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. 2025 Sep;66(9):1400–1405. doi: 10.2967/jnumed.125.270167

CD24 as a Novel Radiopharmaceutical Target for Hepatocellular Carcinoma

Hima Makala 1, Julia Sheehan-Klenk 1, Woonghee Lee 1, Joon-Yong Chung 1, Kwamena E Baidoo 1, Divya Nambiar 1, Peter L Choyke 1, Freddy E Escorcia 1,2,✉
PMCID: PMC12410296  PMID: 40744694

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Keywords: immuno-PET, hepatocellular carcinoma, CD24, 89Zr, liver

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. After the use of liver-directed therapies, it is challenging to differentiate between nonviable tumor and viable residual or recurrent disease using conventional imaging techniques. Targeted radiopharmaceutical imaging agents with specificity for HCC-selective molecules may address this unmet need. CD24, a glycosylated plasma membrane protein, is overexpressed in HCC. Here, we describe a CD24-targeted antibody-based PET (immuno-PET) tracer for the noninvasive detection of CD24-positive (CD24+) tumors. Methods: CD24 expression was assessed at the messenger RNA, total protein, and cell membrane levels across 4 HCC cell lines (Huh7, Hep3B, SNU182, SNU449), 1 hepatoblastoma line (HepG2), and a CD24+ colorectal cancer–positive control line (HT29) using quantitative reverse transcription polymerase chain reaction, Western blot testing, and flow cytometry. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9–mediated CD24 gene knockout was performed in SNU449, Hep3B, and HT29 cells to yield otherwise isogenic cell lines as CD24-negative (CD24−) controls. The binding affinity of a humanized monoclonal anti-CD24 antibody (α-hCD24) for recombinant CD24 protein was confirmed by biolayer interferometry and enzyme-linked immunosorbent assay. We then synthesized the immuno-PET tracer ([89Zr]Zr-desferrioxamine [DFO]-α-hCD24) and assessed target engagement in vivo using PET/CT imaging and ex vivo by evaluating the biodistribution of xenograft models using paired CD24+ and CD24− HCC and HT29 isogenic cell lines in athymic nude mice. Results: Huh7, Hep3B, SNU449, and HT29 cell lines demonstrated high total and plasma membrane expression of CD24. The α-hCD24 antibody exhibited good binding affinity to recombinant human CD24 (dissociation constant, 2.4 nM) and was unaffected by DFO conjugation of α-hCD24 (dissociation constant, 2.7 nM). [89Zr]Zr-DFO-α-hCD24 was efficiently produced at high radiochemical yield (75% ± 5%) and radiopurity (99% ± 1%). PET/CT imaging and biodistribution studies confirmed specific uptake of [89Zr]Zr-DFO-α-hCD24 in Hep3B CD24+ tumors (8.7 ± 1.2 %IA/g) and much lower accumulation in Hep3B CD24− tumors (2.3 ± 0.6 %IA/g) at 144 h after injection. Conclusion: Our findings establish CD24 as a promising radiotheranostic target for HCC. Future work will optimize [89Zr]Zr-DFO-α-hCD24 to improve tumor-to-liver signal and facilitate CD24-targeted radiopharmaceutical therapy.

Hepatocellular carcinoma (HCC) accounts for 75%–85% of primary liver cancer (1). In 2022, it was the sixth most commonly diagnosed cancer globally and the third leading cause of cancer-related deaths, with 865,269 new cases and 757,948 deaths (2). Despite recent advances in immunotherapy for patients with HCC, the overall 5-y survival rate remains less than 20%.

Liver-directed therapies, such as stereotactic body radiation therapy and transarterial radioembolization, have shown efficacy in treating HCC, highlighting its radiosensitivity and the potential of radiotherapy to improve clinical outcomes (3). After such treatments, however, current imaging modalities often fail to differentiate viable tumors from necrotic tissue. Indeed, conventional imaging methods, such as MRI and CT, have limited sensitivities for persistent disease (53% and 62%, respectively) (4). Similarly, [18F]FDG PET detects only 50% of HCC lesions and has limited specificity because of the similar uptake of this radiotracer by the normal liver (5). These limitations underscore the urgent need for new functional imaging modalities to accurately identify viable HCC and guide clinical decision-making. The identification of molecules overexpressed in HCC, but not in normal tissue, and engineering of imaging agents specific to these molecules could yield valuable tools to address this unmet clinical need.

Glypican-3 (GPC3), a glycosylphosphatidylinositol-anchored proteoglycan, is a well-established biomarker for HCC, expressed in approximately 70%–80% of cases (6). Notably, multiple studies, including our own, have demonstrated the potential of GPC3-targeted radiopharmaceuticals for diagnostic and therapeutic (e.g., radiotheranostic) applications in cancers with high GPC3 expression, including preclinical models of liver cancer (7–11) and neuroendocrine prostate cancers (12). However, because GPC3 is absent in 20%–30% of HCC cases, the identification of alternative biomarkers is essential.

The current study investigates CD24, a highly glycosylated cell surface protein, as a novel radiopharmaceutical target. CD24 contributes to tumor progression, metastasis, and immune evasion by suppressing macrophage-mediated phagocytosis (13–15). Its expression in normal adult tissues is relatively restricted, primarily found on immune cells, such as T and B lymphocytes and granulocytes (16). In contrast, CD24 is overexpressed in approximately 70% of human tumors (14,16), including HCC (17,18), and is associated with a poor prognosis (18). These features have made CD24 an attractive therapeutic target, as evinced by CD24-specific monoclonal antibodies (e.g., ATG-031, IMM47) that are currently undergoing clinical evaluation for immunotherapy in solid tumors (19,20). Preclinical studies with antibody–drug conjugates have also demonstrated promise in reducing tumor burden and metastasis in several malignancies, including HCC (21–23). Notably, CD24 has not yet been explored as a target for radiopharmaceutical applications. Our study aims to bridge that gap by evaluating a full-length humanized anti-CD24 antibody (α-hCD24) for its potential in CD24-targeted imaging and therapy.

MATERIALS AND METHODS

Cell Culture

Four HCC cell lines (Huh7, Hep3B, SNU182, SNU449), 1 hepatoblastoma line (HepG2), and a CD24-positive (CD24+) colorectal cancer–positive control line (HT29) (American Type Culture Collection) were used for in vitro and in vivo experiments. Huh7 HCC cells were obtained from Mitchel Ho (Bethesda, MD). HepG2 and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific), whereas SNU449, SNU182, and HT29 cells were maintained in RPMI 1640 medium (ThermoFisher Scientific). Hep3B cells were grown in Eagle’s minimal essential medium (American Type Culture Collection). All media were supplemented with 10% fetal bovine serum (Gibco; ThermoFisher Scientific) and filtered before use. Notably, although HT29 is not an HCC cell line, it is a well-established CD24 expression model, which we leveraged as a positive control. Genetically engineered CD24 knockout (CD24-negative [CD24−]) cell lines were maintained in the same media as parent wild-type cell lines.

More information on the methods used is available in the supplemental materials (available at http://jnm.snmjournals.org), which also describe the construction of CD24− cell lines, quantitative reverse transcription polymerase chain reaction analysis, Western blot testing, Alexa Fluor 647 conjugation, flow cytometry, enzyme-linked immunosorbent assay, biolayer interferometry, α-hCD24 antibody–lysine conjugation, synthesis of [89Zr]Zr-desferrioxamine [DFO]-α-hCD24, murine subcutaneous xenograft models and imaging studies, PET/CT imaging with [89Zr]Zr-DFO-α-hCD24, ex vivo biodistribution studies, and immunohistochemistry (24).

Statistical Analysis

All statistical analyses were performed using Prism version 10.4.1 (GraphPad). Data are presented as mean ± SD or SEM. Comparisons between 2 groups were conducted using either paired or unpaired 2-tailed t-tests. For comparisons involving more than 2 groups, 1-way ANOVA was used. P values of less than 0.05 were considered statistically significant.

RESULTS

CD24 Expression in Liver Cancer Cell Lines

To confirm CD24 expression in HCC, we evaluated 4 primary HCC cell lines (SNU449, Hep3B, Huh7, SNU182) and a hepatoblastoma line (HepG2) at the messenger RNA level using quantitative reverse transcription polymerase chain reaction (Fig. 1A), total protein level by Western blot testing (Fig. 1B), and membrane protein level by flow cytometry (Figs. 1C and 1D). HT29, a colorectal cancer cell line with well-characterized CD24 expression, was used as a positive control to validate tracer specificity before evaluation in HCC models. Both messenger RNA and total protein analyses showed CD24 overexpression in the HT29 and Huh7 cell lines compared with other HCC models (Figs. 1A and 1B). However, assessment of membrane protein expression by flow cytometry showed abundant CD24 expression in SNU449 and Hep3B cells, with a 3.5-fold higher mean fluorescence intensity than in Huh7 cells (Figs. 1C and 1D). Given their high total and membrane protein expression, we selected Huh7, SNU449, Hep3B, and HT29 cell lines (positive controls) for further testing and engineered CD24-knockout lines of each as negative controls. Using a fluorescently labeled α-hCD24 antibody, we confirmed its binding specificity to CD24 by comparing CD24+ and CD24− cell lines (Fig. 1E).

FIGURE 1.

FIGURE 1.

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CD24 expression confirmed at translational and cell membrane levels in HCC cells. (A) Quantitative reverse transcription polymerase chain reaction analysis validating CD24 gene expression in HCC cell lines, with β-actin (ACTB) as internal control. (B) Western blot analysis confirming CD24 protein expression in HCC cells, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control. (C) Flow cytometry histogram demonstrating α-hCD24 monoclonal antibody binding to CD24 on HCC cells, with HT29 colorectal cancer cells as reference and human IgG1 as isotype control. (D) Mean fluorescence intensity (MFI) analysis comparing relative CD24+ expression levels, showing SNU449 > Hep3B > HT29 > Huh7 > SNU182, with no detectable expression in HepG2 cells. (E) Flow cytometry analysis of CD24+ (SNU449, Hep3B, and HT29) versus CD24− cell lines, with CD24− cells exhibiting negligible CD24 expression, using human IgG1 as isotype control. (F) Immunofluorescence staining of CD24+ and CD24− in both HT29 and Hep3B cells, showing CD24 expression (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue). CD24 expression is prominent in parental cells but absent in CD24− clones. Bottom panel displays immunofluorescence results using isotype-matched negative control. Scale bars represent 20 μm. KO = knockout; mRNA = messenger RNA.

Finally, the specificity of α-hCD24 binding was confirmed by the absence of detectable antibody binding using paired parental and otherwise isogenic CD24− HCC (SNU449 and Hep3B) and colorectal (HT29) cell lines (Fig. 1E). We further confirmed CD24 knockout through immunofluorescence analysis. Fixed cells were stained with a primary antibody targeting CD24 as well as an isotype control in both parental HT29-CD24+ colorectal cancer and Hep3B-CD24+ HCC cells, as well as in CD24− cell cultures. CD24 expression was absent in CD24− cells, whereas strong staining was observed in HT29-CD24+ and Hep3B-CD24+ cells (Fig. 1F). These findings confirmed that knockout of CD24 was successful.

Binding Affinity of α-hCD24-DFO Conjugate for CD24

After reacting with p-SCN-Bn-DFO, the DFO-α-hCD24 product showed an average chelator-to-antibody ratio of 1.5:1 (Fig. 2A). High-performance liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis verified the purity and structural integrity after conjugation (Figs. 2B and 2C). The binding affinities of α-hCD24 and DFO-α-hCD24 for recombinant CD24 protein were assessed through multiple binding assays. Enzyme-linked immunosorbent assay results demonstrated that both unconjugated and conjugated antibody exhibited nanomolar affinity (dissociation constant [KD], 0.99 ± 0.1 nM) for human CD24 protein (Fig. 3A). Biolayer interferometry analysis further confirmed nanomolar affinity of both the unconjugated α-hCD24 (KD, 2.4 nM) (Fig. 3B) and the DFO-α-hCD24 conjugate (KD, 2.7 nM) (Fig. 3C), supporting its suitability for radiolabeling and subsequent in vivo testing.

FIGURE 2.

FIGURE 2.

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α-hCD24-DFO conjugation preserves structural integrity and enables efficient radiolabeling. (A) Mass spectrum of α-hCD24 (top) and DFO-α-hCD24 (bottom), confirming stable conjugation with average final chelator-to-antibody ratio of 1.5:1. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis demonstrating that α-hCD24-DFO maintains structural integrity after conjugation, with reduced (R) and nonreduced (NR) conditions indicated. (C) Size-exclusion high-performance liquid chromatography (top) confirming purity of α-hCD24-DFO before radiolabeling. Radio–thin-layer chromatography (bottom) verifying efficient [89Zr]Zr-DFO-α-hCD24 radiolabeling.

FIGURE 3.

FIGURE 3.

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α-hCD24-DFO conjugates retain subnanomolar affinity for human CD24. (A) Enzyme-linked immunosorbent assay–based binding affinity analysis of α-hCD24 and DFO-α-hCD24, yielding KD of 0.99 ± 0.1 nM. Biolayer interferometry results comparing α-hCD24 (B) and DFO-α-hCD24 (C), confirming preserved binding characteristics after conjugation.

Radiochemical Yield and Purity of [89Zr]Zr-DFO-α-hCD24

The radiochemical yield of [89Zr]Zr-DFO-α-hCD24 was 75% ± 5%, and the specific activity was 259 MBq/mg. The radiochemical purity of [89Zr]Zr-DFO-α-hCD24 was found to be at least 99% ± 1%, using instant thin-layer chromatography (Fig. 2C).

[89Zr]Zr-DFO-α-hCD24 Uptake in CD24+ and CD24− Tumors

In HT29-CD24+ tumor–bearing mice, PET/CT imaging demonstrated specific and sustained accumulation of [89Zr]Zr-DFO-α-hCD24 in the tumors at 72 h after injection (Fig. 4A). Minimal signal was observed in mice bearing HT29-CD24− tumors, confirming the target specificity of the tracer (Fig. 4B). Ex vivo biodistribution corroborated imaging findings, showing 19.1 ± 0.1 %IA/g in HT29-CD24+ tumors versus 9.3 ± 0.9 %IA/g in HT29-CD24− tumors at 72 h after injection (Fig. 4C). Signals at bilateral hind joints were attributed to free, unchelated 89Zr. Tumor-to-liver ratios indicated moderate signal-to-background contrast, with values of 1.9 ± 0.1 and 0.8 ± 0.1 at 72 h after injection in HT29-CD24+ and HT29-CD24− tumors, respectively (Fig. 4C). Tumor-to-muscle ratios further demonstrated favorable signal contrast (25.4 ± 2.14 vs. 13.2 ± 1.8 in HT29-CD24+ and HT29-CD24− tumors, respectively) (Fig. 4C). Detailed biodistribution results are presented in Supplemental Table 1.

FIGURE 4.

FIGURE 4.

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[89Zr]Zr-DFO-α-hCD24 specifically localizes to CD24+ human colorectal tumors. (A) Maximum-intensity-projection (MIP) PET/CT images of [89Zr]Zr-DFO-α-hCD24 in HT29-CD24+ xenografts at 72 h after injection (n = 3). Tumors are outlined in white dotted circles. (B) MIP PET/CT images of [89Zr]Zr-DFO-α-hCD24 in HT29-CD24− xenografts at 72 h after injection (n = 3), with tumors marked by white dotted circles. (C) Biodistribution analysis of [89Zr]Zr-DFO-α-hCD24 across major organs in HT29-CD24+ and HT29-CD24− xenografts at 72 h after injection, comparing tumor-to-muscle and tumor-to-liver ratios. Data are presented as mean ± SE (n = 3 per group), with radioactivity uptake shown as %IA/g. Statistical significance was determined using paired t-test. **P < 0.001. L. = large; S. = small.

Similarly, PET/CT imaging showed clear tracer uptake in Hep3B-CD24+ tumors at 72 h after injection, with negligible uptake in Hep3B-CD24− tumors (Figs. 5A and 5B; Supplemental Fig. 2). Quantitative ex vivo biodistribution at 144 h after tracer injection confirmed uptake values of 8.7 ± 3.2 %IA/g and 2.3 ± 0.5 %IA/g in Hep3B-CD24+ and Hep3B-CD24− tumors, respectively (Fig. 5C). At 144 h after injection, Hep3B-CD24+ tumors exhibited an SUVmax of 2.7 ± 0.8, whereas Hep3B-CD24− tumors had an SUVmax of 0.7 ± 0.1 (Fig. 5D). The SUVmean at 144 h after injection was 1.1 ± 0.3 in Hep3B-CD24+ tumors and 0.1 ± 0.04 in Hep3B-CD24− tumors (Fig. 5E). Tumor-to-liver ratios continued to show moderate signal-to-background contrast at 144 h after injection (0.8 ± 0.1 and 0.2 ± 0.1 in Hep3B-CD24+ and Hep3B-CD24− tumors, respectively) (Fig. 5C). Tumor-to-muscle ratios for Hep3B-CD24+ tumors were significantly higher, further confirming specificity (P < 0.05) (Fig. 5C). Significant tracer accumulation was also observed in the spleen (19.4 ± 2.3 %IA/g in Hep3B-CD24+ tumors and 16.4 ± 3.8 %IA/g in Hep3B-CD24− tumors) and liver (11.8 ± 0.8 %IA/g in Hep3B-CD24+tumors and 11.1 ± 2.1 %IA/g in Hep3B-CD24− tumors). Full biodistribution data for Hep3B-CD24+ and Hep3B-CD24− tumor groups are presented in Supplemental Table 3.

FIGURE 5.

FIGURE 5.

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[89Zr]Zr-DFO-α-hCD24 exhibits time-dependent accumulation in CD24+ human HCC tumors. (A) Representative maximum-intensity-projection (MIP) PET/CT images of [89Zr]Zr-DFO-α-hCD24 in Hep3B-CD24+ xenografts at 48, 72, 96, 120, and 144 h after injection (n = 8). Tumors are outlined with white dotted circles. (B) Representative MIP PET/CT images of [89Zr]Zr-DFO-α-hCD24 in Hep3B-CD24− xenografts (n = 5) at 48, 72, 96, 120, and 144 h after injection. Tumors are indicated with white dotted circles. (C) Biodistribution analysis of [89Zr]Zr-DFO-α-hCD24 across major organs in Hep3B-CD24+ and Hep3B-CD24− xenografts at 144 h after injection, comparing tumor-to-blood and tumor-to-muscle ratios. SUVmax (D) and SUVmean (E) values were compared between Hep3B-CD24+ and Hep3B-CD24− xenografts. (F) Representative hematoxylin and eosin and CD24 immunohistochemistry images of Hep3B-CD24+ and Hep3B-CD24− xenograft tumors. Scale bars represent 50 µm. Data are expressed as mean ± SE. Statistical significance was determined using paired t-test. *P < 0.05; **P < 0.001.

Notably, despite the high expression of CD24 found in messenger RNA and total protein analyses, Huh7 tumors exhibited low uptake compared with background organs, likely due to modest CD24 surface (vs. cytoplasmic) expression (Figs. 1C and 1D). Ex vivo analysis showed a tumor-to-blood ratio of 0.7 ± 0.1, with high uptake in the spleen (16.9 ± 5.7 %IA/g) and liver (12.1 ± 1.1 %IA/g), consistent with antibody catabolism (Supplemental Fig. 1). Detailed biodistribution data for Huh7 tumors are provided in Supplemental Table 2. We also assessed CD24 expression using immunohistochemistry after biodistribution studies, confirming that membrane expression of CD24 was observed exclusively in Hep3B-CD24+ tumors (Fig. 5F).

DISCUSSION

Our CD24-targeted immuno-PET agent, [89Zr]Zr-DFO-α-hCD24, showed target binding in CD24+ HCC tumors and maintained high uptake in xenografts throughout the study period. These findings suggest that [89Zr]Zr-DFO-α-hCD24 is a promising immuno-PET agent for HCC, offering a noninvasive approach for detecting CD24+ tumors.

GPC3 is a well-established target in HCC; however, its expression is heterogeneous and absent in 20%–30% of tumors. Notably, Hep3B and Huh7 models exhibit moderate to low GPC3 expression, limiting the effectiveness of GPC3-targeted imaging and therapeutic approaches. Kelada et al. (25) evaluated a GPC3-targeted humanized 89Zr-labeled full-length antibody immuno-PET tracer in Hep3B and Huh7 xenografts and found relatively modest tumor uptake at 144 h after injection (6.03 ± 2.34 %IA/g and 6.53 ± 1.67 %IA/g, respectively). In contrast, our CD24-targeted immuno-PET agent, [89Zr]Zr-DFO-α-hCD24, demonstrated higher tumor uptake in Hep3B-CD24+ xenografts, with 8.7 ± 1.2 %IA/g at the same time point. These results highlight CD24 as a promising alternative imaging biomarker in GPC3-low HCC.

CD24-targeted imaging remains an emerging field, as prior studies primarily focused on fluorescence-based approaches. In HCC, CD24-targeted monoclonal antibodies labeled with near-infrared fluorophores have demonstrated the potential for use in imaging and therapy (26). However, such approaches are limited to superficial lesions because of signal attenuation. In contrast, PET imaging offers superior sensitivity and quantitative assessment of molecular targets and enables whole-body tumor detection for disease staging (27,28).

CD24 is also implicated in several oncogenic processes, suggesting its potential as a therapeutic target or biomarker across various histologies. In 2018, Ooki et al. (29) reported that CD24 was associated with cancer stemness in a cohort of patients, and detection of CD24 expression in patient urine led investigators to propose CD24 as a noninvasive urinary biomarker for bladder cancer detection. Preclinically, CD24 expression has been linked to chemotherapy resistance in triple-negative breast cancer, with CD24+ cells exhibiting lower susceptibility to taxane-based treatments (30).

Emerging CD24-targeted therapeutic antibodies, such as ATG-031 and IMM47, have shown promise in enhancing antitumor effects via macrophage-mediated phagocytosis in early-phase trials (NCT05985083 and NCT06028373) (19,20). To the best of our knowledge, the use of these antibodies as radioconjugates has not explored. We choose to use α-hCD24 as opposed to either ATG-031 and IMM47 for both pragmatic and performance reasons. Although ATG-031 is commercially available, its reported affinity (KD, 8.0 nM) is poorer than that of α-hCD24 (KD, 2.4 nM). IMM47, despite exhibiting an affinity (KD, 0.8 nM) higher than that of α-hCD24, it is not widely available for preclinical research.

Beyond studies evaluating the therapeutic efficacy of CD24-targeted antibodies, antibody–drug conjugates have demonstrated reduced tumor growth in preclinical HCC and colorectal cancer xenograft models (22,23). Given the results of our study, CD24-specific PET imaging could serve as a compelling noninvasive biomarker to select patients who may benefit from CD24-targeted therapies, such as antibody–drug conjugates and, perhaps, radiopharmaceutical therapy.

This work underscores the importance of distinguishing total protein expression from membrane protein expression in translational studies that use orthogonal in vitro and in vivo assays. For example, although the Huh7 cell line exhibited high total CD24 expression, its moderate membrane expression resulted in minimal uptake in vivo. Conversely, the Hep3B cell line, which had relatively low total CD24 expression, exhibited high membrane expression and significantly greater tracer accumulation in vivo. These findings underscore the importance of designing radiopharmaceuticals that target highly expressed membrane proteins and highlight the potential value of tissue biopsies in HCC patients to assess for membrane-specific expression of targets of interest.

Despite the promising results of this proof-of-concept study, some limitations must be acknowledged. Although SNU449 tumor cells exhibited high CD24 membrane expression, we were unable to establish viable tumor models in mice using subcutaneous or orthotopic implantation. Further optimization is needed to generate these models for future testing. Additionally, whereas [89Zr]Zr-DFO-α-hCD24 showed high localization in CD24+ tumors by PET visualization and ex vivo biodistribution, we also observed notable uptake in bone, particularly at the bilateral hind joints. This distribution pattern aligns with the known behavior of free 89Zr, which exhibits a high affinity for phosphate and accumulates in bone (31,32). Work to improve 89Zr chelation strategies to reduce off-target skeletal uptake is ongoing in our laboratory and elsewhere (33,34). We also observed high uptake in the liver and spleen, typical clearance organs for full-length antibodies. Furthermore, Fc receptors in liver resident macrophages likely contributed to the elevated liver signal, which could be mitigated by blocking with unlabeled (cold) antibody or modifying the Fc portion of the antibody (35). Alternative CD24-targeting strategies, such as with peptides or antibody fragments (35), or the use of pretargeting approaches that decouple the antibody circulation time from radionuclide delivery are worth exploring to optimize the tumor-to-background ratio for radiotheranostics (36).

CONCLUSION

Our CD24-targeted immuno-PET agent, [89Zr]Zr-DFO-α-hCD24, exhibited good target engagement in murine models of HCC and maintained high uptake in xenografts throughout the study period. These findings suggest that [89Zr]Zr-DFO-α-hCD24 can localize to viable HCC and may serve as a functional imaging agent to help distinguish viable from nonviable HCC, especially after liver-directed therapy. Although these initial results are encouraging, because of the high uptake observed in the liver and spleen in vivo, current efforts are underway to optimize the agent’s performance before considering the further development of a radiopharmaceutical therapy with this antibody.

DISCLOSURE

This work was supported by the Intramural Cancer Institute of the National Institutes of Health, including the National Cancer Institute, Center for Cancer Research, and the Molecular Imaging Branch. The projects funded under this support are ZIA BC011800 and ZIA BC010891. Biolayer interferometry work was supported by the Biophysics Core at the National Institutes of Health. No other potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENT

We thank Dr. Diane Milenic for reading and providing edits.

KEY POINTS

QUESTION: Can we develop a CD24-specific immuno-PET imaging agent to detect HCC?

PERTINENT FINDINGS: [89Zr]Zr-DFO-α-hCD24 can selectively detect CD24+ tumors and has the potential to serve as a functional imaging agent for HCC.

IMPLICATIONS FOR PATIENT CARE: [89Zr]Zr-DFO-α-hCD24 has potential as a noninvasive diagnostic tool for detecting CD24+ HCC and other CD24+ cancers.

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