CAR T-cell therapy has revolutionized treatment for patients with hard-to-treat large B-cell lymphoma, but not all CAR T products are created equal. Two of the most widely used therapies, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), both target CD19, but they perform differently in patients. Axi-cel often shows stronger responses, but it also causes more side effects. What accounts for these differences?

To answer that, researchers at the Moffitt Cancer Center used single-cell RNA sequencing to analyze 57 CAR T-cell infusion products from patients treated with either axi-cel or tisa-cel. They wanted to understand how the individual cells in each product differ and how those differences might influence outcomes.

Single-cell RNA sequencing of axicabtagene ciloleucel and tisagenlecleucel infusion products
identifies CAR-expressing T cells

(A) Overview of study design, patient sample numbers, and manufacturing differences between axi-cel and tisa-cel. (B) UMAP demonstrating single-cell clustering based on RNA expression of 105,326 CAR-T cell infusion product cells from patients with standard care axi-cel or tisa-cel treated large B-cell lymphoma. Cluster designation is based on gene expression patterns that are known to be associated with select differentiation states (C) Total percentage of CD4+, CD8+, or myeloid cells by product type. Each dot represents a different patient/product (pink=axi-cel; olive=tisa-cel). Shown are mean+SEM. Two-sided Wilcoxon rank-sum test was used to calculate p value followed by FDR correction. (D) UMAP showing cells from axi-cel (top, pink/maroon) and tisa-cel (bottom, olive/green) infusion products that either do not express the respective CAR transgene (left, axi-cel pink, tisa-cel olive) or do express the CAR transgene (right, axi-cel maroon, tisa-cel green). (E) Correlation of tisa-cel percentage of T cells expressing CAR protein by flow cytometry (conducted by the manufacturer) and the percentage of tisa-cel product T cells expressing the CAR transgene as quantified by scRNA-seq. Each dot represents a different tisa-cel patient infusion product (n=18). (F) Stacked bar graphs demonstrating the proportion of cells within each cluster for axi-cel (left), and tisa-cel (right) products, separated by CAR+ and CAR− cells. axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; DR, durable responder; FDR, false discovery rate; IACs, immune effector cell-associated neurologic toxicity-associated myeloid cells; mregDC, mature immunoregulatory dendritic cells; NDR, non-durable responder; PFS, progression-free survival; scRNA-seq, single-cell RNA sequencing; tisa-cel, tisagenlecleucel; Treg, regulatory T cells; UMAP, uniform manifold approximation and projection.

What they found was striking. Axi-cel and tisa-cel are made up of very different types of T cells. Axi-cel products contained more “central memory” and “effector” T cells, the types known to mount strong immune responses. These cells were also more active in producing proteins and carrying out immune functions. In contrast, tisa-cel products had more cells that were in a state of high proliferation but were less activated.

Even more interestingly, the gene expression patterns of the CAR-positive cells in axi-cel were clearly different from other T cells in the product, suggesting strong CAR-driven effects. In tisa-cel, on the other hand, the CAR-positive cells looked very similar to normal T cells, suggesting a weaker impact of the CAR engineering.

The team also replicated the manufacturing processes used for both products in the lab. They discovered that longer expansion times, like those used in tisa-cel production, caused the loss of valuable T-cell subtypes and reduced the cells’ functional potential.

In summary, the research suggests that how the cells are grown during manufacturing, not just the CAR design, may play a big role in how effective the final product is. This could explain why axi-cel performs better in the clinic, but also why it may trigger stronger immune-related side effects.