Abstract

Background In recent years, tumor immunotherapy, exemplified by immune checkpoint inhibitors, has achieved remarkable success in certain patients. However, the efficacy of such therapies remains minimal in the majority of colorectal cancer patients. Overcoming the suppression of the tumor immune microenvironment and developing novel immunotherapeutic strategies have become focal points of research in this field. Adoptive cell therapy demonstrates immense potential, with chimeric antigen receptor macrophages exhibiting inherent advantages in combating solid tumors. GPA33, a surface antigen that is expressed in nearly all colorectal cancers, is a promising target for immunotherapy in colorectal cancer. This study aims to evaluate the potential therapeutic effects of GPA33-targeted CAR-macrophages in colorectal cancer. Methods Using single B cell isolation technology and single-cell PCR technology, we cloned the complete variable region sequences of mouse antibody light and heavy chains, which were subsequently employed to construct the scFv fragment of GPA33 CAR. CAR-Ms were generated by transfecting THP-1 cells with lentivirus, followed by PMA induction. The targeted phagocytic activity of CAR-Ms was observed using fluorescence microscopy, and their targeted phagocytosis, polarization trends, and tumoricidal effects were validated through flow cytometry. Cytokine secretion was quantified using ELISA assays. The in vitro and in vivo anti-tumor effects of CAR-Ms were assessed through bioluminescence measurements, while the safety of CAR-Ms was evaluated using HE staining and a small animal biochemical analyzer. Results Based on the successful development of a novel GPA33 antibody with high affinity and specificity, we constructed CAR-Ms targeting GPA33. For the in vitro experiments, GPA33-specific CAR-Ms were able to mediate antigen-dependent M1-like polarization of macrophages, enhancing their tumor phagocytic activity and pro-inflammatory cytokine secretion. In in v