the following sequence: "QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCMGGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSS".

[0074] SEQ ID NO: 2 has the following sequence: "DVQLQESGGGSVQAGGSLKLTCAASGYIFNSCMGGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSS".

[0075] In one implementation, the immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of: a. SEQ ID NO: 1 or SEQ ID NO: 2 b. A polypeptide having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2, c. A polypeptide that differs from SEQ ID NO: 1 or SEQ ID NO: 2 by 1, 2 or 3 amino acids.

[0076] The immunoglobulin single variable domain that targets or specifically binds to HER2 and contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO:1 or SEQ ID NO:2 exhibits high specificity, strong antigen-binding affinity, low immunogenicity, and favorable pharmacokinetics.

[0077] In SEQ ID NO: 1, the N-terminal glutamine (Q) is replaced with aspartic acid (D) in SEQ ID NO: 2. This allows for the avoidance of N-pyroglutamic acid formation, which would result in two distinct protein types. When using an immunoglobulin single variable domain containing N-terminal glutamine (Q), there is a risk of deamidation, leading to a random and uncontrollable process, resulting in an undefined product that requires additional purification. This additional purification step results in significant product loss and reduced yield. Figures 5 to 7 The reversed-phase high-performance liquid chromatography (RP-HPLC) of a protein batch is shown, which contains SEQ ID NO: 1 ( Figure 5 (GMP batch) or SEQ ID NO: 2 ( Figure 6 Research and development batches and Figure 7The immunoglobulin single variable domains (GMP batches) were compared in Table 1 below. A significant back peak (RRT = 1.05 = 38%) was observed in the protein batch containing the immunoglobulin single variable domain of SEQ ID NO:2 compared to the batch containing the immunoglobulin single variable domain of SEQ ID NO:1. This is because the N-terminal glutamine present in the immunoglobulin single variable domain of SEQ ID NO:1 generates N-pyroglutamic acid after production and purification. This variant is more hydrophobic and elutes as a back peak in RP-HPLC. In SEQ ID NO:2, the N-terminal amino acid is converted to aspartic acid, thus preventing N-terminal cyclization.

[0078] This can also be confirmed by RP-HPLC-UV-MS data. The UV 280 nm spectrum of the protein batch containing the immunoglobulin single variable domain of SEQ ID NO:1 showed a distinct back peak. This peak was identified as a variant with glutamine N-terminus cyclized to pyroglutamic acid. In the protein batch containing the immunoglobulin single variable domain of SEQ ID NO:2, this variant was absent (N-terminal D-cyclization did not occur) (data not shown).

[0079] In addition, in the case of SEQ ID NO: 1 ( Figure 5 ) or SEQ ID NO: 2 ( Figures 6 to 7 Among batches of immunoglobulin monovariable domain proteins, only minor differences were observed in other product-related variants.

[0080] Table 1: RP-HPLC results of protein batches containing immunoglobulin single variable domains of SEQ ID NO: 1 or SEQ ID NO: 2

[0081] In one embodiment, the immunoglobulin single variable domain is a recombinant protein produced by the expression host. In one embodiment, the expression host is a bacterial host, such as *Escherichia coli*. E. coli ) or Bacillus subtilis ( Bacillus subtilis In another embodiment, the expression host is yeast, such as *Saccharomyces cerevisiae*. Saccharomyces cerevisiae ) or Pichia pastoris ( Komagataella phaffii In other embodiments, the 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), or a plant host (e.g., Nicotiana benthamiana (Nicotiana benthamiana)). Nicotiana benthamiana ) or Arabidopsis thaliana ( Arabidopsis thaliana or algal hosts (e.g., Chlamydomonas reinhardtii, Chlamydomonas reinhardtii In other embodiments, the recombinant protein is produced by a cell-free expression system.

[0082] As used herein, the terms “specific recognition” or “specific binding” or simply “specific” refer to the ability of an immunoglobulin monovariable domain to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens, and do not necessarily imply high affinity (as further defined herein). In some embodiments, specific binding interactions will distinguish between desired and undesired antigens in a sample, and in some embodiments, the fold of distinction is more than about 10 to 100 times or more (e.g., more than about 1,000 times or 10,000 times). The terms “specific binding,” “selective binding,” “preferential binding,” and their grammatical equivalents are used interchangeably herein. As used herein, the term “affinity” refers to the degree to which an immunoglobulin monovariable domain binds to an antigen such that the equilibrium between the antigen and the immunoglobulin monovariable domain shifts toward the presence of the complex formed by their binding. Thus, for example, in the case of an antigen and antibody (fragment) combined at approximately equal concentrations, a high-affinity antibody (fragment) will bind to the available antigen, thereby shifting the equilibrium toward a higher concentration of the complex. The dissociation constant is commonly used to describe the affinity between an antibody (fragment) and an antigen target. Typically, the dissociation constant is below 10. -5 M. Preferably, the dissociation constant is less than 10. -6 M, more preferably, less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M.

[0083] It can specifically bind to a particular antigen or antigenic determinant (e.g., an epitope) and / or an immunoglobulin monovariable domain that has an affinity for it, referred to as “targeting” or “targeting” the antigen or antigenic determinant.

[0084] In one embodiment, the radiolabel comprises a radionuclide selected from the group consisting of: fluorine-18 ( 18 F), Indium 111 ( 111 In), Copper 61 ( 61 Cu), Copper 64 ( 64 Cu), Gallium-67 ( 67 Ga), gallium 68( 68 Ga), Technetium 99m( 99m Tc).

[0085] As previously mentioned, immunoglobulin single variable domains can be labeled with short-lived radionuclides (e.g., gallium-68 or fluorine-18), which reduces the radiation dose to the patient compared to the longer-lived radionuclides required for radiolabeling monoclonal antibodies (Abs). In a preferred embodiment, the immunoglobulin single variable domain is labeled with gallium-68 (e.g., gallium-68 or fluorine-18).68 Marked with Ga). Uses Gallium-68 (Ga). 68 Ga68-labeled immunoglobulin single variable domains provide rapid, uniform, and deep tissue penetration (within minutes), rapid clearance of unbound fragments (within hours), and exhibit low blood circulation time, high signal-to-noise ratio, and high stability even under harsh chemical and physicochemical conditions. Furthermore, Ga68 is optimal for PET imaging, offering low radiation burden on the subject (attenuation t1 / 2 = 68 minutes) and high spatial resolution for imaging analysis. Additionally, multiple scans can be performed in a short period of time, and... 68 Ga generators are flexible, convenient, and widely available.

[0086] As will be apparent to those skilled in the art, specific modifications to the tracer may involve the introduction of chelating groups, for example, for chelating one of the aforementioned metals or metal cations. Suitable chelating groups include, for example, but not limited to, DTPA (diethylenetriaminepentaacetic acid) and its derivatives (including 1B4M-DTPA derivatives and CHX-A''-DTPA derivatives), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its derivatives (including DOTA-GA derivatives, DOTAM derivatives, DO3A and its derivatives, DO2A and its derivatives, CB-DO2A derivatives and DO3AM derivatives), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and its derivatives (including NODA derivatives, NODA-GA derivatives, NO2A derivatives, NOOTAM derivatives, NOPO derivatives and TRAP derivatives), HBED (N,N--bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid) and its derivatives (including HBED-CC derivatives, HBED-CI derivatives, HBED-CA derivatives, HBED-AA derivatives and SHBED derivatives), DEPA (7-[2-(biscarboxymethylamino)ethyl]-4,10-biscarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetic acid) and its derivatives, pyridinecarboxylic acid (PA)-based chelating agents and their derivatives (including H2dedpa, H4octapa, H2azapa and H5decapa, and their derivatives), HEHA (1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid) and its derivatives, TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) and its 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-[1,4,7]triazacyclononane-1-yl-ethyl]-2-carbonylmethyl-amino]-tetraacetic acid) and its derivatives (including C-NETA derivatives and NE3TA derivatives), AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazacyclotetraacetic acid) ) and its derivatives, DATA (6-amino-1,4-diazatriacetic acid) and its derivatives, TCMC (1,4,7,10-tetraazatriacetic acid) and its derivatives -1,4,7,10-tetra(2-aminoacylmethyl)cyclododecane) and its derivatives, PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15), 11,13-trien-3,6,9-triacetic acid) and its derivatives, Macropa (6-[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl]methyl]-4-isothiocyanate-pyridine-2-carboxylic acid) and its derivatives, THP (tris(hydroxypyridinone) and its derivatives, DFO (Deferoxamine) and its derivatives, BCPA (N,N'-1,4-butadiylbis[3-(2-chlorophenyl)acrylamide]) and its derivatives, MAG-2 (2-mercaptoacetyldiglycine) and its derivatives, MAG-3 (2-mercaptoacetyltriglycine) and its derivatives, MAS-3 (mercaptoacetyltriserine) and its derivatives, HYNIC (hydrazinenicotinic acid) and its derivatives, and RESCA (restricted complexing agent). In one embodiment, functional groups such as maleimide, NCS, and NHS are introduced into the chelating agent to allow for various conjugation methods. For example, the isothiocyanate functional group (R-NCS) allows for the formation of stable thiourea bonds with free amines at alkaline pH. NHS is another example of an amine reactive linker.

[0087] In a preferred embodiment, the tracer is [ 68 [Ga]Ga-NOTA-anti-HER2-immunoglobulin single variable domain, wherein the immunoglobulin single variable domain has the sequence of SEQ ID NO:1 or SEQ ID NO:2. A phase I study confirmed that using the [Ga-NOTA-anti-HER2-immunoglobulin single variable domain...] 68 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, and optimal image quality is achieved 90 minutes post-injection.

[0088] PET / CT imaging, combining positron emission tomography (PET) and X-ray computed tomography (CT), has become a standard component of diagnosis and staging in oncology. Beyond cancer detection and staging, PET / CT imaging is increasingly important as a tool for quantitative monitoring of individual treatment responses and for evaluating new drug therapies. Several methods exist for measuring the rate and / or total amount of tracer accumulation in tumors. PET scanners are designed to measure the concentration of radioactivity in vivo [kBq / ml], which is directly related to tracer concentration. However, the relative tissue uptake of the tracer is of general interest. In practice, the two most important sources of variation are the amount of radionuclide injected and patient size. To correct for these differences, at least to the first order, the standard uptake (SUV) is commonly used as a relative measure of tracer uptake. In one embodiment of the invention, the uptake of the tracer is quantified by the SUV. The basic formula for calculating the SUV is shown in Equation 1 below: (Equation 1) Where "r" represents the radioactivity concentration [kBq / ml] measured by a PET scanner within the target area (ROI), "a'" is the decay correction for the injected radionuclide [kBq], and "w" is the patient's body weight [g], which is used as a proxy for the tracer's distribution volume. If all injected tracer remains in the body and is uniformly distributed, the total systemic SUV will be 1 g / ml, regardless of the amount of tracer injected or the patient's body size. Under the assumption that 1 ml of tissue weighs 1 gm, the SUV is dimensionless. Both approaches are used in practice. It is also recommended to use lean body mass as w to correct for lower tracer uptake in adipose tissue. Using the SUV as a measure of relative tissue / organ uptake, facilitating comparisons between patients, has been suggested as a basis for diagnosis.

[0089] As described above, in the method of the present invention, the subject undergoes a whole-body positron emission tomography / computed tomography (PET / CT) scan to determine the uptake of the tracer. In one embodiment, a standard uptake (SUV) of the tracer in the lesion is determined.

[0090] In another embodiment, SUV includes "SUV" 峰值 Peak SUV (SUV) 峰值 A region of origin (ROI) is defined as a small, fixed-size target area centered on a high-uptake region of the tumor. 峰值 The average SUV within the range. In another embodiment, SUV includes "SUV 均值 The mean SUV is defined as the average SUV calculated within the target area (ROI). In another implementation, SUV includes the maximum standard intake, which is defined here as "SUVmax SUV max and ROI 峰值 like Figure 4 As shown.

[0091] In a preferred embodiment, the initial lesion classification (whereby each of the one or more lesions is classified as positive or negative) is based on a predetermined standard uptake threshold (SUV), such as SUV 峰值 In one embodiment, the SUV 峰值 The cutoff value is between 1.5 and 5. In a preferred embodiment, the SUV 峰值 The cutoff value is 2, where SUVs 峰值 Lesions with a value below 2 are classified as negative, and SUVs 峰值 Lesions with a value higher than 2 are classified as positive. In one implementation, other or additional parameters may be used to quantify tracer uptake and classify lesions as HER2 positive or HER2 negative. Other or additional parameters may include the number and / or size of lesions.

[0092] Preliminary data (not shown) indicates that, 68 Ga]Ga-NOTA-anti-HER2-immunoglobulin single variable domain comparison [ 18 F]FDG PET / CT has higher sensitivity and higher specificity for determining disease severity.

[0093] In one embodiment, the specificity of the initial lesion classification is 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%, and more preferably at least 95%. In another embodiment, the specificity of the initial lesion classification is 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%, and more preferably at least 95%.

[0094] In a preferred embodiment, the tracer is administered intravenously.

[0095] In one embodiment, the dose of the radiolabeled biomolecule is 50 MBq to 500 MBq, more preferably 50 MBq to 250 MBq, more preferably 80 MBq to 220 MBq, and more preferably 100 MBq to 200 MBq.

[0096] In one embodiment, the dose of the radiolabeled biomolecule is 25 μg to 1 mg, more preferably 50 μg to 500 μg, and even more preferably 100 μg to 300 μg.

[0097] In one embodiment, the method further includes selecting one or more positively classified lesions to obtain one or more biopsy samples. Because the method of the invention can classify lesions as HER2-negative or HER2-positive, one or more positively classified lesions can be selected to obtain biopsy samples. Therefore, the method of the invention can aid in decision-making, avoid unnecessary invasive tissue biopsies, and guide the biopsy to tissue locations where effective diagnostic biopsy samples can be obtained. Thus, the method of the invention allows for reduced sampling errors, thereby reducing the number of false negative results.

[0098] In one embodiment, the method further includes acquiring the one or more biopsy samples and analyzing HER2 expression in the biopsy samples. In one embodiment, HER2 expression in the biopsy samples is analyzed by immunohistochemistry (IHC) and / or in situ hybridization (ISH). IHC measures the expression of the HER2 receptor on the cell surface of the biopsy tissue sample, which can be graded as 0, 1+, 2+, or 3+. In situ hybridization (ISH) measures gene amplification.

[0099] Specifically, IHC is a method for identifying and describing the expression pattern and intensity of HER2 protein on the cell membrane of breast cancer cells, while ISH utilizes HER2 and CEP17 probes to detect the presence of gene amplification. HER2 IHC is scored using a three-tier system (0 to 3+) based on cell integrity, intensity, and percentage. For questionable results (score 2+), ISH is used for further testing. Specifically, a case is HER2+ when ISH shows an average HER2 copy number ≥6.0 signal / unit. In summary, HER2+ breast cancer is defined as an IHC score of 3+, or an IHC score of 2+ and a positive ISH; while tumors with a HER2 IHC score of 1+ or 2+ but no gene amplification shown on ISH are defined as HER2-low expression. Recently, in addition to the significant and definitive efficacy of HER2-targeted therapy in HER2+ breast cancer, the possibility of targeting HER2 in HER2-low expression breast cancer is being explored. A subgroup of breast cancer patients with negative HER2 biomarkers detected by ISH / IHC also benefited from anti-HER2 therapy. A subset of HER2-negative tumors tested in core biopsy samples may respond to targeted therapy due to intratumoral heterogeneity.

[0100] As previously described, the method of the present invention allows for the determination of HER2 expression throughout the body, thereby enabling targeted biopsies of HER2-positive lesions. Furthermore, in a preferred embodiment, the method of the present invention allows for the detection of lesions with extremely low HER2 expression. This category typically remains undetected when tested using prior screening techniques.

[0101] In a preferred embodiment of the method of the present invention, the subject is classified based on HER2 status, wherein the classification is based on the initial lesion classification and the biopsy sample analysis. In a preferred embodiment, the method of the present invention is used to reduce false negative results. For example, false negative results frequently occur in patients with progressive metastatic breast cancer. Therefore, in one embodiment, the method of the present invention is used to determine HER2 expression in one or more lesions of a subject, wherein the subject is a patient with progressive metastatic breast cancer.

[0102] In one embodiment, the method of the present invention allows the subject to be classified as having extremely low HER2 expression.

[0103] In one implementation, the initial lesion classification based on PET / CT scans and analysis of the targeted biopsy samples can classify subjects as HER2 zero expression (or "HER2 negative"), HER2 ultra-low expression, HER2 low expression, or HER2 high expression (or "HER2 positive").

[0104] The presence of HER2 overexpression indicates sensitivity to HER2-specific treatment regimens, such as the administration of HER2-specific antibodies or other binding molecules with affinity for the HER2 receptor. Several such HER2-specific treatment regimens are currently available on the market (e.g., Herceptin®), with others under development. Therefore, in a related aspect, the present invention also provides a method for treating HER2-positive malignancies, comprising i) determining, using the determination method of the present invention, that the malignancy in a subject is HER2-positive, and ii) treating the malignancy with a HER2-specific therapy.

[0105] Therefore, the method of the present invention can, for example, diagnose and / or molecularly characterize cancers selected from the following non-limiting groups: breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary cancer, lung cancer, prostate cancer, esophageal cancer, and gastroesophageal cancer.

[0106] In this context, molecular characterization involves characterizing HER2 expression, such as determining whether any HER2 is expressed, determining the level of HER2 expression, for example before and after treatment, which can be done by obtaining images before and after treatment, and determining the extent or anatomical content of HER2 expression (e.g. for surgical purposes).

[0107] HER2 status in breast cancer is assessed to select patients suitable for targeted therapy with anti-HER2 therapy. According to the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), HER2 positivity is defined as protein overexpression (3+ score) on immunohistochemistry (IHC) and / or gene amplification under in situ hybridization (ISH). However, as previously mentioned, the introduction of novel anti-HER2 compounds is changing this paradigm, as some breast cancers with low protein expression levels (i.e., 1+ / 2+ score, no gene amplification) can benefit from HER2 antibody-drug conjugates (ADCs). Recently, the potential for HER2 targeting in HER2 “ultra-low expression” (i.e., score 0, ≤10% of tumor cells with incomplete or weak staining) has been highlighted. These novel findings are overturning the traditional dichotomy of HER2 status and significantly improving expectations in the field. Recently, HER2-low expressing tumors (i.e., IHC 1+ or 2+, ISH negative, also known as HER2 “low-expressing” breast cancer) have demonstrated impressive response rates and progression-free survival (PFS) after receiving ADC-based therapy. Another increasingly prominent complicating issue is the clinically relevant presence of incomplete, weakly stained tumor cells in a subset of breast cancers with an IHC score of 0. This HER2 “ultra-low expression” phenotype could explain promising evidence for treatment response in some HER2-negative breast cancers.

[0108] In one implementation, the subjects receive different treatments based on the classification.

[0109] Currently, there are targeted therapies for HER2-overexpressing tumors, such as monoclonal antibodies that specifically bind to the extracellular domain of HER2 (trastuzumab and pertuzumab), and specific tyrosine kinase inhibitors that interact with the intracellular domain of HER2 (lapatinib).

[0110] Recently, trastuzumab-mettansine and trastuzumab-detrastuzumab (T-DXd) have emerged as novel treatment options for HER2-overexpressing cancers. These antibody-drug conjugates (ADCs) have also shown efficacy in cancers with lower expression levels (sometimes referred to as "HER2-low expression cancers"). Notably, even in HER2-low expression cancers, where HER2 pathway activation is lower than in HER2+ breast cancer, novel anti-HER2 ADCs can still target it. Because these novel ADCs contain potent chemotherapeutic agents, they can guide these agents to tumor cells even with low HER2 positivity rates.

[0111] As mentioned earlier, patients with "HER2 low expression" have IHC scores of 1+ and 2+ and are ISH negative. Trastuzumab-Detrastuzumab (T-DXd) is a HER2-targeting ADC containing a topoisomerase I inhibitor. According to data from the DESTINY-Breast 03 trial presented at ESMO 2021, this drug demonstrated an impressive progression-free survival benefit in pre-treated HER2-positive breast cancer patients, even at earlier lines of treatment. Progression-free survival (PFS) is defined as the time from randomization to disease progression or death from any cause in a clinical trial.

[0112] T-DXd demonstrated promising activity in a phase Ib trial that included patients with low HER2 expression (overall response rate (ORR) 37%; PFS 11.1 months; median overall survival (mOS) 29.4 months). Similarly, trastuzumab-docarbamazine showed interesting clinical activity in patients with HER2-positive metastatic breast cancer in a phase I study (ORR 28% in HR-positive patients; ORR 40% in HR-negative HER2-low expression 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 treatment; it excludes disease stability and is a direct measure of the drug's tumor-killing activity. Median overall survival (mOS) is the time it takes for 50% of patients to die and 50% to survive.

[0113] ADCs can overcome some of the limitations of monoclonal antibodies by carrying and releasing cytotoxic payloads that can be internalized by peripheral cells that do not express HER2 (bystander effect). From this perspective, ADCs differ primarily in the following aspects: the cytotoxic activity of the payload, the drug-to-antibody ratio (DAR), and the cleavability of the linker connecting the payload and antibody. T-DM1 consists of trastuzumab and DM1 (Mettansin), a microtubule polymerization inhibitor, linked by an incleavable linker, with a DAR of 3.5. T-DM1 requires internalization and antibody degradation to exert its cytotoxic effect. This requirement for internalization prevents it from targeting peripheral cells that do not express HER2. In contrast, T-Dxd conjugates trastuzumab with deruxtecan (a topoisomerase I inhibitor) via a cleavable but stable linker, with a DAR of 8.

[0114] In one implementation, lesion-specific or combined treatments are performed based on the initial lesion classification. For example, non-HER2 lesions can be ablated or surgically removed, while HER2-positive lesions can be treated with HER2-targeted therapy.

[0115] HER2 expression during the disease process is dynamic and exhibits temporal heterogeneity. HER2 status can change, for example, after neoadjuvant chemotherapy or during metastatic progression.

[0116] Therefore, in one embodiment, the HER2 expression in the lesions of the subject is reassessed after a period of time. The reassessment includes repeating steps a to c of the above method to perform subsequent lesion classification based on the uptake value of the tracer, wherein each of the one or more lesions is reclassified as positive or negative.

[0117] For example, tumor response can be assessed after the application of HER2-targeted therapy, and this could potentially allow treating physicians to adjust the therapy more precisely (e.g., introduce treatment intervals, dose reduction, or substitution). Therefore, the method of the present invention allows for the assessment of treatment efficacy. For example, the method of the present invention can be used to confirm treatment response early, thereby allowing for the implementation of treatment dose reduction or treatment intervals (shortening the duration of treatment and reducing associated comorbidities). Alternatively, HER2 treatment resistance can be identified early, thereby allowing for more rapid improvement of treatment regimens.

[0118] Furthermore, HER2 changes following neoadjuvant treatment or throughout tumor progression are potential indicators of poor prognosis. Therefore, detecting temporal heterogeneity can provide more accurate prognoses. In the context of this invention, prognosticating an individual with or suspected of having cancer refers to predicting the survival probability of an individual with cancer, or predicting the risk of recurrence associated with the invasion and metastatic behavior (i.e., malignant progression) of tumor tissue / cells. In one embodiment, the prognosis includes cancer staging. Cancer staging is determined based on the size of the cancer and whether or not it has spread to other parts of the patient's body, or the extent of spread. Compared to other tracers currently known in the field of medical imaging, the tracer of this invention allows for improved pharmacokinetic properties. Therefore, more accurate prognoses (including more accurate cancer staging) can be determined. This allows for the identification of optimal treatment (e.g., participation in specific investigational clinical trials) and improved disease outcomes (e.g., mortality, probability of recovery, chance of recurrence).

[0119] As previously mentioned, HER2 status varies between 3% and 10% of patients with primary and recurrent breast cancer, and inconsistencies in HER2 status even exist between and within individual lesions. Furthermore, a 37.3% temporal heterogeneity has been reported within HER2 0 and HER2-negative tumors, primarily driven by changes in HER2 status between HER2-zero and HER2-low expression tumors.

[0120] Therefore, in another aspect, the method of the present invention is used to assess the heterogeneity of HER2 expression in subjects. In one embodiment, the method of the present invention is used to assess interlesional heterogeneity of HER2 expression in subjects. In one embodiment, the method of the present invention is used to assess intralesional heterogeneity of HER2 expression in subjects. In one embodiment, the method of the present invention is used to assess temporal heterogeneity of HER2 expression in subjects, wherein the HER2 expression in the lesions of the subjects is reassessed after a period of time, the reassessment comprising repeating steps a to c of the above method and performing subsequent lesion classification according to the amount of tracer uptake, wherein each of the one or more lesions is reclassified as positive or negative.

[0121] Beyond cancer detection and staging, PET / CT imaging is becoming increasingly important as a quantitative monitoring tool for individual responses to treatment. As previously mentioned, HER2 expression in the lesions of the subject can be reassessed after a period of time, such as after cancer treatment, to evaluate a specific cancer therapy. This reassessment includes repeating steps a through c of the above-described method and performing subsequent lesion classification based on the tracer uptake, wherein one or more lesions are reclassified as positive or negative.

[0122] In one embodiment, the method of the present invention can therefore be used to evaluate cancer therapies. In one embodiment, the above-described method is used for pre- and post-treatment assessments. Therefore, the method of the present invention enables monitoring of response to treatment, thereby making treatment more personalized and effective. As previously mentioned, tumor response can be assessed, for example, after the application of HER2-targeted therapy, and will potentially allow treating physicians to adjust the therapy more accurately (e.g., introducing treatment intervals, dose reduction, or substitution). Treatment response can be identified early through the method of the present invention, thereby allowing for the implementation of treatment dose reduction or treatment intervals (shortening treatment duration and reducing associated complications), or HER2 treatment resistance can be identified early, thereby allowing for faster improvement of treatment options.

[0123] In one embodiment, the tracer is contained in a pharmaceutical composition, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier, adjuvant, or diluent.

[0124] The tracer or pharmaceutical composition may be administered via any suitable route. Suitable routes of administration include, but are not limited to, oral administration, parenteral administration, intravenous administration, intradermal administration, intramuscular administration, subcutaneous administration, rectal administration, intraperitoneal administration, inhalation administration, oral administration, sublingual administration, or local administration (e.g., percutaneous administration).

[0125] In some embodiments, the tracer or pharmaceutical composition can be administered via the urogenital route to reach the target, such as visceral organs or local lesions, or can be delivered more effectively and efficiently to organs (e.g., the bladder or vagina) via infusion.

[0126] In some embodiments, the pharmaceutical composition comprises a technically acceptable excipient suitable for enabling the composition to be administered orally, parenterally, intravenously, intradermally, intramuscularly or subcutaneously, rectally, intraperitoneally, by inhalation or oral administration, by sublingual route, or topically (e.g., percutaneously).

[0127] The tracer can be mixed or compounded with conventional pharmaceutically acceptable excipients. Those skilled in the art will understand that the pharmaceutical compositions of the present invention can be prepared and administered using conventionally used methods of administration, media, excipients, or carriers that are inert to the tracer. Formulations for use in subjects comprise the tracer, one or more acceptable excipients, and optionally a therapeutic agent. The excipients must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the recipient.

[0128] The pharmaceutical composition can be conveniently in unit dosage form, and such formulations can be prepared by any method conventionally known in the pharmaceutical field. The combination of the tracer and one or more adjuvants is then physically processed to present the formulation in a suitable delivery form (e.g., by encapsulating the drug in liposomes or microemulsions compatible with human tissues to form an aqueous suspension).

[0129] The adjuvants or auxiliary ingredients used in the formulations of this invention may include any pharmaceutical ingredient generally considered acceptable in the art, such as mixtures, buffers, solvents, etc.

[0130] Because parenteral dosage forms are typically administered outside of a patient's natural defenses against contaminants, they can be sterile or can be sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, dry preparations that can be dissolved or dispersed in pharmaceutically acceptable injectable media, injectable suspensions, and emulsions.

[0131] Suitable media for providing parenteral dosage forms of the compounds of the present invention include, but are not limited to: sterile water; USP water for injection; saline; phosphate buffer; glucose solution; aqueous media, such as, but not limited to, sodium chloride injection, Ringer's injection, glucose injection, glucose sodium chloride injection, and lactated Ringer's injection; water-miscible media, such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous media, such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds disclosed herein that alter or modify the solubility of pharmaceutically acceptable salts of the compounds of the present invention are also included in the parenteral dosage forms disclosed herein, including conventional and controlled-release parenteral dosage forms.

[0132] Formulations for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may further contain additional agents such as antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the target subject. In one embodiment, the composition includes additives that reduce undesirable retention in vivo of the immunoglobulin monovariable domain (or conjugates of the invention), such as positively charged amino acids that have been shown to reduce renal retention. Formulations may include aqueous and non-aqueous sterile suspensions containing suspending agents and thickeners.

[0133] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that can be used are water, USP Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0134] Injectable formulations can be sterilized, for example, by filtration through a sterile filter, or by adding a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0135] The present invention also provides a convenient pharmaceutical kit. Such a kit may contain a tracer and a generally pharmaceutically acceptable carrier. The kit may further contain conventional kit components, such as needles for injecting the composition, one or more vials for mixing the composition components, etc., as will be apparent to those skilled in the art. Furthermore, the kit may include instructions (inserts or labels) for indicating the amount of components, guidelines for mixing components, and administration methods.

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

[0137] "Carrier" or "adjuvant," particularly "pharmaceutically acceptable carrier" or "pharmaceutical acceptable adjuvant," refers to any suitable excipient, diluent, carrier, and / or adjuvant that does not induce harmful antibodies in an individual receiving the composition, nor does it provide protection. Therefore, pharmaceutically acceptable carriers are inherently non-toxic and non-therapeutic, as is well known to those skilled in the art. Suitable carriers or adjuvants typically comprise one or more compounds, including, but not exhaustively, large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated viral particles. As a non-limiting example, the carrier or adjuvant may be Ringer's solution, dextro glucose solution, or Hank's solution. Non-aqueous solutions, such as fixed oils and ethyl oleate, may also be used. A preferred excipient is a 5% saline solution of glucose. Excipients may contain small amounts of additives, such as substances for enhancing isotonicity and chemical stability, including buffers and preservatives.

[0138] As used herein, the terms “therapeutic effective amount,” “therapeutic effective dose,” and “effective amount” refer to the amount required to achieve one or more expected results. As used herein, “pharmaceutically acceptable” means that there is no biologically or otherwise undesirable material, i.e., the material can be administered to an individual with the compound without causing any adverse biological effects or harmful interactions with any other component in a pharmaceutical composition containing the material.

[0139] The invention is further described by way of the following non-limiting embodiments, which are intended to illustrate the invention and are not intended to, nor should be construed as, limiting the scope of protection of the invention.

[0140] The invention will now be described in more detail with reference to non-limiting embodiments.

[0141] Embodiments and / or accompanying drawings

[0142] Figure 1 This diagram illustrates an embodiment of the method of the present invention for determining HER2 expression and selecting one or more HER2-positive lesions to obtain one or more biopsy samples. The method of the present invention allows for the selection of the correct medication for the correct patient.

[0143] Figure 2A schematic diagram illustrates traditional, current, and future possible HER2 classifications. Traditional and current classifications rely solely on IHC / ISH, while the method of embodiments of the present invention involves performing a whole-body PET / CT scan on the subject using a tracer comprising a radiolabeled biomolecule conjugated to HER2. The method of embodiments of the present invention classifies each individual lesion as HER2 positive or HER2 negative and allows for targeted biopsy. Unlike traditional and existing classifications, the method of embodiments of the present invention allows differentiation between patients with HER2 O and HER2 ultra-low expression.

[0144] Figure 3 The algorithm for defining HER2 expression profiles according to the ASCO / CAP guidelines is shown. Disease with low HER2 expression is defined as an IHC score of 2+ and ISH negative, or an IHC score of 1+. Breast cancer is classified as a 2+ score if >10% of tumor cells show weak to moderate, intact membrane staining, or strong membrane staining seen in ≤10% of tumor cells. A score of 1+ is defined as weak or barely visible incomplete membrane staining in >10% of tumor cells. Evaluation criteria for gene amplification using ISH are detailed in the text box, depending on the type of probe used. Within the IHC score category, there are two different types of expression states: complete absence of expression and weak or barely visible incomplete membrane staining in ≤10% of tumor cells. The latter type of tumor, despite being HER2 negative, does still exhibit protein expression and can be described as HER2 “ultra-low expression.” Solid and dashed lines represent different levels of evidence in current clinical practice. Abbreviations: IHC: Immunohistochemistry; ISH: In situ hybridization; HER2: Human epidermal growth factor receptor 2. The final result needs to be confirmed using a dual-probe ISH.

[0145] Source: Breast Biomarker Report, CAP Cancer Protocol Template, v1.4.1.1, updated November 2021.

[0146] Figure 4 Images obtained via PET scans are shown, with annotations indicating the maximum standard uptake (SUV) values, defined here as SUV. max Target Area of ​​Interest (ROI) 峰值 And the area of ​​highest intake. Peak SUV (SUV) 峰值 A region of origin (ROI) is defined as a small, fixed-size target area centered on a high-uptake region of the tumor. 峰值 The average SUV within the range. The SUV 峰值 Or SUVs include the maximum standard intake value, which is defined in this article as "SUVs" max This can be used in the method for initial classification of lesions in embodiments of the present invention, wherein each of the one or more lesions is classified as HER2 positive or HER2 negative.

[0147] This invention is not limited to the embodiments described and / or the implementations illustrated in the accompanying drawings. Rather, the method of this invention can be implemented in many different ways without departing from the scope of the invention.

Claims

1. A method for determining HER2 expression in one or more lesions of a subject, the method comprising: a. Administering a tracer to the subject, wherein the tracer comprises a radiolabeled biomolecule conjugated to HER2; b. Perform whole-body positron emission tomography / computed tomography (PET / CT) on the subject; c. Determine the amount of tracer absorbed in the lesion; d. Initial lesion classification based on the amount of tracer taken up, wherein each of the one or more lesions is classified as HER2 positive or HER2 negative.

2. The method of claim 1, wherein, The method further includes selecting one or more lesions classified as HER2 positive and obtaining one or more biopsy samples from the one or more positive lesions.

3. The method according to claim 2, further comprising analyzing HER2 expression in the one or more biopsy samples by immunohistochemistry (IHC) and / or in situ hybridization (ISH).

4. The method according to any of the preceding claims, wherein, The HER2-binding biomolecule contains an immunoglobulin single variable domain that targets or specifically binds to HER2.

5. The method of claim 4, wherein, The immunoglobulin single variable domain that targets or specifically binds to HER2 contains an amino acid sequence that has at least 80% amino acid sequence identity with SEQ ID NO:1 or SEQ ID NO:

2.

6. The method according to any one of the preceding claims, wherein, The radiolabel contains a radionuclide selected from the following: fluorine-18 ( 18 F), Indium 111 ( 111 In), Copper 61 ( 61 Cu), Copper 64 ( 64 Cu), Gallium-67 ( 67 Ga), gallium 68( 68 Ga), Technetium 99m( 99m Tc).

7. The method of any of the preceding claims, wherein, The HER2-binding biomolecule is coupled with a chelating agent selected from the group consisting of DTPA and its derivatives, DOTA and its derivatives, Nota and its derivatives, HBED and its derivatives, DEPA and its derivatives, pyridinecarboxylic acid-based chelating agents and their derivatives, HEHA and its derivatives, TETA and its derivatives, NETA and its derivatives, AAZTA and its derivatives, DATA and its derivatives, TCMC and its derivatives, PCTA and its derivatives, Macropa and its derivatives, THP and its derivatives, DFO and its derivatives, BCPA and its derivatives, MAG-2 and its derivatives, MAG-3 and its derivatives, MAS-3 and its derivatives, HYNIC and its derivatives, and RESCA.

8. The method according to any of the preceding claims 3 to 7, wherein, Based on the initial lesion classification and the biopsy sample analysis, the subjects were classified according to their HER2 status.

9. The method of claim 8, wherein, The subjects could be classified as having very low HER2 expression.

10. The method according to any one of claims 8 to 9, wherein, Based on the classification of the subjects, the subjects received different treatments.

11. The method according to any one of the preceding claims, wherein, Based on the initial lesion classification, lesion-specific treatment or combination therapy is performed.

12. The method according to any one of the preceding claims, wherein, The PET / CT scan was performed between 30 and 300 minutes after the administration of the tracer.

13. The method according to any one of the preceding claims, wherein, The HER2 expression in the lesions of the subject is reassessed after a period of time, the reassessment comprising repeating steps a to c of the method of claim 1, and classifying the lesions based on the uptake of the tracer, wherein each of the one or more lesions is reclassified as HER2 positive or HER2 negative.

14. The use of the method of any one of claims 1 to 13 in assessing interlesional heterogeneity, intralesional heterogeneity and / or temporal heterogeneity of HER2 expression in subjects.

15. The use of the method of any one of claims 1 to 13 in evaluating cancer therapies.

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