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. 2026 Jun 8;11:56. doi: 10.1186/s41181-026-00460-0

Development of NOTA-based antibody-fragment labeling agent that reduces early renal radioactivity levels

Shunsuke Katamoto 1, Hiroyuki Suzuki 1, Madoka Kobayashi 1, Shota Kise 1, Kento Kannaka 1, Nobuki Kazuta 1, Tomoya Uehara 1,✉
PMCID: PMC13469042  PMID: 42258063

Abstract

Background

Gallium-68 (68Ga) and copper-64 (64Cu) are radiometals used for nuclear medicine imaging and therapy. The compound 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms stable complexes with these radiometals. However, 68Ga/64Cu-labeled-low molecular weight proteins show high radioactivity levels in the kidneys. To address this problem, a NOTA-conjugated Met-Val-Lys (MVK)-maleimide (Mal) (NOTA-MVK-Mal), was recently designed to reduce renal radioactivity levels. The agent achieved this by releasing radiometal-NOTA-methionine as a radiometabolite via the action of renal brush border enzymes. As 68Ga and 64Cu have short half-lives, it is crucial to reduce renal radioactivity from the early post-injection-period. Based on this molecular strategy, this study designed and synthesized a new NOTA-based radiometal-labeling agent, designated NOTA-conjugated Phe-Gly-Lys (FGK)-Mal (NOTA-FGK-Mal), which reduced renal radioactivity in the earlier post-injection period.

Results

Considering that the FGK sequence is a substrate of enzymes on the renal brush border, isothiocyanatobenzyl-NOTA was conjugated to an antibody Fab fragment through a FGK linkage to produce [67Ga]Ga-NOTA-FGK-Fab. Enzymatic recognition of the linkage was evaluated using a low-molecular-weight [67Ga]Ga-NOTA-FGK derivative. The biodistribution of radioactivity after the injection of [67Ga]Ga-NOTA-FGK-Fab into mice was compared with that of [67Ga]Ga-NOTA-MVK-Fab. The FGK linkage was recognized by brush border membrane enzymes to liberate [67Ga]Ga-NOTA-Phe-Gly. This indicated that the recognition of the FGK sequence in the [67Ga]Ga-NOTA-FGK derivative was the GK sequence, not the expected FG sequence. When injected into mice, [67Ga]Ga-NOTA-FGK-Fab resulted in lower renal radioactivity levels than those induced by [67Ga]Ga-NOTA-MVK-Fab without a reduction in tumor radioactivity levels.

Conclusion

These results suggest that NOTA-FGK(Mal) is a useful radiometal labeling agent for low-molecular-weight proteins that reduces renal radioactivity levels from as early as post-injection. This molecular design is not dependent on the type of antibody or its constructs, it is expected that NOTA-FGK(Mal) will also be applicable to a variety of target molecules.

Supplementary Information

The online version contains supplementary material available at 10.1186/s41181-026-00460-0.

Keywords: Radiotheranostics, NOTA, Renal brush border, 68Ga, 64Cu

Introduction

In recent years, various radionuclides have been used for molecular imaging and radiotherapy (Zhang et al. 2025). Among these, gallium-68 (68Ga) is one of the most useful and widely used radionuclides owing to its ability to be eluted from 68Ge/68Ga generators (Tanwar and Pandey 2025). Copper-64 (64Cu) is a radionuclide that has also attracted attention as it emits low-energy β+ rays and is produced by cyclotrons (Krecisz et al. 2025, Wu et al. 2016). The emission of β− rays by 64Cu also holds promise for therapeutic applications. Therefore, the radionuclide pair of 68Ga and 64Cu or 64Cu alone, can be used for radiotheranostics as a combined diagnostic and radiotherapeutic procedure (IAEA 2022, Fournier et al. 2012, Persson et al. 2016).

The half-lives of 68Ga and 64Cu are short (68 min and 13 h, respectively); therefore, the rapid accumulation of radiotracer in the target tissues after administration into the body is desirable. In addition, as radiotracers constantly emit radiation, the rapid biodistribution of radiotracers in the body is effective in mitigating myelotoxicity. Accordingly, low-molecular-weight compounds are preferred carriers of these radioisotopes. In recent years, a wide variety of antibodies targeting various antigens have been developed. Specifically, antibody fragments, such as Fab and Fv, are promising carriers of these radionuclides (Maloney et al. 2020). Furthermore, low-molecular-weight antibody mimics, such as affibodies and minibodies, are also promising carriers of these radionuclides (Wei et al. 2024). In general, these antibody fragments or mimics do not contain radiometal-binding moieties. Therefore, bifunctional chelating agents have been used to produce radiolabeled antibody fragments and mimics. In particular, the compound 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) formed stable complexes with both 68Ga and 64Cu (Wu et al. 2016, Suzuki et al. 2021). Therefore, bifunctional chelating agents based on NOTA, such as p-isothiocyanatobenzyl (SCN-Bn)-NOTA have been employed for the radiolabeling of antibody fragments and mimics (Suzuki et al. 2021, Uehara et al. 2014, Uehara et al. 2018). However, radiometal-labeled antibody fragments and mimics show high radioactivity levels in the kidney, thereby hindering diagnosis around the kidney and causing side effects during radiotherapy (Suzuki et al. 2024, Barta et al. 2023, Vegt et al. 2010).

To circumvent this problem, radionuclide-labeled agents that reduce renal radioactivity level by releasing urinary excreted radiometabolites via enzymes in the renal brush border membrane (BBM; brush border strategy) have been proposed (Suzuki et al. 2021, Uehara et al. 2018, Suzuki et al. 2024, Suzuki et al. 2023, Arano 2021). Among these, a NOTA-based radiometal-labeling agent for low-molecular-weight proteins, designated NOTA-Met-Val-Lys (MVK)-maleimide (Mal) was developed (Fig. 1A) (Suzuki et al. 2021, Uehara et al. 2018). This agent demonstrated low renal radioactivity levels in the kidney due to the release of a urinary radiometabolite (metal-NOTA-Met) by renal brush border enzymes. The efficacy of this reagent has been demonstrated for other low-molecular-weight peptides and in clinical studies (Trachsel et al. 2023, Valpreda et al. 2022, Peng et al. 2025, Zhang et al. 2024). The 67Ga/64Cu-labeled Fabs prepared using this reagent reduced renal radioactivity levels in tumor-bearing mice at 1 h post-injection compared with that in mice treated with 67Ga/64Cu-labeled Fabs prepared by a conventional method. The half-life of 68Ga is 68 min, and when using this radionuclide, it is desirable to further reduce renal radioactivity levels by 1 h post-injection. In this study, a novel NOTA-based radiolabeling agent, known as NOTA-Phe-Gly-Lys (FGK)(Mal), was synthesized using a brush border strategy (Fig. 1B). This reagent was designed to reduce renal radioactivity levels by releasing urinary radiometabolites via cleavage of the FGK sequence by BBM enzymes. In this study, 67Ga was used as a substitute for 68Ga because the study facility was unable to utilize 68Ga.

Fig. 1.

Fig. 1

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Structures of A M-NOTA-MVK-Fab, B M-NOTA-FGK-Fab and C In-DO3AiBu-FGK-Fab. M-NOTA-MVK-Fab and In-DO3AiBu-FGK-Fab release M-NOTA-Met and In-DO3AiBu-Phe as radiometabolites, respectively. M-NOTA-FGK-Fab is designed to release M-NOTA-Phe and/or N-NOTA-Phe-Gly as radiometabolites

Results

Preparation of 67Ga-labeled compounds

A new bifunctional chelating agent with a renal brush border enzyme-cleavable linkage, NOTA-FGK(Mal), was synthesized by reacting SCN-Bn-NOTA with FGK(Mal) in dry dimethylformamide (DMF), followed by reversed-phase high-performance liquid chromatography (RP-HPLC) purification (Scheme S1). In this study, Fab fragments of the anti-EGFR were used. This NOTA derivative was conjugated with thiolated Fab fragments using the standard 2-iminothiolane method (Uehara et al. 2018, Jendryczko et al. 2022). The number of NOTA-derivatives introduced per molecule of Fab fragment were estimated to be 0.7 by calculating the number of thiol groups in the Fab fragment before and after the conjugation reaction. As a control, NOTA-MVK(Mal) was conjugated to Fab fragments using the same method used to produce NOTA-MVK-Fab. Both NOTA-conjugated Fab fragments were labeled with 67Ga in the presence of acetate, and the resulting radiolabeled Fab fragments were analyzed using thin layer chromatography (TLC) and size exclusion high performance liquid chromatography (SE-HPLC). The radiochemical yield and purity of both 67Ga-labeled Fab fragments were > 90% (Fig. S1).

Enzymatic recognition of the cleavable linkages

To assess the recognition of cleavable linkages by renal BBM enzymes, a model benzoyl (Bzo) conjugate, NOTA-FGK(Bzo), was synthesized and labeled with 67Ga. After removing free NOTA-FGK(Bzo) via RP-HPLC, [67Ga]Ga-NOTA-FGK(Bzo) was incubated with BBM vesicles (BBMVs) to estimate the release of radiometabolites (Fig. 2). Figure S2 shows the percentage of radioactivity in the fraction of radiometabolites after incubation of [67Ga]Ga-NOTA-FGK(Bzo) and [67Ga]Ga-NOTA-MVK(Bzo) with BBMVs at 37 ˚C for 2 h, respectively. The rate of radiometabolite ([67Ga]Ga-NOTA-Phe-Gly and [67Ga]Ga-NOTA-Phe) release from [67Ga]Ga-NOTA-FGK(Bzo) was 6.8% and markedly lower than the release of [67Ga]Ga-NOTA-Met release from [67Ga]Ga-NOTA-MVK(Bzo) (14.8%).

Fig. 2.

Fig. 2

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Radiochromatogram of [67Ga]Ga-NOTA-FGK(Bzo) after incubation with BBMVs for 2 h (Top). [67Ga]Ga-NOTA-Phe (middle) and [67Ga]Ga-NOTA-Phe-Gly (bottom) are standard samples

Biodistribution studies in normal and tumor-bearing mice

The biodistribution of radioactivity after the injection of 67Ga-labeled Fab fragments in normal mice is summarized in Fig. 3 and Table S1. When injected into normal mice, 67Ga-labeled Fab fragments showed similar elimination rates of radioactivity from circulation. [67Ga]Ga-NOTA-FGK-Fab showed much lower renal radioactivity levels from 1 h to 6 h than that of [67Ga]Ga-NOTA-MVK-Fab. The radioactivity levels in urine after injection of [67Ga]Ga-NOTA-FGK-Fab tended to be higher than that after [67Ga]Ga-NOTA-MVK-Fab injection (Fig. 3). RP-HPLC analyses of urine samples collected at 6 h post-injection of [67Ga]Ga-NOTA-FGK-Fab showed that the major radiometabolite was [67Ga]Ga-NOTA-Phe-Gly (62%) (Fig. 4).

Fig. 3.

Fig. 3

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Biodistribution of radioactivity after injection of [67Ga]Ga-NOTA-FGK-Fab (●) and [67Ga]Ga-NOTA-MVK-Fab (○) in normal mice. *: Significance was determined by an unpaired Student’s t-test. p < 0.05

Fig. 4.

Fig. 4

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The radiochromatograms of urine sample collected for 6 h after injection of [67Ga]Ga-NOTA-FGK-Fab analyzed by RP-HPLC. The top graph is the urine samples after proteins were removed, and the bottom is the [67Ga]Ga-NOTA-Phe-Gly standard sample

The biodistribution of radioactivity at 1 and 3 h post-injection of the two 67Ga-labeled Fab fragments into nude mice bearing U87MG cell xenografts is summarized in Fig. 5. Detailed results are listed in Table S2. Although no notable differences were observed in tumor uptake 1 h post-injection between the two 67Ga-labeled Fab fragments, [67Ga]Ga-NOTA-FGK-Fab showed substantially lower radioactivity than that of [67Ga]Ga-NOTA-MVK-Fab in the kidney. As a result, the tumor/kidney ratio of [67Ga]Ga-NOTA-FGK-Fab (0.11 ± 0.02) was 1.66-fold higher than that of [67Ga]Ga-NOTA-MVK-Fab (0.07 ± 0.01) at 1 h post-injection. At 3 h post-injection of [67Ga]Ga-NOTA-FGK-Fab, the radioactivity levels in the tumors were maintained at levels comparable with those of [67Ga]Ga-NOTA-MVK-Fab (Table S2).

Fig. 5.

Fig. 5

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Biodistribution of radioactivity after 1 h a or 3 h b injection of [67Ga]Ga-NOTA-FGK-Fab (white bar) and [67Ga]Ga-NOTA-MVK-Fab (back bar) in tumor bearing mice. *: Significance was determined by an unpaired Student’s t-test. p < 0.05

Discussion

The characteristics of the radiometabolites are important for the brush border strategy. Radiometabolites were released not only on the BBMs but also within vesicles, such as coated pits and coated vesicles (Suzuki et al. 2021, Suzuki et al. 2023). Membrane permeability is considered an essential characteristic of radiometabolites. An enzyme-cleavable FGK linkage-conjugated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivative was previously reported as an antibody fragment-labeling agent, known as isobutyl 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3AiBu-Bn)-FGK(Mal) (Fig. 1C) (Suzuki et al. 2023). The indium-111 (111In)-labeled Fab fragments prepared using DO3AiBu-Bn-FGK(Mal) reduced renal radioactivity levels by releasing [111In]In-DO3AiBu-Bn-F as a radiometabolite. Although the precise mechanism underlying the rapid excretion of [111In]In-DO3AiBu-Bn-F remains unclear, the mechanism is thought to involve transporters. In contrast, different chelating moieties can lead to pronounced differences in the biodistribution of radiometal-labeled chelate-conjugated amino acids, even when the same amino acid is bound to a chelate (Uehara et al. 2018, Akizawa et al. 2001). Therefore, in this study, NOTA-FGK(Mal) was designed and synthesized using the FGK sequence as the enzyme-cleavable moiety and NOTA as the chelating moiety, and its usefulness as a 68Ga/64Cu-labeling agent was evaluated for antibody Fab fragments.

To assess the recognition of cleavable linkages by renal BBM enzymes, [67Ga]Ga-NOTA-FGK(Bzo) was incubated with BBM vesicles (BBMVs) to estimate the release of [67Ga]Ga-NOTA-Phe, which was expected to be a radiometabolite. Because 111In-labeled Fab fragments prepared using DO3AiBu-Bn-FGK(Mal) reduced renal radioactivity levels by releasing [111In]In-DO3AiBu-Bn-F as a radiometabolite (Suzuki et al. 2023). However, [67Ga]Ga-NOTA-FGK(Bzo) released little [67Ga]Ga-NOTA-Phe and instead released [67Ga]-NOTA-Phe-Gly (Fig. 2). BBM enzymes cleave both Phe-Gly and Gly-Lys sequences, as indicated by [111In]In-DO3AiBu-Bn-FGK-Fab. The precise reason that more [67Ga]Ga-NOTA-Phe-Gly than [67Ga]Ga-NOTA-Phe was released remains unclear; however, it may be attributable to steric hindrance at the Ga-NOTA moiety, which consequently inhibited the recognition of the Phe-Gly sequence. As indicated in Figure S2, the rate of radiometabolite ([67Ga]Ga-NOTA-Phe-Gly and [67Ga]Ga-NOTA-Phe) release from [67Ga]Ga-NOTA-FGK(Bzo) was markedly lower than the release of [67Ga]Ga-NOTA-Met release from [67Ga]Ga-NOTA-MVK(Bzo). Previous reports have shown no correlation between the amount of radiometabolites produced in in vitro studies using BBMVs and the reduction in renal radioactivity in vivo (Suzuki et al. 2021, Uehara et al. 2007). Therefore, studies using BBMVs are considered to be experiments to confirm whether or not recognition occurs by BBMVs. In this study, [67Ga]Ga-NOTA-FGK(Bzo) releases radiometabolites through the action of BBM enzymes; therefore, [67Ga]Ga-NOTA-FGK(Mal) and 67Ga-labeled Fab fragments were prepared using this agent.

When injected into normal mice, 67Ga-labeled Fab fragments showed similar elimination rates of radioactivity from circulation (Fig. 3 and Table S1). Thus, similar portions of the two radiolabeled Fab fragments were filtered through the glomerulus and transported to the proximal renal tubules after administration. Indeed, renal radioactivity levels at 10 min post-injection were similar (Fig. 3). Generally, 67Ga-labeled Fab prepared using p-SCN-Bn-NOTA without a cleavable linker has been shown to increase renal radioactivity over 3–6 h post-injection(Suzuki et al. 2021, Uehara et al. 2018). However, in this study, both 67Ga-labeled Fabs prepared with cleavable linkers showed reduce renal radioactivity levels within 1 h post-injection. In addition, [67Ga]Ga-NOTA-FGK-Fab showed much lower renal radioactivity levels from 1 h to 6 h than that of [67Ga]Ga-NOTA-MVK-Fab. When using short-half-life radioisotopes, such as 68Ga, reducing the radioactivity level in the kidney from early post-injection time is highly advantageous. The radioactivity levels in urine after injection of [67Ga]Ga-NOTA-FGK-Fab tended to be higher than that after [67Ga]Ga-NOTA-MVK-Fab injection (Fig. 3). These results also supported the high renal clearance of [67Ga]Ga-NOTA-FGK-Fab. RP-HPLC analyses of urine samples collected at 6 h post-injection of [67Ga]Ga-NOTA-FGK-Fab showed that the major radiometabolite was [67Ga]Ga-NOTA-Phe-Gly (62%) (Fig. 4). In a previous study using [67Ga]Ga-NOTA-MVK-Fab, the proportion of [67Ga]Ga-NOTA-Met in the urine was only 50%. These results suggest that [67Ga]Ga-NOTA-FGK-Fab releases the targeted radiometabolite more selectively, thereby leading to an earlier reduction in renal radioactivity levels.

In the studies using tumor bearing, no notable differences were observed in tumor uptake 1 h post-injection between the two 67Ga-labeled Fab fragments, while [67Ga]Ga-NOTA-FGK-Fab showed substantially lower radioactivity than that of [67Ga]Ga-NOTA-MVK-Fab in the kidney (Fig. 5). As a result, the tumor/kidney ratio of [67Ga]Ga-NOTA-FGK-Fab (0.11 ± 0.02) was 1.66-fold higher than that of [67Ga]Ga-NOTA-MVK-Fab (0.07 ± 0.01) at 1 h post-injection (Table S2). At 3 h post-injection of [67Ga]Ga-NOTA-FGK-Fab, the radioactivity levels in the tumors were maintained at levels comparable with those of [67Ga]Ga-NOTA-MVK-Fab (Table S2). Previous studies have showed that the tumor uptake of [67Ga]Ga-NOTA-MVK-Fab was similar to that of 67Ga-labeled Fab prepared using p-SCN-Bn-NOTA without a cleavable linker (Suzuki et al. 2021, Uehara et al. 2018). Furthermore, in a clinical study of affibodies using NOTA-MVK(Mal), radioactivity in the tumor was maintained, whereas renal radioactivity levels decreased (Zhang et al. 2024). These results suggest that a similar effect can be expected with NOTA-FGK(Mal). Hence, this agent is a useful radiolabeling agent for antibody fragments and low-molecular-weight antibody mimics that reduce renal radioactivity levels without compromising tumor radioactivity levels.

Many compounds have employed brush border strategies (Trachsel et al. 2023, Valpreda et al. 2022, Peng et al. 2025, Bendre et al. 2020, Lundmark et al. 2024). However, the efficacy of these compounds in reducing renal radioactivity levels has been inconsistent, with variations observed among the different reagents. These compounds contain a substrate sequence for brush border enzymes; however, few studies have referred to the structure of the radiometabolites generated following enzymatic cleavage. As previously reported, the properties of radiometabolites are very important for brush border strategies (Suzuki et al. 2021, Suzuki et al. 2023, Arano 2021). As shown in this study, even identical substrate sequences, such as FGK, exhibit distinct cleavage sites, specifically, when DOTA and DO3AiBu are conjugated, the FG sequence is cleaved (Suzuki et al. 2023), whereas when NOTA is conjugated, the GK sequence is cleaved. These results suggest that the metabolic sequence introduced into a compound does not necessarily lead to the predicted metabolic progression at the time of design. In this study, [67Ga]Ga-NOTA-Phe-Gly was unexpectedly released as a radiometabolite. Furthermore, [67Ga]Ga-NOTA-Phe-Gly showed higher hydrophilicity than that of [67Ga]Ga-NOTA-Phe, according to the RP-HPLC retention time (Fig. 2). This indicates that [67Ga]Ga-NOTA-Phe-Gly is a highly urinary compound. As a result, the pairing of [67Ga]Ga-NOTA-Phe-Gly as a radiometabolite and the FGK sequence as a cleavable linker may have been useful because the release of [67Ga]Ga-NOTA-Phe-Gly by the action of brush border enzymes reduced renal radioactivity levels early after injection.

Conclusion

This study demonstrated that NOTA-FGK(Mal), which was developed for radiogallium and radiocopper, showed low renal radioactivity levels without impairing tumor radioactivity levels in the early post-injection period. This represents the earliest reduction in renal radioactivity levels observed among agents employing the brush border strategy. Given the applicability of NOTA-MVK(Mal) to diverse target molecules, NOTA-FGK(Mal) is also expected to be applicable to various target molecules.

Methods

General

[67Ga]Ga-citrate was purchased from PDRadiopharma, Inc. (Tokyo, Japan). NOTA-MVK(Bzo), NOTA-MVK(Mal), and the renal BBMVs were prepared as described previously (Uehara et al. 2018, Fujioka et al. 2005). The analytical methods are described in the Supporting Information. All commercial chemicals were of analytical grade and were used without further purification.

Synthesis of Boc-FGK-OH

Boc-FGK-OH was synthesized by manual Fmoc solid-phase peptide synthesis using Cl-Trt(2-Cl)-resin (Watanabe Chemical Industries, Ltd., Hiroshima, Japan) as the solid phase and the protected acids Fmoc-Lys(Dde), Fmoc-Gly-OH, and Boc-Phe-OH. The peptide chain was constructed manually according to a previously published method. The strategy entailed (i) 2 h of coupling with the protected amino acid derivative (2.5 equivalent) in the presence of N,N'-diisopropylcarbodiimide (2.5 equivalent) and 1-hydroxybenzotriazole (2.5 equivalent) in dimethylformamide (DMF, ca. 3 mL), and (ii) 20 min of Fmoc-deprotection with 20% piperidine-DMF. After constructing the peptide sequence, the peptide resin was treated with 2% hydrazine/DMF (approximately 3 mL) for 1 h to cleave the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group of Lys. The peptide resin was then treated with a mixture of acetic acid/2,2,2-trifluoroethanol/dichloromethane (3/1/6; ca. 2 mL) for 2 h to cleave the peptide from the resin. After filtration, the filtrate was treated with diethyl ether (~ 3 mL) to precipitate Boc-FGK-OH as a white solid; electrospray ionization mass spectrometry (ESI-MS) m/z [M + H]+ 451, found 451.

Synthesis of FGK(Mal)-OH

N-(Methoxycarbonyl)maleimide (33 mg, 0.21 mmol) was added to a solution of Boc-FGK-OH (65 mg, 0.14 mmol) in 5 mL of saturated aqueous NaHCO3 at 0 °C with vigorous stirring for 30 min. After 30 min of mixing at room temperature, the pH of the solution was adjusted to 3 using 1 M H2SO4 at the same temperature. The solution was extracted with ethyl acetate (10 mL × 3) and dried over an anhydrous Na2SO4. After removing the solvent in vacuo, Boc-FGK(Mal)-OH was purified using preparative TLC (CHCl3/MeOH = 5/1) to obtain a white solid (41 mg, 55%); ESI-MS m/z [M + H]+ 531, found 531. Boc-FGK(Mal)-OH (41 mg, 77 µmol) was dissolved in 4 N HCl/ethyl acetate (3 mL), and the mixture was stirred at room temperature for 1 h. After removing the solvent in vacuo, the HCl salt of Gly-Lys(Mal)–OH was obtained as a white solid (36 mg, quant.). ESI-MS m/z [M + H]+ 431, found 431.

Synthesis of NOTA-FGK(Mal)-OH (NOTA-FGK(Mal))

The HCl salt of FGK(Mal)-OH (10 mg, 21 µmol) was dissolved in dry DMF (200 µL), and triethylamine (12 µL) was added. Thereafter, SCN-Bn-NOTA (10 mg, 22 µmol) was added to the solution, and the mixture was stirred for 2 h at room temperature. The compound was purified using preparative RP-HPLC to obtain NOTA-FGK(Mal) as a white solid (1.6 mg, 8.7%); ESI-MS m/z [M + H]+ 881, found 881.

Synthesis of FGK(Bzo)

FGK(Bzo) was synthesized using methods similar to those described above, using Fmoc-Phe-OH and Fmoc-Lys(Bzo) instead of Boc-Phe-OH and Fmoc-Lys(Dde)-OH. The FGK(Bzo) was obtained as a white solid (9.7 mg, 69%); ESI-MS m/z [M + H]+ 455, found 455.

Synthesis of NOTA- FGK(Bzo) (NOTA-FGK(Bzo))

The SCN-Bn-NOTA (5 mg, 11 µmol) was dissolved in 0.1 M borate buffer (pH 9.0) adjusted to pH at 8–9 with 0.1 N NaOH aqueous (aq), after which FGK(Bzo) (5.0 mg, 11 µmol) was added to the solution and stirred at room temperature for 2 h. The desired compound was purified using preparative RP-HPLC, and NOTA-FGK(Bzo) was obtained as a white solid (7.7 mg, 77%); ESI-MS m/z [M + H]+ 905, found 905.

Synthesis of NOTA-Phe-Gly and NOTA-Phe

The SCN-Bn-NOTA (5 mg, 11 µmol) was dissolved in 0.1 M borate buffer (pH 9.0) adjusted to pH at 8–9 with 0.1 N NaOH aq, after which each amino acid (Phe-Gly or Phe, 11 µmol) was added to the solution and stirred at room temperature for 2 h. The desired compound was purified using preparative RP-HPLC; ESI-MS m/z NOTA-Phe-Gly: [M + H]+ 673, found 673; NOTA-Phe: [M + H]+ 616, found 616.

Preparation of Ga-complexes

NOTA-conjugated peptide derivatives (3 µmol) were added to a solution of non-radioactive GaCl3 (1.1 mg, 6.5 µmol) in 0.25 M acetate buffer (pH 5.5, 50 µL). After stirring for 1 h, the Ga complex was purified using preparative RP-HPLC to obtain a white solid; ESI-MS m/z Ga-NOTA-FGK(Bzo): [M + H]+ 971, found 971; Ga-NOTA-Phe-Gly: [M + H]+ 739, found 739; and Ga-NOTA-Phe: [M + H]+ 682, found 682.

Preparation of 67Ga-complexes

A 10-µL solution of [67Ga]GaCl3 in 0.05 N HCl was mixed with 0.25 M acetate buffer (pH 5.5, 10 µL). After 5 min, a solution of NOTA-conjugated peptide derivatives (2 × 10− 4 M, 10 µL) in 0.1 M acetate buffer (pH 5.5) was added, and the mixture was incubated at 40 ˚C for 1 h. Radiochemical purity was analyzed via RP-HPLC equipped with an on-line UV/Vis detector and radioactivity detector. Identification of the radiochemical species was verified by the similarity of the RP-HPLC retention times of the reaction solution and its non-radioactive Ga-NOTA counterpart.

Monoclonal antibody and cells

Monoclonal antibody against epidermal growth factor receptor (EGFR; PC-61.53) was purchased from Merk Biopharm (Tokyo, Japan). Fab fragments of the anti-EGFR antibody were prepared using a Fab Preparation Kit (Thermo Fisher Scientific, Yokohama, Japan). U87MG cells were cultivated in Dulbecco’s Modified Eagle Medium (Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Nippon Bio-Supply Center, Tokyo, Japan), 2 mM L-glutamine (Sigma Aldrich Japan, Tokyo, Japan), and 1% penicillin-streptomycin (10,000 units/mL and 10,000 µg/mL, Nacalai Tesque Inc.) in a humidified atmosphere containing 5% CO2 at 37 °C.

Preparation of 67Ga-labeled fab fragments

The 67Ga-labeled Fab fragments were prepared as previously described. A 10-µL [67Ga]GaCl3 solution was added to 0.1 M ammonium acetate buffer (pH 5.5, 10 µL). After 5 min, each NOTA-conjugated Fab (10 µL, 2 mg/mL, 0.1 M ammonium acetate buffer pH 5.5) was added to the solution, which was gently incubated at 37 °C for 1 h. Thereafter, 20 µL of 20 mM ethylenediaminetetraacetic acid solution was added, and each 67Ga-labeled Fab fragment was purified using a centrifuge column procedure with Sephadex G-50 Fine, equilibrated, and eluted with Dulbecco’s phosphate-buffered saline (D-PBS).

Enzymatic recognition of [67Ga]Ga-NOTA-FGK(Bzo)

Enzymatic recognition of [67Ga]Ga-NOTA-FGK(Bzo) was determined as previously described. A solution of BBMVs (10 µL, 10 mg/mL) was preincubated at 37 ˚C for 10 min, followed by the addition of RP-HPLC-purified [67Ga]Ga-NOTA-MVK(Bzo) (10 µL) in D-PBS. After 2 h of incubation at 37 ˚C, aliquots of the sample were taken from the solution and analyzed immediately using reversed-phase TLC (0.1 M ammonium acetate/methanol = 1/1). The samples were also treated with ethanol (40 µL) to precipitate proteins, centrifuged at 15,000 × g for 1 min, and analyzed using RP-HPLC.

In vivo study

All animal studies were approved by the Chiba University Animal Care Committee (No. 4-183). The number of animals in each group was empirically determined based on previous biodistribution and imaging studies. Three to five animals were used in each group to determine statistically significant differences. Separate groups of six-week-old male ddy mice (~ 25 g, Japan SLC Inc., Shizuoka, Japan) were injected via the tail vein with each of the 67Ga-labeled Fab fragments (100 µL, 11.1 kBq, 5 µg). The animals were sacrificed, and the organs were dissected at 10 min, 1 h, 3 h, and 6 h post-injection. Each tissue of interest was excised and weighed, and radioactivity counts were determined using a gamma-well counter. Urine and feces were collected 6 h post-injection, and radioactivity counts were determined using a gamma well counter. Each value is expressed as the mean percent injected dose/g of tissue ± standard deviation (SD) for a group of 3–5 animals, except for the stomach and intestine.

Five-week-old male BALB/c-nu/nu mice were xenografted in their right hind legs via subcutaneous injection of U87MG cells suspended in BD Matrigel (3 × 106 cells). When the tumor reached approximately 10 mm in diameter, these mice were used for in vivo biodistribution studies (body weight ≈ 25 g). Biodistribution studies were conducted using male BALB/c-nu/nu mice bearing U87MG tumor xenografts at 1 h and 3 h post-injection of each 67Ga-labeled Fab fragment (100 µL, 11.1 kBq, 5 µg). Tissues of interest were dissected and weighed, and radioactivity counts were determined using a gamma-well counter. Values are expressed as the mean percent injected dose/g of tissue ± SD for a group of five animals.

Analysis of radiometabolites in urine

The urine samples were collected from six-week-old ddy mice at 6 h post-injection of 67Ga-labeled-Fab (100 µL, 222 kBq, 5 µg). The samples were then filtered through a polycarbonate membrane (0.45 μm) and analyzed using SE-HPLC. After precipitation of proteins with ethanol, urine samples were analyzed via RP-HPLC using online radioactivity detectors.

Statistics

Quantitative data are expressed as means ± SD. Statistical analysis was performed using one-way analysis of variance followed by an unpaired Student’s t-test.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (120.1KB, docx)

Acknowledgements

This study was supported, in part, by a Grant-in-Aid for Scientific Research (B) (20H03619) from the Japan Society for the Promotion of Science (JSPS). We would like to thank Editage (www.editage.jp) for English language editing.

Abbreviations

NOTA

1,4,7-triazacyclononane-1,4,7-triacetic acid

SCN-Bn

p-isothiocyanatobenzyl

BBM

Brush border membrane

MVK

Methionine-valine-lysine

Mal

Maleimide

FGK

Phenylalanine-glycine-lysine

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

DO3AiBu-Bn

Isobutyl 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid

DMF

Dimethylformamide

RP-HPLC

Reversed-phase high-performance liquid chromatography

TLC

Thin layer chromatography

SE-HPLC

Size exclusion high performance liquid chromatography

Bzo

Benzoyl

BBMV

Brush border membrane vesicle

GK

Glycine-lysine

CpTR-GK(Boc)

tert-butyloxycarbonyl organorhenium complex

SPECT

Single photon emission computed tomography

Boc

tert-butoxycarbonyl

Fmoc

9-fluorenylmethoxycarbonyl

Dde

1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl

ESI-MS

Electrospray ionization mass spectrometry

EGFR

Epidermal growth factor receptor

SD

Standard deviation

Author contributions

S. K., H. S., M. K., S. K., and K. K. synthesized non-radioactive Ga- and 67Ga-labeled Fabs, performed in vitro and in vivo experiments, and analyzed the results. N. K. analyzed the results and edited the manuscript. T. U. designed the study and wrote the manuscript with the support of all authors. All the authors approved the final version of the manuscript.

Data availability

Data and materials are available from the corresponding authors upon reasonable request.

Declarations

Ethics approval and consent to participate

Animal studies were conducted in accordance with the institutional guidelines approved by the Chiba University Animal Care Committee. Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (120.1KB, docx)

Data Availability Statement

Data and materials are available from the corresponding authors upon reasonable request.

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