18 F-radiolabeled biomolecule

Abstract

This application relates to <supgt;18< / supgt;F radiolabeled residues and biomolecules, and a method for radiolabeling biomolecules with radioactive fluorine atoms. The biomolecule has an affinity for a specific type of cell and can specifically bind to certain cells, such as cancer cells. Related biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. This application also provides a composition comprising such a labeled biomolecule, and a method for using the labeled biomolecule and / or composition in imaging applications.

Description

Technical Field

[0001] The present invention relates to methods for preparing compounds that can be used for radiolabeling biomolecules and methods for preparing such radiolabeled biomolecules. The present disclosure also provides precursors of radiolabeled biomolecules and the corresponding radiolabeled biomolecules. These compounds can effectively retain the radioactivity from intracellularly internalized biomolecules, making such compounds useful for diagnosing diseases, especially cancer. Background Art

[0002] Many monoclonal antibodies (mAbs), mAb fragments, and peptides have been labeled with different radionuclides and then used for the detection and treatment of cancer. Many of the most clinically relevant molecular targets, such as HER2, epidermal growth factor receptor (EGFR), and the tumor-specific mutant EGFRvIII, are rapidly internalized into tumor cells. From a labeling perspective, this is a major problem because when radiolabeled biomolecules bind to tumor-associated receptors or antigens, they are transported into the cells and taken up by endosomes / lysosomes, where they are rapidly degraded. The difficulty is that these radioactive degradation products can then rapidly escape from the tumor cells. As a result, there is no longer sufficient radioactivity within the tumor cells to allow for tumor imaging or treatment.

[0003] For example, consider an mAb that reacts with EGFRvIII labeled with radioactive iodine, where the standard labeling method used is direct electrophilic substitution. In such a case, due to extensive internalization, the radioactivity remaining in the tumor is very low after receptor binding and subsequent proteolytic degradation. This is due to the rapid washout of the major catabolite, iodotyrosine. To circumvent this problem, "residualizing agents" have been developed, which attempt to trap the radioactivity within the tumor cells after the labeled mAb has been internalized. Such residualizing agents for radioactive iodine include N-(4-guanidinomethyl-3-iodobenzoyl)-Lys-N-maleimidyl-Gly-Geeek, where e and k represent residues of D-glutamic acid and D-lysine, respectively, also known as N ε -(3-iodobenzoyl)-Lys 5 -N α -maleimidyl-Gly 1 -Geeek, where e and k represent residues of D-glutamic acid and D-lysine, respectively, also known as N 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 IB-MalGeeek; and 2,2',2''-(10-(2-((6-(3-(((N-succinimidyloxy)carbonyl)-5-iodobenzamido)hexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (SIB-DOTA). Compared to directly labeled biomolecules, a substantial enhancement in the retention of radioactivity in tumors has been seen when the same biomolecules are labeled with one of these residual auxiliaries.

[0004] Positron emission tomography (PET), as a state-of-the-art imaging technique, is very sensitive and has excellent quantitative capabilities. The most widely used positron-emitting radionuclide worldwide is fluorine 18, which has a half-life of 110 min. To combine the advantages of this imaging technique with the targeting properties of internalized molecules, it is necessary to develop residual agents with which these biomolecules can be labeled with fluorine 18. In addition, additional efficient methods for preparing such labeled biomolecules are needed. Summary of the Invention

[0005] The present invention relates to methods, compounds, and compositions for radiolabeling biomolecules (also referred to as macromolecules) with radioactive halogen atoms, particularly with 18 F. Advantageously, after in vivo administration, such methods, compounds, and compositions minimize the loss of radioactive halogen 18 F due to in vivo dehalogenation, maintain the biological activity of the biomolecule, maximize retention in diseased cells such as cancer cells, and minimize radioactive retention in normal tissues. Biomolecules have an affinity for specific types of cells. That is, biomolecules can specifically bind to specific cells, such as cancer cells. The compositions of the present invention include radiolabeled biomolecules. Such biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. Such examples of biomolecules for the purposes of the present invention include bispecific antibodies, 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, larger molecules, such as proteins >50 kDa, including antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and F(ab’) 2Fragment. In addition, nanoparticles having a size less than 50 nm can be used in the methods disclosed herein. In some embodiments, the principles disclosed herein are particularly relevant to VHH molecules and other types of small protein constructs, as will be described more thoroughly herein.

[0006] The methods of the present invention utilize a prosthetic compound that is effective for radiolabeling. Accordingly, the present disclosure provides such radiolabeled compounds and precursors that provide such prosthetic compounds. The present disclosure further provides radiolabeled macromolecules (e.g., biomolecules) comprising such compounds / free radicals and one or more macromolecules. In some such embodiments, these radiolabeled macromolecules are targeted radiotherapy agents. The prosthetic compounds and radiolabeled compounds of the present invention can be used, for example, to diagnose diseases.

[0007] In one aspect, the present disclosure provides a method for preparing 18 an 18F-labeled biomolecule, comprising: providing a functionalized biomolecule comprising a dienophile; providing an 18F-containing reagent comprising a diene; and reacting the functionalized biomolecule with the 18 18F-containing reagent by an inverse electron demand Diels-Alder cycloaddition reaction to provide the 18 18F-labeled biomolecule. The reagent and the resulting 18 18F-labeled biomolecule can vary. In one specific non-limiting embodiment of the above method, the 18 18F-containing reagent comprises 6- 18 18F]fluoronicotinoyl-PEG 4 -methyltetrazine. In one specific non-limiting embodiment, the functionalized biomolecule comprises a biomolecule derivatized with TCO-GK-PEG 4 -NHS. In one specific embodiment, the foregoing method can be used to provide an 18 18F-labeled biomolecule of the following formula: 18 18F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -biomolecule, such as including but not limited to 18 18F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7.

[0008] In another aspect of the present disclosure, a method for preparing 18 a 18F-labeled biomolecule is provided, comprising: providing a functionalized biomolecule comprising a diene; providing an 18 18F-containing reagent comprising a dienophile; reacting the functionalized biomolecule with the 18The reagent of F reacts through an inverse electron demand Diels - Alder cycloaddition reaction to provide the 18 F - labeled biomolecule. Similarly, the reagents and the resulting 18 F - labeled biomolecules associated with such methods can vary. In one particular non - limiting embodiment of the method just mentioned above, the reagent containing 18 F includes 6 - 18 F] fluoronicotinoyl - PEG 4 -GK - TCO. In one particular non - limiting embodiment, the functionalized biomolecule includes a biomolecule derivatized with –Mal - PEG 4 -Tz. In one particular embodiment, the foregoing method can be used to provide an 18 F - labeled biomolecule of the following formula: 18 F]FN - PEG 4 -GK - TCO - Tz - PEG 4 -Mal - biomolecule, such as including but not limited to 18 F]FN - PEG 4 -GK - TCO - Tz - PEG 4 -Mal5F7GGC.

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

[0010] The present disclosure further provides certain 18 F - labeled biomolecules, including biomolecules conjugated with a 18 F]FN - PEG 4 -Tz - TCO - GK - PEG 4 - and 18 F]FN - PEG 4 -GK - TCO - Tz - PEG 4 -Mal - 18 F - labeled residue conjugate, and certain non - limiting examples of such labeled biomolecules have the following formula:18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 and 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC.

[0011] In another aspect of the present disclosure, a method for preparing 18 F-labeled residual agent is provided, including: providing a first compound including a guanidine moiety and an alkyne moiety; providing a second compound including a fluoroalkyl azide and a PEG linker; reacting the first compound and the second compound by click chemistry to obtain 18 F-labeled residual agent. In some embodiments, the click chemistry is catalyzed by, for example, a copper catalyst. The reagents of this method can vary. In a specific non-limiting embodiment, the first compound is N-succinimidyl 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and the second compound is 1-azido-2-(2-(2-(2- 18 F]fluoroethoxy)ethoxy)ethoxy)ethane. The present disclosure further provides a method for preparing 18 F-labeled biomolecule, including: performing the method just mentioned above, and reacting the labeled moiety with the biomolecule. The present disclosure additionally provides specific 18 F-labeled residual agents, for example, including but not limited to 3-(1-(2-(2-(2-(2- 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid N-succinimidyl ester and corresponding 18 F-labeled biomolecules, including biomolecules conjugated with such 18 F-labeled residual agents.

[0012] In yet another aspect, the present disclosure provides a method for preparing 18 F-labeled residual agent, the method including: providing a borate ester precursor including a guanidine moiety; and reacting the borate ester precursor with a 18 F-containing reagent by 18 F-fluorodeboronation. The reagent can vary. In some embodiments, the borate ester precursor further includes a TFP ester. In some embodiments, the 18 F-containing reagent is 18 F]tetraethylammonium fluoride. In some embodiments, the reaction step can be carried out in the presence of a catalyst, for example, including but not limited to a copper catalyst. A method for preparing 18Method for labeling biomolecules with 18 F-labeled residual agent; and reacting the 18 F-labeled residual agent with a biomolecule. The present disclosure further provides a 18 F-labeled residual agent, which comprises 3- 18 F]fluoro-5-guanidinomethylbenzoic acid tetrafluorophenyl ester, and a 18 F-labeled biomolecule, including a biomolecule conjugated with such a reagent.

[0013] In addition, the present disclosure provides a method for imaging cancer cells, comprising using any one of the 18 F-labeled biomolecules described herein.

[0014] Suitably labeled and incorporated into the 18 F-labeled biomolecules provided herein, the biomolecules can vary widely. In various embodiments, the biomolecules labeled by the above method are nanobodies. Exemplary nanobodies include but are not limited to HER2-specific nanobodies. Specific HER-2 specific nanobodies include but are not limited to 5F7, 5F7GCC, 2Rs15d and their variants. Description of the Drawings

[0015] To provide an understanding of the embodiments of the present invention, reference is made to the accompanying drawings, which are not necessarily drawn to scale and in which reference numerals refer to the components of the exemplary embodiments of the present invention. The drawings are merely exemplary and should not be construed as limiting the present invention.

[0016] Figure 1A is the 18 F]AlF-NOTA-PEG 4 -Tz-TCO-GK-2Rs15d structure;

[0017] Figure 1B is an exemplary reaction scheme for synthesizing 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7;

[0018] Figure 2A is the 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC structure;

[0019] Figure 2Bare the hetero-in tumor, kidney, blood and muscle obtained from paired-label biodistribution of athymic mice with BT474M1 xenografts- 125 I]SGMIB-5F7 (black) and 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC (black / white stripes) uptake graphs;

[0020] Figure 2C are obtained by administering 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC in mice with BT474M1 xenografts: a series of maximum intensity projection (MIP) images:

[0021] Figure 3 is an exemplary reaction scheme for the synthesis of 18 F]RL-III-2Rs15d;

[0022] Figure 4A are MicroPET / CT images obtained 2 hours after administering 18 F]RL-III-5F7 in mice with BT474M1BrM3-Fluc intracranial xenografts;

[0023] Figure 4B are Figure 2A brain sections of the mice in, stained with H&E;

[0024] Figure 4C are autoradiography images of adjacent sections of the mice (where the images in Figure 4B and Figure 4C are not of the same size);

[0025] Figure 5A is the structure of SGMIB; and

[0026] Figure 5B is an exemplary reaction scheme for the synthesis of 18 F]TFPFGMB. DETAILED DESCRIPTION

[0027] The present disclosure generally provides certain 18 F-labeling methods for biomolecules, and certain precursors and products provided thereby. The disclosed methods mainly focus on certain 18Preparation of F-labeled residues and certain exemplary labeled biomolecules; however, such methods have broader applicability in certain cases, e.g., the preparation of other labeled biomolecules 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 entirety. The present disclosure further provides certain 18 F-labeled residues and certain 18 F-labeled biomolecules, and compositions comprising the same, and methods of using such labeled biomolecules and / or compositions for imaging purposes.

[0028] Certain abbreviations are used throughout this application and are used in a manner recognized in the art; for convenience, their definitions are provided below.

[0029] 2Rs15d: A nanobody, as 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.

[0030] 5F7: A nanobody, as described, for example, in M. Pruszynski et al., Nuclear Medicine and Biology 40(2013)52–59;

[0031] 5F7-GCC: A 5F7 nanobody variant containing cysteine at its C-terminus, as described, for example, in M. Pruszynski et al. / Nuclear Medicine and Biology 40(2013)52–59.

[0032] Boc: tert-Butoxycarbonyl protecting group

[0033] DMA: Dimethylacetamide

[0034] FN: Fluoronicotinoyl

[0035] GK: Glycine lysine

[0036] HER-2: Human epidermal growth factor receptor 2 protein

[0037] HPLC: High performance liquid chromatography

[0038] IEDDAR: Inverse electron demand Diels-Alder cycloaddition reaction

[0039] iso- 131 I]SGMIB: 3-guanidinomethyl-5- 131 I]N-succinimidyl 3-iodobenzoate

[0040] Mal: Maleimidyl

[0041] PEG: Poly(ethylene glycol)

[0042] RBBE: Renal brush border enzyme

[0043] RCY: Radiochemical yield

[0044] sdAbs: Single domain antibody fragments

[0045] [*I]SGMIB: N-succinimidyl 4-guanidinomethyl-3-iodobenzoate

[0046] TCO: A moiety containing trans-cyclooctene

[0047] TFA: Trifluoroacetic acid

[0048] TFP: Tetrafluorophenyl

[0049] Tz: A moiety containing a tetrazine (including but not limited to tetrazine, 3-methyl-6-phenyl-1,2,4,5-tetrazine, and additional derivatives).

[0050] VHH: The variable domain of a heavy chain only antibody (also known as sdAb, nanobody).

[0051] According to one embodiment of the present disclosure, there is provided a 18 18F-labeling method that can be particularly useful for labeling sdAbs and other types of small protein constructs. Such a method involves an inverse electron demand Diels-Alder cycloaddition reaction (IEDDAR). Such a method involves the following steps: a) providing a reagent containing 18 18F; and b) using IEDDAR to couple the reagent containing 18 18F with a functionalized biomolecule (such as an sdAb or other small protein construct) to provide a 18 18F-labeled biomolecule.

[0052] The disclosed method can provide non-site-specific labeling or site-specific labeling. Advantageously, such a method can allow for high product yields as well as retention of affinity and / or immunoreactivity. Generally, in addition to the 18 18F moiety, the reagent containing 18The reagent of F also includes a moiety suitable for IEDDAR (i.e., a diene or dienophile functional group). Similarly, in addition to the biomolecule, the functionalized biomolecule includes a complementary moiety suitable for IEDDAR such that when 18 the moiety of F includes a diene, the functionalized biomolecule includes a dienophile, and when 18 the moiety of F includes a dienophile, the functionalized biomolecule includes a diene. In some embodiments, the selection and / or preparation of such reagents can provide site-specific or non-site-specific labeling of biomolecules.

[0053] In one embodiment, in addition to the label ( 18 F), the reagent containing 18 F (which is provided and then coupled to the functionalized biomolecule) also includes a diene suitable for IEDDAR for step b) above. An exemplary diene is a tetrazine-containing moiety, which can advantageously be included in the reagent containing 18 F. Advantageously, in some such embodiments, the reagent containing 18 F includes a fluoronicotinyl moiety, wherein the fluoronicotinyl moiety includes 18 F. Various other functional groups can be present within the reagent containing 18 F, as long as such other functional groups do not negatively interfere with the desired IEDDAR. For example, the reagent containing 18 F can further include linkers of different lengths (e.g., PEG). A specific exemplary reagent containing 18 F (diene) that can be effectively used in the disclosed methods is 6- 18 F] fluoronicotinyl-PEG 4 -methyltetrazine.

[0054] When the reagent containing 18When the reagent of F includes a diene applicable to IEDDAR in step b), the functionalized biomolecule used in this method includes a dienophile. An exemplary dienophile applicable to IEDDAR disclosed herein is an octene moiety (e.g., within the TCO functional group). Such "functionalized biomolecules" can generally include various sdAbs or other small protein constructs. In a specific embodiment, the biomolecule includes an anti-HER2 sdAb. An exemplary biomolecule that has been effectively demonstrated for this method is 5F7; however, the method is not limited thereto. In some such embodiments, the functionalized biomolecule can 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. Similarly, in some embodiments, various other functional groups can be included within the functionalized biomolecule, as long as such other functional groups do not negatively interfere with the desired IEDDAR. In a specific embodiment useful for the disclosed method, the functionalized biomolecule includes TCO-GK-PEG 4 -NHS linker. The reaction between the functionalized biomolecule (through its dienophile) and the reagent containing 18 F (through its diene) can provide the desired labeled biomolecule that competes for IEDDAR.

[0055] In other embodiments, the moiety associated with the functionalized biomolecule and the reagent containing 18 F is converted (e.g., such that the diene associates with the functionalized biomolecule (instead of the reagent containing 18 F as described above) and the dienophile associates with the reagent containing 18 F (instead of the functionalized biomolecule as described above). Advantageously, in some embodiments, the reagent containing 18 F includes a fluoronicotinyl moiety, where the fluoronicotinyl moiety includes 18 F. In such embodiments, in addition to the label ( 18 F), the reagent containing 18 F (which is provided and then coupled to the functionalized biomolecule) also includes a dienophile applicable to IEDDAR in step b) above. An exemplary dienophile applicable to IEDDAR disclosed herein is an octene moiety (e.g., within the TCO functional group). Various other functional groups can be present within the reagent containing 18 F, as long as such other functional groups do not negatively interfere with the desired IEDDAR. For example, the reagent containing 18 F can further include linkers of different lengths, where the linker includes, for example, PEG and / or a renal brush border enzyme (RBBE) cleavable linker. A specific exemplary reagent containing 18The F(dienophile) reagent is 6- 18 F] fluoronicotinoyl-PEG 4 -GK-TCO.

[0056] When the reagent containing 18 F includes a dienophile suitable for IEDDAR in step b), the functionalized biomolecule used in the method includes a diene. An exemplary diene is a tetrazine-containing moiety, which can advantageously be included within the functionalized biomolecule. Such "functionalized biomolecules" can generally also include various sdAbs or other small protein constructs. In a particular embodiment, the biomolecule includes an anti-HER2 sdAb. An exemplary biomolecule for which the method has been effectively demonstrated is 5F7-GGC; however, the method is not limited thereto. Various other functional groups can be present within the functionalized biomolecule, provided that such other functional groups do not negatively interfere with the desired IEDDAR. For example, the functionalized biomolecule can further include linkers of different lengths (e.g., PEG). In a further embodiment, the reagent containing 18 F includes an RBBE-cleavable linker. A particular exemplary functionalized biomolecule (diene) reagent that can be effectively used in the disclosed method is 5F7-GGC-Mal-PEG 4 -Tz. The reaction between the functionalized biomolecule (through its diene) and the reagent containing 18 F (through its dienophile) can provide the desired labeled biomolecule for IEDDAR.

[0057] In one embodiment, the reference method provides 18 F-labeled biomolecules (e.g., prepared according to one of the methods referenced above). Two exemplary such 18 F-labeled biomolecules are shown below as Formulas I and II.

[0058]

[0059] Formula I: 18 F]FN-PEG 4 -Tz-TCOGK-PEG 4 -biomolecule

[0060]

[0061] Formula II: 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC

[0062] In another embodiment, a method for preparing18 Method for radiolabeling a residual agent and 18 a biomolecule with 18 F, the method comprising a click reaction (e.g., a copper-catalyzed click reaction). The disclosed reaction comprises the steps of: a) providing a compound having a guanidine and comprising an alkyne moiety; b) providing a compound comprising a fluoroalkyl azide and comprising a PEG linker; c) subjecting the compounds of steps a) and b) to click chemistry; and optionally, in order to form a radiolabeled biomolecule, 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 a higher radiochemical yield than similar reactions previously reported in which the azide moiety is present on the compound having a guanidine and the compound reacts with an alkyne (6- 18 F] fluorohex-1-yne). See Glaser, Bioconjugate Chem. 2007, 18, 989-993, which is incorporated herein by reference in its entirety.

[0063] The compound comprising a fluoroalkyl azide and comprising 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 it reacts (i.e., the compound having a guanidine) can vary. An exemplary such compound is 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoic acid N-succinimidyl ester).

[0064] The 18 F-labeled residual agent provided by the above steps a)-c) can be reacted with various biomolecules to obtain 18 F-labeled biomolecules. The biomolecules employed in the method can generally include various sdAbs or other small protein constructs (e.g., the types of biomolecules outlined above). In one particular embodiment, the biomolecule comprises an anti-HER2 sdAb. Two exemplary biomolecules for which the method has been effectively demonstrated are 2Rs15d and 5F7; however, the method is not limited thereto. The method can provide a simpler synthetic operation than previous click chemistry methods for preparing such compounds and can provide a higher overall radiochemical yield in some embodiments. The present disclosure further provides 18 F-labeled biomolecules and intermediates (including 18 F-labeled residual agents) provided by such reactions. An exemplary 18 F-labeled biomolecule is shown in Formula III below.

[0065]

[0066] Formula III: 18 F]RL-III-2Rs15d

[0067] The present disclosure provides additional methods for producing 18 F-labeled residual agents and 18 F-labeled biomolecules, which employ fluorodeboronation. In certain embodiments, the method involves providing a boronic acid ester precursor comprising a TFP ester and a guanidine moiety, wherein the nitrogen atom on the guanidine moiety is protected (e.g., with a Boc group or other suitable protecting group, which can be introduced / removed under conditions that do not negatively impact the desired reaction). By treating with a suitable 18 F-containing reagent (e.g., 18 F]tetraethylammonium fluoride 18 F]TEAF), the boronic acid ester precursor undergoes 18 F-fluorodeboronation. This fluorodeboronation is advantageously catalyzed by, for example, a copper reagent (including but not limited to Cu(Py) 4 (OTf) 2 ). The resulting compound can then be treated to deprotect the nitrogen atom on the guanidine moiety (e.g., when the protecting group is Boc, these groups can be removed by treatment with TFA).

[0068] The deprotected 18 F-labeled residual agent can then be conjugated to a biomolecule, which can include any of the biomolecules described above. In one particular non-limiting embodiment, the biomolecule is a nanobody, e.g., a variant such as 5F7 or 5F7. The present disclosure further provides 18 F-labeled biomolecules and intermediates provided by such reactions. An exemplary such 18 F-labeled biomolecule is shown in Formula IV below.

[0069]

[0070] Formula IV: 18 F]TFPFGMB-5F7

[0071] The present disclosure further provides a composition comprising a radiolabeled biomolecule as disclosed herein (e.g., those described / illustrated above) in combination with a pharmaceutically acceptable adjuvant, diluent, or carrier 18F-labeled biomolecules, e.g., those including Formulas I, II, III, and / or IV). In another aspect of the present disclosure, provided is a method of diagnosing cancer, comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule as disclosed herein and / or an effective amount of a pharmaceutical composition as disclosed herein.

[0072] The following examples are provided by way of illustration and not by way of limitation.

[0073] Example 1: Fluorine-18 labeling of anti-HER2 sdAb with 6-fluoronicotinoyl moiety: via inverse electron demand Diels-Alder reaction (IEDDAR) involving a renal brush border enzyme-cleavable linker

[0074] Objective:

[0075] Single domain antibody fragments (sdAb) are now considered useful platforms for labeling with short-lived positron emitters such as 18 F, which can result in rapid tumor uptake and rapid whole body clearance. However, high renal activity levels from labeled sdAb are a major problem. Previously, we labeled HER2-specific sdAb, 2Rs15d, with 18 F]AlF-NOTA moiety via 18 F]AlF-NOTA-PEG 18 -Tz-TCO-GK-PEG 4 -2Rs15d; see 4 -2Rs15d; see Figure 1A and Zhou et al., Bioconjugate Chem. 2018, 29, 12, 4090-4103, which is incorporated herein by reference in its entirety). Although 18 F]AlF-NOTA-PEG 4 -Tz-TCO-GK-PEG 4 -2Rs15d achieved significantly (>15-fold) lower renal activity levels compared to a control containing a non-BBE-cleavable linker, 18 F]AlF-NOTA residue was not very high. To investigate whether the fluoronicotinoyl moiety leads to higher tumor uptake, we modified the above method by replacing 18 F]fluoronicotinoyl (FN) group. 18 F]AlF-NOTA with

[0076] Method:

[0077] Another HER2-specific sdAb, 5F7, was derivatized with TCO-GK-PEG 4 -NHS and then conjugated with 6- 18 F]fluoronicotinoyl-PEG 4 -methyltetrazine ( 18 F]2) via IEDAR coupling as Figure 1B shown ( 18 F]2 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)ethoxy)ethyl)carbamoyl)guanidinium-2-trifluoromethanesulfonate (1) (see Figure 1B ). For comparison, the validated residual agent N-succinimidyl 3-guanidinomethyl-5- 125 I]iodobenzoate (iso- 125 I]SGMIB; see Choi et al., Nucl. Med. Biol. 2014, 41, 10, 802-812, which is incorporated herein by reference in its entirety) was also used to label 5F7. Radiochemical purity (RCP) was determined by SDS-PAGE and immunoreactive fraction (IRF) by the Lindmo method. HER2 binding affinity and paired labeling ( 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.

[0078] Results:

[0079] Intermediate 18 F]2 was synthesized from precursor 1 with a yield of 44.8 ± 3.5%. For IEDDAR, 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 was obtained in 76.0% RCY with an RCP of >99% by SDS-PAGE; K D and IRF were 5.4 ± 0.7 nM and 77.5%, respectively. 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 uptake in SKOV-3 cells in vitro at 1, 2, and 4 h was 2.4 ± 0.2%, 2.3 ± 0.3%, and 2.6 ± 0.1% of the input activity, respectively. Co-cultured iso- 125I]SGMIB-5F7 achieved significantly higher values (25.9 ± 1.5%, 32.6 ± 2.3%, and 40.8 ± 0.8%). Different from the in vitro results, 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 uptake in SKOV3 xenografts (2.6 ± 2.0% ID / g and 3.7 ± 1.4% ID / g at 1 h and 3 h) was not significantly different from that of co-injected iso- 125 I]SGMIB-5F7 {2.0 ± 2.2% ID / g and 6.5 ± 2.6% ID / g, respectively, (P > 0.05)}. Because the 18 F level in the kidney is one-seventh of that, 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 tumor-to-kidney ratio (T:K) at 1 h and 3 h was 0.6 ± 0.2 and 4.6 ± 2.0, respectively, which was significantly higher (P < 0.05) than those observed for co-injected iso- 125 I]SGMIB-5F7 (0.1 ± 0.1 and 1.3 ± 1.2). Despite the different sdAbs, in this study for 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7, the obtained T:K values were much higher than those previously reported for 18 F]AlF-NOTA-PEG 4 -Tz-TCO-GK-PEG 4 -2Rs15d (0.4 ± 0.1 and 2.1 ± 0.8 at 1 h and 3 h, respectively; P < 0.05 at 3 h).

[0080] Conclusion:

[0081] Although the tetrazine moiety is generally considered unstable for standard 18 F-labeling conditions of SNAr, we obtained up to 47% RCY by reducing the amount of base. The sdAb 5F7 modified with a TCO moiety and a brush border enzyme-cleavable linker was labeled with 18 F]2 by IEDDAR using 18 F, with high yield while retaining the affinity and immunoreactivity to HER2. This 18 F-labeling method requires further investigation for application to sdAbs and other types of small protein constructs.

[0082] Example 2:Fluorine-18 Labeling of Anti-HER2 sdAb with 6-Fluoro Nicotinoyl Moiety: Via Inverse Electron Demand Diels-Alder Reaction (IEDDAR) Involving a Renal Brush Border Enzyme Cleavable Linker

[0083] Objective:

[0084] The HER2-specific sdAb, 5F7 was derivatized as follows. First, Michael addition of 5F7-GGC with maleimide-PEG 4 -Tz was performed and the 1:1 conjugate of 5F7-GGC-Mal-PEG 4 -Tz was separated by SE-HPLC. For 18 F labeling using IEDDAR, a reagent containing 18 F-labeled TCO was synthesized, which also contained a renal brush border enzyme (RBBE) cleavable linker, PEG 4 linker and 6- 18 F]fluoro nicotinoyl moiety ( 18 F]FN-PEG 4 -GK-TCO). A similar reagent (precursor) with ammonium trimethyltrifluoromethanesulfonate in place of F was also synthesized. 18 The 18 F-labeled agent 4 F]FN-PEG 4 -GK-TCO was obtained from the precursor with a radiochemical yield (RCY) of 47.8 ± 9.4% (n = 10). It conjugated with 5F7-GGC-Mal-PEG 4 -Tz with a yield of 27.3 ± 8.2% (n = 5). The overall decay-corrected yield of 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC ( Figure 2A ) was 7 - 8% and the labeled nanobody retained its affinity for HER2. The uptake values in tumor, kidney, blood and muscle of the paired-labeled biodistribution of 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-5F7GGC and 125 I]SGMIB-5F7 are shown as Figure 2B . Significantly higher tumor / kidney and tumor / blood ratios of 18 F compared to 125 I were obtained. The MIP images of mice with BT474M1 xenografts are shown as Figure 2CAs shown, as hypothesized, very little uptake was observed in the hepatobiliary organs at 3 h after intraperitoneal injection, and very high contrast images with uptake were observed essentially only in the tumor and bladder.

[0085] Example 3: With 3-(1-(2-(2-(2-(2- 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid N-succinimidyl ester, an alternative residual coagent for fluorine-18 labeling of single-domain antibody fragments

[0086] Objective:

[0087] Single-domain antibody fragments (sdAbs) are attractive vectors for immunoPET. Earlier, we used the residual coagent 3-((4-(4- 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoic acid N-succinimidyl ester ( 18 F]SFBTMGMB or 18 F]RL-I to label anti-HER2 sdAbs; Vaidyanathan et al., J. Nucl. Med., 2018, 115, 171306, which is incorporated herein by reference in its entirety; and Zhou et al., Mol. Imag. Biol., 2018, 19, 867 - 877, which is incorporated herein by reference in its entirety). The coagent was synthesized by a copper-catalyzed click reaction between a molecule with azide and guanidine and 6- 18 F]fluorohex-1-yne (FH). However, one drawback of FH is its extreme volatility, making the synthesis operation difficult. To overcome this problem, we developed a similar agent by reverse click ligand—a molecule with guanidine containing an alkyne moiety that was clicked with a fluoroalkyl azide including a PEG linker. 18 F]RL-I was used to label anti-HER2 sdAbs; Vaidyanathan et al., J. Nucl. Med., 2018, 115, 171306, which is incorporated herein by reference in its entirety; and Zhou et al., Mol. Imag. Biol., 2018, 19, 867 - 877, which is incorporated herein by reference in its entirety). The coagent was synthesized by a copper-catalyzed click reaction between a molecule with azide and guanidine and 6-

[0088] Method:

[0089] 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoic acid N-succinimidyl ester (6; Figure 3 ) was synthesized in three steps from 3-(hydroxymethyl)-5-iodobenzoic acid 2-(trimethylsilyl)ethyl ester (3; also see Choi et al., Nucl. Med. Biol. 2014, 41, 10, 802 - 812, which is incorporated herein by reference in its entirety). It was reacted with 1-azido-2-(2-(2-(2- 18(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethane (see Michel et al., J. Med. Chem. 2011, 54, 4, 939 - 948, which is incorporated herein by reference in its entirety) was clicked and the Boc group of the resulting intermediate 7 was removed to obtain 3-(1-(2-(2-(2-(2- 18 (2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoic acid N-succinimidyl ester (8; 18 (SFETGMB; 18 (RL-III; see 3). The anti-HER2 sdAb 2Rs15d was labeled with 18 F and 125 I as follows: It was conjugated with 18 (RL-III and 4-guanidinomethyl-3- 125 (Iodo)benzoic acid N-succinimidyl ester ( 125 (SGMIB; see Vaidyanathan and Zalutsky, Nat. Protocols, 2007, 2, 282 - 286, which is incorporated herein by reference in its entirety), respectively. 18 (The purity of RL-III-2Rs15d was evaluated by TCA precipitation, SDS PAGE, and size exclusion HPLC. Its HER2 binding affinity was determined in a saturation binding assay using HER2-expressing BT474M1 human breast cancer cells, and its immunoreactive fraction (IRF) was evaluated by the Lindmo method. The 18 (paired labeling internalization of RL-III-2Rs15d and 125 (SGMIB-2Rs15d was performed on BT474M1 cells in vitro. The 18 (bio-distribution of RL-III-2Rs15d and 125 (SGMIB-2Rs15d in athymic mice bearing subcutaneous HER2-expressing SKOV3 human ovarian cancer xenografts was compared.

[0090] Results:

[0091] Boc 2 - 18 (SFETGMB was synthesized with a total radiochemical yield of 22.1 ± 2.4% (n = 5) in 125 min. 18 (RL-III was conjugated with 2Rs15d (2 mg / mL) with a yield of 37.5 ± 13.5%. 18 (The radiochemical purity of RL-III-2Rs15d > 96%. Kd And IRF were 5.7 ± 0.3 nM and 81.5 ± 1.0%, respectively. The initial binding radioactive percentages of 18 F]RL-III-2Rs15d internalized in BT474M1 cells were 10.8 ± 1.0%, 10.6 ± 0.4%, and 9.8 ± 0.7% at 1, 2, and 4 h, respectively; 125 the corresponding values of 18 I]SGMIB-2Rs15d were 10.2 ± 0.6%, 10.0 ± 0.4%, and 9.1 ± 0.4%. 125 The uptake of 125 I]SGMIB-2Rs15d in SKOV3 xenografts were 5.5 ± 0.8% ID / g, 6.4 ± 3.1% ID / g, and 3.8 ± 0.7% ID / g at 1, 2, and 3 h, respectively (P < 0.05 except at 2 h). Compared with 18 I]SGMIB-2Rs15d,

[0092] the uptake of 18 F]RL-III-2Rs15d in some normal tissues was much higher (2 - 4 times in the kidney; 25 - 43 times in the liver; 14 - 19 times in the spleen). 18 F]RL-III-5F7 in mice with intracranial tumors. As shown in Figure 4, the intracranial BT474M1 tumors were clearly visualized with 18 F]RL-III-5F7 ( Figure 4A ). Histology and autoradiography of brain sections confirmed the presence and location of the tumors ( Figure 4B and C).

[0093] Conclusion:

[0094] The co - adjuvant 18 F]RL-III was synthesized with a radiochemical yield approximately 3 - fold higher than that previously obtained for 18 F]RL-I. sdAb2Rs15d was labeled with 18 F]RL-III with a similar yield as that obtained for 18 F]RL-I. Regarding the RCY, 18 F]RL-III-2Rs15d gave a considerable advantage. 18In vitro and in vivo tumor uptake of FRL-III-2Rs15d with co-incubation / injection of 125 was similar to that of ISGMIB-2Rs15d, demonstrating 18 the residual ability of FRL-III. 18 Normal tissue uptake of FRL-III-2Rs15d was similar to that previously seen for 18 FRL-I-2Rs15d, although in a different model. These results suggest that 18 FRL-III is a better co-agent than 18 FRL-I and requires further investigation with further structural modifications to reduce the active uptake of the labeled sdAb in some normal tissues. Use of this co-agent-labeled sdAb in the context of intracranial tumors demonstrated 18 that FRL-III-sdAb is a good imaging agent.

[0095] Example 4: Preparation of 3- 18 F-fluoro-5-guanidinomethylbenzoic acid tetrafluorophenyl ester ( 18 F]TFPFGMB).

[0096] Objective:

[0097] We set out to prepare an F-labeled residual agent ( 18 ) similar to SGMIB. In particular, we targeted a reagent similar to iso-SGMIB but containing a tetrafluorophenyl (TFP) ester instead of an N-hydroxysuccinimide (NHS) ester (( Figure 5A ) with an acceptable PCY. 18 F]TFPFGMB; Figure 5B )

[0098] Method:

[0099] To this end, a boronic acid ester precursor (9, as Figure 5B shown) containing a TFP ester was synthesized, in which all nitrogens in the guanidine group were protected by Boc groups and subjected to 18 F-fluorodeboronation: treated with 18 F]tetraethylammonium fluoride ( 18 F]TEAF), Cu(Py) 4 (OTf) 2 in DMA at ~100 °C for 5 - 10 min. The product ( Figure 5BThe intermediate 2) was separated by normal-phase HPLC and had an RCY of 18.0 ± 7.0% (n = 4). The resulting labeled intermediate 10 was deprotected by treatment with TFA. The nanobody variant of 5F7 (“5F7-variant”) was conjugated to compound 11 (as Figure 5B shown) in a yield of 4.7 ± 0.2%; n = 2) by incubating a solution of 5F7 in borate buffer pH 8.5 with 3 at 37 °C for 20 min.

[0100] Results:

[0101] Individual experiments showed that uptake of the input dose was 30.2 ± 1.7%, 40.1 ± 1.5%, and 45.5 ± 1.6% at 1, 2, and 4 h, respectively, after incubation of HER2-expressing BT474M1 cells with 18 F]TFPFGMB-5F7-variant at 37 °C. Nonspecific binding, determined at 2 h, was 6.9 ± 0.3%. The activities captured intracellularly at these three time points were 13.8 ± 0.3%, 17.1 ± 1.0%, and 24.0 ± 1.4%, respectively.

[0102] All publications, patents, and patent applications mentioned in the specification represent the level of those skilled in the art to which the present invention pertains. All publications, patents, and patent applications are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be made within the scope of the embodiments.

Claims

1. A method, comprising: providing one of the following: 1) A functionalized biomolecule comprising a dienophile and a reagent comprising a diene containing 18 F, or 2) A functionalized biomolecule comprising a diene and a reagent comprising a dienophile containing 18 F, or 3) a first compound comprising a guanidine moiety and an alkyne moiety and a second compound comprising a fluoroalkyl azide and a PEG linker; Reacting the functionalized biomolecule comprising the dienophile with the F-containing reagent comprising the diene by inverse electron demand Diels-Alder cycloaddition reaction to provide the 18 F-labeled biomolecule, or 18 F-labeled biomolecule, or Reacting the diene-containing functionalized biomolecule with the F-containing reagent comprising a dienophile by inverse electron demand Diels-Alder cycloaddition reaction to provide the 18 F-labeled biomolecule, or 18 F-labeled biomolecule, or React the first compound comprising a guanidine moiety and an alkyne moiety with the second compound comprising a fluoroalkyl azide and a PEG linker by click chemistry to provide the 18 F-labeled residue agent, Wherein the reagent containing 18 F includes 18 F] nicotinoyl group.

2. The method according to claim 1, wherein, The reagent containing 18 F includes 6- 18 F] fluoronicotinoyl-PEG 4 -methyltetrazine.

3. The method according to claim 1, wherein, the dienophile comprises one or more of an octene moiety, a trans-cyclooctene (TCO) moiety, and a tetrazine (Tz) moiety.

4. The method according to claim 1, wherein, the functionalized biomolecule further comprises a linker.

5. The method according to claim 4, wherein, the linker comprises a renal brush border enzyme-cleavable linker.

6. The method according to claim 1, wherein, The functionalized biomolecule includes a biomolecule derivatized with TCO-GK-PEG 4 -NHS or a biomolecule derivatized with –Mal-PEG 4 -Tz.

7. The method according to claim 1, wherein, the biomolecule is a nanobody, wherein the nanobody is a HER2-specific nanobody.

8. The method according to claim 1, wherein, The 18 biomolecule labeled with F is 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -biomolecule or 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-biomolecule, where FN is fluoronicotinoyl, Tz is a tetrazine-containing moiety, TCO is a trans-cyclooctene-containing moiety, GK is glycine lysine, and Mal is a maleimidyl group.

9. The method according to claim 1, wherein, The reagent containing 18 F includes 6- 18 F] fluoronicotinoyl-PEG 4 -GK-TCO.

10. The method according to claim 4, wherein, the linker comprises PEG.

11. The method according to claim 1, wherein, the click chemistry is catalyzed by a copper catalyst.

12. The method according to claim 1, wherein, The first compound is N-succinimidyl 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and the second compound is 1-azido-2-(2-(2-(2- 18 F]fluoroethoxy)ethoxy)ethoxy)ethane.

13. A 18 biological molecule labeled with F, comprising a biological molecule conjugated to a residual agent labeled with F selected from the following: 18 F]FN-PEG 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 - and 18 F]FN-PEG 4 -GK-TCO-Tz-PEG 4 -Mal-;​ where FN is fluoronicotinoyl, Tz is a tetrazine-containing moiety, TCO is a trans-cyclooctene-containing moiety, GK is glycine lysine, and Mal is a maleimidyl group.

14. The 18 biomolecule labeled with F, wherein, the biomolecule is a nanobody.

15. The 18 biomolecule labeled with F wherein, the nanobody is a HER2-specific nanobody.

16. Use according to any one of claims 13-15 of 18 a biomolecule labeled with F in the preparation of a reagent for imaging cancer cells.

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