In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specific CAR-T Cells in B Cell Non-Hodgkin's Lymphomas

Abstract

Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy.

Keywords: CAR-T cells; CD19; chimeric antigen receptor; immunotherapy; second-generation CAR; third-generation CAR.

Figure 1

In Vivo Expansion and Persistence of Infused CD19.CARTs in Peripheral Blood Expansion was assessed by qPCR and is shown after one infusion of CD19.CARTs in patients with active disease (A), after lymphodepleting chemotherapy, and in remission post-autologous stem cell transplant (B), without lymphodepleting chemotherapy. (C) Representative flow cytometry data at peak (week 1 to 2) CD19.CART expansion in four patients (data were acquired in two instruments and thus axes’ scales differ slightly). The flow cytometry staining for the CD19.CARs cannot distinguish between 2G and 3G CARs, and thus the data represent the combined 2G and 3G circulating CARTs. (D) Summary of expansion data after the first (with lymphodepleting chemotherapy) and second (without preceding lymphodepleting chemotherapy) infusions for those patients who received two CD19.CART infusions. Data points represent critical post-infusion intervals after infusion of CD19.CARTs. The lines summarize the mean expansion and persistence (error bars, SEM). (E) Cytokine levels at baseline and during peak CART expansion in patients who had clinical evidence of possible CRS.

Figure 2

Patient #2 Achieved Durable CR after Infusion of CD19.CARTs Patient #2 is a 67-year-old male who had primary refractory DLBCL arising in a background of follicular lymphoma and was unable to proceed to ASCT after two salvage regimens. He was given lymphodepletion followed by CART infusion and, 10 days later, he developed fever (A) and tachypnea and was admitted to the hospital, where all cultures remained negative. On admission, inflammatory markers, including CRP (peaking at 12.2 mg/dL on day 11) and IL-6 (peaking at 91.2 pg/mL on day 11 from 6.3 pg/mL at baseline) were elevated, consistent with mild CRS. At this point, we documented a 5-log expansion of his CART cells by genomic qPCR, corresponding to 25% of circulating T cells by flow cytometry (B). In contrast to all other patients, the 2G CAR-T cells ultimately expanded more. His symptoms resolved spontaneously in less than a week while receiving conservative treatment and a repeat PET scan obtained 6 weeks after CART infusion showed CR (C), which has been sustained for more than 18 months.

Figure 3

Patients #4 and #16 Achieved CR after Infusion of CD19.CARTs (A) Patient #4 is 64-year-old female with DLBCL transformed from ENMZL, which had relapsed a few months post-high-dose-therapy and ASCT. At this point, she received lymphodepleting chemotherapy followed by CART cells. Notably, she did not develop any clinically apparent CRS, but a repeat PET scan 6 weeks after infusion was consistent with CR, which has been maintained for more than 12 months. (B) Patient #16 is a 52-year-old male with de novo DLBCL, which relapsed 3 months after high-dose therapy and ASCT and was refractory to two second-line chemotherapy regimens and led to gastric outlet obstruction syndrome. The patient received lymphodepleting chemotherapy followed by CART cells, developed mild CRS within 1 week, and a repeat PET scan 6 weeks after infusion demonstrated a dramatic CR. Expansion data for each patient is shown on the bottom row.