Construction of Novel GPC3 Antibodies in Diagnosis and Immunotherapy/Targeted Therapy of Hepatocellular Carcinoma: Results and Prospects Based on in Vitro and in Vivo Experiments

Abstract

Background: Primary liver cancer (PLC) is the sixth most common malignant tumor in the world. Hepatocellular carcinoma is the most common form (85–90%) of PLC. We aimed to explore the construction of a new type of Glypican-3 (GPC3) antibody and determining its value in the diagnosis and immunotherapy/targeted therapy of hepatocellular carcinoma. Methods: Patients with or with hepatic benign tumors were recruited. Preoperative peripheral blood and postoperative paraffin samples of patients with HCC and peripheral blood samples of patients with benign liver tumors were collected. The expression of GPC3 protein was measured by immunohistochemical assay and enzyme-linked immunosorbent assay (ELISA). The Laser scanning confocal technique was used to observe the phenomenon of endocytosis induced by antibodies and the localization of endocytosis bodies. The viral plasmid targeting GPC3 chimeric antigen receptor (CAR-GPC3) was constructed using BMK antibody and the gene sequences of # 5m and # 49m scFv. The antibody-radionuclide conjugate ¹³¹I-anti-GPC3 Conjugate (¹³¹I-aGPC3) was prepared by labeling # 49GPC3-mAb with ¹³¹I. A mouse model of subcutaneous xenograft tumor was established using 7721 cells without GPC3 and Hep3b cells with high expression of GPC3. The targeting effect of ¹³¹I-aGPC3 on tumors in mice was observed by single photon emission computed tomography (SPECT). Results: GPC3 protein was mainly expressed in the tumor cell membrane and cytoplasm. The antibody content in the peripheral blood of patients with positive expression of GPC3 protein was significantly higher than in patients with negative GPC3 protein and benign tumors (p < 0.01). The radiation signals were gradually enriched in the tissue of GPC3-positive tumors. 72 hours after injection, the background signal in vivo gradually disappeared, whereas the radiation signal in the tumor site concentrated significantly concentrated and could last longer. In addition, ¹³¹I-aGPC3 was gradually enriched in the tumor site of the Hep3b transplanted tumor model. The radiation signal was significantly enhanced, while in the 7721 transplanted tumors, the antibody was metabolically cleared, and there was no obvious radiation signal. Conclusion: CAR-GPC3-T, constructed with new antibody sequences of # 5GPC3-mAb and # 49GPC3-mAb, kills HCC tumor cells in vitro.