Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.

Experimental Design:67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab).

Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images.

Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309–16. ©2018 AACR.

Translational Relevance

Antibody fragments have been used as vehicles to deliver radiation to tumors for molecular imaging and targeted radionuclide therapy in combination with metallic radionuclides of appropriate nuclear properties. However, these radiolabeled antibody fragments exhibit high and persistent localization of radioactivity in the kidney after injection, which has hindered tumor visualization and limited therapeutic effectiveness. We herein describe newly designed gallium-67 (67Ga)-labeled antibody Fab fragments that liberate a 67Ga chelate through urinary excretion by the action of enzymes present on the brush border membrane lining the lumen of the renal proximal tubule. The 67Ga-labeled Fab exhibited low renal radioactivity levels from early postinjection time onward without decreasing tumor radioactivity levels. This amplified the tumor-to-kidney ratios of radioactivity and provided clear tumor images in a nude mouse model. Because many polypeptides share similar metabolic fates in the kidney, the present procedure may be applicable to a variety of polypeptides of interest.

Low-molecular-weight antibody fragments/constructs (LMW Abs) such as Fab and single-chain Fv fragments, diabody, and nanobody have been applied as vehicles to deliver radiation to tumors for molecular imaging and targeted radionuclide therapy, due to their faster elimination rates from circulation and more homogeneous distribution in tumor tissues than intact antibodies. Indeed, radiolabeled LMW Abs provided higher tumor-to-nontarget ratios of radioactivity (1, 2). However, radiolabeled LMW Abs exhibit high and persistent localization of radioactivity in the kidney when they are labeled with metallic radionuclides, which has hindered tumor visualization near the kidney regions and limited therapeutic effectiveness since their emergence in 1980s (3–6).

The mechanism underlying the undesirable radioactivity levels in the kidney has been elucidated; the renal radioactivity is caused by the long residence time of radiometabolite(s) generated after lysosomal proteolysis of radiolabeled LMW Abs, following glomerular filtration and subsequent reabsorption in renal cells (7–9). Numerous efforts have been made to reduce the renal radioactivity levels; blockage or reduction of tubular reabsorption of radiolabeled LMW Abs by pre- or co-injection of basic amino acids such as L-lysine or a plasma expander (1, 3), or facilitated excretion of radiometabolite(s) from the renal lysosomal compartment to the urine (10, 11). Despite these efforts, further reduction in renal radioactivity from early postinjection time is still needed to fully exploit the pharmacokinetics of radiolabeled LMW Abs for molecular imaging and targeted radionuclide therapy.

To establish a strategy to solve the problem, we previously developed 3′-131I-iodohippuryl Nϵ-maleoyl-lysine (HML) to prepare 131I-HML-labeled Fab fragments (131I-HML-Fab in Fig. 1A) as a prototype compound of our molecular design (6). In this design, 131I-iodohippuric acid is liberated from covalently conjugated 131I-HML-Fab by the action of enzymes present on the brush border membrane (BBM) lining the lumen of the proximal renal tubule, and the resulting 131I-iodohippuric acid is excreted into urine whereas the antibody molecules are reabsorbed into renal cells. Indeed, 131I-HML-Fab significantly reduced renal radioactivity levels shortly after injection without impairing the radioactivity levels in the tumor (6, 12). In our follow-up study, 188Re-tricarbonyl(cyclopentadienyl-carbonate)rhenium (188Re-CpTR) was prepared via a five-step radiosynthesis, and was coupled to a Fab fragment via a glycyl-lysine linkage (188Re-CpTR-GK-Fab: Fig. 1B; ref. 13), considering similar in vivo behaviors of glycine conjugate of 188Re-CpTR (188Re-CpTR-Gly) and 131I-iodohippuric acid (14). 188Re-CpTR-GK-Fab also exhibited low renal radioactivity levels from early postinjection time onwards without reducing tumor accumulation, due to a release and subsequent urinary excretion of 188Re-CpTR-Gly. These results suggested that the molecular design of HML would be applied to metallic radionuclides of clinical relevance if an appropriate combination of a radiometal chelate and a linkage structure are designed.

Figure 1.

Chemical structures of radiolabeled Fab fragments. A,131I-HML-Fab. B,188Re-CpTR-GK-Fab. Both radiolabeled Fab fragments exhibited low renal radioactivity levels by liberating 131I-iodohippuric acid (A) or 188Re-CpTR-Gly (B) from the Fab fragments by the action of enzymes on renal brush border enzymes. C,67Ga-NOTA-MVK-Fab. The peptide bond between methionine and valine is designed to be cleaved by enzymes on renal BBM to liberate 67Ga-NOTA-Met metabolite that is excreted in the urine. D,67Ga-NOTA-MI-Fab. The peptide bond between methionine and isoleucine is cleaved by enzymes in renal lysosomes to liberate 67Ga-NOTA-Met. E,67Ga-NOTA-Fab. 67Ga-NOTA was directly conjugated to Fab fragments via thiourea bond. 67Ga-labeled Fab fragments (D) and (E) were used as references to estimate 67Ga-NOTA-MVK-Fab (C).

Figure 1.

Chemical structures of radiolabeled Fab fragments. A,131I-HML-Fab. B,188Re-CpTR-GK-Fab. Both radiolabeled Fab fragments exhibited low renal radioactivity levels by liberating 131I-iodohippuric acid (A) or 188Re-CpTR-Gly (B) from the Fab fragments by the action of enzymes on renal brush border enzymes. C,67Ga-NOTA-MVK-Fab. The peptide bond between methionine and valine is designed to be cleaved by enzymes on renal BBM to liberate 67Ga-NOTA-Met metabolite that is excreted in the urine. D,67Ga-NOTA-MI-Fab. The peptide bond between methionine and isoleucine is cleaved by enzymes in renal lysosomes to liberate 67Ga-NOTA-Met. E,67Ga-NOTA-Fab. 67Ga-NOTA was directly conjugated to Fab fragments via thiourea bond. 67Ga-labeled Fab fragments (D) and (E) were used as references to estimate 67Ga-NOTA-MVK-Fab (C).

Close modal

Among the routinely available metallic radionuclides, isotopes of gallium are of interest for molecular imaging, since gallium-67 (67Ga) is a gamma-emitter (T1/2=3.3 d) useful for immunoSPECT, whereas gallium-68 (68Ga) is a positron emitter (T1/2 = 68 min) suitable for immunoPET. Furthermore, 68Ga is available from a long-lived 68Ge/68Ga generator system that potentially allows for the cost-effective production and the use of 68Ga-labeled probes far from cyclotron facilities (15). Indeed, lots of studies have shown that 67/68Ga-labeled LMW Abs and peptides exhibit rapid tumor targeting and provide tumor images in short postinjection time (16–20). However, these 67/68Ga-labeled LMW Abs and peptides exhibit high and persistent radioactivity levels in the kidney. Prior studies by ourselves and others have shown that 67Ga-labeled NOTA-conjugated methionine (67Ga-NOTA-Met) is rapidly excreted from the renal lysosomes to the urine when the radiolabeled compound is generated after lysosomal proteolysis of the parental 67Ga-labeled LMW Abs (10, 21) where NOTA represents 1,4,7-triazacyclononane-N,N',N”-triacetic acid. These results suggested that 67/68Ga-labeled LMW Abs that liberate 67/68Ga-NOTA-Met by the action of enzymes on the BBM of proximal renal tubules would reduce renal radioactivity levels shortly after injection.

In this study, we used a Fab fragment of an mAb against c-kit (4) as a model LMW Ab, because many LMW Abs and peptides share similar metabolic fates in the kidney (21–23). We designed and synthesized 67Ga-labeled Fab with an enzyme-cleavable Met-Val-Lys (MVK) linkage to liberate 67Ga-NOTA-Met (67Ga-NOTA-MVK-Fab; Fig. 1C). To evaluate the recognition of the MVK sequence in 67Ga-NOTA-MVK-Mal by the enzymes on renal BBM, a low molecular weight model compound, 67Ga-NOTA-MVK(Benzoyl)-OH (67Ga-NOTA-MVK-Bzo), was synthesized, where the maleimide group in NOTA-MVK-Mal was substituted with a benzoyl group to prevent maleimide-mediated reaction with enzymes. The feasibility of the present molecular design was assessed through the comparative biodistribution studies of 67Ga-NOTA-MVK-Fab in normal and tumor-bearing nude mice with 67Ga-NOTA-conjugated Fab fragments through a conventional thiourea linkage (Fig. 1E) and through a Met-Ile linkage (Fig. 1D) that liberates 67Ga-NOTA-Met in the renal lysosomes (10).

General

The synthesis methods used to prepare the chemical compounds and the details of the analytical methods using RP-HPLC and TLC are described in the Supporting Information. 67GaCl3 was supplied by FUJIFILM RI Pharma Co., Ltd. The renal brush border membrane vesicles (BBMV) were prepared as described previously (24). All commercially available chemicals were of analytical grade and used without further purification.

Monoclonal antibody and cells

The monoclonal antibody against c-kit (12A8) and non–small cell lung cancer SY cells were obtained from Immuno-Biological Laboratories (Takasaki, Japan). The Fab fragments of the 12A8 antibody were prepared by standard procedure using papain. SY cells were cultivated in RPMI-1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS (Nippon Bio-Supply Center, Tokyo) and 1% penicillin–streptomycin (5,000 U; 5,000 μg/mL, Invitrogen, Life Technologies Japan Ltd.,) in a humidified atmosphere containing 5% CO2 at 37°C. The cell was grown to 80% to 90% confluence before trypsinization and formulation into an equal volume of RPMI-1640 medium and Matrigel (BD Biosciences) for implantation into mice.

Preparation of 67Ga-labeled Fab Fragments

67Ga-labeled Fab fragments were prepared according to the procedure as described previously (10). The detailed procedures for preparing the NOTA-Fab conjugates are described in the Supporting Information. Briefly, a 5 μL solution of 67GaCl3 was added to 0.25 mol/L acetate buffer (pH 5.5, 5 μL). After 5 minutes, each NOTA-Fab conjugate (10 μL, 2 mg/mL, 0.25 mol/L acetate buffer pH 5.5) was added to the solution, and the solution was gently incubated at 37°C for 1 hour. A 20 μL solution of 20 mmol/L EDTA was then added, and the mixture was incubated for 30 minutes at the same temperature. Each 67Ga-labeled Fab fragment was purified by a centrifuged column procedure using Sephadex G-50 fine, equilibrated and eluted with Dulbecco's Phosphate-Buffered Saline (D-PBS).

Plasma stability of 67Ga-NOTA-Met-Val-Lys(Benzoyl)-OH (67Ga-NOTA-MVK-Bzo)

67Ga-NOTA-MVK-Bzo (20 μL) was added to a freshly prepared murine plasma (230 μL), and the solution was incubated at 37°C. Aliquots of samples were collected after incubation for 1, 6, and 24 hours, and were analyzed by TLC.

Enzymatic recognition of 67Ga-NOTA-MVK-Bzo

The enzymatic recognition of 67Ga-NOTA-MVK-Bzo was determined according to the procedure as described before (24). A solution of BBMVs (10 μL, 10 mg/mL) was preincubated at 37°C for 10 minutes, followed by the addition of RP-HPLC–purified 67Ga-NOTA-MVK-Bzo (10 μL) in D-PBS. After 30 minutes, 1 and 2 hours incubation at 37°C, aliquots of the sample were taken from the solution and analyzed immediately by RP-TLC. The samples were also treated with ethanol (40 μL) to precipitate proteins, centrifuged at 15,000 × g for 1 minute, and were analyzed by RP-HPLC. Similar experiments were performed in the presence of inhibitors (D,L-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MGTA), Captopril, Cilastatin, and Phosphoramidon) for brush border enzymes at a final concentration of 1 mmol/L. All experiments were carried out in triplicate.

In vivo study

Animal studies were conducted in accordance with the institutional guidelines approved by the Chiba University Animal Care Committee. The number of animals in each group was empirically determined based on prior biodistribution and imaging studies. As such, 5 animals were used in each group to provide statistically significant data between the groups studied. Separate groups of 6-week-old male ddY mice (ca. 35 g, Japan SLC Inc., Shizuoka, Japan) were injected via the tail vein with each of the 67Ga-labeled Fab (100 μL, 11.1 kBq, 5 μg). Animals were sacrificed and organs dissected at 10 minutes, 1, 3, 6, and 24 hours postinjection. The tissues of interest were excised, weighed, and the radioactivity counts in each tissue were determined with a gamma well-counter. Urine and feces were collected for 6 and 24 hours postinjection and the radioactivity counts were determined with a gamma well-counter. Each value was expressed as the mean percent injected dose/g of tissue ± (SD) for a group of 5 animals except for stomach and intestine.

Five-week-old male BALB/c nu/nu mice (Japan SLC, Inc.) were xenografted subcutaneously in their right hind legs via injection of SY 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 and SPECT/CT tumor imaging studies (body weight: ca. 22 g). Biodistribution studies were conducted using male BALB/c nu/nu mice bearing SY tumor xenografts at 3 hours postinjection of each 67Ga-labeled Fab fragments (100 μL, 11.1 kBq, 5 μg). Tissues of interest were dissected out, weighed, and the radioactivity counts were determined using a gamma well-counter. Values were expressed as the mean %injected dose/g of tissue ± (SD) for a group of 5 animals.

Analyses of radiometabolites in urine

The urine samples were collected from 6-week-old ddY mice by 6 hours postinjection of 67Ga-NOTA-MVK-Fab (100 μL, 222 kBq, 5 μg). The samples were then filtered through a polycarbonate membrane (0.45 μm) and analyzed by SE-HPLC. After the ethanol precipitation of proteins, the urine samples were also analyzed by RP-HPLC using on-line radioactivity detectors.

Small animal SPECT/CT imaging studies

SPECT/CT imaging studies were conducted at 2.5 hours after tail vein injection of each of the 67Ga-labeled Fab (100 μL, 3.7 MBq, 20 μg) to male BALB/c nu/nu mice bearing SY tumor xenografts (n = 2). For SPECT/CT imaging studies, the mice were anesthetized with 1.2% (v/v) isoflurane (DS Pharma Animal Health, Osaka), positioned on the animal bed, and kept under anesthesia via a nose cone anesthesia system. The small animal SPECT/CT imaging system (SPECT4/CT, Trifoil Inc.) was equipped with a five pinhole (1.0 mm) collimator. Data acquisition was performed at 60 seconds per projection with the stepwise rotation of 64 projections over 360°. SPECT Triumph-RECON software (Trifoil Inc.) was used to obtain the SPECT images and data was reconstructed using a 3D-ordered subset expectation maximization (3D-OSEM) algorithm using 2 subsets and 8 iterations.

Statistical analysis

Quantitative data were expressed as means ± SD. Statistical analysis was done by comparison using a one-way analysis of variance followed by Tukey's multiple-comparison test (Graph Pad Prism).

Preparation of 67Ga-labeled Fab fragments

A new bifunctional chelating agent with a renal brush border enzyme-cleavable linkage, NOTA-MVK-Mal, was obtained by reacting SCN-Bn-NOTA with Met-Val-Lys-Mal in dry DMF and subsequent RP-HPLC purification (Supplementary Scheme S1). NOTA-MVK-Mal was then conjugated with thiolated Fab fragments using a standard 2-iminothiolane method. The number of NOTA-MVK introduced per molecule of Fab fragment was estimated to be 0.81 to 1.67 by calculating the thiol groups in the Fab fragment before and after the conjugation reaction. NOTA-MI-Fab and NOTA-Fab were also prepared as references (Fig. 1D and E), and the number of the chelating groups introduced in the Fab fragments was determined to be 0.7 to 1.67. All NOTA-conjugated Fab fragments were labeled with 67Ga in the presence of acetate, and the resulting radiolabeled Fab fragments were analyzed by TLC, CAE, and SE-HPLC. The radiochemical yields and purities of all 67Ga-labeled Fab fragments were over 95%.

Enzyme recognition and plasma stability of the MVK linkage

The recognition of the MVK sequence in 67Ga-NOTA-MVK-Mal by the enzymes on renal BBM was evaluated with a low molecular weight compound, 67Ga-NOTA-MVK-Bzo, where the maleimide group in NOTA-MVK-Mal was substituted with a benzoyl group to prevent the maleimide-mediated reactions with enzymes on BBMV (Supplementary Scheme S2). After removing free NOTA-MVK-Bzo by RP-HPLC, 67Ga-NOTA-MVK-Bzo was incubated with BBMVs (24) to estimate the release of 67Ga-NOTA-Met. The incubation of 67Ga-NOTA-MVK-Bzo with BBMVs at 37°C for 2 hours resulted in the cleavage and release of 67Ga-NOTA-Met of ca. 19% (Fig. 2A). The release of 67Ga-NOTA-Met from the 67Ga-NOTA-MVK-Bzo was inhibited by phosphoramidon, an inhibitor of neutral endopeptidase (NEP; Fig. 2B). When 67Ga-NOTA-MVK-Bzo was incubated in freshly prepared mouse plasma for 24 hours at 37°C, >90% of radioactivity remained as the intact compound (Fig. 2C).

Figure 2.

Characterization of the MVK linkage in 67Ga-NOTA-MVK-Bzo. A, Formation of the 67Ga-NOTA-Met radiometabolite from 67Ga-NOTA-MVK-Bzo after the incubation with BBMVs at 37°C. The amount of 67Ga-NOTA-Met increased with time upon exposure to BBMVs. These results indicated that the Met-Val sequence was recognized and cleaved by enzymes on BBMs. B, The formation of 67Ga-NOTA-Met metabolite from 67Ga-NOTA-MVK-Bzo after incubation with BBMVs at 37°C for 2 hours in the absence (Control) or presence of an inhibitor of carboxypeptidase (MGTA), angiotensin-converting enzyme (captopril), dipeptidase (cilastatin), and neutral endopeptidase (phosphoramidon). Bars show the means ± SD of three experiments. The formation of 67Ga-NOTA-Met was significantly inhibited by phosphoramidon, indicating that the Met-Val sequence of 67Ga-NOTA-MVK-Bzo was predominantly recognized and cleaved by neutral endopeptidase (NEP). C, Stability of 67Ga-NOTA-MVK-Bzo in murine plasma at 37°C. The 67Ga-NOTA-MVK-Bzo remained stable despite the presence of hydrolytic enzymes in plasma, suggesting that the Met-Val linkage would remain stable and would not impair tumor accumulation when conjugated to Fab fragments.

Figure 2.

Characterization of the MVK linkage in 67Ga-NOTA-MVK-Bzo. A, Formation of the 67Ga-NOTA-Met radiometabolite from 67Ga-NOTA-MVK-Bzo after the incubation with BBMVs at 37°C. The amount of 67Ga-NOTA-Met increased with time upon exposure to BBMVs. These results indicated that the Met-Val sequence was recognized and cleaved by enzymes on BBMs. B, The formation of 67Ga-NOTA-Met metabolite from 67Ga-NOTA-MVK-Bzo after incubation with BBMVs at 37°C for 2 hours in the absence (Control) or presence of an inhibitor of carboxypeptidase (MGTA), angiotensin-converting enzyme (captopril), dipeptidase (cilastatin), and neutral endopeptidase (phosphoramidon). Bars show the means ± SD of three experiments. The formation of 67Ga-NOTA-Met was significantly inhibited by phosphoramidon, indicating that the Met-Val sequence of 67Ga-NOTA-MVK-Bzo was predominantly recognized and cleaved by neutral endopeptidase (NEP). C, Stability of 67Ga-NOTA-MVK-Bzo in murine plasma at 37°C. The 67Ga-NOTA-MVK-Bzo remained stable despite the presence of hydrolytic enzymes in plasma, suggesting that the Met-Val linkage would remain stable and would not impair tumor accumulation when conjugated to Fab fragments.

Close modal

Biodistribution studies in normal mice

The biodistribution of radioactivity after injection of the three 67Ga-labeled Fab fragments to normal mice is summarized in Fig. 3A and B and Supplementary Table S1. Although the radioactivity levels of 67Ga-NOTA-MVK-Fab in the blood were similar to those of 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab, significant differences were observed in renal radioactivity levels of the three. The lowest renal radioactivity levels were achieved with 67Ga-NOTA-MVK-Fab as early as 30 minutes to 24 hours postinjection, whereas 67Ga-NOTA-MI-Fab displayed much lower renal radioactivity levels from 3 to 24 hours compared with 67Ga-NOTA-Fab. The RP-HPLC analyses of the urine samples collected by 6 hours postinjection of 67Ga-NOTA-MVK-Fab showed that the major radiometabolite had a retention time of 15.5 min identical to that of 67Ga-NOTA-Met standard (Fig. 3C).

Figure 3.

Time–activity curves of radioactivity in the blood and kidney after injection of the three 67Ga-labeled Fab fragments in normal mice. A, All three 67Ga-labeled Fab fragments displayed similar rates of elimination from the blood. B,67Ga-NOTA-MVK-Fab exhibited significantly lower renal radioactivity levels than 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab from 10 minutes to 24 hours after injection. C, The radiochromatograms of urine samples collected by 6 hours after injection of 67Ga-NOTA-MVK-Fab analyzed by RP-HPLC after removing proteins (C, top) and the 67Ga-NOTA-Met standard sample (C, bottom, 15.5 minutes). The radiochromatograms of urine samples showed that the majority of radioactive fractions had a retention time of 15.5 minutes, similar to that of 67Ga-NOTA-Met standard.

Figure 3.

Time–activity curves of radioactivity in the blood and kidney after injection of the three 67Ga-labeled Fab fragments in normal mice. A, All three 67Ga-labeled Fab fragments displayed similar rates of elimination from the blood. B,67Ga-NOTA-MVK-Fab exhibited significantly lower renal radioactivity levels than 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab from 10 minutes to 24 hours after injection. C, The radiochromatograms of urine samples collected by 6 hours after injection of 67Ga-NOTA-MVK-Fab analyzed by RP-HPLC after removing proteins (C, top) and the 67Ga-NOTA-Met standard sample (C, bottom, 15.5 minutes). The radiochromatograms of urine samples showed that the majority of radioactive fractions had a retention time of 15.5 minutes, similar to that of 67Ga-NOTA-Met standard.

Close modal

Biodistribution studies in tumor-bearing mice

The biodistribution of radioactivity after administration of the three 67Ga-labeled Fab fragments in nude mice bearing SY cell xenografts is summarized in Fig. 4. Detailed results are shown in Supplementary Table S2. Although no significant differences were observed in tumor uptake 3 hours postinjection among the three 67Ga-labeled Fab fragments, 67Ga-NOTA-MVK-Fab registered significantly lower radioactivity levels in the kidney than did either 67Ga-NOTA-MI-Fab or 67Ga-NOTA-Fab. As a result, the tumor/kidney ratio of 67Ga-NOTA-MVK-Fab was 4- and 7-fold higher than that of 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab, respectively, at 3 hours postinjection. The SPECT/CT imaging studies conducted with all 67Ga-NOTA-labeled Fab fragments in nude mice bearing SY tumor xenografts displayed clear tumor visualization at 3 hours postinjection of the 67Ga-labeled Fab fragments (Fig. 4B). 67Ga-NOTA-MVK-Fab provided a much higher contrast tumor image when compared with 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab.

Figure 4.

Comparative tumor and kidney accumulation of radioactivity in nude mice after 3-hour injection of the three 67Ga-labeled Fab fragments. A, The quantitative measurement of radioactivity in the tumor (% injected dose/g of tissue) and the tumor/kidney ratios after injection of 67Ga-NOTA-Fab (SCN), 67Ga-NOTA-MI-Fab (MI), and 67Ga-NOTA-MVK-Fab (MVK) in nude mice bearing SY tumors. All three 67Ga-labeled Fab fragments delivered similar amounts of radioactivity to tumors. Because of much lower renal radioactivity levels, 67Ga-NOTA-MVK-Fab registered significantly higher tumor/kidney ratios of the radioactivity than 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab (P < 0.05). B, SPECT/CT images of nude mice. The SPECT/CT images clearly reflected the tissue distribution studies (A), and 67Ga-NOTA-MVK-Fab provided a significantly higher-contrast tumor image.

Figure 4.

Comparative tumor and kidney accumulation of radioactivity in nude mice after 3-hour injection of the three 67Ga-labeled Fab fragments. A, The quantitative measurement of radioactivity in the tumor (% injected dose/g of tissue) and the tumor/kidney ratios after injection of 67Ga-NOTA-Fab (SCN), 67Ga-NOTA-MI-Fab (MI), and 67Ga-NOTA-MVK-Fab (MVK) in nude mice bearing SY tumors. All three 67Ga-labeled Fab fragments delivered similar amounts of radioactivity to tumors. Because of much lower renal radioactivity levels, 67Ga-NOTA-MVK-Fab registered significantly higher tumor/kidney ratios of the radioactivity than 67Ga-NOTA-MI-Fab and 67Ga-NOTA-Fab (P < 0.05). B, SPECT/CT images of nude mice. The SPECT/CT images clearly reflected the tissue distribution studies (A), and 67Ga-NOTA-MVK-Fab provided a significantly higher-contrast tumor image.

Close modal

The key concept of the present molecular design consists of the release and urinary excretion of a hippurate-like radiometal chelate from covalently conjugated radiolabeled LMW Abs by the action of enzymes on the BBM lining the lumen of the proximal renal tubule. Of the two hippurate-like 67/68Ga complexes we have evaluated so far, 67Ga chelate of succinyldeferoxamine (67Ga-SDF; refs. 25, 26) and 67Ga-NOTA-Met (10), we selected 67Ga-NOTA-Met considering its higher stability than 67Ga-SDF (27) and its potential application to beta-emitting radiocoppers (64Cu and 67Cu) for targeted radionuclide therapy (28, 29). In addition, the development of a 67/68Ga-NOTA-labeled LMW Abs with low renal radioactivity levels would provide an insight to design another polyaminopolycarboxylate-based macrocyclic ligand, DOTA, derivative for labeling with indium-111, 90Y, lutetium-177 and actinium-225, where DOTA represents 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (30, 31).

We then designed a cleavable linkage that liberates 67Ga-NOTA-Met from the covalently conjugated parental Fab molecule by the action of the BBM enzymes. In our previous study, we selected Gly-Lys (GK) sequence as the cleavable linkage to liberate iodohippuric acid from the covalently conjugated Fab fragments because the GK sequence is a substrate of carboxypeptidase M abundant on the renal BBM (6, 32). We used the similar approach to liberate 67Ga-NOTA-Met from covalently conjugated Fab fragments. NEP is highly expressed on the renal BBM (33, 34), and the enzyme cleaves the peptide linkage between Met and Val (35). In addition, the presence of a free carboxylate group of Lys next to the substrate sequence at the C-terminus enhances the recognition by the enzyme (36). In light of these findings, we selected a tripeptide sequence, Met-Val-Lys (MVK), as a cleavable linkage, and the ϵ-amino group of Lys was converted to a maleimide group for antibody conjugation. The N-terminus amino group of the tripeptide was then coupled with SCN-Bn-NOTA to prepare NOTA-MVK-Mal (Supplementary Scheme S1).

The MVK sequence in 67Ga-NOTA-MVK-Bzo was cleaved by the presence of BBMVs (24) to liberate 67Ga-NOTA-Met (Fig. 2A), which was partially inhibited by phosphoramidon (Fig. 2B), indicating that NEP is involved in the cleavage of the MV sequence in 67Ga-NOTA-MVK-Bzo. High plasma stability of the cleavable linkage constitutes another criterion for in vivo applications because the use of a plasma-labile ester or disulfide bond as the cleavable linkage resulted in decreased tumor radioactivity levels of radiolabeled antibodies (37, 38). As shown in Fig. 2C, the MVK linkage in 67Ga-NOTA-MVK-Bzo remained stable murine plasma despite the presence of hydrolytic enzymes, suggesting that 67Ga-NOTA-MVK-Fab would not impair tumor radioactivity levels delivered by the antibodies.

To evaluate the ability of NOTA-MVK-Mal to produce 67Ga-labeled Fab fragment of low renal radioactivity while preserving tumor radioactivity levels, the Fab fragments against c-kit were conjugated with NOTA-MVK-Mal and labeled with 67GaCl3 to prepare 67Ga-NOTA-MVK-Fab (Fig. 1C). For comparison, 67Ga-NOTA-MI-Fab (Fig. 1D) and 67Ga-NOTA-Fab (Fig. 1E) were prepared. The former liberates 67Ga-NOTA-Met after lysosomal proteolysis in the renal cells (10) whereas the latter generates 67Ga-NOTA-conjugated lysine (67Ga-NOTA-Lys) after lysosomal proteolysis. The high plasma stability of the three 67Ga-labeled Fab fragments reinforced that 67Ga-NOTA chelates, MVK and MI linkages, and the thiourea bonds remained stable in plasma (Supplementary Table S3).

When injected into mice, all the 67Ga-labeled Fab fragments showed similar elimination rates of the radioactivity from circulation (Fig. 3, Supplementary Table S1). These three 67Ga-labeled Fab fragments were prepared at similar specific activities, and all the 67Ga-labeled Fab fragments remained stable in murine plasma (Supplementary Table S3). Thus, similar mass amounts and radioactive portions of the three 67Ga-labeled Fab fragments were filtered through glomerulus and transported to proximal renal tubules after administration. However, significant differences were observed in the renal radioactivity levels among the three 67Ga-labeled Fab fragments. The lower renal radioactivity levels of 67Ga-NOTA-MI-Fab than those of 67Ga-NOTA-Fab were attributable to the different elimination rates of the two final radiometabolites (67Ga-NOTA-Met and 67Ga-NOTA-Lys) from the lysosomal compartment of the kidney, as discussed previously (10, 21). 67Ga-NOTA-MVK-Fab demonstrated significantly lower radioactivity levels in the kidney even after 10 minutes postinjection onwards than did 67Ga-NOTA-MI-Fab (P < 0.05). Because both 67Ga-NOTA-MVK-Fab and 67Ga-NOTA-MI-Fab liberated 67Ga-NOTA-Met as the major radiometabolite, the differences in renal radioactivity levels between the two 67Ga-labeled Fab fragments indicated that 67Ga-NOTA-MVK-Fab liberated 67Ga-NOTA-Met at an earlier stage of antibody metabolism in the kidney (reabsorption process vs. lysosomal proteolysis) and subsequent elimination into the urine. Moreover, 67Ga-NOTA-MVK-Fab reduced the renal radioactivity levels without impairing the tumor radioactivity levels (Fig. 4 and Supplementary Table S2). Because many LMW Abs and peptides share the similar metabolic fates in the kidney (21–23), the present procedure would be applicable to a variety of 67/68Ga-labeled LMW Abs and peptides of interest without the addition of any adjuvants. The combination of the present procedure and an inhibitor of tubular reabsorption (e.g., L-lysine or a plasma expander, Gelofusine) may constitute a more effective way to reduce renal radioactivity levels of a variety of 67/68Ga-labeled LMW Abs.

The present procedure may be limited to LMW Abs and peptides that undergo no or slow rates of internalization to target cells, since 67/68Ga-NOTA-Met may also be generated and eliminated from the target cells following intracellular metabolism. The findings in this study also suggest the application of NOTA-MVK-Mal to 64/67Cu-labeling of LMW Abs for target radionuclide therapy, which would reduce renal absorbed dose without impairing tumor absorbed dose. However, further studies are needed, since different physicochemical properties between the two radiometal chelates (Cu-NOTA vs. Ga-NOTA) may affect enzyme recognition of the cleavable MVK linkages to which each radiometal chelate is conjugated.

In conclusion, the present study shows a solution to the long-unsettled problem of high and persistent renal radioactivity levels after injection of LMW Abs labeled with metallic radionuclides as exemplified with 67Ga-labeled Fab fragments. This labeling procedure may be applied to a variety of LMW Abs of interest for SPECT and PET imaging. The findings in this study would also provide a good basis to develop radiolabeled LMW Abs of low renal radioactivity levels using a variety of metallic radionuclides of interest.

No potential conflicts of interest were disclosed.

Conception and design: T. Uehara, Y. Arano

Development of methodology: T. Uehara, Y. Arano

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Uehara, M. Yokoyama, H. Suzuki, H. Hanaoka

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Uehara, M. Yokoyama, H. Suzuki, Y. Arano

Writing, review, and/or revision of the manuscript: T. Uehara, M. Yokoyama, H. Suzuki, Y. Arano

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Arano

Study supervision: T. Uehara, Y. Arano

The authors thank Dr. Ashfaq Mahmood for editorial assistance with the article. The authors are grateful to FUJIFILM RI Pharma Co. Ltd. for providing 67GaCl3. This work was supported in part by a Grant-in-Aid for Young Scientists (A) (no. 25713045, to T. Uehara) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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