We previously treated B-cell malignancies with CAR-19 T cells preceded by treatment with fludarabine and high-dose cyclophosphamide. 21 This high-dose chemotherapy regimen combined with an infusion of CAR-19 T cells resulted in many long-term remissions of B-cell malignancies, but it also caused significant toxicity. 20 , 21 We hypothesized that administering a low-dose chemotherapy regimen before infusion of CAR-19 T cells would promote their antilymphoma activity and would cause less hematologic toxicity than high-dose chemotherapy. Use of low-dose chemotherapy would also allow a clear determination of the antilymphoma activity of CAR-19 T cells because the low-dose chemotherapy has limited direct antilymphoma activity.

Recipient leukocyte depletion by radiation therapy or chemotherapy enhanced the antitumor activity of adoptively transferred T cells in multiple murine models, 29 - 31 and most clinical trials of adoptive T-cell therapy include chemotherapy before T-cell infusions. 12 , 14 - 16 , 20 , 21 In mice, one way that recipient lymphocyte depletion enhances the antitumor activity of adoptively transferred T cells is by increasing serum levels of cytokines such as interleukin-15 (IL-15). 30 IL-15 is a glycoprotein in the 4-α-helix bundle family of cytokines. 32 - 34 IL-15 is primarily produced by dendritic cells, monocytes, and macrophages, 32 , 34 and it induces T-cell proliferation and enhances T-cell function. 32 - 34

Chimeric antigen receptors (CARs) are artificial proteins that incorporate an antigen recognition domain and T-cell signaling domains. 1 - 8 T cells genetically modified to express a CAR recognize and kill malignant cells expressing the antigen targeted by the CAR. 9 - 11 T cells expressing CARs that target the B-cell antigen CD19 (CAR-19) have potent activity against acute lymphoid leukemia (ALL). 12 - 16 Lymphoma is much more common than ALL, 17 , 18 but compared with ALL, fewer cases of effective treatment of lymphoma with CAR-19 T cells have been published. 19 - 22 Diffuse large B-cell lymphoma (DLBCL) that is either refractory to chemotherapy 17 , 18 , 23 - 25 or relapsed after autologous stem-cell transplantation (ASCT) 26 , 27 carries a grim prognosis. New therapies are needed for advanced-stage B-cell lymphomas. CAR T cells cause adverse events including but not limited to hypotension, cardiac toxicity, and neurologic toxicities. These toxicities are thought to be caused by cytokines directly released by CAR T cells or released by other cells in response to cytokines produced by CAR T cells. 12 , 13 , 15 , 21 , 28

A fludarabine and cyclophosphamide chemotherapy regimen 37 was administered before CAR-19 T-cell infusions to enhance the activity of adoptively transferred T cells. 29 - 31 Both chemotherapy agents were given intravenously once per day for 3 days on the same days ( Fig 1A ). Treatment responses were defined according to standard international criteria. 38

The CAR used in this work was encoded by a gamma-retroviral vector and contained an anti-CD19 single-chain variable fragment derived from a murine monoclonal antibody, hinge and transmembrane regions from human CD28, the CD28 costimulatory domain, and the CD3ζ T-cell activation domain. 35 Anti-CD19 CAR T cells were cultured for 6 to 10 days by adding the anti-CD3 monoclonal antibody OKT3 directly to whole (unsorted) peripheral blood mononuclear cells suspended in culture medium containing IL-2 and transducing the cells as described in the Data Supplement. 20 , 36 CAR T-cell doses were administered as CD3 + CAR + cells per kilogram of body weight ( Table 1 ). The percentage of CAR + T cells was determined by flow cytometry and was used to calculate the number of cells to infuse. Flow cytometry, immunohistochemistry, cytokine assays, and quantitative polymerase chain reaction are described in the Data Supplement. 11 , 20 , 35

We assessed serum levels of 41 proteins in patients with grade 3 or 4 neurologic toxicities and patients with only < grade 3 neurologic toxicities (Data Supplement). Peak serum granzyme B levels were higher in patients with grade 3 or 4 neurologic toxicities compared those who had only < grade 3 neurologic toxicities ( Fig 4C-E ). In addition, peak levels of serum IL-10 and IL-15 were higher in patients with grade 3 or 4 neurologic toxicities compared with those who had only < grade 3 neurologic toxicities ( Fig 4F-G ).

Patients who experienced grade 3 or 4 neurologic toxicity had higher levels of blood CAR + cells compared with patients who had only < grade 3 neurologic toxicities ( Fig 4A ). We performed lumbar punctures to collect cerebrospinal fluid (CSF) from 11 patients with neurologic toxicity. The median number of white blood cells in the CSF of these patients was 22/μL (range, 1 to 215/μL). Flow cytometry was performed on the CSF of nine of the 11 patients who underwent CSF collection, and CAR-19 T cells were detected in the CSF of all nine patients ( Fig 4B ; Data Supplement).

IL-10 is a cytokine that is known to have immunosuppressive properties 43 ; however, IL-10 can also promote T-cell antitumor activity. 44 , 45 Peak serum IL-10 levels were significantly higher among patients who achieved a remission of lymphoma compared with those who did not achieve a remission ( Fig 3J-L ). Aside from IL-15, IL-10 was the cytokine with the clearest difference in peak serum levels when comparing patients who did or did not achieve a remission.

We assessed serum levels of 41 different proteins in patients who achieved a remission (CR or PR) and patients who did not achieve a remission (SD or progressive disease; Data Supplement). IL-15 was the cytokine associated most closely with remissions. Patients who achieved a remission of lymphoma had higher IL-15 area-under-the-curve levels from day –5 to day 14 and higher peak serum IL-15 levels than patients who did not achieve a remission ( Fig 3F-I ). On the day of CAR T-cell infusion, the median serum IL-15 level of patients who achieved a remission was higher than that of patients who did not achieve a remission (30 v 16 pg/mL; P = .010; Data Supplement). This suggests that the environment that CAR T cells enter upon infusion is a determinant of treatment outcomes.

The low-dose chemotherapy conditioning regimen administered before CAR-19 T-cell infusions caused an increase in serum IL-15 ( Fig 1C ). Because IL-15 is known to induce T-cell proliferation, 32 - 34 we reasoned that IL-15 might be associated with peak blood levels of CAR-19 T cells after infusion. We measured serum IL-15 levels at several time points before and after CAR-19 T-cell infusion, and the median day of peak IL-15 levels was day 2 after CAR T-cell infusion. We found a correlation between peak serum IL-15 levels and peak blood CAR + cell numbers ( Fig 3E ).

The number of blood CAR + cells peaked at a median of 8.5 days (range, 6 to 35 days) after infusion ( Fig 3A-B ). CAR + cell numbers dropped rapidly after peaking. Blood CAR + cell numbers decreased to 0 or 1/μL by 3 months after infusion in all patients. Peak blood CAR + cell numbers were higher in patients who achieved lymphoma responses of CR or PR compared with those who achieved SD or progressive disease ( Fig 3A-C ). There was no statistically significant difference in the peak number of blood CAR + T cells in patients who received infusions of CAR-19 T cells at 1 × 10 6 /kg versus 2 × 10 6 /kg. CAR + T cells collected from the blood of patients after infusion primarily had phenotypes of effector memory or effector memory-RA T cells ( Fig 3D ); in comparison, CAR-19 T cells at the time of infusion included more T cells with phenotypes of naïve and central memory T cells ( Fig 1D ). There was an association between the percentage of central memory T cells among the infused CAR T cells and peak blood CAR T-cell levels ( P < .001; Spearman r = 0.7; Data Supplement).

Depletion of normal B cells is an expected toxicity of anti-CD19 CAR T cells. 8 , 12 , 19 , 20 Because of extensive prior treatment, B-cell counts were already low in most patients at the time of protocol enrollment. The median blood B-cell count at enrollment was 1/μL (range, 0 to 123/μL). Only patient 23 had a normal pretreatment B-cell count. Six patients in CR have recovered normal blood B-cell counts, and five remain in CR (Data Supplement). This demonstrates that patients can remain in CR after recovery of normal B-cell counts.

Two patients had notable delayed adverse events. Patient 23 developed myelodysplastic syndrome (MDS) 20 months after CAR T-cell infusion, but the CAR-19 gene was not detectable in the patient’s bone marrow at the time of MDS diagnosis. MDS is common in lymphoma patients with histories of chemotherapy and ASCT, so the extensive treatment received by patient 23 before protocol enrollment likely contributed to his MDS. 41 Patient 27 developed vision loss 3 months after CAR T-cell infusion. Although the etiology of this vision loss is not definitively known, the clinical course and electroretinography were consistent with fludarabine toxicity. 42

All toxicities greater than grade 1 are listed in Table 2 . The most prominent toxicities in this trial were neurologic; 55% of patients had grade 3 or 4 neurologic toxicities. In contrast, only four (18%) of 22 patients had grade 3 or 4 hypotension; three patients required brief courses of vasopressor drugs to treat it. One patient required mechanical ventilation for severe dyspnea; another patient required mechanical ventilation for airway control during severe neurologic toxicity. Common grade 3 and 4 neurologic toxicities included dysphasia, confusion, and tremor. All acute toxicities resolved completely, and no patients died as a result of toxicity. Three patients received specific immunosuppressive drugs: patient 32 received the corticosteroid dexamethasone for severe neurologic toxicity, and patients 36 and 40 received the IL-6 receptor antagonist tocilizumab. Aside from those three patients, all other patients received only supportive treatment without any immunosuppressive drugs, so most patients on this trial had acute CAR T-cell toxicities that resolved spontaneously within a limited period of time.

For all 22 patients treated, there was a 73% remission rate with 55% complete remissions (CRs) and 18% partial remissions (PRs). Among patients with DLBCL, the overall remission rate was 68% with 47% CRs and 21% PRs. The duration of responses currently ranges from 7 months to 24 months ( Fig 2A ). Twelve of 22 patients achieved CRs, 11 of 12 CRs are ongoing, and the current median duration of all CRs is 12.5 months ( Table 1 ). No patient with an ongoing remission received any further antilymphoma therapy after CAR T-cell infusion. In contrast to CRs, no PRs or outcomes of stable disease (SD) are ongoing. Patient 40 underwent allogeneic hematopoietic stem-cell transplantation after achieving a PR ( Table 1 ). The 12-month progression-free survival of all patients was 63.3% ( Fig 2B ), and overall survival is provided in the Data Supplement. Figure 2C-D shows two examples of positron emission tomography/computed tomography scans from patients who achieved CR of chemotherapy-refractory DLBCL.

A median of 97.6% (range, 90.6% to 99.5%) of the infused cells were CD3 + ; a median of 74.4% (range, 39.4% to 93.6%) of the infused CD3 + cells expressed CAR-19. A median of 43.4% of CD3 + CAR + cells expressed CD4 (range, 1.9% to 82.1%), and 54% of CD3 + CAR + cells expressed CD8 (range, 13.4% to 92.1%). C-C chemokine receptor-7 (CCR7) and CD45RA were used to divide T cells into four different subsets ( Fig 1D ). Naïve and central memory T cells have greater proliferative capacity than effector memory and effector memory-RA T cells. 40 Infused CAR-19 T cells included T cells with the phenotypes of all four of these T-cell subsets, including a substantial fraction of cells with the phenotype of central memory T cells. 40 The CAR T cells secreted a variety of proteins in an antigen-specific manner ( Fig 1E-H ; Data Supplement).

Nineteen of the 22 treated patients had one of the various types of DLBCL, two patients had follicular lymphoma, and one patient had mantle cell lymphoma ( Table 1 ). Eleven of 19 patients with DLBCL had chemotherapy-refractory lymphoma. Five other patients with DLBCL had lymphoma that had relapsed 10 months or less after ASCT as their last treatment prior to protocol enrollment. Eleven patients with DLBCL were high risk by second-line, age-adjusted International Prognostic Index. 39 The median number of unique lymphoma therapies received before protocol enrollment was four (range, one to seven). Therapies received before protocol enrollment and lymphoma chromosome rearrangements are provided in the Data Supplement.

DISCUSSION Section: Choose Top of page Abstract INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION << REFERENCES

An infusion of CAR-19 T cells preceded by a low-dose conditioning chemotherapy regimen of cyclophosphamide and fludarabine induced remissions of advanced-stage lymphoma. The low-dose chemotherapy regimen used in this study could not directly induce the remissions of lymphoma reported here because of the documented resistance of these lymphomas to antilymphoma chemotherapy regimens (Data Supplement). Importantly, our CAR T-cell production process was simple and robust. No patient failed to receive treatment because of cell production issues.

We recently treated patients who had had an allogeneic hematopoietic stem-cell transplantation with human leukocyte antigen–matched allogeneic T cells expressing the same CAR-19 used with autologous T cells in this study.36,46 We did not administer chemotherapy before infusions of the allogeneic CAR-19 T cells. We administered allogeneic CAR+ T cells at up to 8.2 × 106/kg with acceptable levels of toxicity.46 In stark contrast to this report of patients treated with autologous CAR-19 T cells at 1 to 2 × 106/kg, neurologic toxicity in the trial of allogeneic anti-CD19 CAR T cells without chemotherapy was rare and mild.36,46 These results suggest that lowering or eliminating the conditioning chemotherapy administered before CAR T-cell infusions might lessen the severity of toxicity; however, lowering chemotherapy intensity might also lead to lower remission rates.46

We compared hematologic toxicities in patients who received CAR-19 T cells after our current low-dose chemotherapy regimen and patients who received our previously reported high-dose chemotherapy regimen, which included a total dose of cyclophosphamide of 60 to 120 mg/kg and a total dose of fludarabine of 125 mg/m2.21 Two of 22 patients treated with the new low-dose chemotherapy required platelet transfusions; 10 of 15 patients receiving high-dose chemotherapy required platelet transfusions (P < .001). The median length of time that patients had severe neutropenia with an absolute neutrophil count of less than 500/μL was 0 days (range, 0 to 6 days) with the low-dose chemotherapy versus 5 days (range, 0 to 16 days) with the previously used high-dose chemotherapy (P < .001). In this study, we increased the daily cyclophosphamide dose from 300 mg/m2 to 500 mg/m2 in four patients in an attempt to increase the CR rate. Because all four of these patients experienced substantial toxicity (Table 2), we reduced the cyclophosphamide dose back to 300 mg/m2; however, because we had a small number of patients treated with the 500 mg/m2 dose of cyclophosphamide, we cannot draw firm conclusions.

Our data indicate that IL-15 is associated with the efficacy of CAR-19 T cells. In agreement with prior work,47 IL-15 was one of the serum proteins most prominently increased after the low-dose chemotherapy regimen. Serum IL-15 levels were strongly associated with peak levels of CAR+ cells, and blood IL-15 levels were higher in patients who achieved a remission of lymphoma than in patients who did not achieve a remission. IL-15 causes T-cell (including CAR T-cell) proliferation and activation,32-34,48-50 so the mechanism of IL-15 improving lymphoma treatment outcomes by increasing activated CAR-19 T-cell levels is quite plausible. Other investigators have performed preclinical experiments that demonstrated the ability of IL-15 to enhance adoptive T-cell therapies in mice.9,48,50-53 To the best of our knowledge, our work is the first to show an association in humans between serum IL-15 levels and remission of lymphoma after adoptive T-cell therapy. Clinical trials that evaluate the ability of IL-15 agonists to enhance CAR T-cell antimalignancy activity should be considered to formally assess the role of IL-15 in T-cell therapy.32,33 It must be kept in mind that IL-15 might worsen toxicity in clinical trials combining CAR T cells and IL-15.

We undertook experiments to better understand the important problem of neurologic toxicity with CAR-19 T-cell therapies.12,14-16,21 Neurologic toxicity was closely associated with high peak blood CAR+ cell levels, and by using flow cytometry, we found CAR T cells in the CSF of every patient with neurologic toxicity. Interestingly, peak serum granzyme B levels were closely associated with neurologic toxicity (Fig 4C-E). High granzyme B levels were associated with grade 3 or 4 neurologic toxicity but not with a remission of lymphoma (Data Supplement). Granzyme B has been previously linked to neuronal toxicity.54 Notably, high serum IL-10 and IL-15 levels were associated with both remission of lymphoma and neurologic toxicity. Other investigators have previously shown an association of interferon-γ and IL-6 with neurologic toxicity.16 We also found that serum levels of interferon-γ were higher in patients with grade 3 or 4 neurologic toxicities than in those with only < grade 3 neurologic toxicities (P = .04; Data Supplement), but this difference was not as consistent as the differences for granzyme B, IL-10, and IL-15. We observed a trend that was not statistically significant for higher peak serum IL-6 levels in patients with grade 3 or 4 neurologic toxicity (Data Supplement). Development of new approaches to prevent or treat neurologic toxicity caused by CAR T cells is an important need.

Patients with DLBCL that is refractory to chemotherapy or relapsed less than 12 months after ASCT generally survive less than 9 months.23,25-27 Our results from treating advanced-stage lymphoma with CAR-19 T cells compare favorably to established treatment regimens.17,18,23-25 Induction of CRs in patients with large masses of chemotherapy-refractory lymphoma is especially notable (Fig 2). Our results should encourage further research aimed at developing CAR T-cell therapies with less toxicity and higher remission rates.