Comparison of axicabtagene ciloleucel and tisagenlecleucel patient CAR-T cell products by single-cell RNA sequencing

Abstract

Background: Autologous CD19 chimeric antigen receptor (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 versus 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 used 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 used 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 with 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.

Keywords: Chimeric antigen receptor - CAR; Lymphoma; T cell.

Figure 1. 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 (online supplemental figure S2A-D. See online supplemental methods for the full name of each cluster. (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.

Figure 2. Axi-cel and tisa-cel CAR T cells have different phenotype and gene expression profiles. (A) Cell clusters of CAR+ CD4+ T cells between axi-cel and tisa-cel products. (B) % of CD4 CAR T cells that fall within specific clusters comparing axi-cel and tisa-cel on a patient level basis (each dot represents a different patient’s cell type as a % of total CAR+ CD4 cells). (C) Cell clusters of CAR+ CD8+ T cells between axi-cel and tisa-cel products. (D) % of CD8 CAR T cells that fall within specific clusters comparing axi-cel and tisa-cel on a patient level basis (each dot represents a different patient’s cell type as a % of total CAR+ CD8 cells). (E) Evaluation of Hallmark gene-set expression within T-cell clusters between axi-cel and tisa-cel. Red=higher in axi-cel within that cluster, blue=higher in tisa-cel within that cluster. Cells are independently clustered with Hallmark categories shown vertically on the left. Shown are normalized enrichment scores. (F) Evaluation of Gene Ontology Cellular Component pathways. Shown are gene sets with significantly different expression between axi-cel and tisa-cel within T-cell clusters. (G, H) Meta-analysis identified genes with differential expression across clusters in single-cell RNA sequencing data, between axi-cel and tisa-cel products. Genes are labeled on the volcano plot with differential expression between axi-cel and tisa-cel with a meta |log2FC|>=2, meta log(p value)>300, and expressed in >15% of CAR+ cells in axi-cel. (G) CD4+ CAR+ cells. (H) CD8+ CAR+ cells. For B and D, shown are mean+SEM; two-sided Wilcoxon rank-sum test was used to calculate p value followed by FDR correction. axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; FDR, false discovery rate; tisa-cel, tisagenlecleucel.

Figure 3. ScRNA-seq reveals major differences in phenotype and gene expression profiles between CAR-expressing and non-CAR-expressing T cells within axi-cel, but not tisa-cel, products. (A) Percentage of cells that fall within specific clusters comparing axi-cel or tisa-cel product cells that do (CAR+) or do not (CAR−) express the CAR transgene by scRNA-seq. Each dot represents a different patient’s cells in a cluster within that category. Shown are mean+SEM. Two-sided Wilcoxon rank-sum test was used to calculate p value followed by FDR correction. FDR-adjusted p value: *≤0.05; **≤0.01; ***≤0.001, ****≤0.0001. (B, C) Evaluation of HALLMARK and Gene Ontology gene sets across clusters, comparing CAR+ to CAR− cells, revealed a significant difference in expression levels in many more gene sets for axi-cel products (pink) than tisa-cel products (olive). (B) Each dot represents the number of significantly different gene sets between CAR+ and CAR− cells within one cluster, for one product. (C) Each column shows a different cluster and the number of significantly different gene sets in aggregate across all patient products. (D) Previously validated gene score that quantifies tonic signaling of the CAR, comparing axi-cel (pink) to tisa-cel (olive) CAR+ cells (left), and CAR+ (red) and CAR− cells (gray) for both axi-cel (right, top) and tisa-cel (right, bottom) products. axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; scRNA-seq, single-cell RNA sequencing; tsa-cel, tisagenlecleucel.

Figure 4. Prolonged ex vivo CAR expansion during manufacture contributes to similarity in phenotype between CAR+ and CAR− cells. Experimentally manufactured CAR-T cell products from healthy donors were evaluated for cell surface protein expression of key differentiation and exhaustion markers by flow cytometry across time. (A) Shown is the experimental schema evaluating three different manufacturing conditions: both 4-1BBz and 28z CARs made from selected T cells stimulated with antiCD3/antiCD28 coated beads, and 28z CARs from PBMCs stimulated with antiCD3 antibody. Each of the three conditions was then expanded for a short 3–4 days duration (early) or a long 12–14 days duration (late). At each time point, the geometric mean fluorescent intensity (GMFI) for different differentiation and exhaustion markers was calculated for CD8+ cells that do (CAR+) or do not (CAR−), express the CAR protein. The delta GMFI for each manufacture condition, with both early and late expansion, was created by taking the difference between the GMFI for CD8+CAR+ and the GMFI for CD8+CAR−. (B) Shown is the delta GMFI for the indicated marker for each of the manufacture conditions with early (short 3–4 days) expansion (blue circle) and with late (long 12–14 days) expansion (red triangle). Each dot represents a different healthy donor's delta GMFI at the early (blue circle) or late (red triangle) timepoint. P values represent a paired t-test. Manufacture conditions were only significantly different for TCF7. Shown are CD8 T cells, with CD4 T cells from the same experiment displayed in online supplemental figure S9A. (C) In a separate experiment, the protein expression at four time points of ex vivo expansion was evaluated by flow cytometry. Shown is GMFI on the y axis and time in days from stimulation after viral transduction on the x axis. CAR-T cells were manufactured using a gamma-retroviral vector to express either a second generation CD19-CD28z CAR, or a CD19-41-BB, CAR construct. Cells were manufactured with PBMC as starting material stimulated with soluble anti-CD3 Ab and IL-2 (similar to axi-cel) or using a CD19-CD28z magnetically selected T cells as starting material stimulated with anti-CD3/anti-CD28 beads (similar to tisa-cel). Flow cytometry on the CAR+ (pink for CD28z construct, olive for 41-BBz construct) or CAR− (black) CD8 T cells allowed comparison of phenotype across time and by different constructs and manufacturing conditions. Shown are CD8 T cells with CD4 T cells from the same experiment shown in online supplemental figure S9B. CAR, chimeric antigen receptor; PBMC, peripheral blood mononuclear cells.

Figure 5. Different scRNA-seq gene sets and genes, independent of phenotypic clusters, associate with efficacy of axi-cel. (A) Stacked bar graphs of CAR+ cells, demonstrating the proportion of cells within each CD4 (left) and CD8 (right) cluster, separated into durable responder (DR) and non-durable responder (NDR) for axi-cel. The number of CAR+ single cells analyzed in each group is indicated in parentheses. (B) Evaluation of Hallmark gene set expression within T-cell clusters between DR and NDR patients for axi-cel. Red=higher in DR and blue=higher in NDR. Cells are independently clustered with Hallmark categories shown vertically on the left. Shown are normalized enrichment scores (NES). (C) Evaluation of Gene Ontology Cellular Component gene-set expression within T-cell clusters between DR and NDR patients for axi-cel. Red=higher in DR and blue=higher in NDR. Shown are NES. (D) Meta-analysis was performed to identify genes showing differential expression between DR and NDR CAR+ cells across clusters identified in scRNA-seq data, for axi-cel. axi-cel, axicabtagene ciloleucel; CAR, chimeric antigen receptor; scRNA-seq, single-cell RNA sequencing.