Comparison of axicabtagene ciloleucel and tisagenlecleucel patient CAR-T cell products by single cell RNA sequencing
Background: Autologous CD19 CAR T cell therapy leads to durable responses and improved survival in patients with relapsed or refractory large B cell lymphoma (R/R LBCL). Among approved CAR T cell products, axicabtagene ciloleucel (axi-cel; CD19/CD28) has greater real-world efficacy and cytokine-associated toxicity than tisagenlecleucel (tisa-cel; CD19/4-1BB), for reasons that are poorly understood. Methods: Here we report single cell RNA sequencing (scRNA-seq) of 57 pre-infusion CAR T cell products from axi-cel (n=39) and tisa-cel (n=18) patients treated as standard-of-care for R/R LBCL, and their biological associations with clinical outcomes. In vitro CAR manufacturing conditions mimicking those known for axi-cel and tisa-cel were performed using CD19/CD28z or CD19/4-1BBz constructs. Results: ScRNA-seq revealed that axi-cel and tisa-cel are markedly different products. Axi-cel is comprised of more CD4 central memory, CD8 central memory, and CD8 effectors, whereas tisa-cel is comprised of more proliferative CD4 and CD8 cells. Across multiple T cell subsets, axi-cel had greater expression of immune response pathways and protein synthesis and trafficking pathways vs. tisa-cel. On comparison of infusion product CAR transgene-positive (CAR+) cells to CAR transgene-negative (CAR-) T cells, axi-cel CAR+ cells had vastly different gene expression than axi-cel CAR- cells. Unexpectedly, tisa-cel CAR+ cells were highly similar to tisa-cel CAR- cells. Under recapitulated CAR-T manufacturing conditions known to be utilized for axi-cel and tisa-cel, we found that CAR+ cells differed from CAR- cells early after manufacturing yet became more similar to CAR- cells after prolonged expansion. Prolonged time in expansion culture, as utilized during tisa-cel manufacturing, greatly decreased naïve and central memory T cell subsets. Conclusions: Following manufacture, axi-cel is less differentiated and has greater immune activation compared to tisa-cel, potentially accounting for its greater efficacy and toxicity in patients. Our data support the conclusion that tisa-cel is adversely affected by its manufacturing rather than by the CAR construct. Overall design: Patients and samples: All patients were consented to prospective sample collection and research database protocols approved by the Institutional Review Board (IRB) of the University of South Florida (USF) or by Advarra. All patient samples were collected at Moffitt Cancer Center. Patient CAR T-cells were obtained by washes from commercial product infusion bags after patient infusion, and cryopreserved for batch analysis.