Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels

Abstract

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum interleukin-15 (IL-15). Patients who achieved a remission had a median peak blood CAR⁺ cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR⁺ cell level of 15/μL ( P = .027). High serum IL-15 levels were associated with high peak blood CAR⁺ cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-15 levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

Fig 1.

Low-dose chemotherapy depletes lymphocytes and modulates serum proteins. (A) Schematic of the clinical protocol. Fludarabine and cyclophosphamide were administered on days –5 to –3. Fludarabine dose was 30 mg/m² per day for all patients. Cyclophosphamide dose was 300 mg/m² for 18 patients and 500 mg/m² for four patients. A single dose of chimeric antigen receptor (CAR) targeting CD19 (CAR-19) T cells was administered on day 0. (B) The blood lymphocyte counts for each patient are shown before and after chemotherapy. Each patient’s lymphocyte counts are connected by a line (P < .001 for the paired comparison of before and after chemotherapy). The after time point was the day of CAR T-cell infusion. The median blood lymphocyte count just before the start of chemotherapy was 635/μL (range, 150 to 1,660/μL). The median blood lymphocyte count on the day of CAR T-cell infusion was 40/μL (range, 0 to 310/μL). The normal range for blood lymphocytes was 1,320 to 3,570/μL. (C) Interleukin-15 (IL-15), IL-7, and monocyte chemoattractant protein-1 (MCP-1) all increased after chemotherapy, and perforin decreased. The protein levels before and after chemotherapy for each patient are connected by a line. Chemotherapy samples for the after time point were all drawn on the day of CAR T-cell infusion. For all four proteins, P < .001 when paired before and after chemotherapy levels for each patient were compared. (D) CAR⁺ T cells from the time of infusion were stained for C-C chemokine receptor-7 (CCR7) and CD45RA to identify T cells with phenotypes of these subsets: naïve (CCR7⁺CD45RA⁺), central memory (CM; CCR7⁺CD45RA^–), effector memory (EM; CCR7^–CD45RA^–), and effector memory RA subsets (EMRA; CCR7^–CD45RA⁺). Plots were gated on CD3⁺CAR⁺ lymphocytes. Means and standard error of the mean are shown. (E-H) Anti-CD19 CAR T cells secreted a variety of proteins in an antigen-specific manner. Graphs show the levels of proteins in culture supernatants after CAR-19 T cells from the time of infusion were cultured overnight with either CD19^–NGFR-K562 cells or CD19⁺CD19-K562 cells. Results for each patient are connected by a line. CD19-specific production of interferon-γ (IFN-γ), granzyme B, IL-10, and tumor necrosis factor alpha (TNF-α) occurred.

Fig 2.

Chimeric antigen receptor (CAR) targeting CD19 (CAR-19) T cells eradicated large masses of chemotherapy-refractory lymphoma. (A) Graphical representation of the types of antilymphoma responses and the durations of responses. (B) Progression-free survival starting at the day of cell infusion and ending at the day of disease progression is shown for all patients. Red marks indicate censored patients with ongoing complete remissions (CRs) at the time of last follow-up with one exception: the red mark at 4 months after CAR T-cell infusion indicates the time point when patient 40 underwent allogeneic stem-cell transplantation while in partial remission. Two patients were censored at the 13-month time point and three patients were censored at the 14-month time point, but there is only one red mark on the graph for each of these time points. (C) Patient 35 had received four types of lymphoma therapy, and his diffuse large B-cell lymphoma was chemotherapy refractory at the time of protocol enrollment. After CAR-19 T-cell infusion, his lymphoma entered an ongoing CR. (D) At the time of protocol enrollment, patient 38 had received five types of prior lymphoma therapy, and her diffuse large B-cell lymphoma was refractory to chemotherapy. After CAR-19 T-cell infusion, the lymphoma entered an ongoing CR. For (C) and (D), the white arrows indicate sites of lymphoma. Residual red-colored areas in the after-treatment images are normal findings in the brain, heart, kidneys, and bladder. PD, progressive disease.

Fig 3.

Clinical remissions of lymphoma were associated with high peak blood levels of chimeric antigen receptor–positive (CAR⁺) cells and interleukin-15 (IL-15). The absolute number of CAR⁺ peripheral blood mononuclear cells (PBMCs) in patients who achieved clinical antilymphoma responses of either (A) complete remission (CR) or partial remission (PR) or (B) stable disease (SD) or progressive disease (PD) were quantified by polymerase chain reaction. (C) Patients who achieved remission (CR + PR) had higher levels of CAR⁺ PBMCs than patients who did not (SD + PD; P = .027). Horizontal lines represent the medians in panels C, F, I, and L. (D) CAR⁺ T cells from patient blood samples collected between 4 and 15 days after CAR T-cell infusion were stained for C-C chemokine receptor-7 (CCR7) and CD45RA. The graph shows the fraction of T cells with phenotypes of four different T-cell subsets: naïve (CCR7⁺CD45RA⁺), central memory (CM; CCR7⁺CD45RA^–), effector memory (EM; CCR7^–CD45RA^–), and effector memory-RA (EMRA; CCR7^–CD45RA⁺) subsets. Plots were gated on CD3⁺CAR⁺ cells. For each T-cell subset, mean and standard error of the mean are shown. (E) The peak level of blood CAR⁺ cells correlated with the peak level of IL-15 (Spearman correlation r = 0.7; P = .001). (F) Patients who achieved remission of lymphoma (CR + PR) had higher serum IL-15 area-under-the-curve (AUC) levels from day –5 to day 14 than patients who did not (SD + PD; P < .001). Serum levels of IL-15 for (G) all patients who achieved remission (CR + PR) or (H) patients who did not (SD + PD) are shown. Day 0 is the day of CAR T-cell infusion. (I) Patients who achieved remission (CR + PR) after CAR T-cell infusion had higher peak serum IL-15 levels than patients who did not (P = .001). The serum levels of IL-10 for (J) all patients who achieved remission (CR + PR) or (K) patients who did not (SD + PD) are shown. (L) Patients who achieved remission (CR + PR) had higher peak serum levels of IL-10 than patients who did not achieve remission (SD + PD; P = .010). A total of 41 serum proteins were assessed (panels I and L), and the results of the two proteins with the most impressive differences are shown. Results for all 41 serum proteins are provided in the Data Supplement. Statistical correction for multiple comparisons was not performed.

Fig 4.

Neurologic toxicity was associated with high peak blood levels of chimeric antigen receptor–positive (CAR⁺) cells and increased levels of certain serum proteins. (A) The number of blood CAR⁺ cells in patients with grade 3 or 4 neurologic toxicities was higher than that in patients with only < grade 3 neurologic toxicities (P = .003). (B) Flow cytometry revealed CAR targeting CD19 (CAR-19) T cells in the cerebrospinal fluid (CSF) in all assessed patients with neurologic toxicity. The flow cytometry result of CSF from patient 31 from 9 days after CAR-19 T-cell infusion is shown as a representative example. The plot is gated on CSF mononuclear cells. Granzyme B serum levels of (C) patients with grade 3 or 4 neurologic toxicities and (D) those with only < grade 3 neurologic toxicities are shown. Patients with grade 3 or 4 neurologic toxicities had higher peak serum levels of (E) granzyme B (P = .002), (F) interleukin-10 (IL-10; P = .006), and (G) IL-15 (P = 0.014) than patients with only < grade 3 neurologic toxicities. A total of 41 serum proteins were assessed (panels E, F, and G), and the results of the three proteins with the most impressive differences are shown. Results for all 41 serum proteins are provided in the Data Supplement. Statistical correction for multiple comparisons was not performed.