Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Gallium-67/68-labeled antibody fragment for immuno-SPECT/PET shows low renal radioactivity without loss of tumor uptake Authors: Tomoya Uehara1*, Miki Yokoyama1, Hiroyuki Suzuki1, Hirofumi Hanaoka2, Yasushi Arano1. Affiliations: 1 Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 Japan. 2 Graduate School of Medicine, Gunma University, 3-39-15 Showa-chou, Maebashi, 371-8511 Japan. Running title: 67/68Ga-labeled Fab of low renal radioactivity levels Keywords: gallium-67/68, renal radioactivity, antibody fragment, renal brush border enzymes, immunoPET/SPECT. *To whom correspondence should be addressed: Tomoya Uehara Ph.D. Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 Japan. TEL: +81-43-226-2898 E-mail: [email protected] 1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The word count: 5511 The total number of figure and tables: 4 The authors declare no potential conflicts of interest. 2 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 150-word statements 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 a 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 onwards without decreasing tumor radioactivity levels. This amplified the tumor-to-kidney ratios of radioactivity and provided clear tumor images in a nude mice model. Since many polypeptides share similar metabolic fates in the kidney, the present procedure may be applicable to a variety of polypeptides of interest. 3 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract: 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. Since antibody fragments and 4 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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. Introduction 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 1980’s (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. 5 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2018; DOI: 10.1158/1078-0432.CCR-18-0123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 while 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) (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. Among the routinely available metallic radionuclides, isotopes of gallium are of interest for 67 molecular imaging, since gallium-67 ( Ga) is a gamma-emitter (T1/2=3.3 d) useful for 68 immunoSPECT, while gallium-68 ( Ga) 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 6 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer