177Lu Anti-angiogenic Radioimmunotherapy Targeting ATP synthase in Gastric Cancer Model

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate syn-thase (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 se-rum. 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 sig-nificantly 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, en-hanced 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 ap-proach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs.

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Medicine and Pharmacology - Oncology and Oncogenics

1. Introduction

Angiogenesis, the formation of new blood vessels, is a crucial process for normal tissue growth and tumor expansion. Disrupting this process has become a major focus for researchers aiming to develop effective cancer therapies. Tumors hijack angiogenesis to fuel their growth and metastasis [1]. This uncontrolled vessel formation is countered by endogenous inhibitors like angiostatin. Angiostatin maintains a balanced angiogenic environment by suppressing the effects of pro-angiogenic factors like vascular endothelial growth factor and fibroblast growth factor [2]. While typically residing within the mitochondrial inner membrane, adenosine triphosphate synthase (ATPS), an enzyme responsible for ATP generation, has been found on the surface of endothelial and tumor cells, called "ectopic" ATPS [3]. Specific subunits (α/β) of this ectopic ATPS serve as binding sites for angiostatin [4]. Antibodies developed against specific ATPS subunits can directly inhibit the enzyme's activity on the endothelial cell surface, which, in turn, disrupts the formation of new blood vessels and directly hinders the proliferation and migration of cancer cells [5,6,7]. We also have shown that radiolabeled anti-ATPS antibodies are promising tools for radioimmunotherapy and immuno-positron emission tomography (PET) imaging. Radioiodine (¹³¹I) labeled anti-ATPS antibody effectively suppressed the tumor growth by 2.5-fold in a gastric cancer model [8]. Anti-ATPS antibodies labeled with Zirconium-89 (⁸⁹Zr) demonstrated significantly higher targeting specificity for MDA-MB-231 tumors with abundant ectopic ATPS expression, compared to PC-3 tumors with low expression [9]. Given its role in tumor angiogenesis, ectopic ATPS emerges as a novel target for developing effective anti-angiogenic therapies.

Since the landmark report in 1981 on a successful radiolabeled antibody targeting carcinoembryonic antigen [10], radioimmunotherapy has remained a beacon of hope in the fight against cancer, offering a powerful tool for targeted therapy. Zevalin™ and Bexxar™, being used for the treatment of non-hodgkin’s lymphoma, are prime examples of radioimmunotherapy successfully employed in modern medicine. Lutetium-177 (¹⁷⁷Lu) has gained widespread popularity as a preferred radioisotope in recent years. This radioisotope demonstrates its versatility in cancer treatment. Studies have shown its effectiveness in treating two distinct cancers: unresectable metastatic neuroendocrine tumors [11] and metastatic castration-resistant prostate cancer [12]. In both cases, it is used as a targeted therapy approach (peptide receptor radionuclide therapy and radioligand therapy). ¹⁷⁷Lu has favorable characteristics for radioimmunotherapy. It emits both high-energy β-ray (Emax=761 KeV) and γ-rays (113 in 6.4% and 208 KeV in 13%) and decays with a half-life of 6.7 days [13], which is favorable for radioimmunotherapy.

This study aims to develop a new radioimmunotherapy approach that specifically targets tumor angiogenesis. We synthesized ¹⁷⁷Lu labeled anti-ATPS antibody to achieve this and evaluated its anti-tumor efficacy in a gastric cancer animal model.

2. Materials and Methods

2.1. Radiosynthesis of ¹⁷⁷Lu-DOTA-ATPS mAb

The ATPS mAb was purchased from Abcam (ab14730, MW 52 kDa; Cambridge, USA) and stored as aliquots at -78°C. ¹⁷⁷LuCl₃ was obtained from PLATOM (National Centre for Nuclear Research, Poland). For conjugation, a 50-fold molar excess of p-SCN-Bn- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, Futurechem, Seoul, Korea) in 30 µL dimethyl sulfoxide was added to the ATP mAb (100–200 µg in 20 µL 0.1 N NaHCO₃ buffer), and the reaction mixture was incubated at 37°C for 30 min. After incubation, the conjugation mixture was purified by using Slide-A-Lyzer™ Dialysis Cassettes (2K MWCO, Thermo Fisher Scientific, Rockford, IL, USA) to remove unconjugated p-SCN-Bn-DOTA. ¹⁷⁷LuCl₃ (37-111 MBq/10uL) was buffered with 0.5M NH₄OAc (pH 5.5), followed by 100–200 µg DOTA-mAb. The reaction mixture was incubated at 37°C for 30 min [14]. After completion of synthesis, the reaction mixture is purified on a size-exclusive PD-10 column (GE Healthcare), and ¹⁷⁷Lu-DOTA-ATPS mAb is separated by eluting with PBS (Figure 1). For analysis of labeled ¹⁷⁷Lu-DOTA-ATPS mAb, it is spotted on silica gel impregnated aluminum sheets (Merck, Darmstadt, Germany), developed with 0.02M citrate buffer, and confirmed by using radio-thin-layer chromatography (radio-TLC) (Bioscan, Eckert & Ziegler Radiopharma Inc. Wilmington, MA, USA). In vitro stability was measured in triplicates on the 2 hr, 24 hr, and 7th day, in PBS or serum, at 4°C, room temperature, or 37°C.

2.2. Cancer Cell Culture and Xenograft Tumor Model

All cancer cell lines, human breast adenocarcinoma (MDA-MB-231), human fibrosarcoma (HT-1080), human lung adenocarcinoma (A549), human follicular thyroid carcinoma (FTC-133), human prostate adenocarcinoma (PC-3), and human gastric adenocarcinoma (MKN-45) were purchased from the Korean Cell Line Bank (Seoul, Korea).

All cells except FTC-133 (FTC-133 cells were cultured in DMEM/F-12, 1:1 mixture, 10% fetal bovine serum, WelGENE Inc., Daegu, Korea) were cultured with RPMI-1640 medium (WelGENE) supplemented with 10% FBS and 1% penicillin/streptomycin (WelGENE) at 37^oC and 5% fully humidified CO₂. Animal experiments were performed according to protocols approved by the Care of Experimental Animals Committee (IACUC No. 2021-0067). 6-week-old female Balb/c nude mice (Orient Bio, Seongnam, Korea) were maintained under specific pathogen-free conditions [15]. To create a tumor xenograft model, 5 x 10⁶ tumor cells were mixed with phenol red-free Matrigel (Corning^®, Bedford, MA, USA), and injected subcutaneous inoculation into the right shoulder of each mouse. Experiments were performed about 10 to 14 days after injection of cells, when tumors reached a diameter of approximately 5 to 10 mm.

2.3. Cellular Uptake of ¹⁷⁷Lu-DOTA-ATPS mAb in Various Cancer Cells

Cellular uptake of ¹⁷⁷Lu-DOTA-ATPS mAb was measured in the six human cancer cell lines as described previously [8]. In brief, 5 × 10⁵ cells were seeded per well in 12-well plates and cultured for 24 hrs. Upon attachment, 37 kBq ¹⁷⁷Lu-DOTA-ATPS mAb was added to freshly replaced culture media, followed by incubation of the cells for 1, 4, or 24 hrs at 37^oC and 5% CO₂. After incubation, the cells were washed twice with cold PBS and harvested with 0.1N NaOH. Radioactivity of the cells was counted using a Gamma-HEs gamma counter (Shinjin Medics Inc., Goyang, Korea) and normalized to the cell protein content obtained using the Bradford method [16]. Data are presented as the percentage of 1-hr uptake.

2.4. Specific binding of ¹⁷⁷Lu-DOTA-ATPS mAb in MKN-45 Cells

MKN-45 cells were cultured in 12-well plates and incubated with 111 kBq free ¹⁷⁷Lu, ¹⁷⁷Lu-DOTA-ATPS mAb or ¹⁷⁷Lu-DOTA-IgG for 24 hrs at 37^oC and 5% CO₂. After incubation, the cells were washed twice with cold PBS, harvested with 0.1N NaOH, and the radioactivity was counted using a gamma counter. The cell protein content was determined using the Pierce 660^TM Protein Assay Kit for normalization (Thermo Fisher Scientific, Rockford, IL, USA). Cellular uptake was expressed as a percentage of free ¹⁷⁷Lu uptake.

2.5. Competitive Binding of ¹⁷⁷Lu-DOTA-ATPS mAb in MKN-45 Cells

Competitive inhibition of ¹⁷⁷Lu-DOTA-ATPS mAb binding was examined using unlabeled ATPS mAb in MKN-45 cells. Cells were cultured in 12-well plates and treated with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2 (as described previously). The cells were pretreated with 6.4 μM unlabeled ATPS mAb for 1 hr, while control cells were incubated with the vehicle. Then 111 kBq ¹⁷⁷Lu-DOTA-ATPS mAb was added to the cells and incubated for 4 or 24 hrs under the same conditions. After incubation, cellular uptake was calculated and expressed as a percentage relative to that of untreated control. Cellular uptake, specific binding, and inhibition experiments were all performed in triplicate.

2.6. ¹⁷⁷ Lu-DOTA-ATPS mAb Radioimmunotherapy in MKN-45 Cells

MKN-45 cells were cultured in 96-well plates and treated with: 3.7 or 7.4MBq ¹⁷⁷Lu-DOTA-ATPS mAb, unlabeled ATPS mAb, or left untreated for 24 hrs at 37^oC and 5% CO₂. To investigate the effect of combination therapy, MKN-45 cells were treated with either 7.4MBq ¹⁷⁷Lu-DOTA-ATPS mAb or 5mg/ml sunitinib (LC Laboratories^®, MA, USA), both, or left untreated for 24 hrs at 37^oC and 5% CO₂ [17]. After incubation, cell viability was measured using an XTT assay kit (Cayman Chemical, Ann Arbor, MI, USA) with a further 2-hr incubation at 37°C. Absorbance was read at 450 nm using a microplate reader (Bio-Rad Laboratories Inc.).

2.7. Biodistribution Study of Wild-Type Mice and MKN-45 Tumor Xenograft Models

Wild-type mice and MKN-45 tumor-bearing mice (n=4-5 per time point) were intravenously injected with 3.7 MBq ¹⁷⁷Lu-DOTA-ATPS mAb, ¹⁷⁷Lu-IgG, or free ¹⁷⁷Lu. The mice were then anesthetized, sacrificed, and dissected for organ radioactivity analysis at 1, 2, 4, and 7 days after injection. Major organs (heart, lung, liver, spleen, stomach, kidneys, intestine, muscle, bone), blood, and tumors were dissected, weighed, and counted for radioactivity using a gamma counter. Uptake in the organs and tumors was expressed as the percentage of the injected dose per gram of tissue (%ID/g).

2.8. Blocking Study of ¹⁷⁷Lu-DOTA-ATPS mAb in MKN-45 Tumor-Bearing Mice

To investigate blocking efficacy, 50 µg of unlabeled ATPS mAb was co-injected with 3.7 MBq ¹⁷⁷Lu-DOTA-ATPS mAb (1 µg as mAb) through the tail vein (n=2). Tumors and organs were then removed at 1 and 7 days after injection for subsequent radioactivity analysis. The organs were weighed and counted for radioactivity using a gamma counter. Results were expressed as %ID/g.

2.9. Radioimmunotherapy, Immunohistochemical Staining and ¹⁸F-FDG-PET Imaging in MKN-45 Tumor-bearing Mice

To investigate radioimmunotherapy, tumor models were intravenously injected with 18.5 MBq ¹⁷⁷Lu-DOPA-ATPS mAb, 30 μg unlabeled ATPS mAb (10 times larger than the therapeutic dosage of ¹⁷⁷Lu-DOPA-ATPS mAb), 30 μg unlabeled IgG, and vehicle (normal saline), respectively, once a week for 4 weeks (n=4 for each group) [8]. Tumor size was measured twice a week in two dimensions (length and width). Tumor volume was calculated using the formula, V = (length x width²)/2, and compared among the groups [18]. Tumor growth inhibition (TGI, %) was calculated using the formula, TGI = (1 - mean volume of treated tumors/mean volume of control tumors) x 100.

The effect of combination therapy of ¹⁷⁷Lu-DOTA-ATPS mAb and sunitinib was evaluated in mice bearing MKN-45 tumors. Mice were divided into four groups: vehicle (0.9% NaCl), 18.5 MBq ¹⁷⁷Lu-DOTA-ATPS mAb alone, 40 mg/kg sunitinib alone, or a combination of both (n=6 for each group). Mice received 18.5 MBq ¹⁷⁷Lu-DOTA-ATPS mAb and 40 mg/kg sunitinib once a week for 4 weeks [19]. Tumor size was measured twice a week as described above. Body weights were measured once a week.

For imaging, PET images were acquired 1 hr after intravenous injection of 18.5 MBq 2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) using a PET scanner (SimPET, BRIGHTONIX IMAGING^®, Seoul, Korea) before and after therapies. Mice were scanned for 20 minutes under anesthesia with isoflurane inhalation.

For immunohistochemistry, tumors were dissected immediately after the PET imaging at 4^th week. Slides were stained using an anti-CD31 antibody (ab28364, abcam) according to the manufacturer’s standard procedure.

2.10. Statistical analysis

All data are presented as means ± standard errors. The statistical comparison of cellular uptake and tumor size was evaluated by Student’s t-test and Kruskal-Wallis test using statistical software (R, version 3.1.2), and the difference was considered significant at p < 0.05.

3. Results

3.1. Labeling Efficiency and In Vitro Stability of ¹⁷⁷Lu-DOTA-ATPS mAb

The ¹⁷⁷Lu-DOTA-ATPS mAb was successfully synthesized according to the schematic representation. The radiochemical yield of ¹⁷⁷Lu-DOTA-ATPS mAb was 99.0% (Figure 2A). The in vitro stabilities of ¹⁷⁷Lu-DOTA-ATPS mAb in serum were at least 95% on the 2^nd day and 85% on the 7^th day at all temperature conditions (Figure 2B). Similarly, the in-vitro stabilities of ¹⁷⁷Lu-DOTA-ATPS mAb in PBS were at least 94% on the 2^nd day, regardless of temperature. However, on the 7^th day, the stability dropped significantly to 65%, 68%, and 91% at 4^oC, room temperature, and 37^oC, respectively (all p < 0.005).

3.2. Cellular Uptake, Specific Binding, and Inhibition of ¹⁷⁷Lu-DOTA-ATPS mAb

Six cancer cell lines were evaluated for their cellular uptake of ¹⁷⁷Lu-DOTA-ATPS (Figure 3A). MKN-45 cells exhibited a time-dependent increase in uptake reaching 189.3% ± 9.8% and 450.8% ± 13.3% of the 1-hr uptake at 4 and 24 hrs, respectively (p < 0.0005 and < 0.0001, respectively). PC-3 also showed increased uptake at both 4 hr (121.9% ± 4.9%, p < 0.05) and at 24 hr (190.7% ± 8.8%, p < 0.0005). Similarly, MDA-MB-231 (186.4% ± 7.7%, p < 0.001), HT-1080 (158.7% ± 9.5%, p < 0.005), A549 (128.0% ± 9.9%, p < 0.05), and FTC-133 (186.2% ± 4.9%, p < 0.001) showed a significant increase in cellular uptake of ¹⁷⁷Lu-DOTA-ATPS mAb at 24 hr, while their 4-hr uptake remained unchanged (p > 0.05). Notably, MKN-45 cells demonstrated significantly higher uptake compared to other cell lines at 24 hr (all p < 0.0005). This finding prompted further cellular and animal studies using MKN-45 cells.

In MKN-45 cells, the uptake of ¹⁷⁷Lu-DOTA-ATPS mAb was compared with that of free ¹⁷⁷Lu (¹⁷⁷LuCl) and ¹⁷⁷Lu-DOTA-IgG at 24 hr (Figure 3B). The cellular uptake of ¹⁷⁷Lu-DOTA-ATPS mAb (128.7% ± 3.6%) was significantly higher than that of free ¹⁷⁷Lu and ¹⁷⁷Lu-DOTA-IgG (88.5% ± 7.8%) (all p < 0.05). There was no significant difference in cellular uptake between free ¹⁷⁷Lu and ¹⁷⁷Lu-DOTA-IgG (p > 0.05).

The uptake of ¹⁷⁷Lu-DOTA-ATPS mAb was inhibited by a high dose of unlabeled ATPS mAb at both 4 hr (81.0% ± 5.9%, p < 0.005) and at 24 hr (62.2% ± 5.4%, p < 0.005) (Figure 3C). The inhibitory effect of unlabeled ATPS mAb was more pronounced at 24 hr compared to 4 hr (p < 0.05).

3.3. ¹⁷⁷ Lu-DOTA-ATPS mAb Radioimmunotherapy in MKN-45 Cells

Radioimmunotherapy with ¹⁷⁷Lu-DOTA-ATPS mAb significantly reduced cell viability (Figure 4A) compared with vehicle-treated controls. Treatment with 3.7 MBq (78.9% ± 1.2%, p < 0.005) and 7.4 MBq (70.4% ± 1.7%, p < 0.01) of ¹⁷⁷Lu-DOTA-ATPS mAb resulted in a dose-dependent decrease in viable cells (p < 0.005). Unlabeled ATPS mAb also significantly reduced cell viability (89.8% ± 0.5%, p < 0.001 vs. control), however the therapeutic effect of either dose of ¹⁷⁷Lu-DOTA-ATPS mAb was significantly greater (all p < 0.0001).

An anti-angiogenic therapy with sunitinib (5mg/ml) significantly decreased the number of viable MKN-45 cells (73.8% ± 1.0% of controls, p < 0.00005) (Figure 4B). Combination therapy with 7.4 MBq of ¹⁷⁷Lu-DOTA-ATPS mAb and sunitinib showed a greater reduction in cell viability (46.5% ± 1.5%) compared to single therapy with ¹⁷⁷Lu-DOTA-ATPS mAb (52.5% ± 0.2%, p < 0.05) or sunitinib (p < 0.00005). Notably, among single therapies, 7.4 MBq of ¹⁷⁷Lu-DOTA-ATPS mAb exhibited a greater therapeutic effect than 5mg/ml sunitinib (p < 0.00005).

3.4. Biodistribution of ¹⁷⁷Lu-DOTA-ATPS mAb in Wild-Type Mice and MKN-45 Tumor Xenograft Models

The biodistribution of ¹⁷⁷Lu-DOTA-ATPS mAb was evaluated in wild-type mice (Figure 5) and mice bearing MKN-45 tumors (Figure 6) on days 1, 2, 4, and 7. In wild-type mice, bone marrow uptake of ¹⁷⁷Lu-DOTA-ATPS mAb reached 20.4 ± 1.3%ID/g on day 1 and remained stable from day 2 to day 7. Renal uptake was highest on day 1 (25.4 ± 0.6%ID/g) and decreased slightly from day 2 to day 7 (13.0 to 17.4%ID/g). Hepatic uptake ranged from 17.9 to 20.6%ID/g.

In contrast, ¹⁷⁷LuCl primarily accumulated in the bone marrow (68.1 ± 1.2%ID/g, 70.4 ± 1.6%ID/g, 75.6±1.7%ID/g, and 72.7 ± 2.2%ID/g on days 1, 2, 4, and 7, respectively). Renal uptake peaked at 11.8 ± 0.7%ID/g on day 1 and decreased slowly thereafter. The liver uptake ranged from 5.6 to 6.2%ID/g.

For ¹⁷⁷Lu-DOTA-IgG, bone marrow uptake remained lower than that of ¹⁷⁷Lu-DOTA-ATPS mAb and ¹⁷⁷LuCl throughout the study (all < 10%ID/g). Uptake of ¹⁷⁷Lu-DOTA-IgG was similar among the liver, spleen and kidneys. Renal uptake reached a peak on day 1, while hepatic and splenic uptake increased over time.

Tumor uptake of ¹⁷⁷Lu-DOTA-ATPS mAb reached 16.0 ± 1.5%ID/g on day 7, which was significantly higher than that of ¹⁷⁷LuCl (7.6 ± 0.5%ID/g, p < 0.05) and ¹⁷⁷Lu-DOTA-IgG (8.9 ± 0.5%ID/g, p < 0.05). Similarly, on day 4, ¹⁷⁷Lu-DOTA-ATPS mAb showed greater tumor uptake (12.4 ± 0.4%ID/g) compared to ¹⁷⁷LuCl (4.2 ± 0.5%ID/g, p < 0.005) and ¹⁷⁷Lu-DOTA-IgG (8.8 ± 0.5%ID/g, p < 0.00005). There was no significant difference in tumor uptake between ¹⁷⁷LuCl and ¹⁷⁷Lu-DOTA-IgG on day 7. The biodistribution patterns of ¹⁷⁷Lu-DOTA-ATPS mAb, ¹⁷⁷LuCl, and ¹⁷⁷Lu-DOTA-IgG in the liver, spleen, kidney, and bone marrow of tumor-bearing mice mirrored those observed in wild-type mice.

In an inhibition study, a high dose of unlabeled ATPS mAb significantly reduced the tumoral uptake of ¹⁷⁷Lu-DOTA-ATPS mAb from 6.08 ± 1.0%ID/g on day 1 to 3.8 ± 1.1%ID/g (p < 0.05).

3.5. Radioimmunotherapy, Immunohistochemical Staining and ¹⁸F-FDG-PET Imaging in MKN-45 Tumor-bearing Mice

All animals survived until the end of the experiment regardless of treatment (single agent or combination). The therapeutic efficacy of single agents is shown in Figure 7A. No significant difference in initial tumor volume was observed among the groups (¹⁷⁷Lu-DOTA-ATPS mAb, 92.0 ± 15.5 mm³; unlabeled ATPS mAb, 93.6 ± 3.5 mm³; IgG, 104.6 ± 6.2 mm³; vehicle, 97.1 ± 8.6 mm³, p > 0.05). After four weeks, tumors treated with ¹⁷⁷Lu-DOTA-ATPS mAb (269.1 ± 130.4 mm³) were significantly smaller than those treated with unlabeled ATPS mAb (836.4 ± 53.1 mm³, p < 0.01), IgG (1117.1 ± 364.5 mm³, p < 0.05), or vehicle (1561.4 ± 420.4 mm³, p < 0.05). While tumors treated with unlabeled ATPS mAb or IgG displayed smaller volumes compared to controls, these differences were not statistically significant. %TGI after four weeks of treatment was 82.8% for ¹⁷⁷Lu-DOTA-ATPS mAb, 46.4% for unlabeled ATPS mAb, and 28.5% for IgG treatment. Tumor volume in the ¹⁷⁷Lu-DOTA-ATPS mAb treated group did not significantly change from baseline to 4th week (p > 0.05)

Immunohistochemistry using an anti-CD31 antibody revealed moderate to strong staining in the small vessels of the tumors treated with unlabeled ATPS mAb, IgG, or vehicle (Figure 7B). Conversely, tumors treated with ¹⁷⁷Lu-DOTA-ATPS mAb exhibited minimal staining.

Representative ¹⁸F-FDG PET images of mice after four weeks of treatment are shown in Figure 7C. Tumor volume increased in mice treated with unlabeled ATPS mAb, IgG, or vehicle at 4th week. These tumors also displayed central metabolic defects, indicative of necrotic change. In contrast, tumor volume remained stable in mice treated with ¹⁷⁷Lu-DOTA-ATPS mAb.

The efficacy of combination therapy is shown in Figure 8A. Initial tumor volume did not differ significantly among groups (¹⁷⁷Lu-DOTA-ATPS mAb + sunitinib, 209.3 ± 31.3 mm³; ¹⁷⁷Lu-DOTA-ATPS mAb, 210.3 ± 4.2 mm³; sunitinib, 207.6 ± 22.9 mm³; vehicle, 203.7 ± 18.9 mm³, p > 0.05). After four weeks, tumors treated with ¹⁷⁷Lu-DOTA-ATPS mAb (2644.4 ± 703.7 mm³, p < 0.05), sunitinib (3619.3 ± 1114.0 mm³, p < 0.05) or the combination (1727.6 ± 793.5 mm³, p < 0.01) were significantly smaller than those in the vehicle group. Combination therapy with ¹⁷⁷Lu-DOTA-ATPS mAb and sunitinib demonstrated a greater therapeutic effect compared to either single agent (¹⁷⁷Lu-DOTA-ATPS mAb, p < 0.05; sunitinib, p < 0.005). %TGI after four weeks of treatment was 70.3% for the combination, 54.6% for ¹⁷⁷Lu-DOTA-ATPS mAb alone, and 37.8% for sunitinib alone.

Immunohistochemistry using an anti-CD31 antibody revealed strong staining in the small vessels of tumors from the vehicle group only (Figure 8B). In contrast, tumors treated with ¹⁷⁷Lu-DOTA-ATPS mAb, sunitinib or the combination exhibited minimal staining. These findings indicate an anti-angiogenic effect of the therapeutic approaches.

Representative ¹⁸F-FDG PET images of mice after four weeks of treatment are shown in Figure 8C. Tumor volume increased in all groups at 4th week. Tumors treated with single agents or vehicle were larger than those treated with the combination and displayed central metabolic defects.

4. Discussion

In the present study, we aimed to develop a radioimmunotherapy agent targeting tumor angiogenesis. We achieved this by coupling anti-ATPS mAb to ¹⁷⁷Lu using DOTA as a chelator. Based on cellular uptake results, MKN-45 gastric cancer cells were selected for further development and the evaluation of the therapeutic efficacy of ¹⁷⁷Lu-DOTA-ATPS mAb. The uptake of ¹⁷⁷Lu-DOTA-ATPS mAb was specific and inhibited by unlabeled ATPS mAb in both in vitro and in vivo experiments. ¹⁷⁷Lu-DOTA-ATPS mAb demonstrated a superior therapeutic effect compared to unlabeled ATPS mAb against MKN-45 cells, both in vitro and in vivo. Furthermore, the combination of ¹⁷⁷Lu-DOTA-ATPS mAb with sunitinib significantly enhanced the therapeutic effect in mice bearing MKN-45 tumors. This enhanced efficacy was also evident in ¹⁸F-FDG PET imaging and immunohistochemistry analysis. These results suggest that radioimmunotherapy using ¹⁷⁷Lu-DOTA-ATPS mAb has potential for application in cancer therapy targeting tumor angiogenesis.

¹⁷⁷Lu is a long-lived (half-life of 6.7 days), β-ray emitting (Emax = 0.49 MeV, range = 670 µm in soft tissue) radioisotope suitable for therapy. The success of ¹⁷⁷Lu-based radioligand therapy and peptide receptor radionuclide therapy has contributed to the growing popularity of ¹⁷⁷Lu as a radioisotope for radioimmunotherapy [20]. ¹⁷⁷Lu-PSMA has shown promising results as a treatment option for metastatic castration-resistant prostate cancer following chemotherapy and hormonal therapy [21]. ¹⁷⁷Lu-[DOTA0,Tyr3]-octreotate effectively reduces tumor growth and stabilizes disease in patients with gastroenteropancreatic well-differentiated neuroendocrine tumors, leading to its establishment as a second- or third-line treatment option [22]. ¹⁷⁷Lu-labeled trastuzumab, an antibody that targets the HER2 receptor protein, exemplifies radioimmunotherapy using ¹⁷⁷Lu. This approach demonstrates specific uptake in HER2-positive primary breast cancers and their metastatic sites [23]. Radioimmunotherapy with ¹⁷⁷Lu has also been explored in anti-angiogenesis cancer treatment. ¹⁷⁷Lu-labeled TRC105, an antibody targeting CD105, demonstrated tumor uptake in mice with breast cancer. The uptake was 14.3 ± 2.3 %ID/g on day 1 and 11.6 ± 6.1 %ID/g on day 7, similar to our findings. This approach also inhibited tumor growth and improved survival [24]. These results provide strong support for the use of ¹⁷⁷Lu-labeled radiopharmaceuticals in radioimmunotherapy.

¹⁷⁷Lu, a radiometal isotope, requires a chelating agent to form a stable complex with antibodies. DOTA, diethylenetriamine pentaacetate (DTPA), and ethylene-diamine-tetraacetic acid (EDTA) are the most common chelators for radiometal isotopes [25,26]. In our preliminary study, ¹⁷⁷Lu-DOTA-ATPS mAb exhibited excellent labeling efficiency (around 99.0%) and stability in repeated experiments. Conversely, the labeling efficiency of ¹⁷⁷Lu-DTPA-ATPS mAb was significantly lower (data not shown). This aligns with previous reports [27,28]. We opted for DOTA based on these findings.

DOTA, a macrocyclic chelator offers greater in vivo stability compared to acyclic chelators like DTPA and EDTA (‘macrocyclic effect’) [25]. This translates to its recommendation for labeling ¹⁷⁷Lu. As shown in this study, free ¹⁷⁷Lu accumulates significantly in bone marrow (72.7% for wild-type mice and 69.5% for tumor-bearing mice on day 7). In vivo dissociation of ¹⁷⁷Lu-DOTA-ATPS mAb can decrease therapeutic efficacy and increases bone marrow toxicity. Despite maintaining high in vitro stability (85.5% at 37ºC on day 7) in serum, ¹⁷⁷Lu-DOTA-ATPS mAb exhibited significant bone marrow uptake of free ¹⁷⁷Lu (27.0% for wild-type mice and 39.3% for tumor-bearing mice on day 7). This can be explained by two factors: first, the presence of various blood proteins like transferrin and albumin that strongly bind to ¹⁷⁷Lu, and furthermore, the dilution of ¹⁷⁷Lu-DOTA-ATPS mAb in vivo [26]. Overcoming this limitation is crucial, as researchers are actively developing new chelators [29]. Further studies to improve the in vivo stability of ¹⁷⁷Lu-DOTA-ATPS mAb are required.

ATPS is normally located in the inner mitochondrial membranes as part of the mitochondrial respiratory complex. It participates in ATP production using a proton gradient generated by mitochondrial respiratory complex I-IV [30]. Interestingly, ATPS can also be found on the surface of some cancer and endothelial cells, known as ectopic ATPS. This ectopic ATPS can be a binding site for angiostatin [3]. Therefore, ATPS could serve as a novel target for anti-angiogenic cancer therapies. As previously demonstrated, the anti-ATPS mAb used in this study can target tumor vasculature and cancer cells [9]. In this study, ¹⁷⁷Lu-DOTA-ATPS mAb showed a significant inhibitory effect on MKN-45 gastric cancers. TGI of ¹⁷⁷Lu-DOTA-ATPS mAb (82.8%) was greater than that of unlabeled ATPS mAb (46.6%). Additionally, immunohistochemistry with an anti-CD31 antibody revealed minimal staining in tumors treated with ¹⁷⁷Lu-DOTA-ATPS mAb (Figure 7B). Similarly, minimal anti-CD31 staining was observed in tumors treated with either single-agent sunitinib or combination therapy (Figure 8B). These findings indicate that the anti-cancer efficacy of these therapeutic strategies is likely mediated through targeting tumor angiogenesis.

Combination therapy offers a significant advantage by enhancing anti-cancer effects while potentially reducing side effects compared to single-agent treatment. In this study, combination therapy demonstrated a synergistic increase in therapeutic efficacy (TGI = 70.3%) compared to sunitinib alone (37.8%). This finding suggests promising potential for the clinical application of ¹⁷⁷Lu-DOTA-ATPS mAb in the future.

¹⁷⁷Lu decays by emitting two γ-rays (208 and 113 KeV), which are ideal for gamma camera imaging. While we employed ¹⁸F-FDG PET for tumor visualization in this study, whole-body gamma camera imaging could directly visualize or predict the biodistribution of ¹⁷⁷Lu-DOTA-ATPS mAb. A limitation of this study is the lack of a small animal gamma camera, which prevented us from performing this complementary imaging modality.

Despite encouraging preclinical results, this study represents early-stage research. Further technical refinements are necessary before clinical trials with ¹⁷⁷Lu-DOTA-ATPS mAb can be initiated.

Author Contributions

Conceptualization, J.Y. and B.P.; methodology, J.Y. and B.P.; formal analysis, Y.P. and B.P.; investigation, B.P. and S.K.; writing—original draft preparation, J.Y. and B.P.; writing—review and editing, Y.A. and S.L.; funding acquisition, B.P. and Y.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A2C109081314).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine (IACUC No. 2021-0067).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data on this study are available in the manuscript. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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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 ¹⁷⁷LuCl 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.

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), ¹⁷⁷LuCl (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), ¹⁷⁷LuCl (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**.

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 4^th 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 4^th 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.

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177Lu Anti-angiogenic Radioimmunotherapy Targeting ATP synthase in Gastric Cancer Model

Abstract

Keywords:

Subject:

1. Introduction

2. Materials and Methods

2.1. Radiosynthesis of 177Lu-DOTA-ATPS mAb

2.2. Cancer Cell Culture and Xenograft Tumor Model

2.3. Cellular Uptake of 177Lu-DOTA-ATPS mAb in Various Cancer Cells

2.4. Specific binding of 177Lu-DOTA-ATPS mAb in MKN-45 Cells

2.5. Competitive Binding of 177Lu-DOTA-ATPS mAb in MKN-45 Cells