1. Basic patients’ characteristics
Ten patients with R/R B-cell lymphoma with a median age of 47.7 years (21-70years) were recruited in this study, including eight patients with diffuse large B-cell lymphoma, of whom P4 also had secondary myelofibrosis, one patient with stage IV mantle cell lymphoma (P2), and one patient with stage IV Burkitt lymphoma (P9). Most of the patients had various degrees of extra-nodal infiltration, except for P1, and 50% had more than two extra-nodal infiltrations, including bone marrow, testis, and central nervous system. 30% of patients had double/triple expression lymphomas evaluated by immunohistochemistry, and 20% of patients had masses greater than 7cm in diameter. One patient (P8) had received prior ASCT. Nine patients (90%) were in a state of relapse before receiving CAR-T cells therapy, and one patient (P6) had temporary stable disease (SD). The baseline characteristics of the enrolled patients are summarized in Table 1 (Table 1).
They all received the lymphocyte depleting regimen by fludarabine and cyclophosphamide (FC) before CXCR5 CD19 CAR-T cells therapy. Among them, P2's white blood cell count remained high after the FC regimen, so Bortezomib and Ibrutinib were added to reduce the tumor load, and P10 received local radiotherapy one month before the return of CAR-T cells because of local higher tumor burden. The median time from leukapheresis to infusion was 23 days (18-31 days), and the median infusion dose of CAR-T cells was 4×106/kg (2×106 - 5.08×106/kg). (Table 2)
2. The efficacy of CXCR5 CD19 CAR-T cell therapy
The objective response (ORR) rate was 80%, and CR rate for CXCR5 CD19 CAR-T cells therapy was 50%, the response of all patients is shown in Fig. 2A. The PFS was 5 months (95% CI 1.02-8.98 months), while the OS was 17.76 months (95% CI 13.54-21.98 months) (Fig. 2B), one patient received ASCT at the 1.5 months (at PR) after infusion of CAR-T cells.
Three patients achieved CR. PET/CT showed that the abnormal metabolic lymph node lesions in multiple sites of the whole body disappeared after treatment in P2 (Fig. 3A), but P5 and P7 relapsed at 11.3 and 3.34 months, respectively.
Three patients achieved partial remission (PR). P1 who was assessed as PR relapsed 5 months after treatment and subsequently received radiotherapy and dual-target CAR-T cells therapy. Unfortunately, he eventually died from disease progression. P4 opted for bridging allo-ASCT 1.5 months after achieving PR and died of disease progression at 7 months follow-up. P10 had disease progression after 3 months of achieving PR, and subsequently received chemotherapy and is still in PR status. P10 with a significant lump in the leg, suffered a new mass in the dorsal right foot during hospitalization, in which the tumor in the patient’s leg and dorsal foot was still increasing gradually, but from day 8, the mass was decreased in size to the extent visible. On day13, the new mass on the dorsum of the patient's right foot had disappeared and only a significantly reduced mass at the base of the right leg was visible at discharge.
Two patients did not response to CAR-T cell therapy. P3 showed a stable disease (SD) after multiple prior chemotherapy treatments until diagnosed with relapse before enrolment in this study. He was still assessed as SD after CAR-T cell therapy and treated with ibrutinib for the following10 months. P8 was enrolled in this trial after relapse post-ASCT. She did not achieve remission even after CAR-T treatment and died of disease progression after 3 months.
3. The Safety of CXCR5 CD19 CAR-T cells
CRS was the most common side effect after CAR-T cells infusion. 7 patients (70%) had grade 1 CRS, and 2 patients (20%) had grade 2 CRS (Fig. 3B). The main symptom of nine patients was fever, which resolved after symptomatic treatment including antipyretic agents. Symptoms of P2 and P10 were relieved with tocilizumab. P6 presented with a red rash on day 12 after CAR-T cells infusion, which resolved after treatment with topical medications. All these patients experienced various degrees of hematologic toxicity after CAR-T cells therapy: all patients experienced various degrees of leukopenia, 3 patients developed mild anemia, and half of the patients experienced neutropenia and thrombocytopenia (Fig. 3C).
In short, all toxicities were manageable and reversible. CXCR5 CD19 CAR-T cells had a relatively reliable safety profile within the follow-up time. No immune effector cell-associated neurotoxicity syndrome (ICANS), or infusion dose-related toxic events occurred.
4. CAR-T cells expansion and biomarker analysis
We used flow cytometry to monitor the proliferation and persistence of CXCR5 CD19 CAR-T cells in the peripheral blood of all patients (Fig. 4A). Significant expansion of CAR-T cells was seen at about 3 days post-infusion, with peak expansion at 7-14 days. The median survival time of CAR-T cells in patients with response was longer than that in patients without response (159 vs 37 days); however, there was no significant difference between the two groups (P=0.3069, Fig. 4B), which may be related to the small sample size. Similar results were observed in the peak of the ratio of CAR-T cells to mononuclear cells (19.18% vs 22.54%, P=0.8655, Fig. 4C). Those data indicated that CAR-T cells expansion in vivo may had little effect on clinical outcome.
There were significant increases in the levels of IL-6, ferritin and C-reactive protein within 2 weeks (Fig. 4D-F). which was consistent with the peak expansion time of CAR-T cells. Among patients who relapsed after treatment, the expansion of CAR-T cells and the cytokine levels tended to be lower and flatter.