CD24 as a Novel Radiopharmaceutical Target for Hepatocellular Carcinoma

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.