Development of INER-PP-F11N as the Peptide-Radionuclide Conjugate Drug Against CCK2 Receptor-Overexpressing Tumors

Abstract

This work aimed to evaluate two albumin affinity structure-containing peptide-radionuclide conjugate drugs, INER-PP-F11N-1 and INER-PP-F11N-2, for the diagnosis/treatment of cholecystokinin receptor subtype 2 (CCK2R)-overexpressing cancers. We developed In-111- and Lu-177-labeled INER-PP-F11N radiopharmaceuticals and compared them with the current PP-F11N to investigate metabolic stability, biodistribution, SPECT/CT imaging, and therapeutic responses in CCK2R-expressing tumor xenograft mice. The metabolic stability of [¹¹¹In]In/[¹⁷⁷Lu]Lu-INER-PP-F11N remained above 90% for up to 144 h after labeling, indicating that the compound is highly stable under in vitro conditions. INER-PP-F11N showed 27% and 11% higher cellular uptake and internalization than PP-F11N, respectively. In vivo SPECT/CT imaging confirmed that INER-PP-F11N could accumulate at the tumor site of mice 24 h after receiving the two radiopharmaceutical agents. Biodistribution analysis revealed a significantly greater tumor uptake and reduced accumulation of INER-PP-F11N in the kidneys compared with PP-F11N. Furthermore, INER-PP-F11N significantly inhibited the growth of the CCK2R-overexpressing tumors in mice. The INER-PP-F11N radiopharmaceutical was superior as a theragnostic agent compared with the current PP-F11N. Our study suggests that INER-PP-F11N may be an innovative radiopharmaceutical agent for CCK2R-overexpressing tumors.

Keywords: CCK2R; INER-PP-F11N; peptide-radionuclide conjugate drug; radiopharmaceutical agent; theragnostic.

Figure 1

Synthesized CCK2R derivatives with different linkers between DOTA and the C-terminal binding sequence. (A) The structure of DOTA-PP-F11N: [DOTA-(Glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2]; (B) the structure of DOTA-INER-PP-F11N-1 [DOTA-Lys(-4-TBA)-6-AMBA-(Glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2]; and (C) the structure of DOTA-INER-PP-F11N-2 [DOTA-Lys(-4-TBA)-AHA-(Glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2].

Figure 2

Evaluation of radiopharmaceutical agent using Radio-HPLC. (A) Quantitation of metabolic stability of affinity-labeled DOTA-PP-F11N, DOTA-INER-PP-F11N-1, and DOTA-INER-PP-F11N-2 in 4 °C PBS solution at individual time points. (B) Quantitation of metabolic stability of In-111-labeled DOTA-PP-F11N, DOTA-INER-PP-F11N-1, and DOTA-INER-PP-F11N-2 in 37 °C FBS-containing solution at individual time points. (C) Quantitation of metabolic stability of Lu-177-labeled DOTA-PP-F11N, DOTA-INER-PP-F11N-1, and DOTA-INER-PP-F11N-2 in 4 °C PBS solution at individual time points. (D) Quantitation of metabolic stability of Lu-177-labeled DOTA-PP-F11N, DOTA-INER-PP-F11N-1, and DOTA-INER-PP-F11N-2 in 37 °C FBS-containing solution at individual time points. HPLC: high-performance liquid chromatography.

Figure 3

Cellular uptake of [¹⁷⁷Lu]Lu-DOTA-PP-F11N, [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1, and [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2. (A) A431-CCK2R (−) cells were treated with Lu-177-labeled CCK2R derivatives and then incubated at 37 °C for 1 or 4 h. Surface binding activity (%IA/106 cells): for [¹⁷⁷Lu]Lu-DOTA-PP-F11N: 3.42 ± 0.21% (1 h) and 3.24 ± 0.15% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1: 1.08 ± 0.49% (1 h) and 0.32 ± 0.10% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2: 0.59 ± 0.01% (1 h) and 0.25 ± 0.04% (4 h). Internalization activity (%IA/106 cells): for [¹⁷⁷Lu]Lu-DOTA-PP-F11N: 1.26 ± 0.06% (1 h) and 1.29 ± 0.01% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1: 0.32 ± 0.10% (1 h) and 0.17 ± 0.03 (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2: 0.25 ± 0.04% (1 h) and 0.15 ± 0.18% (4 h). (B) A431-CCK2R (+) cells were treated with Lu-177-labeled CCK2R derivatives and then incubated at 37 °C for 1 or 4 h. Surface binding activity for [¹⁷⁷Lu]Lu-DOTA-PP-F11N: 6.60 ± 0.17% (1 h) and 11.51 ± 3.16% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1: 5.93 ± 1.68% (1 h) and 14.57 ± 4.06% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2: 5.51 ± 0.45% (1 h) and 8.11 ± 2.65% (4 h). Internalization activity for [¹⁷⁷Lu]Lu-DOTA-PP-F11N: 37.37 ± 3.04% (1 h) and 71.44 ± 3.52% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1: 45.32 ± 6.12% (1 h) and 90.49 ± 5.65% (4 h); for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2: 41.58 ± 3.46% (1 h) and 81.26 ± 7.27% (4 h).

Figure 4

NanoSPECT/CT, biodistribution, and anti-tumor effects of [¹⁷⁷Lu]Lu-DOTA-CCK2R derivatives in mice. The yellow circle indicated the tumor implant site. (A) NanoSPECT/CT images of mice after tail-vein injection of [¹⁷⁷Lu]Lu-DOTA, [¹⁷⁷Lu]Lu-DOTA-PP-F11N, [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1, and [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2. (B) At 72 h after tail-vein injection in tumor-bearing mice, tumor uptake of radiopharmaceuticals (%ID/g) was 0.33 ± 0.02 for [¹⁷⁷Lu]Lu-DOTA-PP-F11N, 1.14 ± 0.92 for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1, and 0.88 ± 0.25 for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2. Radiotracer accumulation in the kidneys (%ID/g) was 14.2 ± 5.9 for Lu-177-DTPA-PP-F11N, 7.4 ± 2.3 for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1, and 7.4 ± 1.5 for [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2. (C) In vivo tumor growth in mice treated with [¹⁷⁷Lu]Lu-DOTA-PP-F11N, [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1, and [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2. Tumor volume was measured from the day after tumor inoculation. The asterisk (p < 0.05) and double asterisks (p < 0.01) indicate the significant changes in tumor size.

Figure 5

Histopathological examination of the mice. The kidney (A), liver (B), and heart (C) of the mice treated with [¹⁷⁷Lu]Lu-DOTA-PP-F11N. The kidney showed moderate/severe renal tubular necrosis (arrow 1), moderate renal tubular casts (arrow 2), slight renal tubular degeneration (arrow 3), and slight infiltration of neutrophils (arrow 4). The kidney (D), liver (E), and heart (F) of the mice treated with [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-1. The kidney (G), liver (H), and heart (I) of the mice treated with [¹⁷⁷Lu]Lu-DOTA-INER-PP-F11N-2 (H&E staining, scale bar = 50 µm).