We conducted a phase II clinical trial that tested the efficacy and toxicities of CAR-T therapy in patients with r/r non-Hodgkin’s lymphoma (NCT03196830) at the First Affiliated Hospital of Soochow University. Among the patients enrolled, a total number of 51 patients with r/r DLBCL who received CAR-T therapy from Jan,2018 to Oct,2021 were drawn and analyzed. The diagnosis was made on the basis of the World Health Organization classification. The follow-up visit was conducted until death or the last visit time June 1, 2022.

Markers related to HBV infection including HBV serology [HBsAg, hepatitis B surface antibody (HBsAb), hepatitis B e antigen (HBeAg), hepatitis B e antibody (HBeAb), hepatitis B core antibody (HBcAb)], HBV-DNA and liver function were closely monitored during lines of therapy in all patients and during follow-up time in chronic and resolved HBV infectious patients. The lower limit quantification of HBV-DNA was 60 international units (IU)/milliliter(mL) based on our laboratory standards. Chronic HBV infection was defined by the detection of HBsAg for more than 6 months. Resolved HBV infection was defined as HBsAg negative, HBcAb positive, HBsAb negative or positive, with undetectable HBV-DNA. HBV reactivation in chronic or resolved HBV infection patients was defined as one of the followings:(1) ≥2 log (100 fold) increase in HBV-DNA compared to the baseline level of HBV-DNA. (2) newly detectable HBV-DNA. (3) reappearance of HBsAg. The hepatitis flare was reasonably defined as alanine aminotransferase (ALT)/aspartate aminotransferase (AST) increase to ≥3 upper limit of normal.

Patients received CAR-T therapy targeting CD19(n=8), CD19 combined with CD20(n=7), CD19 combined with CD22(n=9) or tandem CD19/CD22 (n=27), CAR-T cells were produced and quality controlled by the Unicar-Therapy Bio-medicine Technology Co. (Shanghai, China) as previously described (11). The detailed procedure was described in the supplementary data S1. Prior to CAR-T infusion, patients received FC (fludarabine 30mg/m2/d, day -5, -4, -3; cyclophosphamide 300mg/m2/d, day -5, -4, -3) or decitabine (DAC a total dose of 100mg/m2 was administrated equally intravenously for 3 consecutive days) bridging FC conditioning regimen for lymphodepletion, and then scheduled infusion of CAR-T cells by dose escalation in 1 to 4 days (decided by physical status of patients and tumor burden), detailed in supplementary data S2. The evaluation included complete blood count (CBC), coagulation routine, organ function, inflammatory markers such as ferritin, cytokines involving interleukin(IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor(TNF), interferon(IFN)-γ,IL-17. Expansion and persistence of CAR-T cells in peripheral blood were monitored after infusion of CAR-T cells. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were evaluated based on the widely accepted CRS scoring system (12). The application of corticosteroids or IL-6 receptor antibody tocilizumab and other supportive measures depended on the grade of CRS and ICANS.

The response criteria were defined in accordance with the National Comprehensive Cancer Network (NCCN) B-cell lymphomas guidelines, version 4.2022. Primary refractory disease was defined as refractory to first-line immunochemotherapy. Overall survival (OS) was calculated from the first CAR-T infusion to the date of death, or the last follow-up, while progression-free survival (PFS) was calculated from the day of remission after CAR-T therapy to progression, relapse, death, or the last follow-up. Adverse events were evaluated according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE, Version 4.0).

Patient characteristics were summarized using the median and range for continuous variables, and frequency and percentage for categorical variables. Variables were compared between cohorts using Fisher’s exact or chi-square test for categorical variables and Wilcoxon rank-sum test for continuous variables. OS and PFS were estimated by the Kaplan-Meier method and compared using the log-rank test. Statistical analysis was performed using GraphPad Prism 8. The P value <0.05 was considered statistically significant.

51 patients with r/r DLBCL were enrolled in our study. HBV serology at diagnosis of DLBCL and at the time of CAR-T infusion is shown in Figure 1. These 51 patients were divided into 3 cohorts based on the status of HBV infection before CAR-T cell infusion as follows: chronic HBV infection group (Group A, n=6), resolved HBV infection group (Group B, n=25) and non-HBV infection group (Group C, n=20).

Among the 6 patients in Group A, four had detectable HBV-DNA at diagnosis. The median time from first detection of HBV DNA/HBsAg seropositivity to infusion of CAR T-cells was 19.9 months (range 4.9 to 56.1 months). After antiviral treatment with entecavir 0.5mg/d or combine adefovir 10mg/d with entecavir 0.5mg/d, HBV-DNA was undetectable before CAR-T infusion. The median time between undetectable for HBV-DNA and CAR-T infusion was 9.2 months (range 4.5 to 13.1 months). Antiviral treatment was continued during and after CAR-T therapy until the last follow-up. Among 25 patients in Group B, 17 patients (Group B1) were diagnosed at baseline, but the other 8 patients (Group B2) were all negative for HBsAg and HBcAb at baseline, and were diagnosed seropositive for HBcAb during chemotherapy. The median time from first detection of HBcAb seropositivity to infusion of CAR T-cells was 7.1 months (range 0.2 to 38.8 months). 19 of them received antiviral prophylaxis with entecavir. HBV serology was monitored during and after CAR-T therapy in all patients.

The basic characteristics of the three groups were comparable, with respect to patient age (including older patients ≥65 years), gender, Ann Arbor III-IV stage, lactate dehydrogenase (LDH), Extra nodal or central nervous system(CNS) involvement at diagnosis, number of lines prior to CAR-T, previous autologous stem cell transplantation(ASCT), use of CD20 antibody and primary refractory disease. However, bone marrow involvement was significantly higher in patients with chronic HBV infection(P<0.001). Details are shown in Table 1.

Among the 51 patients, the overall response rate and complete remission(CR) rate of CAR-T therapy were 74.5% and 39.2%, respectively. No significant difference were observed between three groups with different HBV status (P=0.29, described in Figure 2A). We also conducted subgroup analysis based on different CAR-T targets, patients were divided into CD19 group(n=8,five in Group B and three in Group C),CD19/CD20 group(n= 7, three in Group B and four in Group C)and CD19/CD22 group(n=36, six in Group A, seventeen in Group B and thirteen in Group C). It seemed dual targets CD19/CD22 achieved better response rates, but there were no statistic difference observed (P=0.13, described in Figure 2B).

The median OS and PFS were 30.6 and 21.8 months, respectively, with a median follow-up of 21.1 months (range 0.5-57.5 months) post CAR-T therapy (shown in Figure 3). The estimated probabilities of OS and PFS at 12 months were 68.6% (CI95%: 57.8-83.2%) and 50.8% (CI95%: 38.1-65.1%), respectively. The estimated probabilities of OS and PFS at 36 months were 43.4%(CI95%: 27.3-60.7%) and 28.7% (CI95%: 13.2-42.6%), respectively. No significant differences in OS (P=0.38) or PFS (P=0.32) were observed among three cohorts.

The most frequent toxicities associated with CAR-T therapy included CRS, ICANS, organ damage, and hematologic toxicity (Table 2). Grade 3-4 CRS was observed in 8 patients and ICANS occurred in 6 patients. Elevations in alanine transaminase (ALT) and total bilirubin (TBIL)were observed in 13 and 3 patients, respectively. 13 patients showed renal dysfunction and 7 patients had heart dysfunction. 46 out of 51 patients showed grade 3-4 hematologic toxicity. In particular, the HBV-infection group did not observe the occurrence of grade 3-4 CRS/ICANS as well as organ damage. Additionally, there were no significant differences in ferritin max/baseline, IL-6 max/baseline, and grade 3-4 hematologic toxicity between three groups.

In total, 4 patients died from CAR-T therapy, none in Group A, two in Group B (B-17,B-22) and two in Group C(C-33,C-41). Patient B-17 was a 50-year-old female patient who fell into a coma and acute renal failure at day +3 after CAR-T infusion. Unfortunately, she didn’t survive after 6 cycles of continuous renal replacement therapy(CRRT) and died of cardiopulmonary arrest. Patient B-22 was a 72-year-old male patient who developed hypotension and confusion on day +4, and later was associated with recurrent atrial fibrillation, severe pneumonia, and renal failure, and died of multiple organ failure (MOF) 18 days after CAR-T infusion. Patient C-33 was a 40-year-old man who developed hypotension, hypoxemia, and coma on day +5 and recovered with high-dose corticosteroids, tocilizumab, and other supportive management, and he died from septic shock and MOF on day +11. Patient C-41 was a 41-year-old woman, and died of disease progression, recurrent seizures, and MOF on day 14 after CAR-T infusion.

In general, HBV reactivation was revealed in 2 patients, one in the chronic HBV infection cohort (patient A-1) after CAR-T therapy and one in the resolved cohort (patient B-11) without prior antiviral prophylaxis before CAR-T therapy. Patient A-1 had B-cell aplasia for 21.2 months, compared to a median duration of 18.5 months (range 11.2-30.7 months) for patients in Group A. Patient B-11 had B-cell aplasia for 17.2 months before HBV reactivation, while the median duration of B-cell aplasia in Group B was 16.8 months (range 6.1-29.5months).

Patient A-1 was a quite interesting case. The 62-year-old man had a history of hepatitis B virus (HBV) infection for more than 30 years, yet paid no attention. When referred to the hospital, liver cirrhosis was manifested with hyperbilirubinemia, hypoalbuminemia, ascites, splenomegaly, and esophageal varices. The serum HBeAg was positive and the HBV-DNA level reached a high up to 3.59×10⁷ IU/mL with mildly elevated ALT. He received entecavir antiviral therapy and eight cycles of immunochemotherapy including rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and local intensity modulated radiotherapy but still got a rapid disease progression. After anti-CD19 and anti-CD22 CAR-T therapy, he achieved durable DLBCL CR. He remained on antiviral prophylaxis, but no further treatments of DLBCL. But 14 months after CAR-T therapy, his routine check-up by liver enhanced magnetic resonance imaging (MRI) detected newly found nodules with a maximum>2cm in diameter, which led to the diagnosis of hepatic malignant tumor (Supplementary Figure 1). Laboratory tests showed that the HBV-DNA level was 94.4 IU/mL, indicating that HBV reactivation occurred, and there was no HBV-related hepatitis flare. After combining antiviral therapy with entecavir and adefovir, together with several sessions of transcatheter arterial chemoembolization and ablation therapy, he survived until the last follow-up, with slowly progressive liver neoplasm and undetectable level of HBV-DNA.

Patient B-11 was a 62-year-old woman with negative HBsAg, positive HBcAb and positive HBsAb, with absence of HBV-DNA, and no antiviral prophylaxis was administered. She achieved CR after 6 cycles of R-CHOP, but soon relapsed. After 2 cycles of salvage combined chemotherapy, including gemcitabine, dexamethasone, and cisplatin (GDP) and 5 cycles of lenalidomide, the disease evaluation was progression of disease (PD). She developed HBV reactivation with a newly detected HBV-DNA titer of 271 IU/mL but no hepatitis flare. She accepted pre-emptive therapy with entecavir and had a quick HBV DNA fall before CAR-T therapy. She did not experience HBV reactivation or liver damage during and after CAR-T therapy up until the last visit.