Efficacy and Safety of Chimeric Antigen Receptor T Cell Therapy Combined with Zanubrutinib in the Treatment of Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Efficacy and Safety of Chimeric Antigen Receptor T Cell Therapy Combined with Zanubrutinib in the Treatment of Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Approximately 30–50% of diffuse large B-cell lymphoma (DLBCL) patients experience disease progression or relapse after chimeric antigen receptor T (CAR-T) cell therapy. Combination therapy may be a feasible strategy to reduce the risk of relapse. Malignant B cells maintain B-cell receptor (BCR) expression on the cell surface and benefit from the proliferation, survival, and migration pathways triggered by BCR. Bruton tyrosine kinase (BTK) is an important component of the BCR signaling pathway, and the anti-lymphoma function generated by inhibiting the BTK pathway makes it a promising therapeutic target in B-cell malignant tumors and inflammatory diseases. Previous basic research has shown that adding the first-generation BTK inhibitor ibrutinib to cultures significantly increases the number of CAR-T cells, and results in the expression of fewer exhaustion markers, promoting CAR-T cell mobilization and the recognition of lymphoma cells. Moreover, ibrutinib is associated with fewer cytokine release syndrome (CRS)-associated cytokines and lower CRS severity. Nonetheless, adverse events related to off-target effects limit the use of ibrutinib, with approximately 23% of patients in the clinic and 49% of patients in the community interrupting ibrutinib treatment.

Zanubrutinib is a new generation of BTK inhibitor with a more dimensional chemical design structure and optimized pharmacokinetic properties. Research has shown that zanubrutinib has fewer off-target effects, weaker inhibitory effects on immune cells, and less inhibition of phospholipase C-γ1 and interleukin-2 than ibrutinib does. For specific types of genetic mutations, zanubrutinib can be more effective for CXCR4MUT, TP53MUT, and MYD88MUT, with a better clinical response than ibrutinib. With the rearrangement of MYC and BCL-2 or BCL-6, zanubrutinib synergistically increases sensitivity to B-cell lymphoma-2 inhibitors. However, the effects of CAR-T cell therapy combined with zanubrutinib have not been reported. Our center retrospectively analyzed efficacy and safety data from patients with relapsed/refractory (R/R) DLBCL treated with CAR-T cell therapy combined with zanubrutinib.

Based on the downstream signaling pathways of BTK, our study included patients with associated genetic changes, including changes in MYD88 and CD79B. The MCD (with MYD88L265PMUT and CD79BMUT) and BN2 (with BCL-6 fusion and NOTCH2MUT) genetic subtypes, and double-hit or double-expressor lymphomas were also included. Six R/R DLBCL patients with high-risk factors such as double-hit lymphoma, large mass lymphoma (diameter ≥10 cm), and high tumor burden (multiple extranodal lesions, or high lactate dehydrogenase levels), were treated from July 2021 to May 2024. Immunohistochemical staining was performed before CAR-T cell infusion to confirm antigen expression. CD3+ T cells were transduced with a lentiviral CD19, CD20, or CD70 scFv CAR fused with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (dual-target CD19/CD20 or CD19/CD70, fourth-generation CAR, 2/6) or CD4/CD8α/CD28/CD3ζ (CD19, axicabtagene ciloleucel, 4/6), and all patients received pretreatment before CAR-T cell infusion (Fludarabine, 30 mg/m2 d1–3; cyclophosphamide, 500 mg/m2 d1–3). CAR-T cells were infused at a median dose of 1.43 × 108 (1.18–2.36 × 108). The detailed information of the patients is documented in Supplementary Material 1.

All patients were treated with zanubrutinib (160 mg bid), which was maintained for 2 years after one month of CAR-T cell infusion, and then the efficacy and safety were evaluated. Efficacy was evaluated via the Lugano lymphoma efficacy evaluation criteria and was assessed via positron emission tomography-computed tomography (PET-CT) at 1, 3, 6, and 12 months after CAR-T cell infusion, followed by every 6 months. The safety evaluation included CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) was determined via the National Comprehensive Cancer Network (NCCN) guidelines. Other adverse events were evaluated using the commonly used Common Terminology Criteria for Adverse Events (CTCAE) 5.0 standard. The primary endpoint was the objective response rate (ORR) of 3 months after CAR-T cell infusion. The secondary endpoints included overall survival (OS), progression-free survival (PFS), duration of response (DOR) rates, and the duration of CAR-T cell expansion in peripheral blood, and the rates of adverse events included CRS, ICANS, hematological toxicity events, and non-hematological adverse events (including infections, atrial fibrillation/flutter, hypertension, tumor lysis syndrome, and secondary malignant tumors) during treatment. This study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee (No. 2025-KY-047-01). Written informed consent was obtained from all patients in the study.

One month after CAR-T cell infusion, 3/6 of the patients achieved a complete response (CR), whereas 3/6 of the patients achieved a partial response (PR) before treatment with zanubrutinib. After 3 months of CAR-T cell infusion and combined with zanubrutinib for 2 months, the three patients who achieved a PR were transitioned to CR. Overall, all the patients achieved CR, with an ORR of 100% at 3 months after CAR-T cell infusion. Regarding safety after CAR-T cell infusion, four patients (66.7%, 4/6) experienced Grade 2 CRS, and one patient (16.7%, 1/6) experienced Grade 3 CRS (patient 5), which was relieved after the use of tocilizumab and a cumulative dose of 15 mg intravenous dexamethasone. The median time of CRS occurrence was the second day after CAR-T cell infusion, and the median duration was 7 days. Fever was the most common symptom, and four patients (66.7%, 4/6) experienced relief after the use of tocilizumab. One patient (16.7%, 1/6) experienced Grade 3 ICANS with transient systemic convulsions and blurred consciousness, which was subsequently relieved after a dose of 5 mg intravenous dexamethasone and antiepileptic treatment. One patient (16.7%, 1/6) experienced Grade 1 ICANS, with dizziness and tremors in both hands, which were relieved after supportive care. With respect to the occurrence of hematological toxicity events, one patient (16.7%, 1/6) experienced Grade 2 hematotoxicity, one patient (16.7%, 1/6) experienced Grade 3 hematotoxicity, and four patients (66.7%, 4/6) experienced Grade 4 hematotoxicity. The median time for the occurrence of hematological toxicity events was the third day after CAR-T cell infusion, and the median duration was 12 days. The most common hematological toxicity event was leukopenia (83.3%, 5/6), followed by neutropenia (66.7%, 4/6) and thrombocytopenia (50.0%, 3/6). All hematological toxicity events were relieved after hematopoiesis support. Within one month after CAR-T cell infusion, five patients (83.3%, 5/6) experienced infection, and the median time of infection was 10 days after CAR-T cell infusion; three patients (50.0%, 3/6) experienced mixed bacterial and fungal pulmonary infection, one patient (16.7%, 1/6) experienced urinary tract infection caused by John Cunningham polyoma virus, and one patient (16.7%, 1/6) experienced bacterial cheilitis; all infections were relieved after anti-infection treatment. With respect to non-hematological toxicity events, one patient (16.7%, 1/6) experienced a mild increase in creatinine during the first month of zanubrutinib treatment and then returned to normal after one week of follow-up, without other patients experiencing non-hematological toxicity events. There were no treatment-related deaths, and none of the patients reduced or stopped zanubrutinib treatment because of adverse events. All toxicities were manageable and reversible.

After a median follow-up of 22.0 months, five patients (5/6) maintained CR, and one patient (patient 5) relapsed in the 6th month after CD19 CAR-T cell infusion. The 1-year PFS and DOR rates were 83.3% (5/6), and the OS rate was 100%. The median PFS and OS were not reached. The median peak of CAR-T cell expansion was on the 13th day after CAR-T cell infusion, and the median duration of CAR-T cell persistence was 470 days. At the last follow-up, continuous expansion of CAR-T cells in the peripheral blood was detected in all the patients, and continuous low-level amplification in patient 5, which may be related to the low efficacy of CAR-T cells during the manufacturing process and the use of corticosteroids in Grade 3 CRS treatment. Owing to the lack of pathological evidence, the possibility of the escape of the CD19 antigen and other changes in molecular genetics cannot be excluded. Most recently, this patient received salvage treatment with a combination of polatuzumab, lenalidomide, and venetoclax for four courses and had PR. Salvage therapies after CAR-T cell therapy failure need to be further explored.

Our study revealed that CAR-T cell therapy combined with zanubrutinib for the treatment of R/R DLBCL was associated with high response rates and acceptable toxicity without increasing the incidence of serious adverse events, even among patients with high-risk factors in our study. Zanubrutinib also maintains the expansion of CAR-T cells compared with the previous data as 34% of patients had no detectable gene-marked CAR-T cells by 24 months in the ZUMA-1 study. Based on the 2-year OS rate and recurrence rate reported in the previous ZUMA-7 study, we predict that maintaining zanubrutinib for two years after CAR-T cell infusion will more stably reduce the risk of recurrence. However, the timing of CAR-T cell therapy combined with zanubrutinib is crucial. For the consideration of adverse events, we started the combination with zanubrutinib after one month of CAR-T cell infusion. Whether zanubrutinib can be used after CAR-T cell infusion at an earlier time point to increase the expansion and persistence of CAR-T cells, or before CAR-T cell infusion to improve T-cell efficiency and reduce tumor burden remains to be explored. We observed that patient 2 previously received orelabrutinib treatment for 5 months and had no response, but still responded to zanubrutinib and achieved a CR after being evaluated as a PR in the first month of CAR-T cell infusion, CAR-T cell therapy combined with zanubrutinib may be effective for patients whose treatment has previously been unsuccessful for other types of BTK inhibitors. Notably, our study revealed that patients with CD79BMUT responded to zanubrutinib and were turned PR into CR after being combined with zanubrutinib for 2 months. More precise classifications based on genetic and clinicopathological features are needed to identify sensitive patients who may benefit from CAR-T therapy combined with BTK inhibitor therapy.

The sample size of our retrospective analysis was small, and considering the differences in patients’ baseline disease status and histories of prior therapies, the variations in CAR-T cell targets, and intracellular signaling domains, the comparability of this study may be limited. More follow-up data are needed to confirm whether the combination of CAR-T cell therapy and zanubrutinib can improve long-term PFS and OS. Moreover, basic research is needed to confirm the interaction between CAR-T cells and zanubrutinib, thereby clarifying the underlying mechanism and identifying potential influencing factors.

doi.org/10.1097/CM9.0000000000003504

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