177Lu Anti-Angiogenic Radioimmunotherapy Targeting ATP Synthase in Gastric Cancer Model

Abstract

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope ¹⁷⁷Lu using DOTA as a chelating agent. ¹⁷⁷Lu-DOTA-ATPS mAb demonstrated high labeling efficiency (99.0%) and stability in serum. MKN-45 cancer cells exhibited the highest cellular uptake, which could be specifically blocked by unlabeled ATPS mAb. In mice, ¹⁷⁷Lu-DOTA-ATPS mAb accumulated significantly in tumors, with a tumor uptake of 16.0 ± 1.5%ID/g on day 7. ¹⁷⁷Lu-DOTA-ATPS mAb treatment significantly reduced the viability of MKN-45 cells in a dose-dependent manner. In a xenograft tumor model, this radioimmunotherapy strategy led to substantial tumor growth inhibition (82.8%). Furthermore, combining ¹⁷⁷Lu-DOTA-ATPS mAb with sunitinib, an anti-angiogenic drug, enhanced the therapeutic efficacy of sunitinib in the mouse model. Our study successfully developed ¹⁷⁷Lu-DOTA-ATPS mAb, a radioimmunotherapy agent targeting tumor blood vessels. This approach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs.

Keywords: 177Lu; ATP synthase; angiogenesis; gastric cancer; radioimmunotherapy.

Figure 1

Schematic diagram for radiosynthesis of ¹⁷⁷Lu-DOTA-ATPS mAb.

Figure 2

Labeling efficiency (A) and in vitro stability (B) of ¹⁷⁷Lu-DOTA-ATPS mAb. The Rf value of ¹⁷⁷Lu-DOTA-ATPS mAb was between 0.01 and 0.05, while that of ¹⁷⁷LuCl₃ was between 0.6 and 1.0. The in vitro stabilities of ¹⁷⁷Lu-DOTA-ATPS mAb in serum remained unchanged up to 7 days. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody; RT, room temperature; PBS, phosphate-buffered saline.

Figure 3

The cellular uptake (A), specific binding (B), and inhibition study (C) of ¹⁷⁷Lu-DOTA-ATPS mAb. MKN-45 cells showed the highest cellular uptake of ¹⁷⁷Lu-DOTA-ATPS mAb among the tested cancer cell lines. ¹⁷⁷Lu-DOTA-ATPS mAb uptake was specific and inhibited by unlabeled ATPS mAb in MKN-45 cells. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.005 **, ns: not significant.

Figure 4

Radioimmunotherapy with ¹⁷⁷Lu-DOTA-ATPS mAb alone (A) and ¹⁷⁷Lu-DOTA-ATPS mAb in combination with sunitinib (B) in MKN-45 cells. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.005 **, p < 0.001 ***, p < 0.0005 ⁺, p < 0.00005 ⁺⁺.

Figure 5

Biodistribution of ¹⁷⁷Lu-DOTA-ATPS mAb (A), ¹⁷⁷LuCl₃ (B), and ¹⁷⁷Lu-DOTA-IgG (C) in wild-type mice on days 1, 2, 4, and 7. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody.

Figure 6

Biodistribution of ¹⁷⁷Lu-DOTA-ATPS mAb (A), ¹⁷⁷LuCl₃ (B), and ¹⁷⁷Lu-DOTA-IgG (C) in mice bearing MKN-45 tumors on day 1, 2, 4, and 7. Comparison of bone marrow and tumor uptake among radiopharmaceuticals (D). Inhibition of ¹⁷⁷Lu-DOTA-ATPS mAb uptake in tumors by unlabeled ATPS mAb (E). DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.005 **, ns: not significant.

Figure 7

Radioimmunotherapy with ¹⁷⁷Lu-DOTA-ATPS mAb. (A) Tumor growth curve during the 4-week treatment with ¹⁷⁷Lu-DOTA-ATPS mAb, unlabeled ATPS mAb, IgG, and vehicle. (B) Immunohistochemical staining with anti-CD31 antibody for MKN-45 tumors after 4 weeks of treatment. (C) ¹⁸F-FDG PET imaging in mice bearing MKN-45 tumors at baseline and at 4th week of treatment. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.01 **, arrows indicate positive staining.

Figure 7

Radioimmunotherapy with ¹⁷⁷Lu-DOTA-ATPS mAb. (A) Tumor growth curve during the 4-week treatment with ¹⁷⁷Lu-DOTA-ATPS mAb, unlabeled ATPS mAb, IgG, and vehicle. (B) Immunohistochemical staining with anti-CD31 antibody for MKN-45 tumors after 4 weeks of treatment. (C) ¹⁸F-FDG PET imaging in mice bearing MKN-45 tumors at baseline and at 4th week of treatment. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.01 **, arrows indicate positive staining.

Figure 8

Combination chemo-radioimmunotherapy with sunitinib and ¹⁷⁷Lu-DOTA-ATPS mAb. (A) Tumor growth curve during the 4-week treatment with ¹⁷⁷Lu-DOTA-ATPS mAb, sunitinib, combination, and vehicle. (B) Immunohistochemical staining with anti-CD31 antibody for MKN-45 tumors after 4 weeks of treatment. (C) ¹⁸F-FDG PET imaging in mice bearing MKN-45 tumors at baseline and 4th week of treatment. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.01 **, p < 0.005 ***, arrows indicate positive staining.

Figure 8

Combination chemo-radioimmunotherapy with sunitinib and ¹⁷⁷Lu-DOTA-ATPS mAb. (A) Tumor growth curve during the 4-week treatment with ¹⁷⁷Lu-DOTA-ATPS mAb, sunitinib, combination, and vehicle. (B) Immunohistochemical staining with anti-CD31 antibody for MKN-45 tumors after 4 weeks of treatment. (C) ¹⁸F-FDG PET imaging in mice bearing MKN-45 tumors at baseline and 4th week of treatment. DOTA, tetraazacyclododecane-1,4,7,10-tetraacetic acid; ATPS, adenosine triphosphate synthase; mAb, monoclonal antibody. p < 0.05 *, p < 0.01 **, p < 0.005 ***, arrows indicate positive staining.