| Author: | Leung, Wing Hei |
| Title: | Study on the therapeutic efficacy of glypican-3 peptide-link chimeric antigen receptor-macrophage in hepatocellular carcinoma |
| Advisors: | Lee, Kin Wah Terence (ABCT) |
| Degree: | Ph.D. |
| Year: | 2026 |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xx, 173 pages : color illustrations |
| Language: | English |
| Abstract: | Hepatocellular carcinoma (HCC) is a highly aggressive cancer with limited treatment options and poor prognosis. Recent advancements have introduced multi-kinase inhibitors such as sorafenib and Lenvatinib as first-line treatments for unresectable HCC, however, their survival benefits are limited by drug resistance. Immunotherapy has gained traction, particularly with immune checkpoint inhibitors such as nivolumab and atezolizumab. Moreover, combination therapies involving these inhibitors have demonstrated enhanced efficacy and have received FDA approval for advanced HCC. Recently, chimeric antigen receptor (CAR) therapy, particularly CAR-T cell therapy, has been a promising cell-based therapy in hematologic malignancies, with ongoing efforts to adapt it for solid tumors like HCC. However, challenges such as off-target effects and the immunosuppressive tumor microenvironment complicate its application. Chimeric antigen receptor macrophages (CAR-Ms) have emerged as a promising immunotherapy for solid tumors, leveraging the unique abilities of macrophages to infiltrate the tumor microenvironment (TME), engulf cancer cells, and prime adaptive immunity. However, conventional CAR-M therapy relies on viral gene delivery, which is costly, technically challenging, and raises safety concerns regarding genomic integration. To overcome these limitations, we developed a peptidic CAR-Ms (pCAR-Ms) platform using a novel phthalaldehyde-amine capture (PAC) bioconjugation method to link Glypican-3 (GPC3)-targeting peptides directly to macrophages, bypassing genetic engineering. Using an in vitro co-culture system, we demonstrated that pCAR-Ms selectively phagocytose GPC3-high HCC cells while sparing GPC3-low or GPC3-knockdown controls, as quantified by flow cytometry and fluorescence microscopy. To validate therapeutic efficacy in vivo, we employed MHCC-97L xenografts and patient-derived tumor xenografts (PDTXs). Intravenously infused pCAR-Ms exhibited robust tumor homing and significantly suppressed HCC growth compared to unmodified macrophages or no treatment controls, without observable toxicity. To further evaluate therapeutic potential in immunologically intact systems, we utilized two immunocompetent HCC models: MYC-overexpressing/Trp53-knockout (MYCOE/Trp53KO) and the antigen-expressing variant (MYCOELucOS/Trp53KO). pCAR-Ms treatment in these models not only recapitulated the tumor control observed in xenografts but also demonstrated three key immunomodulatory effects: (1) sustained M1 polarization, (2) enhanced T cell activation, (3) induction of adaptive immune responses. To dissect the immune mechanisms underlying pCAR-Ms activity, we performed single-cell RNA sequencing (scRNA-seq) on tumor-infiltrating immune cells. This analysis revealed that pCAR-M treatment remodelled the immunosuppressive TME, with a marked increase in M1-like macrophages and a concomitant reduction in M2-like subsets, consistent to the flow cytometry data showing an elevated M1/M2 ratio. Furthermore, scRNA-seq identified enhanced CD4⁺ and CD8⁺ T cell proliferation and activation, aligning with flow cytometry results demonstrating increased T cell infiltration and effector function in pCAR-M-treated tumors. In summary, we present a unique peptidic CAR-M platform that effectively targets GPC3⁺ HCC, overcoming key limitations of conventional CAR therapies. Our multimodal validation, with in vitro phagocytosis assays, in vivo tumor models, scRNA-seq analysis, and immune correlative analyses, demonstrates that pCAR-Ms remodel the TME into an immunostimulatory niche, synergizing with endogenous immunity. This approach not only provides a scalable and safer alternative to viral CAR-M systems but also opens avenues for pCAR-M therapies against other solid tumors. |
| Rights: | All rights reserved |
| Access: | open access |
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