Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2026 Apr 15;69(9):10531–10544. doi: 10.1021/acs.jmedchem.5c03745

Preclinical Evaluation of 5T4-Targeted [89Zr]Zr-DFO-HB5 Immuno-PET Imaging and Comparison of 161Tb vs 177Lu Radionuclide Therapy for Colorectal Cancer

Yuanyuan Liang †, Xiangyun Li †,⊥, Hui Liang †, Tao Wu †, Yao Huang †, Shoumei Liu †, Xia Yang §,∥, Rui Luo ‡, Fanxin Ma #, Ying Huang #, Xiaoan Li †,*, Yan Zhou †,*
PMCID: PMC13182767  PMID: 41984171

Abstract

The oncofetal antigen 5T4 is highly expressed in colorectal cancer (CRC), making it a promising theranostic target. In this study, we developed the diagnostic tracer [89Zr]­Zr-DFO-HB5 (5T4 monoclonal antibody) and therapeutic agents [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5, establishing a novel theranostic strategy for CRC. We selected two 5T4-positive colorectal cancer cell lines and established subcutaneous xenograft models. Immuno-PET imaging revealed specific tumor targeting of [89Zr]­Zr-DFO-HB5 in 5T4-positive CRC mouse models. Ex vivo biodistribution studies showed high and sustained tumor uptake of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5, with gradually decreasing radioactivity accumulation in normal organs over time. Targeted radionuclide therapy (TRT) further confirmed that [177Lu]­Lu/[161 Tb]­Tb-DOTA-HB5 significantly inhibited tumor growth in both models. Biosafety evaluation revealed only mild hematological toxicity, without obvious short-term organ damage. In conclusion, [89Zr]­Zr-DFO-HB5 enables noninvasive detection of 5T4 expression, while [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 exhibits potent antitumor efficacy and favorable biosafety. The study provides a potential theranostic platform for 5T4-positive CRC and related malignancies.

graphic file with name jm5c03745_0009.jpg

graphic file with name jm5c03745_0007.jpg

Introduction

In recent years, colorectal cancer (CRC) has become increasingly severe, with persistently high incidence and mortality. CRC ranks third in incidence and second in mortality among malignant tumors worldwide. The proportion of early diagnosis of CRC is decreasing, whereas the proportion of advanced-stage patients has increased significantly. The prognosis of patients is closely related to disease stage; the 5-year survival rate of early stage patients exceeds 90%, whereas that of stage IV patients is only approximately 10%, and the overall average 5-year survival rate remains below 60%. Currently, the clinical management of CRC involves a comprehensive system centered on surgical resection combined with radiotherapy and chemotherapy, which is widely applied in clinical practice and has improved survival outcomes for some patients.

The rise of targeted therapy and immunotherapy has brought renewed hope for patients with CRC, and more novel drug targets have been discovered through in-depth studies of tumorigenesis and development mechanisms. Oncoprotein 5T4, also known as trophoblast glycoprotein (TPBG), is a promising target expressed in a variety of malignant solid tumors, such as colorectal, nonsmall cell lung, ovarian, pancreatic, renal, gastric, bladder, cervical, and breast carcinoma, while its expression is low or almost nonexistent in the epithelial cells of normal adult tissues. − High 5T4 expression is closely associated with poor prognosis in patients with CRC, as evidenced by an increased risk of tumor recurrence, enhanced tumor migration and invasiveness, and ultimately shorter survival time. ,, In a clinical study of 72 CRC patients, 40% of CRC patients with 5T4-positive tumors had a 5-year survival rate of 22%, with a median survival of 24 months, whereas 5T4-negative patients had a 5-year survival rate of 75%, with a median survival of more than 90 months, suggesting that the expression status of 5T4 is significantly correlated with CRC prognosis.

Molecular targeted therapies face the clinical challenges of tumor resistance and recurrence. The development of novel diagnostic and therapeutic methods with high sensitivity and specificity has far-reaching clinical significance and application value. Radionuclide drug conjugates (RDCs), as emerging, precise targeted tumor drugs, utilized the tumor-targeting property of ligands to deliver different radionuclides to tumor cells, with significant advantages in tumor theragnostics; the resultant targeted radionuclide therapy (TRT) combines radiotherapy and molecular targeting, exhibiting excellent antitumor efficacy. − Zirconium-89­(89Zr) emits β+ particles (Eβaverage= 390 keV, Eβmax = 890 keV) with a half-life of 78.4 h. It exhibits low positron energy and a short positron mean free path, thereby enabling higher spatial resolution in PET imaging. , Its physical half-life matches the pharmacokinetic half-life of antibodies, and it has been widely used in antibody radiolabeling and positron emission tomography (PET) imaging studies. , Lutetium-177 (177Lu) emits β– particles (Eβaverage = 134 keV, Eβmax = 497 keV) with a half-life of 6.71 days, which offers adequate time for tumor treatment, timely radioactive decay, and subsequent clearance from the body. Thus, it is an ideal radionuclide for tumor therapy and pairing with antibody molecules. , In addition, the average diameter of 177Lu in tissues is approximately 670 μm, with a maximum tissue range of ∼2 mm. The radiation energy is primarily deposited locally within the tumor, and it is less damaging to the surrounding normal tissues. Consequently, 177Lu offers a favorable therapeutic safety. However, its efficacy is insufficient to eliminate micrometastases, thus limiting the achievement of long-term disease control in patients. Similar to 177Lu, terbium-161 (161Tb) (with a half-life T 1/2 of 6.95 days) can also serve as a therapeutic radionuclide. Critically, in addition to decaying via the emission of medium-energy β– particles (Eβaverage = 154 keV) 161Tb releases a large number of conversion and Auger electrons, which exhibit an extremely short range (<5 μm) within tissue. , Their high LET (4–26 keV/μm) can significantly enhance the therapeutic efficacy against individual and clusters of cancer cells. − This additional emission of Auger electrons renders161 Tb superior to177 Lu for the treatment of micrometastases, as it compensates for the limitations of β– particles in energy deposition at the microscopic scale.

The 5T4 monoclonal antibody prepared by our team at the preliminary stage demonstrated excellent targeting capability and high binding affinity, specificity, and internalization efficiency. The 5T4-targeting antibody–drug conjugates (ADCs) exhibit potent therapeutic efficacy, indicating promising clinical potential for CRC diagnosis and treatment. Given the demonstrated utility of this antibody as a targeting ligand for RDCs, we utilized its HB5 clone for the subsequent development of the radiopharmaceutical. In this study, [89Zr]­Zr-DFO-HB5 immunPET imaging was employed to identify 5T4-expressing CRC, followed by [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 for radioimmunotherapy to achieve theragnostic integration, and the antitumor efficacy and in vivo safety of 177Lu- and 161Tb-labeled conjugates were compared (Figure A). The aim of this study was to provide a promising theragnostic strategy for the precise diagnosis and safe treatment of CRC.

1.

1

Open in a new tab

(A) The expression levels of 5T4 in different colorectal cancer cell lines (n = 3). (B) The binding capacity of HB5, DOTA-HB5, and DFO-HB5 in HCT116 and HT29 cells (n = 3).

Result

DOTA/DFO-HB5 Exhibited Favorable Cellular Binding Capacity

As shown by flow cytometry, compared with that in LoVo cells, 5T4 expression was higher in HT29 and HCT116 cells (Figure A). Therefore, these two cell lines were used to construct a 5T4-positive colorectal cancer subcutaneous tumor model. In addition, flow cytometry analysis confirmed that antibody modification with DOTA/DFO did not alter the affinity for target binding (Figure B).

89Zr/177Lu/161Tb-HB5 Possessed Satisfactory Radiochemical Characteristics and In Vitro Stability

The radiolabeling purity of [89Zr]­Zr-DFO-HB5 and [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 determined by radio-iTLC exceeded 99% (Figure A). HB5 was determined to be >99% pure by HPLC analysis. In addition, radio-HPLC analysis showed that the RCP of [89Zr]­Zr-DFO-HB5 and [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 exceeded 99%, indicating that radiolabeling did not affect the structural integrity of the antibody (Figure B). The RCP remained above 99% after incubation in physiological saline or 10% FBS at 25 °C for 14 days, indicating good stability in vitro (Figure C).

2.

2

Open in a new tab

Evaluation in vitro of 89Zr/177Lu/161Tb-HB5. (A) Integration diagram of radio-instant thin-layer chromatography (Radio-iTLC) of 89Zr/177Lu/161Tb-HB5 (n = 3). (B) The high-performance liquid chromatography (HPLC) results of unlabeled HB5 and 89Zr/177Lu/161Tb-HB5 (n = 3). (C) Stability of 89Zr/177Lu/161Tb-HB5 in physiological saline and 10% FBS at different time points detected by Radio-iTLC. Radioactive cell saturation binding assay of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 to determine Bmax, K d and EC50 in HCT116 cells (D, E, F, G), Bmax, K d and EC50 in HT29 cells (H, I, J, K) (n = 3).

[177Lu]­Lu-DOTA-HB5 Showed Higher Binding Affinity and Capacity In Vitro Than [161Tb]­Tb-DOTA-HB5

The cell radioactive binding assay demonstrated that the saturation binding amount (Bmax), half-maximal effective concentration (EC50), and equilibrium dissociation constant (K d) of [177Lu]­Lu-DOTA-HB5 to HCT116 were 284.40 ± 50.65 fmol (Figure D), 40.58 nmol/L (Figure E), and 32.95 ± 7.36 nmol/L (Figure E), respectively. The Bmax, EC50, and K d of [161Tb]­Tb-DOTA-HB5 to HCT116 cells were 73.24 ± 28.00 fmol (Figure F), 42.48 nmol/L (Figure G), and 110.20 ± 32.89 nmol/L (Figure G), respectively. For HT29 cells, the Bmax, EC50, and K d of [177Lu]­Lu-DOTA-HB5 were 159.50 ± 21.90 fmol (Figure H), 15.49 nmol/L (Figure I), and 9.25 ± 2.04 nmol/L (Figure I), respectively, the Bmax, EC50, and K d of [161Tb]­Tb-DOTA-HB5 were 71.09 ± 15.95 fmol (Figure J), 20.52 nmol/L (Figure K), and 31.12 ± 9.73 nmol/L (Figure K), respectively. In summary, [177Lu]­Lu-DOTA-HB5 demonstrated a higher Bmax and lower EC50 and K d values compared to [161Tb]­Tb-DOTA-HB5, suggesting greater binding affinity and capacity to the target in vitro.

[89Zr]­Zr-DFO-HB5 Visualized 5T4-Positive Tumors by PET/CT Imaging

PET/CT imaging of [89Zr]­Zr-DFO-HB5 was conducted in HCT116 tumor-bearing mice at various time points within 8 days subsequent to intravenous injection. As presented in the representative PET/CT images (Figure A), [89Zr]­Zr-DFO-HB5 in the HCT116 tumor model clearly depicted the tumor morphology at 24 h postinjection (p.i.). The radioactive uptake of the tumor gradually augmented over time and persisted for 192 h. Subsequently, regions of interest (ROIs) were delineated, and the radioactive uptake by the heart (blood), tumor, and liver was quantitatively analyzed as in Figure B. Tumor uptake increased over time and reached its peak at 144 h, with a mean standardized uptake value (SUVmean) of 1.73 ± 0.73, followed by a decline, with a SUVmean value of 1.60 ± 0.16 at 192 h. The radioactive uptake in the heart and liver tissues reached their peaks within the initial 4 h after injection, with SUVmean values of 2.98 ± 0.30 and 1.66 ± 0.22, respectively, and then decreased to 1.21 ± 0.19 and 1.33 ± 0.16 at 192 h, respectively. Conversely, dynamic observation of the radioactive uptake of [89Zr]­Zr-DFO-IgG in HCT116 tumor-bearing mice and [89Zr]­Zr-DFO-HB5 in 5T4-negative LoVo tumor-bearing mice indicated that neither probe showed significant radioactive accumulation in the tumors (Figure C, D). The above findings suggested that [89Zr]­Zr-DFO-HB5 could efficiently and specifically target and recognize 5T4-positive tumors, enabling its visualized detection. Meanwhile, the probe exhibited a relatively fast clearance rate from normal organs, which endowed it with potential application as a diagnostic probe targeting 5T4-positive tumors.

3.

3

Open in a new tab

ImmunoPET imaging results. (A, C, D) Representative [89Zr]­Zr-DFO-HB5/IgG PET/CT images in HCT116/LoVo subcutaneous tumor-bearing BALB/c nude mice from 4 to 192 h after injection (n = 3). (B) Quantitative ROI analysis of tumor, heart, and liver in HCT116 tumor-bearing mice after injection of [89Zr]­Zr-DFO-HB5. Data are expressed as mean ± SD (n = 3).

[177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 Exhibited Favorable Biodistribution in 5T4-Positive CRC Mouse Models

To further investigate the pharmaceutical characteristics of radiolabeled HB5 and evaluate whether a change in the radioisotopes could significantly alter the biodistribution of the intact antibody agent, multiple time-point biodistribution studies of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 were conducted in the HT-29 and HCT116 tumor models.

The results of time-dependent biodistribution studies are presented in Figure A-D. [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 showed a similar distribution profile in major organs and tissues between HCT116 and HT29 tumor models. The tumor uptake kinetics of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 in both models exhibited a characteristic “rise-and-fall” pattern. The tumor uptake of [177Lu]­Lu-DOTA-HB5 peaked on day 4 postinjection at 12.97 ± 2.97%ID/g in the HCT116 models (Figure A) and 19.56 ± 10.15%ID/g in the HT-29 models (Figure B), respectively. In contrast, [161Tb]­Tb-DOTA-HB5 exhibited excellent tumor accumulation. In the HCT116 model, tumor uptake peaked at day 3 postinjection (22.74 ± 3.04%ID/g) (Figure C), while in the HT29 model, peak uptake occurred later, on day 5 postinjection, reaching a high value of 35.04 ± 4.35%ID/g (Figure D).

4.

4

Open in a new tab

Ex vivo biodistribution and TRT studies of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 in BALB/c nude mice. (A, B, C, D) Biodistribution of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 in HCT116 and HT29 tumor-bearing mice over time (n = 3). (E, F, G, H) Tumor-to-liver (T/L), tumor-to-spleen (T/S), tumor-to-kidney (T/K) and tumor-to-blood (T/B) ratios at multiple time after injection of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 (n = 3). (I, J, K, L) Comparison of the biodistribution between [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 and block groups of HCT116 and HT29 models at 72 h after injection (n = 3). (M, N) Tumor growth curves after injection of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5, HB5 only, and control in HCT116 and HT29 tumor-bearing mice (n = 8). (N, P) Tumor growth inhibition rates of treatment with [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5, HB5 only, and control in HCT116 and HT29 tumor-bearing mice at the end of treatment (n = 8). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[177Lu]­Lu-DOTA-HB5 and [161Tb]­Tb-DOTA-HB5 exhibited comparable distributions in other normal organs except for the liver. [177Lu]­Lu-DOTA-HB5 exhibited significant nonspecific uptake in normal organs such as the liver and blood, with particularly prominent accumulation in the liver. In the HCT116 tumor-bearing mice, hepatic uptake peaked at day 3 postinjection, reaching 13.88 ± 2.25%ID/g (Figure A). In the HT29 model, peak hepatic uptake occurred earlier, at day 2 postinjection, with a value of 20.95 ± 5.85%ID/g, after which it gradually decreased in both models (Figure B). [161Tb]­Tb-DOTA-HB5 showed obvious nonspecific uptake in the blood, peaking at day 1 postinjection in both models: 20.74 ± 2.81%ID/g and 16.37 ± 9.54%ID/g in HCT116 (Figure C) and HT29 (Figure D), respectively, followed by a gradual decline over time.

The tumor-to-liver (T/L), tumor-to-spleen (T/S), tumor-to-kidney (T/K), and tumor-to-blood (T/B) ratios are shown in Figure E-H. The T/L, T/S, and T/K ratios of [177Lu]­Lu-DOTA-HB5 peaked at 3–4 days. The T/K value was notably higher than the T/L, T/S, and T/B values, reaching 3.88 ± 1.05 in the HCT116 tumor model on day 3 (Figure E) and 6.68 ± 3.86 in the HT29 model on day 4 (Figure F). However, the T/L ratio was extremely low, with peak values of only 1.42 ± 0.52 and 1.79 ± 0.86 in the HCT116 (Figure E) and HT29 (Figure F) models, respectively. In contrast, the tumor-to-normal T/N ratios of [161Tb]­Tb-DOTA-HB5 continued to increase from day 1 to day 9, indicating that the distribution discrepancy between the tumor and other normal organs became increasingly obvious over time. The T/K ratio of [161Tb]­Tb-DOTA-HB5 was high, with maximum T/K values reaching 5.70 ± 0.82 and 7.20 ± 0.46 in the HCT116 (Figure G) and HT29 models (Figure H), respectively. Moreover, compared with [177Lu]­Lu-DOTA-HB5, [161Tb]­Tb-DOTA-HB5 also exhibited more desirable T/L ratios, with the maximum value reaching 3.61 ± 0.34 in the HCT116 model on day 5 (Figure G), and further rising to 6.39 ± 1.52 in the HT29 model on day 9 (Figure H). These findings indicated that [161Tb]­Tb-DOTA-HB5 had a longer tumor retention time in vivo, along with more thorough clearance from normal tissues, demonstrating superior tumor uptake and biodistribution properties.

Additionally, compared with the nonblockade groups, preadministration of an unlabeled antibody in the blockade groups resulted in reduced tumor uptake. For [177Lu]­Lu-DOTA-HB5, the tumor uptakes of the nonblocking group and blocking group were 10.10 ± 1.43%ID/g vs 4.18 ± 0.05%ID/g (p < 0.001) in HCT116 model (Figure I); the tumor uptakes of the nonblocking group and blocking group were 19.00 ± 3.48%ID/g and 4.59 ± 0.25%ID/g (p < 0.001) in HT29 model (Figure J). For [161Tb]­Tb-DOTA-HB5, the tumor uptakes of the nonblocking group and blocking group were 22.74 ± 3.04%ID/g vs 4.72 ± 0.34%ID/g (p < 0.001) in HCT116 model (Figure K); the tumor uptakes of the nonblocking group and blocking group were 28.53 ± 3.91%ID/g and 5.37 ± 1.58%ID/g (p < 0.001) in HT29 model (Figure L), whereas uptake in other nontarget organs remained unaffected by blocking, demonstrating the specificity of radioimmunoconjugates for 5T4-positive tumors.

[177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 Demonstrateed Significant Antitumor Responses in 5T4-Positive CRC Mouse Models

To investigate and compare the broad applicability of [177Lu]­Lu-DOTA-HB5 and [161Tb]­Tb-DOTA-HB5 as TRT agents, we further assessed the antitumor efficacy of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 in HCT116 and HT29 tumor-bearing mice. [177Lu]­Lu-DOTA-HB5 exhibited significant antitumor efficacy in both tumor models. Compared with the control and the HB5 groups, tumor growth in the [177Lu]­Lu-DOTA-HB5 group was significantly slower (p < 0.001). On day 28, the tumor volumes of [177Lu]­Lu-DOTA-HB5 in the HCT116 and HT29 models reached 933.63 ± 350.76 (Figure M) and 375.05 ± 141.00 mm3 (Figure O), respectively, with relative tumor growth inhibition rates of 44.78 ± 20.75% (Figure N) and 63.72 ± 13.64% (Figure P). The antitumor effect of [161Tb]­Tb-DOTA-HB5 was similar to that of [177Lu]­Lu-DOTA-HB5. The tumor growth in the [161Tb]­Tb-DOTA-HB5 group was also significantly slower compared with that in the control and HB5 groups in both models (p < 0.001). By day 28, the HCT116 and HT29 tumor volumes in the [161Tb]­Tb-DOTA-HB5 group reached 684.37 ± 262.12 (Figure M) and 278.58 ± 108.47 mm3 (Figure O), with relative tumor growth inhibition rates of 59.52 ± 15.50% (Figure N) and 73.06 ± 10.49% (Figure P), respectively. Although there was no significant difference in tumor growth inhibition rates and volumes between the 177Lu and 161Tb groups, [161Tb]­Tb-DOTA-HB5 demonstrated superior antitumor efficacy compared with [177Lu]­Lu-DOTA-HB5.

In addition, the IHC staining results revealed that the [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 (at a dose of 11.1 MBq) can effectively inhibit tumor cell proliferation and promote tumor cell apoptosis (Figure ). In conclusion, the above experimental results demonstrate that [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 TRT exhibited persistent antitumor responses in HCT116 and HT29 mouse models.

5.

5

Open in a new tab

Immunohistochemical staining for proliferation marker ki67 and cell apoptosis marker TUNEL in tumor specimens. Representative IHC images (A, C, E, G) and semiquantitative analysis (B, D, F, H) of HCT116 and HT29 tumors after different treatments (n = 8). Scale bars, 50 μm. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 Showed Promising Biosafety

Based on the results of the biodistribution studies, we found that organs such as the heart, liver, spleen, lungs, and kidneys exhibited relatively high nonspecific uptake and were susceptible to toxicity induced by radiation exposure. The intestines, which are typical radiation-sensitive organs, also faced the risk of radiation damage. Therefore, we selected the aforementioned organs for H&E staining to systematically evaluate the degree of radiation-induced damage. The H&E staining results of the two tumor models exhibited no significant histological damage or abnormal cellular morphological changes in vital organs in the [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 injection groups compared with the control groups (Figure A, B). No evident short-term off-target toxicity of the drug to normal organs in vivo was observed, further suggesting that [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 had good safety, demonstrating its potential value for application in subsequent clinical research.

6.

6

Open in a new tab

Biosafety evaluation of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 in HCT116 and HT29 tumor-bearing mice. (A, B) H&E staining of major organs (n = 8). (C, D) WBC, RBC, PLT, and HGB counts of each group at 28 days postinjection (n = 8). (E, F) The body weight curves during treatment (n = 8). Scale bars, 50 μm. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

A complete blood count was performed at the end of the treatment. In both models, compared with the control group and HB5 group, the [177Lu]­Lu-DOTA-HB5 treatment group exhibited a significant decrease in PLT levels; in addition, the treatment group also showed a significant reduction in the levels of WBCs, RBCs, and HGBs, specifically in the HCT116 model (Figure C, D), suggesting that the radiological effects of 177Lu may induce mild hematological toxicity. In contrast, [161Tb]­Tb-DOTA-HB5 showed significantly lower toxicity; compared with the HB5 group, the treatment group had a significant PLT reduction only in the HT-29 model and an RBC reduction only in the HCT116 model (Figure C, D).

During the observation period, no deaths occurred in any of the groups. Moreover, no significant differences were observed in diverse parameters, including diet, activity, and mental state. Although [177Lu]­Lu-DOTA-HB5 and [161Tb]­Tb-DOTA-HB5 induced slight body weight fluctuations in the HT29 and HCT116 models, respectively. By the end of the treatment monitoring period, the body weight returned to normal levels (Figure E, F).

Discussion

CRC is a substantial source of morbidity and mortality worldwide, with over 45% of CRC patients having metastatic disease at initial diagnosis, and the five-year survival rate for patients in advanced stages is below 10%. Patients with advanced disease may experience a high rate of local recurrence after undergoing treatments such as surgery, chemotherapy, radiotherapy, and immunotherapy. Therefore, novel diagnostic and therapeutic strategies must be developed.

In the study, we explored the 5T4-targeting radionuclide theranostics strategy to CRC. First, we identified CRC models suitable for 5T4-targeted radioimmunotherapy by 89Zr-based PET/CT imaging, then employed HB5 antibody as a targeting carrier to deliver 177Lu and 161Tb to 5T4-positive tumor, thereby enabling the local release of ionizing radiation within the tumors and achieving specific targeted killing of tumor cells. Several preclinical studies have demonstrated the superiority of 161Tb over 177Lu, − which can be attributed to the additional emission of internal conversion and Auger electrons of 161Tb, thus delivering higher radiation doses to targeted tumor cells and their vicinity. Therefore, in this study, we conducted a comparative analysis of the therapeutic efficacy of 177Lu/161Tb-labeled HB5 in colorectal cancer and aimed to evaluate the potential application advantages of 161Tb compared with 177Lu.

Interestingly, cell binding assays demonstrated that [177Lu]­Lu-DOTA-HB5 exhibited superior affinity and binding capacity at the cell membrane level compared to [161Tb]­Tb-DOTA-HB5, while subsequent animal studies suggested that [161Tb]­Tb-DOTA-HB5 may possess more potent antitumor efficacy. This seemingly contradictory results actually reflected the differences in physical properties and in vitro/vivo biological effects between the two radionuclides. Although [177Lu]­Lu-DOTA-HB5 showed stronger membrane-binding ability, this did not necessarily imply superior radiation effects following internalization. In contrast, besides emitting β– particles, 161Tb can also release Auger electrons; if it can be internalized and approach the cell nucleus, it may cause more extensive DNA double-strand breaks, thereby exerting a more potent cytotoxic effect. , Furthermore, the high affinity of [177Lu]­Lu-DOTA-HB5 may lead to the “binding site barrier” effect, which involves excessive binding at the periphery of the tumor, which can impede further penetration into deeper tumor regions, thereby compromising its antitumor efficacy. In contrast, [161Tb]­Tb-DOTA-HB5 exhibits a slightly lower binding affinity, which may facilitate its broader diffusion within tumor tissues and enhance its overall therapeutic performance. , Therefore, while in vitro binding assays are important tools for optimizing targeted drugs, they cannot fully simulate the complex pharmacokinetic and radiobiological effects involved in in vivo therapy. This also explains why the antitumor efficacy observed in mouse models may far exceed the expectations based on in vitro binding assays.

Biodistribution studies demonstrated significant tumor uptake of both [177Lu]­Lu-DOTA-HB5 and [161Tb]­Tb-DOTA-HB5 in HCT116 and HT29 xenograft models. Notably, tumor uptake of [161Tb]­Tb-DOTA-HB5 consistently exceeded that of its 177Lu-labeled counterpart. This enhancement is likely attributable to the additional emission of conversion and Auger electrons from 161Tb, which contribute to a localized dose escalation effect. Regarding distribution in normal tissues, both radioconjugates showed notable accumulation in the blood. However, a distinct difference was observed in hepatic uptake: [177Lu]­Lu-DOTA-HB5 exhibited higher liver retention, whereas [161Tb]­Tb-DOTA-HB5 demonstrated more favorable liver uptake profiles. This suggests that the choice of radionuclide influences the hepatic distribution characteristics of the agent. Despite initial concerns about potential hepatotoxicity and hematotoxicity, post-treatment analyses revealed acceptable safety profiles. Histological evaluation showed no significant liver damage in mice treated with [177Lu]­Lu-DOTA-HB5. Hematological analysis indicated that [161Tb]­Tb-DOTA-HB5 exhibited good hematological safety, while [177Lu]­Lu-DOTA-HB5 may induce mild hematotoxicity, indicating a potential bone marrow suppressive effect. It has been reported that mice may exhibit lower sensitivity to adverse effects from radiopharmaceutical therapies compared to humans, which might explain the absence of severe health impacts in this study. , Consequently, these mild toxicities were not investigated further.

Therapeutic efficacy studies confirmed that both [177Lu]­Lu-DOTA-HB5 and [161Tb]­Tb-DOTA-HB5 significantly suppressed tumor growth, accompanied by only minor body weight changes and acceptable hematological toxicity. Although a trend suggesting superior antitumor efficacy for 161Tb over 177Lu was observed, consistent with findings from previous reports, − , the differences in tumor volume progression and relative tumor growth inhibition rates between the two agents did not reach statistical significance in our study. We hypothesize that this difference may be related to the properties of the targeting ligand. Studies have shown that the extent of the discrepancy in tumor growth inhibition between 161Tb- and 177Lu-labeled drugs correlates with the internalization characteristics of the specific PSMA-targeting ligand used. This observation provides a rationale for the future personalized selection of radionuclides based on both the target and ligand characteristics. Furthermore, the unlabeled HB5 control group showed no significant antitumor effect, confirming that the therapeutic efficacy of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 is primarily mediated by the radiation emitted from the radionuclides rather than by the antibody itself.

Currently, researches on 5T4-targeted radiopharmaceuticals are mostly limited to single diagnostic applications and significantly constrained by the inherent characteristics of the carriers. He et al. developed [68Ga]­Ga-NOTA-H006 using a nanobody (VHH) as the targeting vector. Although it has achieved specific PET imaging of 5T4-positive lesions, the relatively short circulation half-life of VHH results in limited drug accumulation at the tumor site. The study currently focuses solely on the evaluation of diagnostic efficacy and has not conducted exploratory development of therapeutic functions. Li et al. reported an 89Zr-labeled PEGylated diabody, while PEG modification prolonged the carrier’s circulation half-life, precise regulation of the molecular weight and configuration of PEG is imperative. Moreover, its antigen-binding affinity is notably lower than that of intact antibodies, with no involvement in therapeutic function development. In this study, we innovatively constructed a 5T4-targeted radionuclide theranostic system, selecting a full-length 5T4 antibody as the carrier: the diagnostic arm employs 89Zr, whose pharmacokinetic properties are highly compatible with the full-length antibody, enabling precise imaging of 5T4 expression in tumors; the therapeutic arm utilizes radionuclides such as 177Lu and 161Tb for labeling. Relying on the high specificity and sustained tumor retention capacity of the full-length antibody, it achieves targeted radiotherapeutic effects, effectively remedying the limitation that most 5T4-targeted radiopharmaceutical studies are confined to diagnostic applications. However, it is crucial to note that the spatial volume of full-length IgG molecules and the interaction between their Fc domains and the neonatal Fc receptor (FcRn) on endothelial cells prolong the serum half-life of radioimmunoconjugates. − This leads to a slowed blood clearance rate and increased radioactivity concentration in healthy tissues, thereby causing a dose ratio imbalance between targeted and nontargeted tissues, which may reduce the contrast of diagnostic images or compromise therapeutic outcomes. In contrast, antibody fragments retain the antigen-binding activity of full-length antibodies and, due to their smaller molecular size, exhibit stronger tumor penetration ability and superior rapid pharmacokinetic profiles, making them a more promising carrier option for the diagnostic arm. , This provides a valuable insight for the subsequent development of theranostic-compatible carriers: the diagnostic segment can employ short half-life radionuclides such as 68Ga to label small-molecule antibody fragments, leveraging their advantages of rapid targeted imaging and in vivo clearance to further enhance diagnostic efficiency and clinical safety. Therefore, our future research will focus on antibody engineering modification, integrating the aforementioned research insights and the application experience of full-length antibodies to optimize the pharmacokinetic characteristics of the HB5-based theranostic carrier, aiming to accelerate the translation of the theranostic system from laboratory research to clinical practice.

Conclusion

We developed the [89Zr]­Zr-DFO-HB5 probe based on the 5T4 target as a noninvasive probe, offering potential for precise screening of patients with CRC who may benefit from 5T4-targeted therapy. The developed TRT agents [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 significantly inhibited tumor growth with favorable safety profiles, whereas 161Tb demonstrated advantages as a novel therapeutic nuclide compared to 177Lu. Moreover, as a broadly expressed gastrointestinal tumor target, 5T4 exhibits high expression in tumors such as gastric and renal cancers. , Therefore, [89Zr]­Zr-DFO-HB5 and [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 held broad clinical application prospects as diagnostic or therapeutic probes across all tumors with high 5T4 expression.

Materials and Methods

Materials

The anti-5T4 monoclonal antibody HB5 was obtained from Sound Biopharmaceuticals (Chengdu, China). The affinity of HB5 was determined to be 1.53 × 10–9 mol/L by Biacore analysis, and its pI was characterized as approximately 7.94 via iCIEF. Additionally, HB5 exhibited excellent thermal, acid, and freeze–thaw stability. All the chelators, including p-SCN-Bn-DFO and p-SCN-Bn-DOTA, were purchased from Macrocyclics (USA). The positron-emitting radionuclide 89Zr was produced via the (p, n) reaction on natural 89Y using the Jiuyuan 11-MTS cyclotron at Mianyang Central Hospital, followed by separation of 89Zr using an automatic 89Zr separation device (China Longevity Beam Technology Corporation). Carrier-free 177Lu and 161Tb were obtained from the Institute of Nuclear Physics and Chemistry at the China Academy of Engineering Physics (Mianyang, China).

Chelator Conjugation and Characterization

HB5 underwent three cycles of buffer exchange and concentration using Na2CO3–NaHCO3 buffer (pH 9.5) in ultrafiltration centrifuge tubes with a cutoff value of 50 kDa. p-SCN-Bn-DOTA and p-SCN-Bn-DFO were dissolved in dimethyl sulfoxide (DMSO). Subsequently, p-SCN-Bn-DOTA or p-SCN-Bn-DFO solutions were added to aliquots of the antibody solution (≥10 mg/mL) with a molar chelator/antibody ratio of 10:1. Then, the mixture was reacted at 37 °C with shaking at 70 rpm for 1 h. Subsequently, the conjugated DFO-HB5 and DOTA-HB5 were each purified using 0.25 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.0) and 0.5 M NaOAc (pH 5.5) buffers in three cycles, respectively. Finally, the concentration of the conjugated antibody was determined with an ultramicro spectrophotometer. The remaining DFO/DOTA-HB5 solution was aliquoted and stored at 4 °C until further use.

Radiolabeling and Quality Control

For 89Zr-labeling, the [89Zr]­Zr­(C2O4)2 solution was mixed with 2 M Na2CO3 buffer solution, resulting in a final reaction pH of 7. Then, DFO-HB5 was added at a labeling ratio of 0.5 μg/μci (1 μci = 37 kBq) to the solution, and it was incubated with gentle shaking at 37 °C for 60 min. For 177Lu labeling, the [177Lu]­LuCl3 solution was neutralized to pH 5.5 with 0.5 M NaOAC buffer and reacted with the purified DOTA-HB5 (at a 0.5 μg/μci labeling ratio) with continuous shaking at 42 °C for 60 min. The 161Tb-labeling protocol was the same as that for 177Lu. The molar specific activities for all radiolabeled agents were 11.1 GBq/μmol. The radiolabeling purity of [89Zr]­Zr-DFO-HB5 and [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 was assessed by radio instant thin-layer chromatography (radio-iTLC) using 0.5 M sodium citrate buffer (pH 5.5) as the unfolding solvent and iTLC-SG chromatography paper as the solid phase. For the radiochemical purity of (RCP) detected by radioactive high-performance liquid chromatography (radio-HPLC; Shimadzu, Japan), with the specific sample parameters as follows, radiolabeled-HB5 group: radioactivity of 1.85 MBq, total volume of 60 μL; HB5 group: concentration of 1 mg/mL, total volume of 60 μL. An injection volume of 25 μL was adopted for all the aforementioned samples. The radiochemical stability was monitored by radio-iTLC after adding [89Zr]­Zr-DFO-HB5 and [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 to either physiological saline or 10% FBS at 25 °C for 14 days at a concentration of 37 MBq/mL.

Cell Culture

The human cancer cell lines LoVo, HCT116, and HT29 were purchased from Procell Life Science & Technology (Wuhan, China). LoVo cells were cultured in F-12K medium, while HCT116 and HT29 cells were maintained in McCoy’s 5A medium, with all medium supplemented with 10% FBS and 1% penicillin–streptomycin solution. All cells were cultured at 37 °C under a 95/5% air/CO2 atm.

Flow Cytometry Binding Assay

Cells were collected in a 96-well cell culture plate at 4.5 × 105 cells per well (100 μL) in PBS containing 1% FBS and centrifuged at 2000 rpm for 3 min to discard the supernatant. The cells were incubated with HB5/DFO-HB5/DOTA-HB5 (5 μg/mL, 200 μL) for 60 min at 25 °C. Subsequently, the cells were pelleted and washed three times with PBS (1% FBS) to remove the unbound antibody, and then incubated with PE-labeled goat antihuman IgG Fc antibody in the dark for 30 min (1:1000, Thermo Fisher Scientific, A18830). Following three rounds of washing, the samples were placed in PBS (1% FBS) and filtered through nylon mesh membrane, and detected by a FACSCelesta flow cytometer (BD Biosciences, USA). FlowJo software (Tree Star) was used for data analysis.

In Vitro Cell Binding

Cell-binding studies with 177Lu/161Tb-radiolabeled antibodies were performed using HCT116 and HT29 cells (5 × 104 cells/well, triplicate). For HT-29 cells, [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 (133 nM) was added to the cell suspension, with a radioactivity of 0.148 MBq in the first well, and sequential halving for wells 2–12 (0.0740, 0.0370, 0.0185, 0.00925, 0.00463, 0.00231, 0.00116, 0.000580, 0.000290, 0.000145, 0.0000725 MBq). The mixture was then incubated at room temperature for 1 h. Following the removal of the supernatant and multiple washes with with PBS (1% FBS), the cells were transferred to counting tubes and measured on a γ counter (Wizard 2470, PerkinElmer, America). For HCT116 cells, the [177Lu]­Lu-DOTA-HB5 concentration in the first well was 166 nM (with a radioactivity of 0.185 MBq), whereas the concentration of [161Tb]­Tb-DOTA-HB5 in the first well remained at 133 nM (with a radioactivity of 0.148 MBq); all other experimental procedures were identical to those for HT29 cells.

Tumor Xenograft Models

Animal experiments were carried out in strict compliance with the ethical guidelines for institutional animal care and were approved by the Laboratory Animal Ethical Committee of Mianyang Central Hospital (File No. S20250316–01). For imaging or TRT studies of CRC xenograft tumors, 5-week-old female BALB/c nude mice were purchased from GemPharmatech (Chengdu, China). Mice were kept in a pathogen-free environment with 40–60% humidity using a 12-h light/dark cycle and provided free access to sterilized food and water. Approximately 5 × 106 HT-29/HCT116/LoVo cells were mixed in PBS (100 μL) and injected subcutaneously into the mice. Subcutaneous xenografts were used for in vivo studies when tumor volume reached 0.05–0.2 cm3.

ImmunoPET Imaging

PET images of 5T4-positive HCT116 tumor-bearing mice were acquired following intravenous injection of [89Zr]­Zr-DFO-HB5/IgG (3.7 MBq, n = 3) in a 100 μL saline solution. 5T4-negative LoVo tumor-bearing mice were acquired following intravenous injection of [89Zr]­Zr-DFO-HB5 (3.7 MBq, n = 3) in a 100 μL saline solution. PET scanning was conducted utilizing a small-animal PET/CT scanner (Supernova Micro PET/CT, Pingseng Healthcare, China). Prior to imaging, mice were placed in a sealed anesthesia chamber and anesthetized with a 2% isoflurane/oxygen mixture. They were then positioned prone in the chamber of the small-animal PET/CT scanner to initiate scanning. All images were reconstructed by the Avatar 3 software (Pingseng Healthcare). The results were expressed as standard uptake value [SUVmean, mean ± standard deviation (SD)].

Biodistribution Studies

HT29/HCT116 tumor-bearing mice were intravenously injected with [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 (0.74 MBq). The mice were sacrificed at multiple time points postinjection (n = 3 per time point per group): 1, 2, 3, 4, and 5 days for the [177Lu]­Lu-DOTA-HB5 group, and 1, 3, 5, 7, and 9 days for the [161Tb]­Tb-DOTA-HB5 group. Following euthanasia, principal organs such as the tumor, heart, liver, spleen, lungs, and kidneys were harvested, weighed, and measured via an automatic γ counter. Radionuclide uptake in each organ was calculated and expressed as the percentage of the injected dose per gram of tissue (%ID/g). A blocking experiment was carried out to evaluate specificity. Mice in the blocked group (n = 3) received 0.5 mg of unlabeled HB5 24 h before the injection of 0.74 MBq [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5. Tissues were harvested at 72 h postinjection for biodistribution analysis (%ID/g).

Targeted Radiopharmaceutical Therapy and Monitoring

For the radiotherapy experiment, HT29/HCT116 tumor-bearing mice were randomly divided into four groups, with eight mice per group. In the radioimmunotherapy group, the mice received single therapeutic doses of [177Lu]­Lu/[161Tb]­Tb-DOTA-HB5 (11.1 MBq). Mice in the HB5 group were intravenously injected with unlabeled HB5 (7.5 mg/kg), and control mice were intravenously injected with physiological saline following the same protocol. Tumor volume and body weight were monitored two to three times weekly throughout the treatment period. Mice were euthanized 28 days after treatment, and major organs and tumors were harvested for histological analysis.

Hematological Analysis and Histological Staining

Venous blood was collected at the end of the treatment and placed in an anticoagulation tube for whole-blood cytometric analysis, including white blood cell (WBC), red blood cell (RBC), platelet (PLT), and hemoglobin (HGB) levels.

After euthanasia, the heart, liver, spleen, lung, kidney, intestine, and tumor tissues were collected, fixed in 4% paraformaldehyde for 24 h, and subsequently washed in 70% ethanol. All tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to the manufacturer’s protocol (G1076, Servicebio). The HE-stained images were observed and recorded using an optical microscope (Olympus) and a digital sectioning scanner (PANNORAMIC DESK/MIDI/250/1000). Immunohistochemistry (IHC) staining of tumor tissue was performed to observe the expression of Ki67 and TUNEL. Rabbit antimouse Ki67 antibody (1:1000, Servicebio) was used as the primary antibody, and the secondary antibody was the S-vision immunohistochemistry polyclonal antibody (goat antirabbit); TUNEL staining was performed following the manufacturer’s protocol (G1507, Servicebio). IHC-stained images were visualized and captured using a digital sectioning scanner (PANNORAMIC DESK/MIDI/250/1000).

Statistical Analysis

SPSS 22 software was used for data analysis. All quantitative data are expressed as the mean ± SD. The unpaired Student’s t-test and repeated-measures ANOVA were used for significance testing, with statistical significance set at p < 0.05.

Supplementary Material

jm5c03745_si_001.csv (207B, csv)

Acknowledgments

The authors sincerely appreciate all the researchers who participated in this study. The authors also acknowledge the use of BioRender in creating the abstract graphic of the manuscript.

Glossary

Abbreviations

TRT

targeted radionuclide therapy

177Lu

Lutetium-177

161Tb

Terbium-161

89Zr

Zirconium-89

RDCs

radioisotope drug conjugates

PET

positron emission tomography

ADCs

antibody–drug conjugates

RCP

radiochemical purity

radio-iTLC

radio instant thin-layer chromatography

radio-HPLC

radioactive high-performance liquid chromatography

FBS

fetal bovine serum

H&E

hematoxylin and eosin

WBC

white blood cell

RBC

red blood cell

PLT

platelet

HGB

hemoglobin.

All the data have been presented here in the manuscript; any additional data can be provided by contacting the corresponding author.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c03745.

  • Molecular formula strings (CSV)

Y. L. and X. L. contributed equally. Y.Z. acquired the funding, conceptualized and supervised the project, in addition to reviewing the manuscript. X.L. invetigted and conceptualized the project. Y.L. designed, performed, and analyzed all experiments in addition to reviewing the manuscript. X.L. wrote the manuscript and performed the experiments. H. L. performed the experiments and acquired the data.T.W. perfomed the experiments and acquired the data.Y.H. kept the animals and collected the information on animals. S.L. perfomed the experiments. X.Y. provided technical guidance. R.L. producted the radionuclide. F.M. andY.H. producted the antibody.

This work was supported by NHC Key Laboratory of Nuclear Technology Medical Transformation (Mianyang Central Hospital) (2021HYX-013) and the Mianyang Science and Technology Bureau (Mianyang Science and Technology Program, 2023ZYDF073, 2023ZYDF070, 2025ZYDF019).

The authors declare no competing financial interest.

References

  1. Arnold M., Abnet C. C., Neale R. E., Vignat J., Giovannucci E. L., McGlynn K. A., Bray F.. Global Burden of 5 Major Types of Gastrointestinal Cancer. Gastroenterology. 2020;159(1):335–349.e15. doi: 10.1053/j.gastro.2020.02.068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bray F., Laversanne M., Sung H.. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-Cancer J. Clin. 2024;74(3):229–263. doi: 10.3322/caac.21834. [DOI] [PubMed] [Google Scholar]
  3. Shi J. F., Wang L., Ran J. C.. et al. Clinical characteristics, medical service utilization, and expenditure for colorectal cancer in China, 2005 to 2014: Overall design and results from a multicenter retrospective epidemiologic survey. Cancer. 2021;127(11):1880–1893. doi: 10.1002/cncr.33445. [DOI] [PubMed] [Google Scholar]
  4. Zeng H., Zheng R., Sun K.. et al. Cancer survival statistics in China 2019–2021: A multicenter, population-based study. J. Natl. Cancer Cent. 2024;4(3):203–213. doi: 10.1016/j.jncc.2024.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Starzynska T., Rahi V., Stern P. L.. The expression of 5T4 antigen in colorectal and gastric carcinoma. Br. J. Cancer. 1992;66(5):867–869. doi: 10.1038/bjc.1992.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Mulder W. M., Stern P. L., Stukart M. J., De Windt E., Butzelaar R. M., Meijer S., Ader H. J., Claessen A. M., Vermorken J. B., Meijer C.J.. et al. Low intercellular adhesion molecule 1 and high 5T4 expression on tumor cells correlate with reduced disease-free survival in colorectal carcinoma patients. Clin. Cancer Res. 1997;3(11):1923–1930. [PubMed] [Google Scholar]
  7. Stern P. L., Harrop R.. 5T4 oncofoetal antigen: An attractive target for immune intervention in cancer. Cancer Immunol., Immunother. 2016;66:415–426. doi: 10.1007/s00262-016-1917-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hole N., Stern P. L.. A 72 kD trophoblast glycoprotein defined by a monoclonal antibody. Br. J. Cancer. 1988;57(3):239–246. doi: 10.1038/bjc.1988.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Starzynska T., Marsh P., Schofield P. F.. et al. Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma. Br. J. Cancer. 1994;69:899–902. doi: 10.1038/bjc.1994.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Tang Y. L., Li D. D., Duan J. Y., Sheng L. M., Wang X.. Resistance to targeted therapy in metastatic colorectal cancer: Current status and new developments. World J. Gastroenterol. 2023;29(6):926–948. doi: 10.3748/wjg.v29.i6.926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Primac I., Tabury K., Tasdogan A., Baatout S., Herrmann K.. The molecular blueprint of targeted radionuclide therapy. Nat. Rev. Clin. Oncol. 2025;22(11):869–894. doi: 10.1038/s41571-025-01069-z. [DOI] [PubMed] [Google Scholar]
  12. Guosheng D.O.N.G., Xin C.H.E.N., Kexin L.I., Tao Z.H.A.N.G.. Research progress and clinical application of radionuclide drug conjugates. Prog. Pharm. Sci. 2023;47:324–336. [Google Scholar]
  13. Gudkov S. V., Shilyagina N. Y., Vodeneev V. A.. et al. Targeted radionuclide therapy of human tumors. Int. J. Mol. Sci. 2016;17(1):33. doi: 10.3390/ijms17010033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wang F., Guo X., Liu T., Ding L., Xia L., Ding J., Meng X., Jiang J., Zhu H., Yang Z.. Production and purification of solid target PET nuclide 89Zr and its antibody labeling. Chin. J. Nucl. Med. Mol. Imaging. 2020:294–297. [Google Scholar]
  15. Dilworth J. R., Pascu S. I.. The chemistry of PET imaging with zirconium-89. Chem. Soc. Rev. 2018;47(8):2554–2571. doi: 10.1039/C7CS00014F. [DOI] [PubMed] [Google Scholar]
  16. Xiuqi L.I., Shupeng L.I.U., Xiaofei W.U., Mengyang Y.U., Hongyun W.A.N.G.. Radionuclide Drug Conjugates: China’s 15-Year Research and Development Process and Latest Policy Support. Med. J. Peking Union Med. Coll. Hosp. 2024;16(4):847–854. [Google Scholar]
  17. Li D., Cheng S., Zou S.. et al. Immuno-PET Imaging of 89Zr Labeled Anti-PD-L1 Domain Antibody. Mol. Pharmaceutics. 2018;15(4):1674–1681. doi: 10.1021/acs.molpharmaceut.8b00062. [DOI] [PubMed] [Google Scholar]
  18. Bhusari P., Vatsa R., Singh G.. et al. Development of Lu-177-trastuzumab for radioimmunotherapy of HER2 expressing breast cancer and its feasibility assessment in breast cancer patients. Int. J. Cancer. 2017;140(4):938–947. doi: 10.1002/ijc.30500. [DOI] [PubMed] [Google Scholar]
  19. Nedjadi Y., Juget F., Durán M. T.. et al. Activity standardisation of 177Lu. Appl. Radiat. Isot. 2023;200:110986. doi: 10.1016/j.apradiso.2023.110986. [DOI] [PubMed] [Google Scholar]
  20. De Araujo E. B., Caldeira Filho J. S., Nagamati L. T., Muramoto E., Colturato M. T., Couto R. M., Pujatti P. B., Mengatti J., Silva C.P.G.. A comparative study of 131I and 177Lu labeled somatostatin analogues for therapy of neuroendocrine tumours. Appl. Radiat. Isot. 2009;67(2):227–233. doi: 10.1016/j.apradiso.2008.09.009. [DOI] [PubMed] [Google Scholar]
  21. Banerjee S., Pillai M. R., Knapp F. F.. Lutetium-177 therapeutic radiopharmaceuticals: Linking chemistry, radiochemistry, and practical applications. Chem. Rev. 2015;115:2934–2974. doi: 10.1021/cr500171e. [DOI] [PubMed] [Google Scholar]
  22. Valkema R., Pauwels S.A., Kvols L. K., Kwekkeboom D. J., Jamar F., de Jong M., Barone R., Walrand S., Kooij P. P., Bakker W. H.. et al. Long-term follow-up of renal function after peptide receptor radiation therapy with 90Y-DOTA0, Tyr3-octreotide and 177Lu-DOTA0, Tyr3-octreotate. J. Nucl. Med. Mol. 2005;46(1 suppl):83S–91S. [PubMed] [Google Scholar]
  23. Duran M. T., Juget F., Nedjadi Y.. et al. Determination of 161Tb half-life by three measurement methods. Appl. Radiat. Isot. 2020;159:109085. doi: 10.1016/j.apradiso.2020.109085. [DOI] [PubMed] [Google Scholar]
  24. Lehenberger S., Barkhausen C., Cohrs S., Fischer E., Grünberg J., Hohn A., Köster U., Schibli R., Türler A., Zhernosekov K.. et al. The low-energy β– and electron emitter 161Tb as an alternative to 177Lu for targeted radionuclide therapy. Nucl. Med. Biol. 2011;38:917–924. doi: 10.1016/j.nucmedbio.2011.02.007. [DOI] [PubMed] [Google Scholar]
  25. Müller C., Zhernosekov K., Köster U.. et al. A unique matched quadruplet of terbium radioisotopes for PET and SPECT and for α– und β–-radionuclide therapy: An in vivo proof-of-concept study with a new receptor-targeted folate derivative. J. Nucl. Med. 2012;53:1951–1959. doi: 10.2967/jnumed.112.107540. [DOI] [PubMed] [Google Scholar]
  26. Bernhardt P., Benjegard S. A., Kolby L.. et al. Dosimetric comparison of radionuclides for therapy of somatostatin receptor-expressing tumors. Int. J. Radiat. Oncol. Biol. Phys. 2001;51:514–524. doi: 10.1016/S0360-3016(01)01663-7. [DOI] [PubMed] [Google Scholar]
  27. Hindie E., Zanotti-Fregonara P., Quinto M. A., Morgat C., Champion C.. Dose deposits from 90Y, 177Lu, 111In, and 161Tb in micrometastases of various sizes: Implications for radiopharmaceutical therapy. J. Nucl. Med. 2016;57:759–764. doi: 10.2967/jnumed.115.170423. [DOI] [PubMed] [Google Scholar]
  28. Champion C., Quinto M. A., Morgat C., Zanotti-Fregonara P., Hindie E.. Comparison between three promising b–emitting radionuclides, 67Cu, 47Sc and 161Tb, with emphasis on doses delivered to minimal residual disease. Theranostics. 2016;6:1611–1618. doi: 10.7150/thno.15132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wang R., Lai Q., Lu Y., Zhou Y., Tang L., Tao Y., Yao Y., Yu L., Liu Y., Wang Y., Zhang R.. et al. Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency. Protein Expression Purif. 2019;158:51–58. doi: 10.1016/j.pep.2018.07.003. [DOI] [PubMed] [Google Scholar]
  30. Wang R., Lai Q., Tang L., Tao Y., Yao Y., Liu Y., Lu Y., Shen C., Lu R., Fan C.. et al. A novel 5T4-targeting antibody-drug conjugate H6-DM4 exhibits potent therapeutic efficacy in gastrointestinal tumor xenograft models. Am. J. Cancer Res. 2018;8(4):610–623. [PMC free article] [PubMed] [Google Scholar]
  31. O’Connell J. B., Maggard M. A., Ko C. Y.. Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging. J. Natl. Cancer Inst. 2004;96(19):1420–1425. doi: 10.1093/jnci/djh275. [DOI] [PubMed] [Google Scholar]
  32. Sadahiro S., Suzuki T., Ishikawa K., Nakamura T., Tanaka Y., Masuda T., Mukoyama S., Yasuda S., Tajima T., Makuuchi H.. et al. Recurrence patterns after curative resection of colorectal cancer in patients followed for a minimum of ten years. Hepatogastroenterology. 2003;50:1362–1366. [PubMed] [Google Scholar]
  33. Müller C., Reber J., Haller S., Dorrer H., Bernhardt P., Zhernosekov K.. et al. Direct in vitro and in vivo comparison of 161Tb and 177Lu using a tumour-targeting folate conjugate. Eur. J. Nucl. Med. Mol. Imaging. 2014;41:476–485. doi: 10.1007/s00259-013-2563-z. [DOI] [PubMed] [Google Scholar]
  34. Müller C., Umbricht C. A., Gracheva N., Tschan V. J., Pellegrini G., Bernhardt P.. et al. Terbium-161 for PSMA-targeted radionuclide therapy of prostate cancer. Eur. J. Nucl. Med. Mol. Imaging. 2019;46:1919–1930. doi: 10.1007/s00259-019-04345-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Borgna F., Haller S., Rodriguez J. M. M.. et al. Combination of terbium-161 with somatostatin receptor antagonists: A potential paradigm shift for the treatment of neuroendocrine neoplasms. Eur. J. Nucl. Med. Mol. Imaging. 2022;49:1113–1126. doi: 10.1007/s00259-021-05564-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rioja-Blanco E., Banz Y., Schlapbach C.. et al. 161Tb Radioimmunotherapy as a Treatment for CD30-Positive Lymphomas. J. Nucl. Med. 2025;66(6):909–915. doi: 10.2967/jnumed.124.268805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Alcocer-Ávila M. E., Ferreira A., Quinto M. A., Morgat C., Hindié E., Champion C.. Radiation doses from 161Tb and 177Lu in single tumour cells and micrometastases. EJNMMI Phys. 2020;7(1):33. doi: 10.1186/s40658-020-00301-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Bavelaar B. M., Lee B. Q., Gill M. R., Falzone N., Vallis K. A.. Subcellular Targeting of Theranostic Radionuclides. Front. Pharmacol. 2018;9:996. doi: 10.3389/fphar.2018.00996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Chen P., Bordeau B. M., Zhang Y., Balthasar J. P.. Transient inhibition of trastuzumabtumour binding to overcome the“binding-site barrier”and improve the efficacy of a trastuzumab-gelonin immunotoxin. Mol. Cancer Ther. 2022;21(10):1573–1582. doi: 10.1158/1535-7163.MCT-22-0192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lu G. L., Nishio N., Berg V. D. N. S., Martin B. A., Fakurnejad S., van Keulen S., Colevas A. D., Thurber G. M., Rosenthal E. L.. Co-administered antibody improves penetration of antibody–dye conjugate into human cancers with implications for antibody–drug conjugates. Nat. Commun. 2020;11(1):5667. doi: 10.1038/s41467-020-19498-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zeng Z., Li L., Tao J.. et al. [177Lu]­Lu-labeled anti-claudin-18.2 antibody demonstrated radioimmunotherapy potential in gastric cancer mouse xenograft models. Eur. J. Nucl. Med. Mol. Imaging. 2024;51(5):1221–1232. doi: 10.1007/s00259-023-06561-1. [DOI] [PubMed] [Google Scholar]
  42. Li C., Liu J., Yang X.. et al. Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models. Eur. J. Nucl. Med. Mol. Imaging. 2022;50(1):168–183. doi: 10.1007/s00259-022-05954-y. [DOI] [PubMed] [Google Scholar]
  43. Kristiansson A., Vilhelmsson Timmermand O., Altai M.. et al. Hematological toxicity in mice after high activity injections of 177Lu-PSMA-617. Pharmaceutics. 2022;14:731. doi: 10.3390/pharmaceutics14040731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Busslinger S.D., Mapanao A. K., Kegler K., Bernhardt P., Flühmann F., Fricke J., Zeevaart J. R., Köster U., Van der Meulen N. P., Schibli R.. et al. Comparison of the tolerability of 161Tb-and 177Lu-labeled somatostatin analogues in the preclinical setting. Eur. J. Nucl. Med. Mol. Imaging. 2024;51(13):4049–4061. doi: 10.1007/s00259-024-06827-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Grünberg J., Lindenblatt D., Dorrer H.. et al. Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model. Eur. J. Nucl. Med. Mol. Imaging. 2014;41(10):1907–1915. doi: 10.1007/s00259-014-2798-3. [DOI] [PubMed] [Google Scholar]
  46. He Y., Tian R., Xu D.. et al. Preclinical evaluation and pilot clinical study of [68Ga]­Ga-NOTA-H006 for non-invasive PET imaging of 5T4 oncofetal antigen. Eur. J. Nucl. Med. Mol. Imaging. 2025;52(2):611–622. doi: 10.1007/s00259-024-06941-1. [DOI] [PubMed] [Google Scholar]
  47. Li Q., White J. B., Peterson N. C.. et al. Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport. J. Controlled Release. 2018;279:126–135. doi: 10.1016/j.jconrel.2018.04.013. [DOI] [PubMed] [Google Scholar]
  48. Ward E. S., Zhou J., Ghetie V.. Ober RJ Evidence to support the cellular mechanism involved in serum IgG homeostasis in humans. Int. Immunol. 2003;15:187–195. doi: 10.1093/intimm/dxg018. [DOI] [PubMed] [Google Scholar]
  49. Ober R. J., Martinez C., Vaccaro C., Zhou J., Ward E. S.. Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J. Immunol. 2004;172:2021–2029. doi: 10.4049/jimmunol.172.4.2021. [DOI] [PubMed] [Google Scholar]
  50. Suzuki T., Ishii-Watabe A., Tada M.. et al. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: A comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR. J. Immunol. 2010;184(4):1968–1976. doi: 10.4049/jimmunol.0903296. [DOI] [PubMed] [Google Scholar]
  51. Vivier D., Sharma S. K., Zeglis B. M.. Understanding the in vivo fate of radioimmunoconjugates for nuclear imaging. J. Labelled Compd. Radiopharm. 2018;61:672–692. doi: 10.1002/jlcr.3628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Orcutt K. D., Adams G. P., Wu A. M.. et al. Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging. Mol. Imaging Biol. 2017;19(5):656–664. doi: 10.1007/s11307-016-1041-y. [DOI] [PubMed] [Google Scholar]
  53. Rodriguez C., Delaney S., Sarrett S. M., Keinänen O. M., Zeglis B. M.. Antibody Engineering for Nuclear Imaging and Radioimmunotherapy. J. Nucl. Med. 2022;63(9):1316–1322. doi: 10.2967/jnumed.122.263861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tschan V. J., Busslinger S. D., Bernhardt P.. et al. Albumin-Binding and Conventional PSMA Ligands in Combination with 161Tb: Biodistribution, Dosimetry, and Preclinical Therapy. J. Nucl. Med. 2023;64(10):1625–1631. doi: 10.2967/jnumed.123.265524. [DOI] [PubMed] [Google Scholar]
  55. Starzynska T., Wiechowska-Kozlowska A., Mariiez K., Bromley M., Roberts S. A., Lawniczak M., Kolodziej B., Zyluk A., Stern P. L.. et al. 5T4 oncofetal antigen in gastric carcinoma and its clinical significance. Eur. J. Gastroenterol. Hepatol. 1998;10(6):479–484. doi: 10.1097/00042737-199806000-00008. [DOI] [PubMed] [Google Scholar]
  56. Griffiths R. W., Gilham D. E., Dangoor A.. et al. Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: A potential target for T-cell-based immunotherapy. Br. J. Cancer. 2005;93(6):670–677. doi: 10.1038/sj.bjc.6602776. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jm5c03745_si_001.csv (207B, csv)

Data Availability Statement

All the data have been presented here in the manuscript; any additional data can be provided by contacting the corresponding author.

Articles from Journal of Medicinal Chemistry are provided here courtesy of American Chemical Society

RESOURCES