Effects of an Initial Anti-CD19 CAR T-cell Therapy on Subsequent Anti-CD22 CAR T-cell Manufacturing and Clinical Outcomes in Patients with Relapsed/Refractory LBCL

Abstract

Late leukapheresis (>6 months after CAR19) resulted in less residual CAR19, higher CAR22 CD4+ naïve T and TCM cells, less TEM cells, and higher CD8+ TCM cells, but similar clinical outcomes to those with early leukapheresis. CAR22 responses were associated with higher transduction efficiency and CD8+ TCM and less CD8+ TEM cells.

Figure 1.. Circulating CAR19 levels following infusion in cohort 1.

(A) Flow cytometry was utilized to assess circulating CAR19 levels in patients with relapsed/refractory large B cell lymphoma who received axicabtagene ciloleucel at Stanford within the first 28 days. By day 28 post-CAR19 infusion, 85.9% patients having CAR19 comprising less than 5% of CD3⁺ T cells. (B) Using qPCR assay, the maximal CAR19 transgene level within 28 days post-CAR19 infusion was a median of 748.5 copies per 50ng DNA, which decreased over time. Six months post-CAR19 infusion, the transgene level decreased to a median of 11.4 copies per 50ng DNA. LOD: 1.7 copies per 50ng DNA; LOQ: 5.1 copies per 50ng DNA. Abbreviations: IQR, interquartile range; LOD, limit of detection

Figure 2.. Overview of CAR T-cell therapies and intervening therapies in cohort 2.

(A) Swimmer plot illustrates the CAR19 therapy history, intervening therapies between CAR19 and CAR22 therapies, and subsequent leukapheresis for CAR22 manufacturing time. The plot uses color-coding to distinguish between CAR T-cell therapies, while the bars depict the duration of treatment response. These are stratified based on the timing of subsequent leukapheresis for CAR22 manufacturing, either within or beyond 6 months post-CAR19 infusion. (B) Progression-free survival after CAR19 therapy. (C) The intervening therapies and treatment responses resulted in an ORR of 16.7%, CR rate of 10.0%. Abbreviations: CD20 Ab, anti-CD20 monoclonal antibody; LAG-3, lymphocyte-activation gene 3; NE, not evaluable; PD-1, programmed cell death protein 1; PR, partial response; SD, stable disease.

Figure 3.. Kinetics of circulating CAR19 assessed by qPCR assay during the initial 28 days post-CAR19 infusion, subsequent CAR22 manufacturing process, and post-CAR22 infusion.

(A) Following the initial expansion post-CAR19 infusion, circulating CAR19 levels decrease over time. The residual CAR19 did not show a significant increase during the CAR22 manufacturing process. After CAR22 infusion, the maximal median CAR19 transgene levels were 544.2-fold lower than maximal median CAR22 transgene level. The CAR19 transgene level decreased to an unquantifiable level within 30 days post-CAR22 infusion in most patients in both groups. (B) Early group patients exhibited higher CAR19 transgene levels in the leukapheresis material. (C) Patients who received intervening therapies between CAR19 and CAR22 therapies demonstrated a trend toward reduced residual CAR19 transgene levels in leukapheresis material. ☒ represents patient who received only local radiotherapy between CAR19 and CAR22 therapies. LOD: 1.7 copies per 50ng DNA; LOQ: 5.1 copies per 50ng DNA. *, P < .05 ^ns, no significance

Figure 4.. Comparison of CAR22 product characteristics and functional assays between early and late groups.

(A) No correlation between the residual CAR19 transgene levels in leukapheresis material and CAR22 transduction rate in either early or late groups. (B) Patients in the late group had higher transduction rates and VCN of CAR22 products compared to those in early group. The CD4 and CD8 ratio of CAR⁺ cells were similar between the two groups. (C) Late group patients exhibited a higher proportion of CD4⁺ naïve T cells (CD45RA⁺, CCR7⁺), CD4⁺ T_CM cells (CD45RA⁻, CCR7⁺), and CD8⁺ T_CM cells, and a lower proportion of CD4⁺ T_EM (CD45RA⁻, CCR7⁻) cells compared to early group patients. (D) Expression of exhaustion markers, including CD39, LAG-3, PD1, and TIM3, showed no significant difference between the 2 groups. (E) In intracellular cytokine staining, CAR22 products cultured with CD22 low or high expression cells showed similar levels of cytokine (IFNγ, IL-2, and TNFα), as well as similar upregulation of activation (CD69) and degranulation (CD107a) markers between early and late groups. Abbreviations: Avg. gag / c-frag, average number of gag protein per cellular fragment; IFNγ, interferon γ; IL-2, interleukin-2; LAG-3, lymphocyte-activation gene 3; IQR, interquartile range; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin mucin-3; TNFα, tumor necrosis factor α.

Figure 5.. Comparison of complete response rates, progression-free survival, and overall survival across CAR22 therapy subgroups

(A) Comparison of CR rates among the three subgroups. Patients with CAR22 product transduction rates above the median exhibit a higher complete response rate to CAR22 therapy. (B) There were no significant differences in CD4⁺ T cell subsets in the CAR22 product between patients who achieved CR and those who did not. CAR22 products from patients who achieved CR had more CD8⁺ T_CM cells and fewer CD8⁺ T_EM cells. (C) Similar progression-free survival observed across the 3 subgroup analyses. (D) Patients with higher CAR22 product transduction rates demonstrate better overall survival. Overall survival are comparable among the other two subgroups. Abbreviations: PR, partial response; SD, stable disease.