Labeling internalizing anti-epidermal growth factor receptor variant III monoclonal antibody with (177)Lu: in vitro comparison of acyclic and macrocyclic ligands

Abstract

Introduction: The monoclonal antibody (mAb) L8A4, reactive with the epidermal growth factor receptor variant III (EGFRvIII), internalizes rapidly in glioma cells after receptor binding. Combining this tumor-specific mAb with the low-energy beta-emitter (177)Lu would be an attractive approach for brain tumor radioimmunotherapy, provided that trapping of the radionuclide in tumor cells after mAb intracellular processing could be maximized.

Materials and methods: L8A4 mAb was labeled with (177)Lu using the acyclic ligands [(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX-A''-DTPA), 2-(4-isothiocyanatobenzyl)-diethylenetriaminepenta-acetic acid (pSCN-Bz-DTPA) and 2-(4-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (1B4M-DTPA), and the macrocyclic ligands S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (C-DOTA) and alpha-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (MeO-DOTA). Paired-label internalization and cellular processing assays were performed on EGFRvIII-expressing U87.DeltaEGFR glioma cells over 24 h to directly compare (177)Lu-labeled L8A4 to L8A4 labeled with (125)I using either iodogen or N-succinimidyl 4-guanidinomethyl-3-[(125)I]iodobenzoate ([(125)I]SGMIB). In order to facilitate comparison of labeling methods, the primary parameter evaluated was the ratio of (177)Lu to (125)I activity retained in U87.DeltaEGFR cells.

Results: All chelates demonstrated higher retention of internalized activity compared with mAb labeled using iodogen, with (177)Lu/(125)I ratios of >20 observed for the three DTPA chelates at 24 h. When compared to L8A4 labeled using SGMIB, except for MeO-DOTA, internalized activity for (125)I was higher than (177)Lu from 1-8 h with the opposite behavior observed thereafter. At 24 h, (177)Lu/(125)I ratios were between 1.5 and 3, with higher values observed for the three DTPA chelates.

Conclusions: The nature of the chelate used to label this internalizing mAb with (177)Lu influenced intracellular retention in vitro, although at early time points, only MeO-DOTA provided more favorable results than radioiodination of the mAb via SGMIB.

Figure 1

Structures of the DOTA and DTPA bifunctional chelates evaluated in this study.

Figure 2

Paired label in vitro internalization of radiolabeled L8A4 mAb by U87MG.ΔEGFR cells. L8A4 was labeled with ¹²⁵I using Iodogen (top) and with ¹⁷⁷Lu via the C-DOTA macrocycle. The percentage of radioactivity initially bound to the cells that was suface bound, internalized and found in the cell culture supernatant as a function of time is shown.

Figure 3

Figure 3a. Paired label comparison of percentage of initially bound counts to U87MG.ΔEGFR cells remaining in the intracellular compartment as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using Iodogen. Figure 3b Paired label comparison of percentage of initially bound counts to U87MG.ΔEGFR cells remaining in the intracellular compartment as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using SGMIB.

Figure 3

Figure 3a. Paired label comparison of percentage of initially bound counts to U87MG.ΔEGFR cells remaining in the intracellular compartment as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using Iodogen. Figure 3b Paired label comparison of percentage of initially bound counts to U87MG.ΔEGFR cells remaining in the intracellular compartment as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using SGMIB.

Figure 4

Figure 4a. Ratio of ¹⁷⁷Lu to ¹²⁵I activity in U87MG.ΔEGFR cells as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using Iodogen. (Data from the experiments illustrated in Figure 3a). For comparison, dashed line indicates ratio of unity. Figure 4b. Ratio of ¹⁷⁷Lu to ¹²⁵I activity in U87MG.ΔEGFR cells as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using SGMIB. (Data from the experiments illustrated in Figure 3b). For comparison, dashed line indicates ratio of unity.

Figure 4

Figure 4a. Ratio of ¹⁷⁷Lu to ¹²⁵I activity in U87MG.ΔEGFR cells as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using Iodogen. (Data from the experiments illustrated in Figure 3a). For comparison, dashed line indicates ratio of unity. Figure 4b. Ratio of ¹⁷⁷Lu to ¹²⁵I activity in U87MG.ΔEGFR cells as a function of time for L8A4 labeled with ¹⁷⁷Lu using different bifunctional chelates and L8A4 radioiodinated using SGMIB. (Data from the experiments illustrated in Figure 3b). For comparison, dashed line indicates ratio of unity.

Figure 5

SDS-Page analysis of radioactivity present in cell culture supernatant at various time points after binding of radiolabeled L8A4 to U87MG.ΔEGFR cells. Left image; ¹⁴C-labeled 155kDa molecular weight standard in the far left lane,¹⁷⁷Lu-labeled MeO-DOTA-L8A4 in the next four lanes followed by 1B4M-DTPA-L8A4 in the last four lanes. Right image; ¹⁴C-labeled 155kDa molecular weight standard in the far left lane followed by L8A4 labeled using [¹²⁵I]SGMIB in the next four lanes.

Figure 6

Size exclusion HPLC analysis of ¹⁷⁷Lu activity present in cell culture supernatant 24 h after binding of ¹⁷⁷Lu-labeled 1B4M-DTPA-L8A4 to U87MG.ΔEGFR cells. Top; UV trace; botton, radioactivity trace. Arrows indicate elution times of molecular weight standards.