Study on Reducing Toxicity of CAR-T Cell Therapy for Solid Tumors

Academic Background

Chimeric antigen receptor T-cell (CAR-T) therapy has made significant progress in treating hematologic malignancies, but its application in solid tumors faces major challenges. Solid tumors often lack tumor-specific antigens (TSAs), and CAR-T cells may attack normal tissues expressing the target antigen, leading to “on-target, off-tumor toxicity.” This toxicity is particularly severe in clinical practice and can be life-threatening. For example, CAR-T cells targeting carbonic anhydrase IX (CAIX) caused severe liver toxicity in treating metastatic renal cell carcinoma, while CAR-T cells targeting HER2 triggered acute respiratory distress syndrome and led to patient death in treating metastatic colorectal cancer. Therefore, effectively reducing the “on-target, off-tumor toxicity” of CAR-T cells has become a key focus of current research.

Source of the Paper

This study was conducted by multiple authors, including Hongye Wang, Zhaorong Wu, Dan Cui, and others, from institutions such as Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai Cancer Institute, and Suzhou Immunofoco Biotechnology Co., Ltd. The paper was published on January 22, 2025, in the journal Science Translational Medicine, titled “Triple knockdown of CD11a, CD49d, and PSGL1 in T cells reduces CAR-T cell toxicity but preserves activity against solid tumors in mice.”

Research Process and Results

1. Research Design and Objectives

The study aimed to reduce the “on-target, off-tumor toxicity” of CAR-T cells while preserving their antitumor activity against solid tumors by simultaneously knocking down the CD11a, CD49d, and PSGL1 genes in T cells. The research team first explored the roles of these molecules in CAR-T cell migration and toxicity through antibody blockade and gene editing techniques, ultimately developing a low-toxicity CAR-T cell therapy.

2. Experimental Process and Results

a) Antibody Blockade Experiment

The research team first discovered through antibody blockade experiments that simultaneously blocking lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) significantly reduced the “on-target, off-tumor toxicity” of CAR-T cells. Experiments were conducted in both immunodeficient mice (NSG mice) and immunocompetent mice (BALB/c mice). The results showed that mice with combined blockade of LFA-1 and VLA-4 had significantly improved survival rates, reduced weight loss, and decreased CAR-T cell infiltration in the liver and lungs.

b) Gene Knockout and Knockdown Experiments

To translate this discovery into clinical application, the research team used CRISPR-Cas9 technology to knock out the CD11a and CD49d genes in CAR-T cells or RNA interference to knock down the CD11a, CD49d, and PSGL1 (P-selectin glycoprotein ligand 1) genes. The results showed that CAR-T cells with these genes knocked out or knocked down exhibited lower toxicity in both in vitro and in vivo experiments while retaining antitumor activity. Additionally, knocking down these genes promoted T-cell memory formation and reduced “tonic signaling” in CAR-T cells.

c) In Vitro and In Vivo Functional Validation

The research team further validated the in vitro and in vivo functions of these low-toxicity CAR-T cells. In vitro experiments showed that CAR-T cells with knockdown of CD11a, CD49d, and PSGL1 exhibited stronger cytotoxicity and lower expression of exhaustion markers. In vivo experiments demonstrated that these CAR-T cells significantly reduced “on-target, off-tumor toxicity” in mouse models while effectively inhibiting tumor growth. Notably, in HCT116 and N87 tumor models, CAR-T cells with gene knockdown showed antitumor activity comparable to the control group and significantly extended the survival of mice.

d) Mechanistic Studies

The research team also conducted mechanistic studies on CAR-T cells with these genes knocked down. RNA sequencing results showed that CAR-T cells with knockdown of CD11a, CD49d, and PSGL1 had reduced expression of genes related to T-cell exhaustion and increased expression of genes related to T-cell memory formation and survival. Gene set enrichment analysis (GSEA) also revealed significant changes in oxidative phosphorylation and T-cell activation-related pathways in CAR-T cells with gene knockdown. Furthermore, knocking down these genes reduced the adhesion and transendothelial migration abilities of CAR-T cells, thereby decreasing their infiltration into normal tissues.

Research Conclusions

By simultaneously knocking down the CD11a, CD49d, and PSGL1 genes in CAR-T cells, the study successfully reduced the “on-target, off-tumor toxicity” of CAR-T cells while preserving their antitumor activity. These low-toxicity CAR-T cells demonstrated significant therapeutic effects in both in vitro and in vivo experiments, providing a new solution for the application of CAR-T therapy in solid tumors.

Research Highlights

1. Innovative Approach: This study is the first to reduce the toxicity of CAR-T cells by simultaneously knocking down multiple cell adhesion and migration-related genes, offering new insights into optimizing CAR-T therapy.
   
2. Significant Clinical Value: These low-toxicity CAR-T cells demonstrated significant therapeutic effects in multiple mouse models, providing potential clinical prospects for CAR-T therapy in solid tumors.
   
3. In-Depth Mechanistic Studies: The study not only validated the effects of low-toxicity CAR-T cells but also deeply analyzed the underlying molecular mechanisms, providing important theoretical foundations for subsequent research.

Research Significance

This study provides important scientific evidence for the application of CAR-T therapy in solid tumors and opens a new pathway for optimizing CAR-T cells through gene editing technology. In the future, these low-toxicity CAR-T cells are expected to be widely used in clinical practice, bringing hope to more cancer patients. Additionally, this study provides new research directions for further exploring the migration, activation, and exhaustion mechanisms of CAR-T cells.