CAR-Mix cell therapies to treat solid tumors by providing synergistic interaction between engineered macrophages and polyclonal T cells.

Abstract

e14543 Background: Chimeric antigen receptor (CAR) T cells have shown remarkable success against hematologic cancers and are now being adapted for solid tumors. However, MSLN-targeting CAR-T cells demonstrated limited efficacy in chemotherapy-refractory metastatic pancreatic ductal adenocarcinoma patients (NCT03323944). Besides, initial clinical trial data for the HER2-targeting CAR-macrophage (CAR-M) showed suboptimal clinical outcomes (NCT04660929). Current CAR-T or CAR-M therapies typically involve either engineered T cells or macrophages alone. Recent studies revealed that T cells cluster with tumor cells and/or antigen-presenting cells (APCs) in tumors, enhancing their tumor-killing ability. A trispecific antibody engaging T cells with both tumor and myeloid cells also improved antitumor immunity, highlighting the potential of T cell–APC cooperation in combating solid tumors. Based on this, we designed a novel adoptive cell therapy leveraging the synergy between T cells and macrophages to activate both adaptive and innate immunity, aiming to improve solid tumor treatment. Methods: We developed a novel MSLN-targeting CAR-Mix or CAR-cluster cell product using a one-step process optimized from CAR-M production. CAR-Mix mainly consists of CAR-M and T cells. After co-culture with tumor cells, clusters of T cell–tumor cell and T cell–macrophage could be detected by flow cytometry. Results: In vitro tumor-killing assays and in vivo studies with the SKOV3 ovarian cancer xenograft model in NSG mice showed that CAR-Mix had significantly enhanced antitumor efficacy compared to CAR-M or T cells alone. CD69 expression on T cells in CAR-Mix was markedly upregulated following co-culture with MSLN-expressing SKOV3 cells. NY-ESO tetramer-positive T cells also appeared after co-culturing CAR-Mix with SKOV3 cells expressing MSLN and NY-ESO, indicating antigen cross-presentation and efficient T cell activation. Bulk TCR sequencing revealed that the diversity of both TRA and TRB genes significantly decreased after CAR-Mix was co-cultured with tumor cells. This result indicates that specific T cell clones were selectively expanded within the CAR-Mix population upon encountering tumor cells. The enhanced anti-solid tumor activity of CAR-Mix is driven by its antigen cross-presentation capability combined with robust polyclonal T cell activation. Toxicity studies in immunodeficient NSG mice showed no damage to major tissues or organs, and serum biochemical indices remained comparable to controls. Conclusions: In summary, this study shows that CAR-Mix achieves significantly greater anti-solid tumor efficacy than CAR-M or T cells alone, primarily through synergistic interactions between CAR-M and T cells. These results strongly support further clinical evaluation of CAR-Mix as a promising therapy for patients with MSLN-positive solid tumors.