Although CD19 CAR-T cell therapy has significantly improved the objective response rate (ORR) in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL), about 46–60% of patients relapse within a year. Antigen loss or mutation, and limited persistence of CAR-T cells in vivo are key factors driving relapse. The sequential infusion of CD19 and CD20 CAR-T cells (CD19-20 CAR-T therapy) has emerged as a potential solution to extend CAR-T persistence and improve long-term outcomes.

Recently, the lymphoma and myeloma team at Beijing GoBroad Hospital, led by Dr. Ke Xiaoyan and Dr. Hu Kai, published an article in the official journal of the International Society for Cell & Gene Therapy (ISCT), Cytotherapy, titled: “Sequential CD19-20 CAR-T Therapy for Refractory/Relapsed Diffuse Large B-cell Lymphoma.”This work provides new insights for improving CAR-T efficacy in r/r LBCL patients.

Study Methods

Study Design:

This was a single-arm, single-center, registered, prospective study conducted at Beijing Gaobo Hospital from March 2019 to January 2022.

Patient Selection:

Eligible patients were aged 18 years or older with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL), including diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS) and transformed follicular lymphoma (tFL).

CAR Construction and CAR-T Cell Preparation:

Lentiviral vectors encoding second-generation CARs targeting either CD19 or CD20 were used. These constructs included a 4-1BB costimulatory domain and a CD3-zeta signaling domain.

Clinical Procedure:

Bridging therapy was permitted. After lymphodepletion chemotherapy, patients received a CAR-T infusion at a dose of 2×10⁶ CAR-T cells per kilogram of body weight.

Safety and Efficacy Assessment:

The primary endpoint was the safety and tolerability of the sequential CD19-CD20 CAR-T therapy. Secondary endpoints included objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS).

Study Results

Clinical Characteristics:

A total of 32 patients were enrolled, among whom 21 (65.6%) completed sequential CD19-CD20 CAR-T therapy. All patients had received at least three prior lines of therapy. The median age was 54 years. Among them, 61.9% had relapsed after chemotherapy, and 38.1% were chemotherapy-refractory. Additionally, 33.3% were over 60 years old, 66.7% had an IPI score ≥3, and 47.6% had tumor masses ≥5 cm. Among those tested, 47.1% harbored TP53 mutations. Bridging therapy was administered to 71.4% of patients. CD20 CAR-T cells were infused after CD19 CAR-T cells became undetectable in peripheral blood (via FCM), with a median interval of 3.72 months between the two infusions.

Safety:
All adverse events (AEs) occurred within 30 days after CAR-T infusion. Following CD19 CAR-T infusion, 81.0% of patients developed cytokine release syndrome (CRS), with 9.5% experiencing grade ≥3 CRS; 4.8% of patients experienced immune effector cell-associated neurotoxicity syndrome (ICANS), all at grade 2. After CD20 CAR-T infusion, 81.0% of patients also experienced CRS, all at grade 1–2, and no ICANS was observed.

Efficacy:
Among the 21 patients who completed treatment, 61.9% achieved partial remission (PR) and 38.1% achieved complete remission (CR) following CD19 CAR-T infusion. After CD20 CAR-T treatment, 10 out of 13 PR patients (76.9%) converted to CR. With a median follow-up of 24.7 months, 71.4% of patients maintained remission at the time of data cutoff. In the intention-to-treat analysis of all 32 patients, the ORR was 65.6%. Among the 21 patients who completed infusions, the median PFS, DOR, and OS were not reached; the 3-year PFS and OS rates were 71.9% and 95.2%, respectively. In the intention-to-treat population, the median PFS after CD19 CAR-T infusion was 23.54 months, while the median OS was not reached, with a 3-year OS rate of 57.3%. Among the six patients who experienced disease progression, three (50%) underwent repeat biopsies and IHC phenotyping at relapse: two showed CD19-negative/CD20-positive relapse, one showed CD19-positive/CD20-negative relapse, and none showed dual CD19/CD20 positivity at relapse.

CAR-T Cell Expansion and Persistence:

Effective expansion of CD19 and CD20 CAR-T cells was detected in peripheral blood. The median peak expansion for CD19 CAR-T cells was 60×10⁶/L, and for CD20 CAR-T cells was 3.61×10⁶/L. Patients who achieved PR or CR after CD19 CAR-T therapy exhibited similar CAR-T expansion levels (PR: 63.6×10⁶/kg vs. CR: 53.9×10⁶/kg, p=0.47). The median persistence of CD19 CAR-T cells in peripheral blood was 90 days, while CD20 CAR-T cells persisted for 28 days. The duration of CAR-T cell persistence was positively correlated with long-term clinical outcomes (DOR r=0.492, p=0.024; PFS r=0.5, p=0.02; OS r=0.58, p=0.006).

Summary

1. Sequential CD19-CD20 CAR-T therapy shows good tolerability in r/r DLBCL and may reduce relapse and improve PFS and OS.
2. Low-grade CRS and ICANS are manageable. High-grade events were reversible, with no treatment-related deaths.
3. Patients achieving PR may benefit from CD20 CAR-T, possibly deepening responses through complementary antigen targeting.
4. Survival outcomes surpass historical data, suggesting multi-antigen CAR-T can mitigate early relapse risk.
5. Antigen loss remains a challenge—sequential CAR-T cannot fully prevent relapse.
6. A correlation exists between CAR-T persistence and long-term efficacy.

Outlook

Dr. Ke Xiaoyan noted that the Lymphoma and Myeloma Department at Beijing GoBroad Hospital has accumulated extensive experience in CAR-T therapy. As real-world cases increase, CD19 CAR-T may cure half of all r/r B-cell lymphoma patients. Future research will focus on consolidation strategies post-CD19 CAR-T to deepen and sustain remissions. The team's experience suggests that sequential CD20 CAR-T therapy can enhance efficacy, prolong survival, and remain safely manageable.