Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts.sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates.[18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97–100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95–99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16–20 %ID/g over 1–3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7.Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the 18F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([18F]SFBTMGMB-Boc2) was synthesized by a click reaction of an azide precursor and [18F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with 18F using [18F]SFBTMGMB ([18F]RL-I), obtained by the deprotection of [18F]SFBTMGMB-Boc2, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62–80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with 18F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

The selection of an appropriate labeling method for a protein or peptide requires careful consideration of the fate of the molecule after its interaction with the biological milieu. For radio-iodinated proteins and peptides, circumventing the action of the deiodinases normally involved in thyroid hormone metabolism is an important general concern. This led to the development of reagents such as N-succinimidyl 3-[*I]iodobenzoate (SIB), which yield proteins that do not undergo appreciable deiodination in vivo based on their Tyr-dissimilar structure1, 2. However, when a labeled protein or peptide undergoes cellular internalization after binding to a cell surface receptor or antigen, then, depending on its intercellular routing, considerable loss of label from the targeted cell can occur3, 4, 5. Although this discussion is focused on mAbs for simplicity of presentation, this problem can affect other proteins and peptides also. For example, loss of label after cellular internalization of conventionally radio-iodinated epidermal growth factor receptor (EGFR) was first demonstrated 30 years ago by Carpenter and Cohen6.The internalization process can subject mAbs to extensive catabolism, primarily reflecting the action of proteases present in the lysosomal compartment of the targeted cells. Labeling of mAbs with radiometals is generally easy, and for mAbs labeled with radiometals such as 90Y and 177Lu, low-molecular-weight labeled catabolites egress from a tumor to only a small degree7. Compared with radio-iodinated mAbs, this results in higher accumulation of radioactivity in the tumor; however, high renal uptake and low tumor/normal tissue ratios are often drawbacks. When mAbs that undergo internalization are labeled with radio-iodine by direct electrophilic substitution on their Tyr residues, in general, the radioactivity is rapidly lost from the tumor cells, primarily in the form of monoiodotyrosine8. No significant advantage with respect to tumor retention of radioactivity was seen even when such mAbs were labeled with SIB, confirming that a process other than deiodination was responsible for loss of label after mAb internalization9.Newer radio-iodination methods are needed that will result in higher retention of radioactivity in tumor cells after the internalization of labeled mAbs. A number of such 'residualizing labels' have been developed. With these methods, the portion of the molecule bearing the label is inert to lysosomal degradation and becomes trapped inside the cell after mAb proteolysis. The first set of reagents for this purpose (e.g., tyramine-cellobiose (TCB) and inulin-tyramine) contained a non-metabolizable carbohydrate moiety in their structure10, 11, 12, 13, 14. Use of these methods to label internalizing mAbs generally resulted in improved tumor retention of radioactivity compared with their directly iodinated counterparts. However, there are a number of disadvantages associated with this labeling approach, including low protein-labeling yields, low immunoreactivity of the labeled mAbs, protein cross-linking and (as a result of the latter) higher liver uptake10, 14, 15, 16. A more successful method for radio-iodination of internalizing mAbs involves the use of oligopeptides composed of D-amino acids3, 17. The unnatural D-amino acids are not substrates for proteases; peptides consisting of more than three D-amino acids are expected to be trapped within the cells because passive transport of peptides of that size is poor. A variation of this tactic that has been described previously is to attach very hydrophilic molecules such as DTPA (diethylenetriamine pentaacetic) to the D-amino acid peptide4, 18. This is based on the rationale that when mAbs are labeled with radiometals using chelating agents such as DTPA, the radioactivity is retained in the cells in the form of radiometal-DTPA-Lys.Our laboratory has been investigating an alternative strategy for labeling internalizing mAbs that is based on the hypothesis that exocytosis of labeled catabolites across cells and lysosomal membranes can be minimized if they are charged species. As the pH of the lysosomal compartment is approximately 5, species containing basic functions are expected to remain positively charged within the lysosomes. To test the feasibility of this approach, studies were performed using N-succinimidyl 5-[*I]iodopyridine-3-carboxylate ([*I]SIPC), a reagent containing a positively charged pyridine ring, to label an internalizing mAb, L8A4, reactive with EGFRvIII, a mutant form of the EGFR9. Up to 65% higher intracellular retention of radioactivity was observed in vitro for L8A4 labeled using [*I]SIPC compared with L8A4 mAb labeled directly or using [*I]SIB; however, this advantage was transient. On the other hand, excellent tumor/tissue ratios were seen with mAb labeled using [*I]SIPC compared with other methods, primarily because of lower normal organ radio-iodine levels.The reagent described here, N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB), is an extension of the cationic labeled catabolite concept. Guanidine has a pKa of approximately 13 and is therefore expected to remain exclusively in the protonated form within the lysosome. We have exploited this property to develop a conjugation labeling agent useful in the radio-iodination of internalizing mAbs19. [*I]SGMIB was derived simply by attaching a guanidinomethyl function to SIB (Fig. 1). An unlabeled standard of SGMIB (4), its Boc-protected derivative (Boc-SGMIB, 2) and the tin precursor N-succinimidyl 4-[N 1, N 2-bis(tert-butyloxycarbonyl)guanidinomethyl]-3-(trimethylstannyl)benzoate (Boc-SGMTB, 3) were obtained from 3-iodo-4-methylbenzoic acid (1) in multiple steps19. An SGMIB analog labeled with an α-particle-emitting heavy halogen, 211At, N-succinimidyl 3-[211At]astato-guanidinomethylbenzoate ([211At]SAGMB), could be synthesized from the same tin precursor 3 (ref. 20). A considerably higher advantage (twofold to fourfold) in the delivery and retention of radioactivity in tumor was obtained both in vitro and in vivo when internalizing anti-EGFRvIII mAb L8A4 was radiolabeled with [*I]SGMIB or [211At]SAGMB compared with the same mAb radio-iodinated using other methods19, 20, 21. Proteins are conjugated with labeled SGMIB by incubating [*I]SGMIB with a solution of the protein in pH 8.5 borate buffer for 15–20 min at room temperature (20 °C). The labeled protein is purified using gravity gel filtration chromatography. Typical yields for the conjugation are 60–65% for protein concentrations of 3 mg ml−1 or higher.With this protocol, it may not be feasible to synthesize [*I]SGMIB at radioactivity levels that are needed for clinical radio-immunotherapy. To accomplish this, the procedure will need to be modified so that it can be performed using a remote-controlled automated synthesizer.Step 1: 10 min; Step 2: 5 min (if no evaporation required)Steps 3–5: 5 min; Step 6: 32 min; Steps 7 and 8: 45–50 minStep 9: 10 min, Step 10: 17 min; Step 11: 10 minTroubleshooting advice can be found in Table 1.

Several neuroendocrine tumors are known to express both the somatostatin receptor subtype 2 (SSTR2) and the norepinephrine transporter (NET), and radiopharmaceuticals directed toward both these targets such as MIBG and octreotide derivatives are routinely used in the clinic. To investigate the possibility of targeting both NET and SSTR2 conjointly, a conjugate of radioiodinated MIBG and octreotate was synthesized. Attempts to synthesize the radioiodinated target compound (MIBG-octreotate; [131I]12a) from a tin precursor were futile; however, it could be accomplished from a bromo precursor by exchange radioiodination in 3–36% (n = 10) radiochemical yields. The total uptake of [131I]12a in SK-N-SH human neuroblastoma cells transfected to express SSTR2 (SK-N-SHsst2) was similar to that for [125I]MIBG at all time points (34.9 ± 2.4% vs 43.8 ± 1.2% at 4 h; p < 0.05), while it was substantially lower (5.4 ± 0.3% vs 35.9 ± 1.2%) in the SH-SY5Y cell line, a subclone of SK-N-SH line that is known to express SSTR2. The NET blocker desipramine reduced the uptake of [131I]12a only to a small extent, further suggesting a limited role of NET in its binding and accumulation. Uptake of [131I]12a in SK-N-SHsst2 cells was 8–10-fold higher (p < 0.05) than that of [125I]I-Gluc-TOCA, an octreotide analogue, at all time points over a 4 h period and was reduced to about 20% by 10 μM octreotide demonstrating that the uptake of [131I]12a in this cell line is predominantly mediated by SSTR2. The intracellularly trapped radioactivity in SK-N-SHsst2 cells was substantially higher for [131I]12a compared to that for [125I]OIBG-octreotate, an isomeric congener of 12a. Because MIBG has more specific NET-mediated uptake than OIBG, this suggests at least a partial role for NET-mediated uptake of [131I]12a in this cell line. While further refinement in the structure of the conjugate—probably interposition of a flexible and/or cleavable linker between the MIBG and octreotate moieties—may be necessary to make it a substrate/ligand for both NET and SSTR2, this conjugate is demonstrated to be much superior than I-Gluc-TOCA with respect to the uptake in SSTR2-expressing cells.

meta-[211At]Astatobenzylguanidine ([211At]MABG), an analogue of meta-iodobenzylguanidine (MIBG) labeled with the α-emitter 211At, targets the norepinephrine transporter. Because MABG has been shown to have excellent characteristics in preclinical studies, it has been considered to be a promising targeted radiotherapeutic for the treatment of tumors such as micrometastatic neuroblastoma that overexpress the norepinephrine transporter. To facilitate clinical evaluation of this agent, a convenient method for the high level synthesis of [211At]MABG that is adaptable for kit formulation has been developed. A tin precursor anchored to a solid-support was treated with a methanolic solution of 211At in the presence of a mixture of H2O2/HOAc as the oxidant; [211At]MABG was isolated by simple solid-phase extraction. By using C-18 solid-phase extraction, the radiochemical yield from 25 batches was 63 ± 13%; however, loss of radioactivity during evaporation of the methanolic solution was a problem. This difficulty was avoided by use of a cation exchange resin cartridge for isolation of [211At]MABG, which resulted in radiochemical yields of 63 ± 9% in a shorter duration of synthesis. The radiochemical purity was more than 90% and no chemical impurity has been detected. The final doses were sterile and apyrogenic. These results demonstrate that [211At]MABG can be prepared via a kit method at radioactivity levels anticipated for initiation of clinical studies.

IntroductionN-succinimidyl 4-guanidinomethyl-3-[⁎I]iodobenzoate ([⁎I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [131I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[131I]iodobenzoate (iso-[131I]SGMIB) wherein this bulky group was moved from ortho to meta position.MethodsBoc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors — trastuzumab (Tras) and a nanobody 5 F7 (Nb) — were labeled using iso-[⁎I]SGMIB and [⁎I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.ResultsWhen the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[131I]SGMIB were significantly higher than those for Boc2-[131I]SGMIB (70.7 ± 2.0% vs 56.5 ± 5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[131I]SGMIB than with [131I]SGMIB (Nb, 33.1 ± 7.1% vs 28.9 ± 13.0%; Tras, 45.1 ± 4.5% vs 34.8 ± 10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5 F7 Nb indicated similar residualizing capacity over 6 h; however, at 24 h, radioactivity retained intracellularly for iso-[131I]SGMIB-Nb was lower than for [125I]SGMIB-Nb (46.4 ± 1.3% vs 56.5 ± 2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[125I]SGMIB and [131I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [131I]SGMIB-Tras.ConclusionGiven the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.

IntroductionRadioiodinated meta-iodobenzylguanidine (MIBG), a norepinephrine transporter (NET) substrate, has been extensively used as an imaging agent to study the pathophysiology of the heart and for the diagnosis and treatment of neuroendocrine tumors. The goal of this study was to develop an 18F-labeled analogue of MIBG that like MIBG itself could be synthesized in a single radiochemical step. Towards this end, we designed 4-fluoropropoxy-3-iodobenzylguanidine (FPOIBG).MethodsStandards of FPOIBG and 4-fluoropropoxy-3-bromobenzylguanidine (FPOBBG) as well as their tosylate precursors for labeling with 18F, and a tin precursor for the preparation of radioiodinated FPOIBG were synthesized. Radiolabeled derivatives were synthesized by nucleophilic substitution and electrophilic iododestannylation from the corresponding precursors. Labeled compounds were evaluated for NET transporter recognition in in vitro assays using three NET-expressing cell lines and in biodistribution experiments in normal mice, with all studies performed in a paired-label format. Competitive inhibition of [125I]MIBG uptake by unlabeled benzylguanidine compounds was performed in UVW-NAT cell line to determine IC50 values.Results[18F]FPOIBG was synthesized from the corresponding tosylate precursor in 5.2 ± 0.5% (n = 6) overall radiochemical yields starting with aqueous fluoride in about 105 min. In a paired-label in vitro assay, the uptake of [18F]FPOIBG at 2 h was 10.2 ± 1.5%, 39.6 ± 13.4%, and 13.3 ± 2.5%, in NET-expressing SK-N-SH, UVW-NAT, and SK-N-BE(2c) cells, respectively, while these values for [125I]MIBG were 57.3 ± 8.1%, 82.7 ± 8.9%, and 66.3 ± 3.6%. The specificity of uptake of both tracers was demonstrated by blocking with desipramine. The 125I-labeled congener of FPOIBG gave similar results. On the other hand, [18F]FPOBBG, a compound recently reported in the literature, demonstrated much higher uptake, albeit less than that of co-incubated [125I]MIBG. IC50 values for FPOIBG were higher than those obtained for MIBG and FPOBBG. Unlike the case with [18F]FPOBBG, the heart uptake [18F]FPOIBG in normal mice was significantly lower than that of MIBG.ConclusionAlthough [18F]FPOIBG does not appear to warrant further consideration as an 18F-labeled MIBG analogue, analogues wherein the iodine in it is replaced with a chlorine, fluorine or hydrogen might be worth pursuing.Advances in knowledge and implications for patient careAn 18F-labeled analogue of the well-known radiopharmaceutical MIBG could have significant impact, potentially improving imaging of NET related disease in cardiology and in the imaging of neuroendocrine tumors. Although 18F-labeled analogues of MIBG have been reported including LMI1195, we undertook this work hypothesizing that based on its greater structural similarity to MIBG, FPOIBG might be a better analogue than LMI1195.

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the 18F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([18F]SFBTMGMB-Boc2) was synthesized by a click reaction of an azide precursor and [18F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with 18F using [18F]SFBTMGMB ([18F]RL-I), obtained by the deprotection of [18F]SFBTMGMB-Boc2, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62–80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with 18F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.