Tandem CD19/CD22 CAR T‐Cell Therapy as First‐Line Treatment for Adult Patients With High‐Risk Philadelphia Chromosome Negative B‐Cell Acute Lymphoblastic Leukemia

Traditional chemotherapy era, the high-risk genetic subsets of acute lymphoblastic leukemia (ALL) were associated with higher rates of induction failure, persistent measurable residual disease (MRD), and dismal prognosis [1]. Furthermore, the occurrence of severe adverse effects (AEs) and mortality during chemotherapy impedes the applicability of intensive chemotherapy, particularly in elderly or frail patients [2]. There is clear evidence that adult patients with end-of-induction and/or end-of-consolidation MRD levels < 0.01% are a major prognostic factor associated with prolonged survival in B-ALL patients [3]. Chimeric antigen receptor (CAR) T-cell therapies specific to CD19 or CD22 have demonstrated manageable safety profiles and significant efficacy in relapsed/refractory B-ALL. However, their potential when applied as part of first-line therapy for B-ALL patients at risk of early chemotherapy failure has not been studied. This study aimed to assess the feasibility of autologous tandem CD19/CD22 CAR T-cell therapy as a first-line treatment option for high-risk B-ALL patients. Anti-apoptotic Bcl-2 family proteins have been identified as crucial pro-survival factors across multiple high-risk ALL subtypes, including KMT2A-rearranged, Philadelphia chromosome-like, and hypodiploid B-ALL [4]. Venetoclax, a selective Bcl-2 inhibitor, has shown anti-leukemic effects in combination with hypomethylating or conventional chemotherapy in both preclinical and clinical studies in ALL [5]. Therefore, a chemotherapy-free induction regimen using the combination of venetoclax and azacitidine (VA) as a pretreatment prior to CAR T-cell therapy has also been investigated in our study. In this single-center, single-arm, phase 2 trial (NCT06078306) conducted in the First Affiliated Hospital of Soochow University, newly diagnosed patients with high-risk Philadelphia chromosome negative B-ALL aged 18–65 years were enrolled. VA regimen consisted of venetoclax (100 mg on day 1, 200 mg on day 2, 400 mg/day on days 3–21), and azacitidine (75 mg/m2/day on days 1–7). Following induction of VA, patients received autologous tandem CD19/CD22 CAR T-cell therapy (target dose 2.0 × 107 CAR-positive viable T cells per kg). The primary endpoint was the MRD negativity after CAR T-cell therapy. Secondary endpoints were AEs, and survival. A minimum of eight doses of triple intrathecal therapy (methotrexate, cytarabine, and dexamethasone) were administered as central nervous system leukemia prophylaxis. From September 2022 to July 2024, a total of 12 patients received VA followed by autologous tandem CD19/CD22 CAR T-cell therapy as first-line induction treatment (Additional file: Figure S1). 50% were adolescents and young adults (15–39 years), with the remaining 50% being older adults (40–65 years). High-risk cytogenetic or molecular factors were detected in 100% of patients. Specifically, four patients had a detectable KMT2A rearrangement, two had Philadelphia chromosome-like ALL characteristics (one with CRLF2 overexpression and one with EBF1::CSF1R rearrangement), and three had TP53 mutations (two with TCF3::PBX1 rearrangement and concomitant deletions in CDKN2A/B and PAX5, and one with hypodiploidy). Additionally, there was one patient each with ZNF384 rearrangement, MEF2D rearrangement, and IKZF1-IK6. Detailed baseline characteristics of the patients were summarized in Additional file: Table S1. Complete remission (CR) was achieved in 8 (66.7%) patients after the initial induction therapy of VA, and MRD negativity was achieved in 5 patients (41.7%). Among those who did not achieve CR, a median reduction of 93% (range 66%–95%) in bone marrow blasts and hematopoietic recovery was observed. Post CD19/CD22 CAR T-cell infusion, the CR rate was 91.7%, and MRD negativity was 83.3% (Table 1). These results are promising and suggest a potential benefit over conventional chemotherapy, which has historically reported a CR rate of 85% and MRD negativity of 51% [1, 6]. However, it is important to note that our study is not a comparative study and the numbers are limited. Randomized trials with larger sample sizes are needed to confirm these findings. Visualization of response of VA-CD19/CD22 CAR T-cell regimen was in Additional file: Figure S2. Subgroup analyses revealed no significant differences in treatment responses based on traditional prognostic factors (sex, age, white blood cell counts at diagnosis), genetic alternations, bone marrow blasts percentage or CD19 status prior to CAR T-cell therapy (Additional file: Figure S3). The expansion of CAR T-cell reached the median peak level of 68 750 copies/μg gDNA at a median of 11 days (range 6–18) (Table 1). During VA induction, Grade ≥ 3 hematological AEs included neutropenia (66.7%) and thrombocytopenia (8%), with median recovery times of 14 and 2 days, respectively. Grade ≥ 3 non-hematological AEs were sepsis (8%) and pneumonia (8%). During CAR T-cell therapy, Grade 1–2 CRS occurred in 58.3% (7/12) patients, and only one patient experienced Grade 3 CRS. Immune effector cell-associated neurotoxicity syndrome was not observed. There were no treatment-related deaths during induction. The treatment-related AEs for VA-CD19/CD22 CAR T-cell regimen are depicted in Table 1. In our study, bridge transplantation was recommended to all patients with suitable donors and no severe comorbidities, regardless of MRD status. Eight patients underwent allogenic hematopoietic stem cell transplantation (HSCT) in first MRD-negative remission, with a median time of 2.37 months (range 1.37–6.07) post CAR T-cell therapy. One of them received two additional VA prior to HSCT as consolidation due to unfitness from severe Covid-19 pneumonia. The one with MRD-positive remission and the one without remission post CAR T-cell infusions received one cycle of inotuzumab Ozogamicin prior to HSCT. Graft-versus-host disease (GVHD) was limited to Grade 1–2 acute GVHD (n = 2), and one case of mild cutaneous chronic GVHD. One patient experienced CMV viremia and developed CMV pneumonia. Of the 10 patients who underwent transplantation, no cases of transplant-related mortality were observed. Another two patients with MRD negativity received hyper-CVAD-based consolidation therapy for personal preference at a median time of 4 months post CAR T-cell therapy. With a median follow-up of 17 months (range 10–22), the estimated 1-year overall survival, 1-year leukemia-free survival, and 1-year cumulative incidence of relapse rate were 81.8% (95% CI: 44.7–95.1), 81.8% (95% CI: 44.7–95.1) and 18.2% (95% CI: 4.9–55.3), respectively (Additional file: Figure S4). Three patients relapsed: two relapsed after HSCT, one without HSCT relapsed at 14 months post CAR T-cell therapy. All were CD19-positive. Two patients died: all for relapses. In conclusion, the chemotherapy-free induction with the combination of VA, followed by tandem CD19/CD22 CAR T-cell therapy, demonstrated favorable safety and profound responses in patients with newly diagnosed high-risk Ph-negative B-ALL. These findings support a promising targeted/immune-based induction strategy for these patients. Due to the single-arm design and small sample size, further investigation in larger, randomized controlled trials with longer follow-up is warranted. Conception and design: Xiaowen Tang and Depei Wu. Provision of study materials or patients: Xiaowen Tang. Collection and assembly of data: Mengyun Li, Qingya Cui, Xuekai Li, Wei Cui, Xiaoqian Chen, Yan Yu, Juan Chen, and Xin Zhang. Data analysis and interpretation: Mengyun Li, Liqing Kang, and Lei Yu. Manuscript writing: Mengyun Li and Qingya Cui. Final approval of manuscript: All authors. The authors have nothing to report. The study protocol was approved by the ethics committee review board of the First Affiliated Hospital of Soochow University, and written informed consent was obtained from patients or guardians in accordance with the Declaration of Helsinki before the initiation of the study. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. ajh27752-sup-001-SupinfoS1. Data S2. ajh27752-sup-0002-SupinfoS2. 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