18F radiolabeled biomolecules

Abstract

The present invention provides 18 The present invention relates to F radiolabeled residualizing agents and biomolecules, as well as methods for labeling biomolecules with radioactive fluorine atoms. The biomolecules have an affinity for specific cell types and can specifically bind to specific cells (e.g., cancer cells). Such biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. The present invention also provides compositions containing such labeled biomolecules and methods for using the labeled biomolecules and / or compositions for imaging applications.

Description

Detailed Description of the Invention

[0001] FIELD OF THEINVENTION The present invention relates to methods for preparing compounds useful for radioactive labeling of biomolecules, and methods for preparing such radioactively labeled biomolecules. The present disclosure also provides radioactively labeled biomolecules and corresponding radioactively labeled biomolecular precursors, which can effectively retain radioactivity from biomolecules that become incorporated into cells, and such compounds are useful in the diagnosis of disease, particularly cancer.

[0002] 〔background〕 Many monoclonal antibodies (mAbs), mAb fragments, and peptides have been labeled with different radionuclides and used for the detection and treatment of cancer. Many of the most clinically important target molecules (e.g., HER2, epidermal growth factor receptor (EGFR), and the tumor-specific mutation EGFRvIII) are rapidly taken up into tumor cells. This is a major problem from the labeling point of view, because when a radiolabeled biomolecule binds to a tumor-associated receptor or antigen, it is transported into the cell and taken up into endosomes / lysosomes, where it is rapidly degraded. The difficulty is that these radioactive degradation products are rapidly lost from the tumor cells. As a result, there is no longer enough radioactivity in the tumor cells to allow imaging or treatment of the tumor.

[0003] As an example, consider labeling a mAb with reactivity to EGFRvIII with radioactive iodine. Note that the standard labeling method used here is direct electrophilic substitution. In this case, extensive internalization results in low radioactivity retention in the tumor after receptor binding and subsequent proteolysis. This is because the major catabolite, iodotyrosine, is rapidly washed out. To circumvent this problem, "residualizing agents" have been developed that attempt to trap radioactivity within tumor cells after the labeled mAb has been internalized. Such residualizing agents for radioactive iodine include N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB); N ε -(3-Iodobenzoyl)-Lys 5 -N α -Maleimido-Gly 1 -Geeek, where e and k represent residues of D-glutamic acid and D-lysine, respectively. 2 -(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)-D-glutamyl-D-glutamyl-D-glutamyl-N'-(3-iodobenzoyl)-D-lysine, or known as IB-MalGeeek; and 2,2',2''-(10-(2-((6-(3-(((N-succinimidyl)oxy)carbonyl)-5-iodobenzamido)hexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (SIB-DOTA). A significant enhancement of retention of radioactivity in tumors is seen when the same biomolecule is labeled with one of these residualizing prosthetic groups, as compared to a directly labeled biomolecule.

[0004] Positron emission tomography (PET) is a state-of-the-art imaging technique, highly sensitive and with excellent quantification capabilities. The most widely available positron-emitting radionuclide worldwide is fluorine-18, which has a half-life of 110 minutes. In order to combine the advantages of this imaging technique with the targeting properties of internalized molecules, it is necessary to develop retention agents capable of labeling these biomolecules with fluorine-18. Furthermore, more efficient methods for the preparation of such labeled biomolecules are needed.

[0005] Summary of the Invention The present invention relates to a method for producing radioactive halogen atoms (especially 18 The present invention relates to methods, compounds, and compositions for radioactively labeling biomolecules (also called macromolecules) with .F. Advantageously, these methods, compounds, and compositions provide for the dehalogenation of radioactive halogens by in vivo dehalogenation. 18The biomolecules have an affinity for a particular type of cell, i.e., they can bind specifically to a particular cell, e.g., a cancer cell. The compositions of the present invention include radiolabeled biomolecules. Such biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. Examples of biomolecules for the present invention include diabodies, scFv fragments, DARPins, fibronectin type III-based scaffolds, affibodies, VHH molecules (also known as single domain antibody fragments (sdAbs) and nanobodies), nucleic acid or protein aptamers, and nanoparticles. In addition, large molecules (e.g., proteins over 50 kDa, including antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and F(ab')2 fragments) can be used in the methods disclosed herein. In addition, nanoparticles having a size of less than 50 nm can be used in the methods disclosed herein. The principles disclosed herein, in some embodiments, are particularly relevant to VHH molecules and other types of small protein constructs, as described in more detail herein.

[0006] The methods of the invention utilize prosthetic compounds that are useful for radiolabeling. As such, the present disclosure provides such radiolabeled compounds and precursors for preparing such prosthetic compounds. The invention further provides radiolabeled macromolecules (e.g., biomolecules) that include such compounds / radicals and one or more macromolecules. In some embodiments, these radiolabeled macromolecules are targeted radiotherapeutic agents. The prosthetic compounds and radiolabeled compounds of the invention are useful, for example, in diagnosing disease.

[0007] In one aspect, the present invention provides a method for preparing a functionalized biomolecule that includes a dienophile, and 18 providing an F-containing reagent; and reacting the functionalized biomolecule with the 18 React with an F-containing reagent via inverse electron demand Diels-Alder cycloaddition reaction, 18 Providing a F-labeled biomolecule; 18 The present invention provides a method for preparing F-labeled biomolecules. 18 The F-labeled biomolecule may vary. In a non-limiting specific embodiment, in the above preparation method, 18 F-containing reagents are 6-[ 18 F]fluoronicotinyl-PEG4-methyltetrazine. In a specific, non-limiting embodiment, the functionalized biomolecule comprises a biomolecule derivatized with TCO-GK-PEG4-NHS. In a specific embodiment, the method comprises the step of: 18 can be used to provide F-labeled biomolecules: 18 [F]FN-PEG4-Tz-TCO-GK-PEG4-biomolecules, e.g., 18 [F]FN-PEG4-Tz-TCO-GK-PEG4-5F7, but is not limited to this.

[0008] In another aspect, the present invention provides a method for preparing a functionalized biomolecule comprising the steps of: providing a functionalized biomolecule comprising a diene; 18 providing an F-containing reagent; and reacting the functionalized biomolecule with the 18 React with an F-containing reagent via inverse electron demand Diels-Alder cycloaddition reaction, 18 Providing a F-labeled biomolecule; 18 A method for preparing F-labeled biomolecules is also provided. Again, the method includes the steps of: 18 The F-labeled biomolecule may vary. In a non-limiting specific embodiment, in the above preparation method, 18 F-containing reagents are 6-[ 18In a specific, non-limiting embodiment, the functionalized biomolecule comprises a biomolecule derivatized with -Mal-PEG4-Tz. In a specific embodiment, the method comprises the step of: 18 can be used to provide a F-labeled biomolecule module: 18 [F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-biomolecules, e.g., 18 [F]FN-PEG4-GK-TCO-Tz-PEG4-Mal 5F7GC, but is not limited to this.

[0009] In some embodiments of the above-described method, 18 F-containing reagents are 18 F] fluoronicotinyl (FN) group. The functionality of the dienophile can vary. In some embodiments, the dienophile includes an octene moiety. For example, one suitable dienophile includes a trans-cyclooctene (TCO) moiety. Similarly, the functionality of the diene can vary. In some embodiments, the diene includes a tetrazine (Tz) moiety. Various other examples of dienes and dienophiles suitable for IEDDAR will be understood by those skilled in the art. In some embodiments, 18 The F-containing reagent and / or the functionalized biomolecule may further comprise a linker. Such linkers may include, for example, PEG and / or renal brush border enzyme-cleavable linkers.

[0010] The present invention further comprises a specific 18 A F-labeled biomolecule is provided. The biomolecule is 18 F]FN-PEG4-Tz-TCO-GK-PEG4-, and 18 F]FN-PEG4-GK-TCO-Tz-PEG4-Mal- 18 Specific, but not limiting, examples of labeled biomolecules include those conjugated to F-labeled residual agents. 18F]FN-PEG4-Tz-TCO-GK-PEG4-5F7, and 18 F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC.

[0011] Another aspect of the invention is a method for producing a fluoroalkyl azide-containing cycloalkyl ether (PEG) linker comprising the steps of: providing a first compound comprising a guanidine moiety and an alkyne moiety; providing a second compound comprising a fluoroalkyl azide and a PEG linker; and reacting the first compound with the second compound by click chemistry to produce a fluoroalkyl ether-containing PEG linker (PEG) ... 18 Obtaining a F-labeled residual agent. 18 A method for preparing a F-labeled residualizing agent is provided. In some embodiments, click chemistry is catalyzed, for example, by a copper catalyst. The reagents of the method can vary. In one specific, non-limiting embodiment, the first compound is N-succinimidyl 3-((2,3-bis(tert-butoxycarbonyl)3uanidine)methyl)-5-ethynylbenzoate and the second compound is 1-azido-2-(2-(2-(2-[ 18 The present invention further comprises carrying out the method referenced immediately above and reacting the labeled moiety with a biomolecule, 18 The present invention further provides a method for preparing F-labeled biomolecules. 18 The F-labeled retention agent is N-succinimidyl 3-(1-(2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid, and the corresponding 18 F-labeled biomolecules, which 18 The residual agent may include, but is not limited to, a biomolecule bound to an F-labeled residual agent.

[0012] In a further aspect, the present invention provides a method for preparing a boronate precursor comprising the steps of:18 and a F-containing reagent, 18 and reacting the resulting mixture with F fluorodeboronation. 18 A method for preparing a F-labeled residual agent is provided. The reagents can vary. In some embodiments, the boronate precursor further comprises a TFP ester. In some embodiments, 18 F-containing reagents are 18 F]tetraethylammonium fluoride. In some embodiments, the reacting step can be carried out in the presence of a catalyst, including, but not limited to, a copper catalyst. The present invention also relates to a method for producing a fluorinated compound, comprising carrying out the method referenced immediately above. 18 preparing an F-labeled residual agent; 18 and reacting the F-labeled residual agent with a biomolecule. 18 The present invention further provides a method for preparing an F-labeled biomolecule, comprising: 18 The F-labeled residual agent is a tetrafluorophenyl 3-[ 18 F] fluoro-5-guanidinomethylbenzoic acid, and 18 F-labeled biomolecules, which include biomolecules that have been conjugated to such reagents.

[0013] The present invention further comprises the steps of: 18 The present invention provides a method for imaging cancer cells, comprising using any one of the F-labeled biomolecules.

[0014] Labeled according to the methods described herein and 18 Suitable biomolecules to be incorporated into the F-labeled biomolecule can vary widely. In various embodiments, the biomolecule labeled by the above-mentioned method is a nanobody. Exemplary nanobodies can include, but are not limited to, HER2-specific nanobodies. Specific examples of HER2-specific nanobodies can include, but are not limited to, 5F7, 5F7GCC, 2Rs15d, and variants thereof.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS To aid in the understanding of embodiments of the present invention, reference is made to the accompanying drawings, which are not necessarily drawn to scale and in which reference numerals indicate components of exemplary embodiments of the present invention. The drawings are merely illustrative and should not be construed as limiting the present invention.

[0016] Figure 1A shows 18 The structure of [F]AlF-NOTA-PEG4-Tz-TCO-GK-2Rs15d; Figure 1B shows 18 1 is an exemplary reaction scheme for the synthesis of [F]FN-PEG4-Tz-TCO-GK-PEG4-5F7; Figure 2A shows 18 F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC; FIG. 2B shows the iso-[ 125 I]SGMIB-5F7 (Black), and [ 18 F] is a plot of the uptake of FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC (black / white stripes); FIG. 2C shows that in mice bearing BT474M1 xenografts, 18 A series of maximum intensity projection (MIP) images obtained after administration of [F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC; Figure 3 shows 18 1 is an exemplary reaction scheme for the synthesis of F]RL-III-2Rs15d; FIG. 4A is a MicroPET / CT image obtained 2 hours after administration of [18F]RL-III-5F7 in a mouse bearing a BT474M1BrM3-Fluc intracranial xenograft; Figure 4B is a cross-section of the mouse brain from Figure 2A stained with H&E; FIG. 4C is an autoradiographic image of an adjacent section of a mouse (the sizes of the images in FIG. 4B and FIG. 4C are not to scale); Figure 5A is the structure of SGMIB; Figure 5B shows 18 1 is an exemplary reaction scheme for the synthesis of [F]TFPFGMB.

[0017] Detailed Description The present invention generally relates to a method for producing a biomolecule. 18 The present invention provides specific methods for F labeling, as well as specific precursors and products obtained thereby. The disclosed methods are primarily directed to 18 The present invention focuses on the preparation of F-labeled residual agents and certain exemplary labeled biomolecules. However, such methods may find broad applicability in some circumstances. Such methods may find applicability in the preparation of other labeled biomolecules, for example, as disclosed in U.S. Patent No. 9,839,704 to Zalutsky et al., or International Patent Application Publication No. WO2018 / 178936 to Zalutsky et al., which are incorporated herein by reference in their entireties. The present invention further provides a method for the preparation of certain labeled biomolecules, such as the preparation of a fluororesin, a fluororeagent, or a fluororeagent. 18 F-labeled residual agents, specific 18 The present invention provides F-labeled biomolecules, compositions containing them, and methods of using these labeled biomolecules and / or compositions for imaging purposes.

[0018] Certain abbreviations are used throughout this application and are used as generally recognized in the art. For convenience, their definitions are set forth below.

[0019] 2Rs15d: Nanobodies described, for example, in "Vaneycken I, et al (2011) Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 25:2433-2446," which is incorporated herein by reference in its entirety; 5F7: Nanobody described in "M. Pruszynski et al., Nuclear Medicine and Biology 40 (2013) 52-59" etc.; 5F7-GCC: a 5F7 nanobody variant containing a cysteine ​​at the carboxyl terminus, as described in, for example, "M. Pruszynski et al. / Nuclear Medicine and Biology 40 (2013) 52-59"; Boc: tert-butyloxycarbonyl protecting group; DMA: dimethylacetamide; FN: fluoronicotinyl; GK: glycine lysine; HER-2: human epidermal growth factor receptor 2 protein; HPLC: high performance liquid chromatography; IEDDAR: Inverse electron demand Diels-Alder cycloaddition; iso-[ 131 I]SGMIB: N-Succinimidyl 3-guanidinomethyl-5-[ 131 I]iodobenzoic acid; Mal: Maleimide; PEG: poly(ethylene glycol); RBBE: renal brush border enzyme; RCY: Radiochemical yield; sdAbs: single domain antibody fragments; [ * I]SGMIB: N-Succinimidyl 4-guanidinomethyl-3-[ * I]iodobenzoic acid; TCO: trans-cyclooctene-containing moiety; TFA: trifluoroacetic acid; TFP: tetrafluorophenyl; Tz: tetrazine-containing moieties (including but not limited to tetrazine, 3-methyl-6-phenyl-1,2,4,5-tetrazine, and further derivatives); VHH: heavy chain only antibody variable domain (aka sdAb, nanobody).

[0020] According to one embodiment of the present invention, 18 A method for F labeling is provided that may find particular application in the context of labeling sdAb and other types of small protein constructs. The method involves an inverse electron demand Diels-Alder cycloaddition reaction (IEDDAR). The method comprises: a) 18 b) preparing an F-containing reagent; and 18 Attaching the F-containing reagent to a functionalized biomolecule (e.g., an sdAb or other small protein construct); 18 and providing an F-labeled biomolecule.

[0021] The disclosed methods can provide site-nonspecific or site-specific labeling. Advantageously, the methods allow for high product yields and retention of affinity and / or immunoreactivity. In general, 18 The F-containing reagent is 18 In addition to the F moiety, the functionalized biomolecule contains a moiety suitable for IEDDAR (i.e., either a diene functionality or a dienophile functionality). Similarly, the functionalized biomolecule contains, in addition to the biomolecule, a complementary moiety suitable for IEDDAR. 18 If the F moiety contains a diene, the functionalized biomolecule contains a dienophile. F Where the moiety comprises a dienophile, the functionalized biomolecule comprises a diene. The selection and / or preparation of such reagents can, in some embodiments, provide either site-specific or non-site-specific labeling of the biomolecule.

[0022] In one embodiment, 18F-containing reagents ( 18 A F-containing reagent is prepared and then coupled to the functionalized biomolecule ( 18 F), in addition to the diene suitable for the IEDDAR of step b) above. One exemplary diene is a tetrazine-containing moiety. The tetrazine-containing moiety is advantageously 18 Advantageously, in some such embodiments, 18 The F-containing reagent comprises a fluoronicotinyl moiety, the fluoronicotinyl moiety being 18 F. Various other functional groups may be present, so long as the other functional groups do not negatively interfere with the desired IEDDAR. 18 F-containing reagents may be present, for example: 18 The F-containing reagent may further comprise a linker (e.g., PEG) of various lengths. One specific exemplary embodiment that can be effectively utilized in the disclosed methods is 18 F-containing (diene) reagents are 6-[ 18 F] fluoronicotinyl-PEG-4-methyletrazine.

[0023] 18When the F-containing reagent contains a diene suitable for the IEDDAR of step b), the functionalized biomolecule used in the method contains a dienophile. One exemplary dienophile suitable for the IEDDAR disclosed herein is an octene moiety (e.g., in a TCO functional group). The biomolecule of the "functionalized biomolecule" may generally include various sdAb or other small protein constructs. In one particular embodiment, the biomolecule includes an anti-HER2 sdAb. One exemplary biomolecule on which the method has been successfully implemented is 5F7, but the method is not limited thereto. In some embodiments, the functionalized biomolecule may be further modified with one or more chemical moieties (e.g., one or more linkers). In certain embodiments, the linker includes a renal brush border enzyme (RBBE) cleavable linker. Again, in some embodiments, various other functional groups may be present in the functionalized biomolecule, so long as the other functional groups do not negatively interfere with the desired IEDDAR. In one particular embodiment useful in the disclosed method, the functionalized biomolecule comprises a TCO-GK-PEG4-NHS linker. 18 Reaction between an F-containing reagent (via its diene) via IEDDAR can provide the desired labeled biomolecule.

[0024] In other embodiments, functionalized biomolecules and 18 The moiety associated with the F-containing reagent is exchanged. For example, dienes can be 18 The dienophile is attached to a functionalized biomolecule (rather than attached to a F-containing reagent) and the dienophile is attached to a functionalized biomolecule (rather than attached to a functionalized biomolecule as described above). 18 Advantageously, in some embodiments, 18 The F-containing reagent comprises a fluoronicotinyl moiety, 18 In such an embodiment, 18 F-containing reagents ( 18A F-containing reagent is prepared and then coupled to the functionalized biomolecule ( 18 In addition to F), the dienophile suitable for the IEDDAR of step b) above is included. One exemplary dienophile suitable for the IEDDAR disclosed herein is an octene moiety (e.g., in the TCO functional group). Various other functional groups may be included, so long as the other functional groups do not negatively interfere with the desired IEDDAR. 18 F-containing reagents may be present, for example: 18 The F-containing reagent may further comprise a linker of various lengths, including PEG and / or renal brush border enzyme (RBBE) cleavable linkers. One specific exemplary linker that may be usefully utilized in the disclosed methods is 18 F-containing (dienophile) reagents are 6-[ 18 F] fluoronicotinyl-PEG4-GK-TCO.

[0025] 18 When the F-containing reagent contains a dienophile suitable for the IEDDAR of step b), the functionalized biomolecule used in the method contains a diene. One exemplary diene is a tetrazine-containing moiety. The tetrazine-containing moiety may advantageously be included in the functionalized biomolecule. The "functionalized biomolecule" biomolecule may generally include various sdAb or other small protein constructs. In one particular embodiment, the biomolecule includes an anti-HER2 sdAb. One exemplary biomolecule on which the method has been effectively implemented is 5F7-GGC, but the method is not limited thereto. Various other functional groups can be present in the functionalized biomolecule, so long as the other functional groups do not negatively interfere with the desired IEDDAR. For example, the functionalized biomolecule may further include a linker (e.g., PEG) of various lengths. In a further embodiment, 18 The F-containing reagent contains an RBBE cleavable linker. One particular exemplary functionalized biomolecule (diene) reagent that can be effectively utilized in the disclosed method is 5F7-GGC-Mal-PEG4-Tz. The functionalized biomolecule (via its diene) and 18Reaction between the F-containing reagent (via its dienophile) via IEDDAR can provide the desired labeled biomolecule.

[0026] In one embodiment, the method referenced comprises: 18 The present invention provides an F-labeled biomolecule (e.g., prepared according to one of the above methods referenced herein). Two exemplary F-labeled biomolecules are shown below in Formula I and Formula II.

[0027] [ka]

[0028] [ka]

[0029] In another embodiment, the reaction comprises a click reaction (e.g., a copper-catalyzed click reaction), 18 F-labeled residual agents and 18 A method for preparing F-labeled biomolecules is provided. The disclosed reaction includes a) preparing a guanidine-containing compound containing an alkyne moiety, b) preparing a compound containing a fluoroalkyl azide and a PEG linker, c) reacting the compound of step a) with the compound of step b) via click chemistry to form an optionally radiolabeled biomolecule, and d) reacting the resulting compound with a biomolecule. When the click reaction is copper catalyzed, the copper catalyst used can vary and can be any copper-containing compound or salt suitable for catalyzing the reaction. In one particular embodiment, the copper catalyst is copper sulfate. Advantageously, in some embodiments, the click reaction-based method can provide higher radiochemical yields than previously reported for similar reactions in which an azide moiety is present on the guanidine-containing compound. It is noted that the guanidine-containing compound can be an alkyne (6-[ 18F]fluorohexyne). See Glaser, Bioconjugate Chem. 2007, 18, 989-993, which is incorporated herein by reference in its entirety.

[0030] The compound containing a fluoroalkyl azide and a PEG linker can vary. In one embodiment, the compound is 1-azido-2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethane. Similarly, the compound with which the compound reacts (i.e., the guanidine-containing compound) can vary. One example of such a compound is N-succinimidyl 3-((2,3-bis(tert-butoxycarbonyl)guanidine)methyl)-5-ethynylbenzoate.

[0031] Provided through the above steps a) to c) 18 The F-labeled residual agent is reacted with various biomolecules to 18 F-labeled biomolecules can be obtained. Biomolecules used in the method can generally include various sdAbs or other small protein constructs (e.g., the types of biomolecules outlined herein). In one particular embodiment, the biomolecule includes an anti-HER2 sdAb. Two exemplary biomolecules for which the method has been effectively implemented are 2Rs15d and 5F7, but the method is not limited thereto. The method may provide simpler synthetic operations than previous click chemistry methods for the preparation of such compounds, and in some embodiments may provide higher overall radiochemical yields. The present invention further provides the following: 18 F-labeled biomolecules and intermediates ( 18 One exemplary embodiment of the present invention includes a method for producing a fluororesin-containing fluororesin comprising the steps of: 18 The F-labeled biomolecule is shown below in Formula III.

[0032] [ka]

[0033] 18 F-labeled residual agents, and 18 A further method for the preparation of F-labeled biomolecules is provided herein, which uses fluorodeborylation. In certain embodiments, the method includes providing a boronate precursor comprising a TFP ester and a guanidine moiety, where the nitrogen atom on the guanidine moiety is protected (e.g., with a Boc group or other suitable protecting group that can be introduced / removed under conditions that do not negatively affect the desired reaction). The boronate precursor is prepared by the appropriate 18 F-containing reagents (e.g., [ 18 F]tetraethylammonium fluoride, [ 18 F]TEAF) treatment. 18 The resulting compound is then subjected to F fluorodeboronation, which is advantageously catalyzed by a copper reagent, including but not limited to Cu(Py)4(OTf)2. The resulting compound can then be treated to deprotect the nitrogen atoms on the guanidine moiety (e.g., if the protecting groups are Boc, these protecting groups can be removed via treatment with TFA).

[0034] The deprotected 18F-labeled residual agent can then be conjugated to a biomolecule, which may include any of the biomolecules described herein. In one specific, non-limiting embodiment, the biomolecule is a nanobody (e.g., 5F7 or a variant of 5F7). The invention further relates to a method for the preparation of a nanobody obtained by such a reaction. 18 F-labeled biomolecules and intermediates are provided. One exemplary such F-labeled biomolecule is shown in Formula IV below:

[0035] [ka]

[0036] The present invention further relates to a method for the preparation of radioactively labeled biomolecules as disclosed herein (e.g., those described / illustrated above). 18 In one embodiment of the present invention, a composition is provided that includes a F-labeled biomolecule, including those of formula I, II, III, and / or IV, in combination with a pharma- ceutically acceptable adjuvant, diluent, or carrier. In a further embodiment of the disclosure, a method of diagnosing cancer is provided, the method comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule disclosed herein and / or an effective amount of a pharmaceutical composition disclosed herein.

[0037] The following examples are offered by way of illustration and not by way of limitation.

[0038] Example 1: Fluorine-18 labeling of an anti-HER2 sdAb with a 6-fluoronicotinyl moiety via inverse electron demand Diels-Alder reaction (IEDDAR) involving a renal brush border enzyme-cleavable linker. (the purpose) Single domain antibody fragments (sdAbs) are currently the most popular positron emitters that are low molecular weight and therefore short-lived (e.g., 18 F) is considered to be a useful platform for labeling. Low molecular weight positron emitters have rapid tumor uptake and rapid systemic clearance. However, high levels of activity in the kidney from the labeled sdAb are a serious problem. Previously, we have reported that [ 18 The [F]AlF-NOTA moiety was used to synthesize the tetrazine (Tz) / trans-cyclooctene (TCO) [4+2] inverse electron-demand Diels-Alder cycloaddition (IEDDAR) reaction containing a renal brush border enzyme (BBE) cleavable linker bearing a prosthetic group. 18 F labeled the HER2-specific sdAb 2Rs15d ([ 18F]AlF-NOTA-PEG4-Tz-TCO-GK-PEG4-2Rs15d; FIG. 1A, and Zhou et al., Bioconjugate Chem. 2018, 29, 12, 4090-4103, which is incorporated by reference in its entirety.) Compared to renal activity levels in controls containing a non-BBE cleavable linker, 18 F]AlF-NOTA-PEG4-Tz-TCO-GK-PEG4-2Rs15d had significantly lower (>15-fold) kidney activity levels but moderate tumor uptake. 18 To investigate whether the fluoronicotinyl moiety results in higher tumor uptake, we performed 18 F]AlF-NOTA to 6-[ 18 The above procedure was modified by substituting [F]fluoronicotinyl (FN) groups.

[0039] (method) Another HER2-specific sdAb, 5F7, was derivatized with TCO-GK-PEG4-NHS and then conjugated to 6-[ 18 F]Fluoronicotinyl-PEG4-methyltetrazine ([ 18 F]2) was combined with 18 5F7 was synthesized from N,N,N-trimethyl-5-((2-(2-(2-(2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamoyl)10uanidin-2-aminium triflate (1) (see Figure 1B). For comparison, 5F7 was synthesized from the effective residualizing agent N-succinimidyl 3-guanidinomethyl-5-[ 125 I] iodobenzoic acid (iso-[ 125I]SGMIB; see Choi et al., Nucl. Med. Biol. 2014, 41, 10, 802-812, which is incorporated herein by reference in its entirety). Radiochemical purity (RCP) was measured by SDS-PAGE and immunoreactive fraction (IRF) by the Lindmo method. HER2 binding affinity and paired label ( 18 F / 125 I) Cellular uptake assays were performed on HER2-expressing SKOV-3 human ovarian cancer cells. Paired label biodistribution was performed in athymic mice bearing SKOV-3 xenografts.

[0040] (result) Intermediate [ 18 [F]2 was synthesized from precursor 1 in 44.8±3.5% yield. 18 F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 was obtained with 76.0% RCY to IEDDAR, and its RCP was >99% by SDS-PAGE. D The IRF and IRF were 5.4±0.7nM and 77.5%, respectively. 18 The uptake of F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 was 2.4±0.2%, 2.3±0.3%, and 2.6±0.1% of the input activity at 1, 2, and 4 h, respectively. 125 For I]SGMIB-5F7, significantly higher values ​​were obtained (25.9±1.5%, 32.6±2.3%, and 40.8±0.8%). In contrast to the in vitro results, 18 SKOV3 xenograft uptake of [F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 (2.6±2.0%ID / g and 3.7±1.4%ID / g at 1 and 3 h, respectively) was significantly higher than that of co-injected iso-[ 125 There was no significant difference from [I]SGMIB-5F7 (2.0±2.2%ID / g and 6.5±2.6%ID / g, respectively, P>0.05).18 F is 7-fold lower in the kidney, so [ 18 The tumor-to-kidney ratios (T:K) of [F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 at 1 and 3 h were 0.6 ± 0.2 and 4.6 ± 2.0, respectively. The ratios were significantly higher than those of the co-injected iso-[ 125 I] was significantly higher (P<0.05) than the ratios in SGMIB-5F7 (0.1±0.1 and 1.3±1.2). 18 The T:K values ​​obtained in this study for [F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 were 18 This was significantly higher than that previously reported for [F]AlF-NOTA-PEG-Tz-TCO-GK-PEG-2Rs15d (0.4 ± 0.1 and 2.1 ± 0.8 at 1 and 3 h, respectively; P < 0.05 at 3 h).

[0041] (Conclusion) The tetrazine moiety is generally a standard 18 Although it is believed to be unstable under F labeling conditions, by decreasing the amount of base, RCYs up to 47% were obtained. sdAb 5F7 modified with a TCO moiety and a brush border enzyme cleavable linker was cleaved via IEDDAR. 18 The IEDDAR was labeled with F in excellent yields. 18 F]2, and 18 F]2 retained its affinity and immunoreactivity for HER2. 18 The F labeling method warrants further investigation for application to the construction of sdAbs and other types of small proteins.

[0042] Example 2: Fluorine-18 labeling of anti-HER2 sdAb with a 6-fluoronicotinyl moiety via inverse electron demand Diels-Alder reaction (IEDDAR) involving a renal brush border enzyme-cleavable linker. (the purpose) HER2-specific sdAb 5F7 was derivatized as follows: 5F7-GGC was first subjected to a Michael addition reaction with maleimide-PEG4-Tz, and the 1:1 conjugate 5F7-GGC-Mal-PEG4-Tz was isolated by SE-HPLC. 18 To perform F labeling, 18 F-labeled TCO-containing reagent was synthesized ([ 18 F]FN-PEG4-GK-TCO). 18 The F-labeled TCO-containing reagents contain a renal brush border enzyme (RBBE) cleavable linker, a PEG linker, and a 6-[ 18 The compound also contained a [F] fluronicotinyl moiety. A similar reagent (precursor) was also synthesized, which had trimethylammonium triflate in place of F. 18 F-labeled substance [ 18 [F]FN-PEG4-GK-TCO was obtained from the precursor with a radiochemical yield (RCY) of 47.8±9.4% (n=10). This material was conjugated to 5F7-GGC-Mal-PEG4-Tz with a yield of 27.3±8.2% (n=5). 18 The overall decay-corrected yield of the synthesis of [F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC (Figure 2A) was 7-8%, and the labeled nanobody retained its affinity for HER2. 18 F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC, and iso-[ 125 Uptake values ​​in tumor, kidney, blood, and muscle obtained from paired label biodistribution of [I]SGMIB-5F7 are shown in Figure 2B. 18 F vs. 125 Substantially high tumor / kidney and tumor / blood ratios were obtained for I. MIP images in mice bearing BT474M1 xenografts are shown in FIG. 2C. As hypothesized, there was very little uptake in the hepatobiliary organs, with very high contrast uptake essentially only in the tumor and bladder at 3 h pi.

[0043] Example 3: N-Succinimidyl 3-(1-(2-(2-(2-(2-[ 18 F]Fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid, another residual prosthetic group for fluorine-18 labeling of single domain antibody fragments. (the purpose) Single domain antibody fragments (sdAbs) are attractive vectors for immunoPET. Previously, we have demonstrated that the residualizing prosthetic agent N-succinimidyl 3-((4-(4-[ 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoic acid ([ 18 F]SFBTMGMB, or [ 18 F]RL-I; Vaidyanathan et al., J. Nucl. Med., 2018, 115, 171306 and Zhou et al., Mol. Imag. Biol., 2018, 19, 867-877, which are incorporated herein by reference in their entireties), 18 The anti-HER2 sdAb was labeled with F. The prosthetic material was 6-[ 18 The compounds were synthesized by copper-catalyzed click reaction between azide-bearing and guanidine-bearing molecules using [F]fluorohexyne (FH). However, one drawback of FH is its extreme instability, making the synthetic procedure difficult. To overcome this problem, we developed a similar agent by reversing the click partner. The guanidine-bearing molecule contains an alkyne moiety, which is clicked by a fluoroalkyl azide containing PEG linker.

[0044] (method) N-Succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate (6; FIG. 3) was synthesized in three steps from 2-(trimethylsilyl)ethyl 3-(hydroxymethyl)-5-iodobenzoate (3; see also Choi et al., Nucl. Med. Biol. 2014, 41, 10, 802-812, which is incorporated by reference in its entirety). N-Succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate was synthesized from 1-azido-2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethane (see Michel et al., J. Med. Chem. 2011, 54, 4, 939-948, which is incorporated by reference in its entirety). The Boc group was removed from the resulting intermediate 7 to give N-succinimidyl 3-(1-(2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid (8; 18 F]SFETGMB;[ 18 F]RL-III; see Figure 3). The anti-HER2 sdAb 2Rs15d was 18 F]RL-III and N-Succinimidyl 4-guanidinomethyl-3-[ 125 Iodobenzoic acid ([ 125 I]SGMIB; see Vaidyanathan and Zalutsky, Nat. Protocols, 2007, 2, 282-286, which is incorporated herein by reference in its entirety. 18 F and 125 I was labeled with each of them. 18 The purity of [F]RL-III-2Rs15d was assessed by TCA precipitation, SDS-PAGE, and size-exclusion HPLC. 18The HER2 binding affinity of [F]RL-III-2Rs15d was determined by saturation binding assay using HER2-expressing BT474M1 human breast cancer cells, and 18 The immunoreactive fraction (IRF) of [F]RL-III-2Rs15d was evaluated by the Lindmo method. 18 F]RL-III-2Rs15d and [ 125 I] Internalization of the counterlabeled SGMIB-2Rs15d was performed in BT474M1 cells in vitro. 18 F]RL-III-2Rs15d, and [ 125 The biodistribution of [I]SGMIB-2Rs15d was compared in athymic mice bearing subcutaneous HER2-expressing SKOV3 human ovarian cancer xenografts.

[0045] (result) Boc2-[ 18 F]SFETGMB was synthesized in 125 min with a total radiochemical yield of 22.1 ± 2.4% (n = 5). 18 F]RL-III was conjugated to 2Rs15d (2 mg / mL) in a yield of 37.5 ± 13.5%. 18 The radiochemical purity of [F]RL-III-2Rs15d was >96%. d The IRF and IRF were 5.7±0.3nM and 81.5±1.0%, respectively. 18 The percentage of initially bound radioactivity from [F]RL-III-2Rs15d was 10.8±1.0%, 10.6±0.4%, and 9.8±0.7% at 1, 2, and 4 hours, respectively. 125 The corresponding values ​​for I]SGMIB-2Rs15d were 10.2±0.6%, 10.0±0.4%, and 9.1±0.4%. 18 The uptake of [F]RL-III-2Rs15d into SKOV3 xenografts was 4.0±0.5%, 4.2±1.4%, and 2.5±0.3%ID / g at 1, 2, and 3 hours, respectively. 125These values ​​for I]SGMIB-2Rs15d were 5.5±0.8%, 6.4±3.1%, and 3.8±0.7%ID / g (P<0.05, except at 2 h). Uptake in several normal tissues was 125 I]SGMIB-2Rs15d (kidney 2-4 times; liver 25-43 times; spleen 14-19 times) compared with 18 F]RL-III-2Rs15d was significantly higher.

[0046] moreover,[ 18 We evaluated this new residual prosthetic agent RL-III by labeling two nanobodies (5F7 and 2Rs15d) with [F]RL-III. We further demonstrated the efficacy of [F]RL-III in imaging in intracranial tumor-bearing mice. 18 As shown in Figure 4, intracranial BT474M1 tumors showed a potent antitumor effect of [F]RL-III-5F7. 18 F]RL-III-5F7 was clearly visualized (Fig. 4A). Histology and autoradiography of brain sections confirmed the presence and location of the tumor (Fig. 4B and C).

[0047] (Conclusion) It is a prosthetic agent. 18 F]RL-III, 18 F]RL-I was synthesized in a radiochemical yield approximately three times higher than that previously obtained. 18 F]RL-I in a yield similar to that obtained for 18 F]RL-III was used to label sdAb 2Rs15d. 18 F]RL-III-2Rs15d showed a significant advantage in terms of RCY. 18 F]RL-III-2Rs15d uptake into tumors in vitro and in vivo was investigated using co-incubated / co-injected [ 125 I] SGMIB-2Rs15d, which is similar to 18 F]RL-III. 18The uptake of [F]RL-III-2Rs15d into normal tissues was previously reported [ 18 These results were similar to those observed for [F]RL-I-2Rs15d. 18 F]RL-III is 18 These results suggest that this is a better prosthetic agent than [F]RL-I and warrants further work with structural modifications to reduce uptake of activity from the labeled sdAb into some normal tissues. The use of prosthetically labeled sdAbs in the context of intracranial tumors has been shown to be 18 F]RL-III-sdAb is a good imaging agent.

[0048] Example 4: Tetrafluorophenyl 3-[ 18 F]Fluoro-5-guanidinomethylbenzoic acid ([ 18 Preparation of [F]TFPFGMB) (the purpose) The inventors have developed a method similar to SGMIB. 18 We set out to prepare a F-labeled residual agent (Figure 5A). In particular, we developed a novel fluorophore that is similar to iso-SGMIB and contains a tetrafluorophenyl (TFP) ester instead of an N-hydroxysuccinimide (NHS) ester with acceptable RCY ([ 18 F]TFPFGMB; Figure 5B), a residual agent of interest.

[0049] (method) To this end, a boronate precursor containing a TFP ester in which all nitrogens in the guanidine group are protected by Boc groups (9, shown in FIG. 5B) was synthesized. 18 It was subjected to F fluorodeboronation. 18 F fluorodeboronation involves the addition of [ 18 F]Tetraethylammonium fluoride ([ 18F]TEAF), Cu(Py)4(OTf)2. The product (intermediate 2 in Figure 5B) was isolated by normal-phase HPLC with a RCY of 18.0 ± 7.0% (n = 4). The resulting labeled intermediate 10 was deprotected by treatment with TFA. The 5F7 mutant was conjugated to compound 11 (as shown in Figure 5B) in a yield of 4.7 ± 0.2% (n = 2) by incubating a solution of the nanobody mutant of 5F7 ("5F7 mutant") in borate buffer (pH 8.5) with "3" for 20 min at 37 °C.

[0050] (result) In a single experiment, HER2-expressing BT474M1 cells were incubated at 37°C [ 18 After 1, 2, and 4 h of incubation with the [F]TFPFGMB-5F7 mutant, uptake of 30.2±1.7%, 40.1±1.5%, and 45.5±1.6% of the input amount was shown, respectively. Nonspecific binding measured at 2 h was 6.9±0.3%. The activity trapped intracellularly at these three time points was 13.8±0.3%, 17.1±1.0%, and 24.0±1.4%, respectively.

[0051] All publications, patents, and patent applications mentioned in this specification are indicative of the level of one of ordinary skill in the art to which this invention pertains. All publications, patents, and patent applications are incorporated herein by reference to the same extent as if each publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. Although the present invention has been described in some detail by way of illustration and example for the purposes of clarity, it is not intended to be limited thereto. It will be apparent that some changes and modifications can be made within the scope of the present embodiments. [Brief description of the drawings]

[0052] [Figure 1A] The structure of [18F]AlF-NOTA-PEG4-Tz-TCO-GK-2Rs15d. [Figure 1B]1 is an exemplary reaction scheme for the synthesis of [18F]FN-PEG4-Tz-TCO-GK-PEG4-5F7. [Figure 2A] The structure of [18F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC. [Figure 2B] Plot of uptake of iso-[125I]SGMIB-5F7 (black) and [18F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC (black / white stripes) in tumor, kidney, blood, and muscle from paired-label biodistribution in athymic mice bearing BT474M1 xenografts. [Figure 2C] A series of maximum intensity projection (MIP) images obtained after administration of [18F]FN-PEG4-GK-TCO-Tz-PEG4-Mal-5F7GGC in mice bearing BT474M1 xenografts. [Diagram 3] 1 is an exemplary reaction scheme for the synthesis of [18F]RL-III-2Rs15d. [Figure 4A] MicroPET / CT images obtained 2 hours after administration of [18F]RL-III-5F7 in a mouse bearing a BT474M1BrM3-Fluc intracranial xenograft. [Figure 4B] FIG. 2B is a cross-section of the mouse brain from FIG. 2A stained with H&E. [Figure 4C] Autoradiographic images of adjacent sections of mice (image sizes in Figures 4B and 4C are not to scale). [Figure 5A] This is the structure of SGMIB. [Figure 5B] 1 is an exemplary reaction scheme for the synthesis of [18F]TFPFGMB.

Claims

1. A step of preparing the following (1) or (2); (1) Functionalized biomolecules containing dienophiles and dienes １８ F-containing reagents (2) Functionalized biomolecules containing dienes and dienophiles １８ F-containing reagents A functionalized biomolecule containing the dienophile and a functionalized biomolecule containing the diene １８ and an F-containing reagent by inverse electron demand Diels-Alder cycloaddition reaction, １８ Providing an F-labeled biomolecule; or The diene-containing functionalized biomolecule and the dienophile １８ and an F-containing reagent by inverse electron demand Diels-Alder cycloaddition reaction, １８ providing an F-labeled biomolecule, the above １８ The F-labeled biomolecule is １８ F]FN-PEG ４ -GK-TCO-Tz-PEG ４ -Mal- is a biomolecule, the biomolecule is a nanobody, The nanobody is a HER2 specific nanobody. １８ Methods for preparing F-labeled biomolecules.

2. 2. The method of claim 1, wherein the dienophile comprises one or more moieties selected from the group consisting of an octene moiety, a trans-cyclooctene (TCO) moiety, and a tetrazine (Tz) moiety.

3. The method of claim 1 , wherein the functionalized biomolecule further comprises a linker.

4. The method of claim 3 , wherein the linker comprises a renal brush border enzyme cleavable linker.

5. The functionalized biomolecule is -Mal-PEG ４ The method of claim 1, comprising a biomolecule derivatized with -Tz.

6. the above １８ The F-containing reagent is 6-[ １８ F] Fluoronicotinyl-PEG ４ The method of claim 1, comprising -GK-TCO.

7. The method of claim 3 , wherein the linker comprises PEG.

8. [ １８ F]FN-PEG ４ -GK-TCO-Tz-PEG ４ -Mal- １８ a biomolecule bound to a F-labeled residual agent; the biomolecule is a nanobody, The nanobody is a HER2 specific nanobody. １８ F-labeled biomolecules.

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