compound

Abstract

The invention relates to radiolabelled compounds, precursor compounds and reference compounds, and pharmaceutical compositions comprising the radiolabelled compounds. The invention also relates to the radiolabelled compounds for use in positron emission tomography (PET). The invention also relates to methods for radiolabelling a precursor compound to form the radiolabelled compound, and methods for making a precursor compound or a reference compound.

Description

[0001] COMPOUND

[0002] TECHNICAL FIELD

[0003] This invention relates to radiolabelled compounds, precursor compounds and reference compounds, as well as pharmaceutical compositions comprising the radiolabelled compounds. Aspects of the invention also relate to the radiolabelled compounds for use in a diagnostic method practised on the human or animal body using positron emission tomography (PET). Further aspects of the invention relate to methods for radiolabelling a precursor compound to form the radiolabelled compound, and methods for making a precursor compound or a reference compound.

[0004] BACKGROUND

[0005] Positron emission tomography (PET) is a non-invasive nuclear imaging technique that uses radiolabelled molecules to either detect the expression of a target or monitor a metabolic process in vivo.

[0006] Single-photon emission computed tomography (SPECT) is a nuclear imaging modality that produces 3-dimensional images of the distribution of a gamma ray emitting radioactive tracer in vivo to assess the functionality and physiology of organs.

[0007] Functional proteins such as antibodies, single chain fragment variables (SCFVs), antigenbinding fragments (Fab), nanobodies, and cytokines etc. are emerging as lead molecules for developing PET / SPECT tracers and radioimmunotherapeutics, because they have excellent binding affinities and high specificities to their receptors. A key challenge, however, in converting such molecules to PET / SPECT tracers and radioimmunotherapeutics is the conjugation of PET / SPECT and therapeutic radionuclides 'site-specifically' at the desired point in the molecules to ensure their affinity and specificity remain unaffected.

[0008] Conventional site specific conjugation of functional proteins is conducted via a cysteine to the C-terminus of the target proteins, using maleimide based radiolabelled prosthetic groups. In this technique, a sortase A substrate, LPXTG motif has to be genetically engineered into the target proteins for cysteine introduction (Morgan, H. E.; Turnbull, W. B.; Webb, M. E.; Challenges in the use of sortase and other peptide ligases for site-specific protein modification; Chem. Soc. Rev. 2022, 51, 4121- 4145), which adds another level of complexity to access the target proteins.

[0009] A need remains for means for site specific radiolabelling compounds for conjugation to native proteins. Native proteins are proteins purified from their natural sources without any chemical and bioengineering alterations. A site specific radiolabelling method for native protein conjugation would avoids expensive and time-consuming genetic engineering approaches. SUMMARY OF THE INVENTION

[0010] An aspect of the invention provides a radiolabelled compound of formula (I): wherein:

[0011] R1and R2are independently selected from a halogen and -SR3, wherein R3is independently selected from C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl and C6-io aryl;

[0012] L represents a direct bond or a linker, wherein the linker is a Ci-20alkylene which is unsubstituted or substituted by one or more substituents selected from hydroxy, -COOH, C1-12 alkoxy, C1-12 alkyl and hydroxy-Ci-i2-alkyl, wherein zero or one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -O-, -S-, -NR4-, -C(O)NR4-, -NR4C(O)-, -C(O)-, -OC(O)-, and -C(O)O-, wherein R4is hydrogen or C1-12 alkyl, and wherein the C6-io arylene moiety is unsubstituted or substituted by one, two, three or four substituents selected from hydroxy, C1-12 alkyl and C1-12 alkoxy;

[0013] X is absent or a PEG2-20 group; and

[0014] Q* represents an18F-labelled moiety; or a pharmaceutically acceptable salt thereof.

[0015] A further aspect of the invention provides a reference compound of formula (II): wherein:

[0016] R1, R2, L and X are as defined above for the radiolabelled compound of formula (I); and

[0017] Q represents a19F-labelled moiety comprising a chelated19F-AI complex; or a pharmaceutically acceptable salt thereof.

[0018] Another aspect of the invention provides a precursor compound of formula (III): wherein:

[0019] R1, R2, L and X are as defined above for the radiolabelled compound of formula (I); and

[0020] Y represents a moiety capable of chelating an18F-AI complex; or a pharmaceutically acceptable salt thereof.

[0021] A further aspect of the invention provides a pharmaceutical composition comprising a radiolabelled compound of formula (I) as defined above and a pharmaceutically acceptable carrier.

[0022] Another aspect of the invention provides a method for making the precursor compound of formula (III), the method comprising the steps of: a) reacting a compound of formula (V) with a compound of formula (VI) via a coupling reaction to form a compound of formula (IV); and b) deprotecting the compound of formula (IV) to form a compound of formula (III); in accordance with the following reaction scheme: wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I);

[0023] Y is as defined above for the precursor compound of formula (III);

[0024] L1and L2are moieties which are capable of being coupled together to form group L; and Ypis a moiety comprising at least one protecting group, which moiety is capable of being deprotected to provide group Y.

[0025] A further aspect of the invention provides a method for making the radiolabelled compound of formula (I), the method comprising radiolabelling the precursor compound of formula (HI).

[0026] Another aspect of the invention provides a method for making the reference compound of formula (II), the method comprising reacting the precursor compound of formula (III) with a source of fluoride-19 and a source of Al3+, to form the reference compound of formula (II), in accordance with the following reaction scheme: source of fluoride-19 source of Al3+ wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I);

[0027] Y is as defined above for the precursor compound of formula (III); and Q is as defined above for the reference compound of formula (II).

[0028] Still another aspect of the invention provides a radiolabelled compound of formula (I) for use in a diagnostic method practised on the human or animal body using positron emission tomography (PET).

[0029] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0030] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0033] Figure 1 shows HPLC purification of the crude [18F]AIF-NOTA-dibromomaleimide reaction mixture.

[0034] Figure 2 shows HPLC co-elution of [18F]AIF-NOTA-dibromomaleimide with its nonradioactive reference compound.

[0035] Figure 3 shows HPLC quality control of the purified [18F]AIF-NOTA-dibromomaleimide.

[0036] Figure 4 shows Tumour cell uptake of the [18F]AIF-NOTA-NM-02. Blocked samples were pre-treated with 100X native NM-02. Data shown are mean ± SD of three independent experiments conducted in triplicate.

[0037] Figure 5 shows cell uptake of the [18F]AIF-NOTA-IL-4DE. Blocked samples were pretreated with 100X native IL-4DE. Data shown are mean ± SD of independent experiments, where n=5 for 4a0-expressing T4-CAR T-cells, n=5 for non-activated non-transduced T-cells, n=5 for PBMCs, n=3 for SKOV3 and 3HN cancer cells.

[0038] Figure 6 shows PET images of mice injected with [18F]AIF-NOTA-HSA, reconstructed at 5, 20, 40, 80, and 120 min. It was found that most of the [18F]AIF-NOTA-HSA was still in blood circulation 120 min after IV injection, indicated by the very high left ventricular chamber radioactivity accumulation in the PET images shown in Figure 6.

[0039] DETAILED DESCRIPTION

[0040] Aspects of the invention provide or utilise a radiolabelled compound.

[0041] Suitably, the radiolabelled compound is a compound of formula (I) : wherein:

[0042] R1and R2are independently selected from a halogen and -SR3, wherein R3is independently selected from Ci-i2alkyl, Ci-i2alkenyl, Ci-i2alkynyl and C6-io aryl;

[0043] L represents a direct bond or a linker, wherein the linker is a Ci-20alkylene which is unsubstituted or substituted by one or more substituents selected from hydroxy, -COOH, Ci-i2alkoxy, Ci-i2alkyl and hydroxy-Ci-i2-alkyl, wherein zero or one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -O-, -S-, -NR4-, -C(O)NR4-, -NR4C(O)-, -C(O)-, -OC(O)-, and -C(O)O-, wherein R4is hydrogen or Ci-i2alkyl, and wherein the C6-io arylene moiety is unsubstituted or substituted by one, two, three or four substituents selected from hydroxy, Ci-i2alkyl and Ci-i2alkoxy;

[0044] X is absent or a PEG2.20group; and

[0045] Q* represents an18F-labelled moiety; or a pharmaceutically acceptable salt thereof.

[0046] The inventors have developed an18F-labelled disubstituted maleimide compound which comprises (i) a disubstituted maleimide moiety, which can allow for site-specific bioconjugation of proteins through the intramolecular disulfide bridge, and (ii) a fluorine-18 label, which can allow for PET imaging. The compound of formula (I) may be referred to as an18F-based bioconjugation reagent.

[0047] The half-life for fluorine-18 is around 110 min, which is compatible with the in vivo biological half-life of small proteins such as nanobodies and cytokines etc.

[0048] In an embodiment, Q* represents an18F-labelled moiety comprising a chelated18F-AI complex.18F-labelled moieties comprising a chelated18F-AI complex are known in the art and can include, for example, chemical groups such as [18F]AIF-NOTA, [18F]AIF-NODA, [18F]AIF-NODAGA, and [18F]AIF-RESCA. In a preferred embodiment, Q* represents an18F-labelled moiety selected from:

[0049] In an embodiment, Q* represents an18F-labelled moiety with the formula (Q*-A).

[0050] In another embodiment, Q* represents an18F-labelled moiety with the formula (Q*-B1) or (Q*-B2). Q* may for example have the formula (Q*-B1). Alternatively Q* may for example have the formula (Q*-B2).

[0051] The compound of formula (I) comprises a disubstituted maleimide moiety. The substituents are indicated by R1and R2. R1and R2are independently selected from a halogen and -SR3, wherein R3is independently selected from Ci-i2alkyl, Ci-i2alkenyl, Ci-i2alkynyl and C6-io aryl.

[0052] It will be appreciated that R1and R2may be the same or may be different.

[0053] Preferably, R3is C6-io aryl. More preferably, R3is phenyl (-Ph).

[0054] In a preferred embodiment, R1and R2are independently selected from -Br, -I and -SPh. More preferably, R1and R2are -Br.

[0055] In an embodiment, L represents a linker, wherein the linker is a Ci-20alkylene which is unsubstituted or substituted by one or more substituents selected from hydroxy, -COOH, Ci-i2alkoxy, Ci-i2alkyl and hydroxy-Ci-i2-alkyl, wherein zero or one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -O-, -S-, -NR4-, -C(O)NR4-, -NR4C(O)-, -C(O)-, -OC(O)-, and -C(O)O-, wherein R4is hydrogen or Ci-i2alkyl, and wherein the C6-io arylene moiety is unsubstituted or substituted by one, two, three or four substituents selected from hydroxy, Ci-i2alkyl and Ci-i2alkoxy.

[0056] Preferably, L represents a linker, wherein the linker is a Ci-20alkylene wherein one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -C(O)NR4-, -NR4C(O)-, -C(0)-, -0C(0)-, and -C(0)0-, wherein R4is hydrogen or Ci-12 alkyl, and wherein the C6-io arylene moiety is unsubstituted.

[0057] More preferably, L represents a linker, wherein the linker is a Ci-20alkylene wherein one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -C(O)NH- and -NHC(O)-, and wherein the C6-io arylene moiety is unsubstituted.

[0058] In an embodiment, L has the formula:

[0059] -(CH2)n-NH-C(O)-(CH2)m- or -(CH2)n-C(O)-NH-(CH2)m- , wherein n is an integer from 0 to 18 and m is an integer from 0 to 18, and n+m<18.

[0060] In an embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. Preferably, n is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 2 to 10, from 2 to 6, from 2 to 4, or from 2 to 3. More preferably, n is 2.

[0061] In an embodiment, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. Preferably, m is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 1 to 3, or from 1 to 2. More preferably, m is 1.

[0062] In another embodiment, L has the formula : wherein p is an integer from 0 to 18 and q is an integer from 0 to 18, and p+q<18.

[0063] In an embodiment, p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0064] Preferably, p is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 2 to 10, from 2 to 6, from 2 to 4, or from 2 to 3.

[0065] In an embodiment, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0066] Preferably, q is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 1 to 3, or from 1 to

[0067] 2. More preferably, q is 1.

[0068] In a further embodiment, L has the formula: wherein r is an integer from 0 to 18 and s is an integer from 0 to 18, and r+s<18.

[0069] In an embodiment, r is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0070] Preferably, r is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 2 to 10, from 2 to 6, from 2 to 4, or from 2 to 3.

[0071] In an embodiment, s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0072] Preferably, s is an integer from 1 to 10, from 1 to 6, from 1 to 4, from 1 to 3, or from 1 to

[0073] 2. More preferably, s is 2.

[0074] In an embodiment, X is absent. In an alternative embodiment, X is present. When X is present, X is a PEG2-20 group. The PEG2-20 group may have the formula :

[0075] -(O-CH2-CH2)X- or -(CH2-CH2-O)X-, wherein x is an integer from 2 to 20.

[0076] In an embodiment, when L is a direct bond, X is present. In a preferred embodiment, the compound of formula (I) is a compound of the formula:

[0077] This compound may be referred to as an [18F]AIF-NOTA-dibromomaleimide.

[0078] A further aspect of the invention provides a reference compound of formula (II):

[0079] wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I); and Q represents a19F-labelled moiety comprising a chelated19F-AI complex; or a pharmaceutically acceptable salt thereof.

[0080] The reference compound of formula (II) can function as a reference compound for the radiolabelled compound of formula (I), the reference compound having a19F atom (more commonly referred to as an F atom) in place of the18F atom (the radiolabel) in formula (I). The reference compound can, for example, help to confirm the identity of the radiolabelled compound.

[0081] Suitably, Q may represent a moiety selected from:

[0082] In an embodiment, Q represents a moiety with the formula (Q-A).

[0083] In another embodiment, Q represents a moiety with the formula (Q-Bl) or (Q-B2). Q may for example have the formula (Q-Bl). Alternatively Q may for example have the formula (Q-B2).

[0084] Preferably, the reference compound of formula (II) is a compound of the formula:

[0085] Another aspect of the invention provides a precursor compound of formula (III): wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I); and Y represents a moiety capable of chelating an18F-AI complex (and / or capable of chelating a19F-AI complex); or a pharmaceutically acceptable salt thereof.

[0086] The precursor compound of formula (III) can function as a precursor compound for the radiolabelled compound of formula (I). The precursor compound of formula (III) can function as a precursor compound for the reference compound of formula (II).

[0087] Y represents a moiety capable of chelating an18F-AI complex. Such a moiety is also capable of chelating a19F-AI complex.

[0088] Suitably, Y may represent a moiety selected from:

[0089]

[0090] In an embodiment, Y represents a moiety with the formula (Y-A).

[0091] In another embodiment, Y represents a moiety with the formula (Y-Bl) or (Y-B2). Y may for example have the formula (Y-Bl). Or Y may for example have the formula (Y-B2). Preferably, the precursor compound of formula (III) is a compound of the formula:

[0092] A further aspect of the invention provides a pharmaceutical composition comprising a radiolabelled compound of formula (I) as defined above and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises a radiolabelled compound of formula (I) wherein Q* has the formula (Q*-A).

[0093] In another embodiment, the pharmaceutical composition comprises a radiolabelled compound of formula (I) wherein Q* has the formula (Q*-B1), a radiolabelled compound of formula (I) wherein Q* has the formula (Q*-B2), or a combination thereof. A radiolabelled compound of formula (I) wherein Q* has the formula (Q*-B1) and a radiolabelled compound of formula (I) wherein Q* has the formula (Q*-B2) may for example be present as a racemic mixture. Another aspect of the invention provides a method for making the precursor compound of formula (III), the method comprising the steps of: c) reacting a compound of formula (V) with a compound of formula (VI) via a coupling reaction to form a compound of formula (IV); and d) deprotecting the compound of formula (IV) to form a compound of formula (III); in accordance with the following reaction scheme: wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I);

[0094] Y is as defined above for the precursor compound of formula (III);

[0095] L1and L2are moieties which are capable of being coupled together to form group L; and Ypis a moiety comprising at least one protecting group, which moiety is capable of being deprotected to provide group Y.

[0096] L1and L2are moieties which are capable of being coupled together to form group L.

[0097] The coupling reaction in step (a) may, for example, be an amide coupling. In such an embodiment, one of L1and L2has an -NH2end group, and the other of L1and L2has a -COOH end group, which are capable of coupling together to form an amide bond.

[0098] In an embodiment, L1has the formula -(CH2)n-NH2, and L2has the formula HOOC-(CH2)m-, wherein n and m are as defined above.

[0099] In another embodiment, L1has the formula -(CH2)n-COOH, and L2has the formula H2N-(CH2)m- , wherein n and m are as defined above.

[0100] Ypis a moiety comprising at least one protecting group, and Ypis capable of being deprotected to provide group Y. For example, group Ypmay comprise at least one protecting group which is protecting a heteroatom, such as e.g. an oxygen atom, which is present in group Y.

[0101] Suitably, Ypmay represent a moiety selected from:

[0102] wherein R10, R11, R12, R13and R14are protecting groups. Any suitable protecting group may be used. Each of the protecting groups may be the same or different. The protecting groups may, for example, be selected from -‘Bu, -Bn, -CMe2Ph, and 4-methoxybenzyl. In an embodiment, YP represents a moiety with the formula (YP-A).

[0103] In another embodiment, YP represents a moiety with the formula (YP-BI) or (YP-B2). YP may for example have the formula (YP-BI). Alternatively YP may for example have the formula (YP-B2).

[0104] A further aspect of the invention provides a method for making the radiolabelled compound of formula (I), the method comprising radiolabelling a precursor compound. Preferably, the precursor compound may be the precursor compound of formula (III).

[0105] The radiolabelled compound of formula (I) contains the18F radioisotope.18F-fluoride is a commonly used PET radioisotope.

[0106] Suitably, the method comprises the step of reacting the precursor compound of formula (III) with radiolabelling reagents comprising a source of fluoride-18, to form the radiolabelled compound of formula (I), in accordance with the following reaction scheme:

[0107] O wherein R1, R2, L, X and Q* are as defined above for the radiolabelled compound of formula (I); and Y is as defined above for the precursor compound of formula (III). In an embodiment, the radiolabelling reagents comprise a source of fluoride-18 and a source of Al3+.

[0108] The source of fluoride-18 may, for example, be K18F, Na18F, Li18F, Cs18F, tBu4N18F, Et4N18F and / or combinations thereof. Preferably, the source of fluoride-18 is K18F.

[0109] The source of Al3+may, for example, be AICI3, AIBr3, AII3, AI(CH3CO2)3and / or combinations thereof. Preferably, the source of Al3+is AICI3.

[0110] Another aspect of the invention provides a method for making the reference compound of formula (II), the method comprising reacting the precursor compound of formula (III) with a source of fluoride-19 and a source of Al3+, to form the reference compound of formula (II), in accordance with the following reaction scheme: source of fluoride-19 source of Al3+ wherein R1, R2, L and X are as defined above for the radiolabelled compound of formula (I);

[0111] Y is as defined above for the precursor compound of formula (III); and Q is as defined above for the reference compound of formula (II).

[0112] Still another aspect of the invention provides a radiolabelled compound of formula (I) defined above for use in a diagnostic method practised on the human or animal body using positron emission tomography (PET).

[0113] Suitably, the diagnostic method comprises labelling a protein with the radiolabelled compound of formula (I).

[0114] Preferably, the protein is a native protein.

[0115] Suitably, the protein may be selected from an antibody, a single chain fragment variable (SCFV), an antigen-binding fragment (Fab), a nanobody, and / or a cytokine.

[0116] In a preferred embodiment, the diagnostic method comprises labelling an HER2 targeting nanobody with the radiolabelled compound of formula (I). Suitably, the HER2 targeting nanobody may be NM-02.

[0117] In an embodiment, the diagnostic method is a method of diagnosing cancer. Preferably, the diagnostic method may be a method of identifying the location of a tumour.

[0118] Definitions

[0119] It is to be understood that the wavy line in any chemical structures or moieties represented herein, such as shown below, indicates the point of attachment of that structure or moiety.

[0120] The term "hydrogen" or "hydrogen atom" as used herein refers to a -H moiety.

[0121] The term "halo", "halogen" or "halogen atom" as used herein refers to a -F, -Cl, -Br or -I moiety.

[0122] The term "hydroxy" or "hydroxyl" as used herein refers to an -OH moiety.

[0123] The prefix "Cx" "Cx-y" denotes the number of carbon atoms, or range of number of carbon atoms present in that group. Thus, the term "C1-12 alkyl" refers to an alkyl group having from 1 to 12 carbon atoms.

[0124] The term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a saturated hydrocarbon compound, for example having from 1 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkyl" includes the subclass cycloalkyl below. Examples of alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9) and decyl (C10). Examples of linear alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6), and n-heptyl (C7). Examples of branched alkyl groups include, but are not limited to, iso-propyl (C3), isobutyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).

[0125] The term "cycloalkyl" refers a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a cyclic saturated hydrocarbon compound, for example having from 3 to 20 carbon atoms. "Cycloalkyl" includes monocyclic and polycyclic rings including bicyclic rings. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclohexyl (C6), cycloheptyl (C7) and methylcyclopropyl (C4). Cycloalkyl includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. Examples of saturated polycyclic hydrocarbon compounds include, but are not limited to, thujane (Ci0), carane (Ci0), pinane (Ci0), bornane (Ci0), norcarane (C7), norpinane (C7), norbornane (C7), adamantane (Ci0) and decalin (Ci0).

[0126] The term "alkenyl" refers to a monovalent hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds, and for example having from 2 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkenyl" includes the subclass cycloalkenyl below. Examples of alkenyl groups include, but are not limited to ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3) and 2-propenyl (allyl, -CH-CH=CH2).

[0127] The term "cycloalkenyl" refers to a monovalent cyclic hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon double bonds, and for example having from 3 to 20 carbon atoms. "Cycloalkenyl" includes monocyclic and polycyclic rings including bicyclic rings. Examples of unsaturated monocyclic hydrocarbon compounds include, but are not limited to cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4) and dimethylcyclopropene (C5). Examples of unsaturated polycyclic hydrocarbon compounds include, but are not limited to camphene (Ci0), limonene (Ci0) and pinene (Ci0).

[0128] The term "alkynyl" refers to a monovalent hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon triple bonds, and for example having from 2 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkynyl" includes the subclass cycloalkynyl below. Examples of alkynyl groups include, but are not limited to, ethynyl (ethinyl, -C=CH) and 2-propynyl (propargyl, -CH2-C=CH).

[0129] The term "cycloalkynyl" refers to a monovalent cyclic hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon triple bonds, and for example having from 2 to 20 carbon atoms. "Cycloalkynyl" includes monocyclic and polycyclic rings including bicyclic rings.

[0130] The term "aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of an aromatic compound, which moiety may for example be a monocyclic or bicyclic group. The aromatic compound which the aryl group is derived from may contain an all-carbon ring structure or may be a heteroaromatic compound containing one or more heteroatoms in the ring structure. Thus the term "aryl" includes the subclass heteroaryl below. An aryl group with an all-carbon ring structure may for example have from 3 to 20 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl (derived from benzene) and naphthyl (derived from naphthalene). The term "heteroaryl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heteroaromatic compound, which moiety may for example be a monocyclic or bicyclic group. The heteroaryl moiety may for example contain one or more N, O, S or P atoms, and may for example contain from 1 to 20 carbon atoms. Examples of heteroaryl groups include, but are not limited to, pyridinyl (or pyridyl, derived from pyridine), pyrimidinyl (derived from pyrimidine), pyrazinyl (derived from pyrazine), pyrrolyl (derived from pyrrole), imidazolyl (derived from imidazole), pyrazolyl (derived from pyrazole), furyl (derived from furan), thiophenyl (derived from thiophene), oxazolyl (derived from oxazole), isoxazolyl (derived from isoxazole), and thiazolyl (derived from thiazole).

[0131] The term "heterocyclyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety may for example be a monocyclic or bicyclic group. The heterocyclyl group may for example contain one or more N, O, S or P atoms, and may for example contain from 1 to 20 carbon atoms.

[0132] The term "alkoxy" or "alkoxyl" refers to an alkyl-oxy group, where the alkyl group is as defined above. Examples of alkoxy groups include, but are not limited to -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O('Pr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (secbutoxy), -O('Bu) (isobutoxy), and -O(lBu) (tert-butoxy).

[0133] The term "acyl" refers to a group represented by the general formula -C(O)-hydrocarbyl, such as -C(O)-alkyl.

[0134] The term "alkylene" refers to a divalent hydrocarbon moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a saturated hydrocarbon compound, for example having from 1 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkylene" includes the subclass cycloalkylene below. Examples of linear alkylene groups include, but are not limited to, -CH2- (methylene), -CH2CH2- (ethylene), -CH2CH2CH2- (propylene), and - CH2CH2CH2CH2- (butylene). Examples of branched alkylene groups include, but are not limited to, -CH(CH3)-, -CH(CH3)CH2-, and -CH(CH3)CH2CH2-.

[0135] The term "cycloalkylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a cyclic saturated hydrocarbon compound, for example having from 3 to 20 carbon atoms. "Cycloalkylene" includes monocyclic and polycyclic rings including bicyclic rings. Examples of cyclic alkylene groups include, but are not limited to, cyclopentylene (e.g. cyclopent-1, 3-ylene) and cyclohexylene (e.g. cyclohex-1, 4-ylene).

[0136] The term "alkenylene" refers to a divalent hydrocarbon moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds, and for example having from 2 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkenylene" includes the subclass cycloalkenylene below. Examples of linear alkenylene groups include, but are not limited to, -CH=CH- (vinylene), -CH=CHCH2-, -CH2-CH=CH2-, and -CH=CHCH2CH2-. Examples of branched alkenylene groups include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CHCH2- and -CH=CHCH(CH3)-.

[0137] The term "cycloalkenylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a cyclic (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds, and for example having from 3 to 20 carbon atoms.

[0138] "Cycloalkenylene" includes monocyclic and polycyclic rings including bicyclic rings. Examples of cycloalkenylene groups include, but are not limited to, cyclopentenylene (e.g. 4- cyclopenten-l,3-ylene) and cyclohexenylene (e.g. 2-cyclohexen-l,4-ylene; 3-cyclohexen- 1,2-ylene; 2,5-cyclohexadien-l,4-ylene).

[0139] The term "alkynylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a (partially) unsaturated hydrocarbon compound having one or more carboncarbon triple bonds, and for example having from 2 to 20 carbon atoms, which may be linear, branched, or cyclic. Thus the term "alkynylene" includes the subclass cycloalkynylene below.

[0140] The term "cycloalkynylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon triple bonds, and for example having from 3 to 20 carbon atoms. "Cycloalkynylene" includes monocyclic and polycyclic rings including bicyclic rings.

[0141] The term "arylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of an aromatic compound, which moiety may for example be a monocyclic or bicyclic group. The aromatic compound which the arylene group is derived from may contain an all-carbon ring structure or may be a heteroaromatic compound containing heteroatoms in the ring structure. Thus the term "arylene" includes the subclass heteroarylene below. An arylene group with an all-carbon ring structure may for example have from 3 to 20 carbon atoms.

[0142] The term "heteroarylene" refers to a divalent moiety obtained by removing two hydrogen atoms, either both from the same ring atom, or one from each of two different ring atoms, of a heteroaromatic compound, which moiety may for example be a monocyclic or bicyclic group. The heteroarylene moiety may for example contain one or more N, O, S or P atoms, and may for example contain from 1 to 20 carbon atoms.

[0143] The term "PEG" refers to a divalent moiety obtained by removing two hydrogen atoms from two atoms of a PEG (polyethylene glycol) chain, for example having from 2 to 20 repeat units of -(O-CH2-CH2)-. The prefix "PEGX" "PEGx y" denotes the number of repeat units, or the range of the number of repeat units present in that group. Thus, the term "PEG2-20 group" refers to a PEG chain having from 2 to 20 repeat units, corresponding to the formula -(O-CH2-CH2)X- or -(CH2-CH2-O)X-, wherein x is an integer from 2 to 20. Examples of PEG groups include, but are not limited to, PEG2(n is 2), PEG3(n is 3), PEG4(n is 4), PEG5(n is 5), PEG6(n is 6), PEG7(n is 7), PEG8(n is 8), PEG9(n is 9), PEGi0(n is 10), PEGn (n is 11), PEG12 (n is 12), PEG13 (n is 13), PEGi4(n is 14), PEGi5(n is 15), PEGi6(n is 16), PEGi7(n is 17), PEGis (n is 18), PEG19 (n is 19) and PEG20(n is 20).

[0144] The term "substituent" refers to a chemical moiety, which is covalently attached to, or if appropriate, fused to, a parent group.

[0145] The phrase "optionally substituted" refers to a parent group which may be unsubstituted or which may be substituted with one or more, for example one or two, substituents. The substituents on an "optionally substituted" group may for example be selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl groups; carboxylic acids and carboxylate ions; carboxylate esters; carbamates; alkoxy groups; ketone and aldehyde groups; amine and amide groups; -OH; -ON; -NO2; and halogens.

[0146] The term "protecting group" as used herein refers to a group capable of protecting a functional group (e.g. a heteroatom such as an oxygen atom), which protecting group may, subsequent to the reaction for which protection is employed, be removed without disturbing the remainder of the molecule. Protecting groups are well known and listed in standard texts such as Kocienski P. J., Protecting Groups, 3rd ed., Georg Thieme Verlag, New York, 2005; and Greene T. W., Wuts P. G. M., Protective Groups In Organic Synthesis, 3rd ed., John Wiley & Sons, New York, 1998.

[0147] The term "small protein" as used herein can refer to proteins such as nanobodies and cytokines etc. A small protein may, for example, be a protein of around 15 kDa.

[0148] Certain compounds may exist in one or more particular geometric, enantiomeric, diastereomeric, tautomeric, or conformational forms. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation and separation of such isomeric forms are known in the art. Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts.

[0149] Examples of pharmaceutically acceptable acidic / anionic salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.

[0150] Examples of pharmaceutically acceptable basic / cationic salts include sodium, potassium, calcium, magnesium, diethanolamine, N-methyl-D-glucamine, L-lysine, L-arginine, ammonium, ethanolamine, piperazine and triethanolamine salts.

[0151] If the compound is anionic, or has a functional group which may be anionic, then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include alkali metal ions, such as Na+and K+, alkaline earth cations, such as Ca2+and Mg2+, and other cations such as Al3+. Examples of suitable organic cations include ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+, where R is an alkyl group).

[0152] If the compound is cationic, or has a functional group which may be cationic, then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.

[0153] If the compound has both a cationic functional group, or a functional group that can become cationic, and an anionic functional group, or a functional group that can become anionic, then the compound may be present as a zwitterion.

[0154] The following non-limiting examples are provided by way of illustration only.

[0155] EXAMPLES Abbreviations

[0156] NOTA stands for l,4,7-triazacyclononane-l,4,7-triacetic acid.

[0157] 1. Synthetic Chemistry

[0158] Precursor compound 5 (N-[2-(NOTA-amidyl)ethyl]-3,4-dibromo-maleimide, abbreviated as NOTA-dibromomaleimide) and its non-radioactive reference compound 6 ([19F]AIF-NOTA- 3,4-dibromomaleimide, abbreviated as [19F]AIF-NOTA-dibromomaleimide) were prepared in accordance with Scheme 1 below.

[0159] Scheme 1 N-(Methoxycarbonyl)-3,4-dibromomaleimide (compound 1)

[0160] 1

[0161] 3,4-Dibromomaleimide (1.00 g, 3.90 mmol) and / V-methylmorpholine (0.43 mL, 3.90 mmol) were dissolved in THF (35 mL). Next, methylchloroformate (0.3 mL, 3.90 mmol) was added to the reaction mixture and stirred at RT for 20 minutes. The crude mixture was then diluted with DCM (40 mL) and partitioned in water. The combined organic layers were dried over MgSO4and concentrated in vacuo, giving the title compound as pink crystalline solid (1.09 g, 90%).XH NMR (400 MHz, CDCI3) 6 4.00 (s, 3H);13C NMR (101 MHz, CDCI3) 6 158.25, 145.94, 130.43, 53.80

[0162] N-(2-Boc-aminoethyl)-3,4-dibromomaleimide (compound 2)

[0163] 2

[0164] N-(Methoxycarbonyl)-3,4-dibromomaleimide (1) (1.00 g, 3.50 mmol) was dissolved in DCM (10 mL). A solution of N-Boc-ethylenediamine (0.50 g, 3.50 mmol) in DCM (10 mL) was added dropwise and the resulting reaction mixture was stirred at RT for 2 hours. Next, the crude reaction was quenched with water (20 mL) and extracted with DCM ( 3 x 50 mL). The combined organic layers were dried over MgSO4, concentrated in vacuo, and purified with flash column chromatography eluting with DCM I MeOH 0 to 10 % of methanol in 20 column volumes to yield the title compound as a white powder (348 mg, 25%).XH NMR (400 MHz, CDCI3) 3.67 (m, 2H), 3.32-3.25 (m, 2H), 1.33 (s, 9H).13C NMR (101 MHz, CDCI3) 6 163, 155, 128, 79, 39, 38, 27.

[0165] N-(2-Aminoethyl)-3,4-dibromo-maleimide (compound 3)

[0166] N-(2-Boc-aminoethyl)-3,4-dibromomaleimide (2) (300 mg, 0.7 mmol) was dissolved in a 1 : 1 solution of DCM / TFA (2 mL) and stirred at RT for 2 hours. Next, the reaction mixture was concentrated in vacuo to yield the title compound as white powder (273 mg, 95%).XH NMR (400 MHz, D2O) 6 3.90 (m, 2H), 3.21 (m, 2H)13C NMR (101 MHz, D2O) 6 166, 129, 38, 37.

[0167] N-{2-[(tButyl)2-NOTA-amidyl]ethyl]-3,4-dibromo-maleimide (compound 4)

[0168] 4

[0169] NOTA(tBu)2(50 mg, 0.12 mmol), EEDQ (35 mg, 0.14 mmol) were dissolved in DMF (300 mL) and stirred at RT for 30 minutes. Next, a solution of N-(2-aminoethyl)-3,4-dibromo-maleimide (3) (40 mg, 0.10 mmol) in DMF (200 mL) was added to the mixture and stirred at RT overnight. The reaction mixture was concentrated in vacuo. The crude mixture was purified with HPLC using a semi preparative Zorbax SB-300 C18using the following method: 5 to 95 % of solvent B in 30 minutes (solvent A: H2O / 0.1% FA, solvent B: MeCN I 0.1% FA), to yield the title compound as yellow powder (31 mg, 45%).XH NMR (400 MHz, CDCI3) 6 9.05 (t, J = 5.8 Hz, 1H), 4.10 (s, 2H), 3.81 - 3.76 (m, 2H), 3.58 (d, J = 3.6 Hz, 4H), 3.47 (d, J = 3.9 Hz, 2H), 3.32 (s, 2H), 3.21 (s, 4H), 3.14 (s, 2H), 2.95 (d, J = 14.0 Hz, 4H), 1.46 (s, 18H);13C NMR (101 MHz, CDCI3) 6 170, 167, 164, 130, 82, 58, 56, 52, 51, 48, 39, 38, 28; HR-MS (El, m / z) [M+H]+: calc. 694.1451 for C26H4279Br2N5O7+; found 694.1465.

[0170] N-[2-(NOTA-amidyl )ethyl]-3,4-dibromo-maleimide (compound 5)

[0171] 5

[0172] N-{2-[(tButyl)2-NOTA-amidyl]ethyl]-3,4-dibromo-maleimide (4) (30.0 mg, 0.04 mmol) was dissolved in a 1: 1 solution of DCM / TFA (2 mL) and stirred at RT for 2 hours. Next, the reaction mixture was concentrated in vacuo. The crude mixture was purified with HPLC using a semipreparative Zorbax SB-300 C18using the following method: 5 to 95 % of solvent B in 30 minutes (solvent A: H2O / 0.1% FA, solvent B: MeCN I 0.1% FA) to yield the title compound as yellow powder (17.4 mg, 75%).^ NMR (400 MHz, methanol-d4) 6 3.83 (s, 4H), 3.76 (s, 2H), 3.45 (s, 2H), 3.40 (s, 2H), 3.14 (s, 8H), 2.86 (s, 4H);13C NMR (101 MHz, methanol-d4) 6 172, 171, 164, 129, 59, 55, 50, 49, 49, 39, 38; HR-MS (El, m / z) [M+H]+: calc. 582.01299 for Ci8H2679Br2N5O7+; found 582.0210. [19F]AIF-NOTA-3,4-dibromomaleimide (compound 6)

[0173] N-[2-(NOTA-amidyl)ethyl]-3,4-dibromo-maleimide (5) (1.0 mg, 1.71 mmol) was dissolved in NaOAc buffer (20 mL, 0.10 M, pH 4.5) and mixed with AICI3»6H2O (5.10 mmol, 51 mL , 0.10 M in a 0.10 M pH 4.5 NaOAc buffer). The resulting mixture was stirred at RT for 5 minutes, following the addition of 51 mL of 157 M of NaF dissolved in a 0.1 M pH 4.5 NaOAc buffer. The reaction was stirred at 100 °C for 30 minutes before cooling to RT. The crude mixture was purified with HPLC using a semi-preparative Zorbax SB-300 C18 using the following method: 5 to 95 % of solvent B in 30 minutes (solvent A: H2O / 0.1% FA, solvent B: MeCN I 0.1% FA) to yield the title compound as white powder (0.3 mg, 30%). HR-MS (El, m / z) [M+H]+: calc. 625.9842 for CI8H24AI79Br219FN5O7+; found 625.9842.

[0174] 2. Radiochemistry

[0175] The radiolabelled compound 7 ([18F]AIF-NOTA-dibromomaleimide) was radiosynthesized in accordance with Scheme 2 below, using AI18F / NOTA chelation chemistry in moderate radiochemical yields of 51±7% (n=3) with a total production time around 60 min.

[0176] Scheme 2

[0177] [18F]AIF-NOTA-3,4-dibromomaleimide (compound 7)

[0178] 18F-Fluoride (~500 MBq) in water was trapped in a QMA cartridge (Waters Sep-Pak light, pretreated with 10 mL of water), and released with 0.9% saline (300 mL) into a 5 mL Wheaton vial. Glacial acetic acid (10 mL) and AICI3»6H2O (0.24 mmol, 48 mL, 5.0 mM in the 0.10 M pH 4.5 NaOAc buffer) were added to the reaction vial. To the resulting solution, N-[2-(NOTA- amidyl)ethyl]-3,4-dibromo-maleimide (5) (0.50 mg, 0.86 mmol) in EtOH (200 mL) was added. The reaction was heated at 70 °C for 25 minutes before cooling to RT. The crude mixture was purified by HPLC with a semi-preparative HPLC column using the following method: 5 to 95 % of solvent B in 30 minutes (solvent A: 0.01 M NH4OAc, solvent B: MeCN). The title compound has a HPLC retention time of 10.45 min. It was collected from the HPLC and diluted to 10 % acetonitrile in water. [18F]AIF-NOTA-3,4-dibromomaleimide was trapped onto a Sep-Pak C-18 light cartridge (pre-activated with 5 mL methanol followed by 5 mL water). The cartridge was washed with 2 mL of water and [18F]AIF-NOTA-3,4- dibromomaleimide was then eluted with 1.0 mL of ethanol. The ethanol was removed by N2and the radioactive residual was dissolved in DMSO for bioconjugation.

[0179] The identity of radiolabelled compound 7 ([18F]AIF-NOTA-dibromomaleimide) was confirmed by the HPLC co-elution with its non-radioactive reference compound 2, as shown in Figures 1-3.

[0180] 3. Bioconiuaation with NM-02

[0181] Subsequently, the HER2 targeting nanobody, NM-02 was reduced with one equivalent of TCEP and then tandemly conjugated with the radiolabelled compound 7 ([18F]AIF-NOTA- dibromomaleimide) and then one equivalent of its non-radioactive reference compound 6. Scheme 3 shows the bioconjugation of radiolabelled compound 7 ([18F]AIF-NOTA- dibromomaleimide) with NM-02.

[0182] Scheme 3 NM-02 (120 p.g, 30 pL, 8.0 nmol) was incubated with TCEP (8.0 nmol, 1.0 equiv.) in DMSO (3.4 pL) at 37 °C for 1 hour. The pH of the reaction was increased to pH 8.0 by addition of borate buffer (0.5 pL, 1.0 M, pH 8.5). [18F]AIF-NOTA-3,4-dibromomaleimide (7) (~10 MBq) was added to the reaction and was incubated for 15 min at 37 °C. The cold reference compound (6) (1.0 equivalents to NM-02) was then added to the reaction and incubated for a final 15 mins. The reaction was then diluted to 500 pL using PBS and then purified through a PD MiniTrap G-25 column. The [18F]AIF-NOTA-NM-02 was eluted in 100 pL fractions in PBS.

[0183] The bioconjugation efficiency for [18F]AIF-NOTA-NM-02 was 40±8% (n=3) after size exclusion purification.

[0184] 4. Tumour cell uptake of r18FlAIF-NOTA-NM-Q2

[0185] Both HER.2 positive SKBR.3 and negative MDA-MB-231 breast cancer cells were treated with [18F]AIF-NOTA-NM-02. Blocking studies were also performed by pre-treating the SKBR3 cells with 100 folds of native NM-02.

[0186] Both SKBR.3 and MDA-MB-231 breast cancer cells (IxlO6) were washed with PBS and resuspended in 200 pL PBS + 3% BSA. [18F]AIF-NOTA-NM-02 (5.0 nM) was added and incubated with cells for 1 h at 37 °C. For blocking studies, NM-02 (500.0 nM) was incubated with the tumour cells for 1 h at 37 °C before addition of [18F]AIF-NOTA-NM-02 (5.0 nM). After incubation with [18F]AIF-NOTA-NM-02, cells were washed twice with PBS and moved to a fresh Eppendorf tube. Activity in cell pellet, wash fractions, and the original Eppendorf were counted in a 1282 CompuGamma OS (LKB-Wallac).

[0187] Figure 4 shows the tumour cell uptake of the [18F]AIF-NOTA-NM-02. Blocked samples were pre-treated with 100X native NM-02. Data shown are mean ± SD of three independent experiments conducted in triplicate.

[0188] The uptake of [18F]AIF-NOTA-NM-02 by the SKBR3 cells was around 14%. Most of the uptake can be blocked by the native NM-02. While, the HER2 negative MDA-MB-231 breast cancer cells have little uptake of [18F]AIF-NOTA-NM-02.

[0189] The novel18F-bioconjugation reagent, [18F]AIF-NOTA-dibromomaleimide (compound 7), has site specifically labelled a HER2 targeting nanobody, NM-02, through its reduced intramolecular disulfide bridge. The [18F]AIF-NOTA-dibromomaleimide labelled NM-02 has shown specific uptake by the HER2 positive SKBR3 human breast cancer cells.

[0190] 5. Bioconiuoation with IL-4DE

[0191] IL-4DE was reduced with one equivalent of TCEP and then tandemly conjugated with the radiolabelled compound 7 ([18F]AIF-NOTA-dibromomaleimide) and then one equivalent of its non-radioactive reference compound 6. Scheme 4 shows the bioconjugation of radiolabelled compound 7 ([18F]AIF-NOTA-dibromomaleimide) with IL-4DE.

[0192] Scheme 4

[0193] The bioconjugation efficiency for [18F]AIF-NOTA-IL-4DE was 53±5% (n=3) after size exclusion purification. The purified [18F]AIF-NOTA-IL-4DE was stable in PBS at 37 °C for at least 4 hours. No oligomer formation was observed during the [18F]AIF-NOTA-IL-4DE production.

[0194] 6. Cell uptake of F18F1AIF-NOTA-IL-4DE

[0195] A study was conducted to investigate the in vitro uptake of [18F]AIF-NOTA-IL-4DE by the 4o[3-expressing T4-CAR T-cells, non-activated non-transduced T-cells, peripheral blood mononuclear cells (PBMCs), ErbB positive IL-4 receptor-expressing SKOV3 ovarian and HN3 head and neck cancer cells.

[0196] 4ap-expressing T4-CAR T-cells, non-activated non-transduced T-cells, PBMCs, SKOV3 or HN3 cancer cells were washed in PBS and resuspended in 200 pL PBS at a concentration of 5xl06cells / mL. [18F]AIF-NOTA-IL-4DE (50 KBq, 1 nM) was added to the cells and incubated in the dark at 37 °C for 30 min. Cells were washed twice with PBS, with the supernatant retained in separate Eppendorf tubes, and resuspended in fresh media before being gamma counted to establish labelling efficiency. Cell labelling efficiency was calculated as [Radioactivity in cell pellet / (Radioactivity in cell pellet + radioactivity in the supernatant)] x 100 %.

[0197] Figure 5 shows the cell uptake of the [18F]AIF-NOTA-IL-4DE. Blocked samples were pretreated with 100X native IL-4DE. Data shown are mean ± SD of independent experiments, where n=5 for 4a0-expressing T4-CAR T-cells, n=5 for non-activated non-transduced T-cells, n=5 for PBMCs, n=3 for SKOV3 and 3HN cancer cells.

[0198] The uptake of [18F]AIF-NOTA-IL-4DE was around 37% for the 4o[3-expressing T4-CAR T- cells. In contrast, non-activated, non-transduced T-cells, PBMCs, SKOV3 and HN3 cancer cells only had neglectable [18F]AIF-NOTA-IL-4DE uptakes. Thus, [18F]AIF-NOTA-IL-4DE showed selective uptake by the 4o[3-expressing T4-CAR T-cells. 7. Bioconiuaation with HSA

[0199] Human serum albumin (HSA) was reduced with one equivalent of TCEP and then tandemly conjugated with the [18F]AIF-NOTA-dibromomaleimide (compound 7) and then one equivalent of its non-radioactive reference compound (compound 6). The bioconjugation efficiency for [18F]AIF-NOTA-HSA was 66±3% (n=5) after size exclusion purification. The [18F]AIF-NOTA-HSA co-elutes with native HSA with a retention time of 6.49 min.

[0200] 8. PET imaaina of r18F!AIF-NOTA-HSA

[0201] [18F]AIF-NOTA-HSA (~10 MBq) was administered by intravenous injection (IV) in healthy BALB / c mice (n=3). Dynamic Positron Emission Tomography (PET) scans were performed for 120 min post-IV injection.

[0202] Healthy BALB / c mice (n=3) were anaesthetised using 2% isoflurane in oxygen. The animals (n=3) were injected with [18F]AIF-NOTA-IL-HSA (~10 MBq) via the lateral tail vein. Dynamic PET scans of 120 min were immediately performed using a NanoScan PET (Mediso, Budapest, Hungary) scanner. All PET data were reconstructed with the Monte Carlo-based full-3D iterative algorithm Tera-Tomo (Mediso Medical Imaging Systems, Budapest, Hungary). Raw PET data were reconstructed using reconstruction settings (4 iterations, 6 subsets, 0.4x0.4x0.4 mm3 voxel size) as well as intercrystal scatter correction. All reconstructed data were analysed with VivoQuant software (v3.0, inviCRO, LLC, Boston, USA). All animals were euthanised by cervical dislocation at the end of the PET scans.

[0203] Figure 6 shows PET images of the mice, reconstructed at 5, 20, 40, 80, and 120 min. It was found that most of the [18F]AIF-NOTA-HSA was still in blood circulation 120 min after IV injection, indicated by the very high left ventricular chamber radioactivity accumulation in the PET images shown in Figure 6.

Claims

CLAIMS1. A radiolabelled compound of formula (I):wherein:R1and R2are independently selected from a halogen and -SR3, wherein R3is independently selected from Ci-i2alkyl, Ci-i2alkenyl, Ci-i2alkynyl and C6-io aryl;L represents a direct bond or a linker, wherein the linker is a Ci-20alkylene which is unsubstituted or substituted by one or more substituents selected from hydroxy, -COOH, Ci-i2alkoxy, Ci-i2alkyl and hydroxy-Ci-i2-alkyl, wherein zero or one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -O-, -S-, -NR4-, -C(O)NR4-, -NR4C(O)-, -C(O)-, -OC(O)-, and -C(O)O-, wherein R4is hydrogen or Ci-i2alkyl, and wherein the C6-io arylene moiety is unsubstituted or substituted by one, two, three or four substituents selected from hydroxy, Ci-i2alkyl and Ci-i2alkoxy;X is absent or a PEG2.20group; andQ* represents an18F-labelled moiety; or a pharmaceutically acceptable salt thereof.

2. The radiolabelled compound of claim 1, wherein Q* represents an18F-labelled moiety comprising a chelated18F-AI complex.

3. The radiolabelled compound of claim 1 or 2, wherein Q* represents an18F-labelled moiety selected from:

4. The radiolabelled compound of claim 3, wherein Q* represents an18F-labelled moiety with the formula (Q*-A).

5. The radiolabelled compound of any of the preceding claims, wherein R1and R2are independently selected from -Br, -I and -SPh.

6. The radiolabelled compound of claim 5, wherein R1is -Br and R2is -Br.

7. The radiolabelled compound of any of the preceding claims, wherein L represents a linker, wherein the linker is a Ci-20alkylene wherein one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -C(O)NR4-, -NR4C(O)-, -C(O)-, -OC(O)-, and -C(O)O-, wherein R4is hydrogen or Ci-i2alkyl, and wherein the C6-io arylene moiety is unsubstituted.

8. The radiolabelled compound of claim 7, wherein the linker is a Ci-20alkylene wherein one to ten carbon atoms in the alkylene chain are replaced by spacer moieties selected from C6-io arylene, -C(O)NH- and -NHC(O)-, and wherein the C6-io arylene moiety is unsubstituted.

9. The radiolabelled compound of any of the preceding claims, wherein L has the formula:-(CH2)n-NH-C(O)-(CH2)m- or -(CH2)n-C(O)-NH-(CH2)m- , wherein n is an integer from 0 to 18 and m is an integer from 0 to 18, and n+m<18.

10. The radiolabelled compound of claim 9, wherein n is 2 and m is 1.

11. The radiolabelled compound of claim any of claims 1-8, wherein L has the formula:wherein p is an integer from 0 to 18 and q is an integer from 0 to 18, and p+q<18.

12. The radiolabelled compound of claim 11, wherein q is 1.

13. The radiolabelled compound of claim any of claims 1-8, wherein L has the formula:wherein r is an integer from 0 to 18 and s is an integer from 0 to 18, and r+s<18.

14. The radiolabelled compound of claim 13, wherein s is 2.

15. The radiolabelled compound of any of claims 1-10, wherein the compound of formula (I) is a compound of the formula:

16. A reference compound of formula (II):wherein:R1, R2, L and X are as defined in any of claims 1-15; and Q represents a19F-labelled moiety comprising a chelated19F-AI complex; or a pharmaceutically acceptable salt thereof.

17. The reference compound of claim 16, wherein Q represents a moiety selected from:

18. The reference compound of claim 17, wherein Q represents a moiety with the formula (Q-A).

19. The reference compound of any of claims 16-18, wherein the compound of formula (II) is a compound of the formula:

20. A precursor compound of formula (III):wherein:R1, R2, L and X are as defined in any of claims 1-15; andY represents a moiety capable of chelating an18F-AI complex; or a pharmaceutically acceptable salt thereof.

21. The precursor compound of claim 20, wherein Y represents a moiety selected from:

22. The precursor compound of claim 21, wherein Y represents a moiety with the formula (Y-A).

23. The precursor compound of any of claims 20-22, wherein the compound of formula (III) is a compound of the formula:

24. A pharmaceutical composition comprising a radiolabelled compound of any of claims 1- 15 and a pharmaceutically acceptable carrier.

25. A method for making the radiolabelled compound of any of claims 1-15, the method comprising radiolabelling the precursor compound of any of claims 20-23.

26. The method of claim 25, the method comprising the step of: reacting the precursor compound of formula (III) with radiolabelling reagents comprising a source of fluoride-18, to form the radiolabelled compound of formula (I), in accordance with the following reaction scheme:O OR]T N— X — L x— L — Y radiolabell — Q*T N— ing reagents(HI) (I) wherein R1, R2, L, X and Q* are as defined in any of claims 1-15; andY is as defined in any of claims 20-23.

27. The method of claim 26, wherein the radiolabelling reagents comprise a source of fluoride-18 and a source of Al3+.

28. A radiolabelled compound of any of claims 1-15 for use in a diagnostic method practised on the human or animal body using positron emission tomography (PET).

29. The radiolabelled compound for use of claim 28, wherein the diagnostic method comprises labelling a protein with the radiolabelled compound of any of claims 1-15.

30. The radiolabelled compound for use of claim 29, wherein the protein is a native protein.

31. The radiolabelled compound for use of claim 29 or 30, wherein the protein is selected from an antibody, a single chain fragment variable (SCFV), an antigen-binding fragment (Fab), a nanobody, and / or a cytokine.

32. The radiolabelled compound for use of any of claims 28 to 31, wherein the diagnostic method comprises labelling an HER.2 targeting nanobody with the radiolabelled compound of any of claims 1-15.

33. The radiolabelled compound for use of claim 32, wherein the HER.2 targeting nanobody is NM-02.

34. The radiolabelled compound for use of any of claims 28 to 33, wherein the diagnostic method is a method of diagnosing cancer.

35. The radiolabelled compound for use of any of claims 28 to 34, wherein the diagnostic method is a method of identifying the location of a tumour.

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