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 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.

Background Recently, antiprogrammed cell death protein 1 (aPD-1) and antiprogrammed death-ligand 1 (aPD-L1) monoclonal antibodies (mAbs) have been approved. Even though aPD-1 and aPD-L1 mAbs target the same PD-1/PD-L1 axis, it is still unclear whether both mAbs exert equivalent pharmacological activity in patients who are sensitive to PD-1/PD-L1 blockade therapy, as there is no direct comparison of their pharmacokinetics (PK) and antitumor effects. Therefore, we evaluated the differences between both mAbs in PK and therapeutic effects in PD-1/PD-L1 blockade-sensitive mouse models. Methods Herein, murine breast MM48 and colon MC38 xenografts were used to analyze the pharmacological activity of aPD-1 and aPD-L1 mAbs. The PK of the mAbs in the tumor-bearing mice was investigated at low and high doses using two radioisotopes (Indium-111 and Iodine-125) to evaluate the accumulation and degradation of the mAbs. Results aPD-1 mAb showed antitumor effect in a dose-dependent manner, indicating that the tumor model was sensitive to PD-1/PD-L1 blockade therapy, whereas aPD-L1 mAb failed to suppress tumor growth. The PK study showed that aPD-L1 mAb was accumulated largely in normal organs such as the spleen, liver, and kidney, resulting in low blood concentration and low distributions to tumors at a low dose, even though the tumors expressed PD-L1. Sufficient accumulation of aPD-L1 mAb in tumors was achieved by administration at a high dose owing to the saturation of target-mediated binding in healthy organs. However, degradation of aPD-L1 mAb in tumors was greater than that of aPD-1 mAb, which resulted in poor outcome presumably due to less inhibition of PD-L1 by aPD-L1 mAb than that of PD-1 by aPD-1 mAb. Conclusion According to the PK studies, aPD-1 mAb showed linear PK, whereas aPD-L1 mAb showed non-linear PK between low and high doses. Collectively, the poor PK characteristics of aPD-L1 mAb caused lower antitumor activity than of aPD-1 mAb. These results clearly indicated that aPD-L1 mAb required higher doses than aPD-1 mAb in clinical setting. Thus, targeting of PD-1 would be more advantageous than PD-L1 in terms of PK.

Copper-64 (⁶⁴Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to ⁶⁴Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to ⁶⁴Cu. The MVK linkage was recognized by the BBM enzymes to liberate [⁶⁴Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [⁶⁴Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [⁶⁷Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [⁶⁴Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the ⁶⁷Ga-labeled counterpart. In addition, [⁶⁴Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from ⁶⁴Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to ⁶⁴Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.