Abstract
Background
Third‑generation CAR-T cells demonstrated promising efficacy and remarkably low toxicity in refractory or relapsed (R/R) B-cell malignancies. However, data on the patients with central nervous system (CNS) involvement are limited due to concerns regarding treatment-related neurotoxicity. This study aimed to evaluate the safety and efficacy of a novel third-generation anti-CD19 CAR T cells in patients with CNS involvement of B-cell malignancies.
Methods
A total of 21 patients with R/R B-cell malignancies with CNS involvement, including 11 with B-cell acute lymphoblastic leukemia (B-ALL) and 10 with B-cell non-Hodgkin lymphoma (B-NHL) were enrolled. Patients derived lymphocytes were collected through apheresis and lentivirally transduced with the third-generation CAR incorporating both CD28 co-stimulation and TLR2-derived stimulatory domains (1928zT2). Patients received a single-dose 1928zT2 CAR-T cell infusion following lymphodepleting regimen. Safety, efficacy and cellular pharmacokinetics were investigated.
Results
Of the 21 patients with CNS involvement, the overall response rate (ORR) was 71% (15/21), with 73% (8/11) in B-ALL and 70% (7/10) in B-NHL. At a median follow-up of 20.4 months, median duration of response (DOR) was 11.1 months (95% CI, 2.9–24.4). 12-months progression-free survival (PFS) and overall survival (OS) estimates were 41.5% and 61.2%, respectively. Cytokine release syndrome (CRS) of any grade occurred in 20 patients (95%; grade ≥ 3 in 3 patients). Immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 9 patients (42.8%; grade ≥ 3 in 6 patients). All CRS and ICANS events were manageable. The outcomes and adverse events are comparable between B-ALL and B-NHL patients. Notably, 1928zT2 CAR-T cells demonstrated blood–brain barrier penetrance, with subsequent detection in patient cerebrospinal fluid (CSF) correlating significantly with improved clinical outcomes.
Conclusions
Third-generation 1928zT2 CAR-T cells are associated with high response rates, manageable safety and durable remissions in R/R B-cell malignancies with CNS involvement.
Trial registration: ClinicalTrials.gov, NCT04605666. Registered 1 May 2020.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12967-025-06608-x.
Keywords: CAR T cell therapy, B-cell malignancies, Central nervous system involvement, Third-generation CAR, TLR2 co-stimulation
Introduction
Refractory or relapsed (R/R) B-cell malignancies with central nervous system (CNS) involvement, including primary and secondary CNS lymphoma as well as CNS leukemia, are associated with poor prognoses. Due to impermeability of the blood–brain barrier (BBB) to systemic therapies including many monoclonal antibodies, the mainstays of treatment of CNS disease remain systemic high-dose methotrexate (HD-MTX), whole brain radiation therapy (WBRT), or high-dose chemotherapy followed by hematopoietic stem cell transplantation (HSCT) [1–3]. The reported median overall survival (OS) of patients with primary CNS lymphoma, secondary CNS lymphoma and CNS involvement by B-cell leukemia are short at 25.3 months [4], 3.9 months [1], and 8.5 months [5], respectively.
Chimeric antigen receptor (CAR) T-cells directed against the B-cell antigen CD19 have shown promising outcomes for patients with refractory or relapsed (R/R) B-cell malignancies, with response rates of up to 83% in patients with B-cell non-Hodgkin lymphoma (B-NHL) [6] and up to 90% with B-cell acute lymphoblastic leukemia (B-ALL) [7]. However, data on outcomes of CAR T cell therapy for patients with CNS involvement are limited, as CNS involvement is an exclusion criteria for most pivotal CAR T-cell trials because of concerns about treatment-related neurotoxicity [8, 9]. Case reports and small trials indicate that CD19-directed CAR T cell therapy is a safe and effective treatment strategy in R/R B-cell malignancies with CNS involvement [10–13]. To date, most of clinical approved CAR T cell products express second-generation CARs incorporating either a CD28 or 4-1BB co-stimulatory domain [14, 15]. Third-generation CARs harbor two costimulatory domains mediating enhanced and faster expansion as well as longer persistence of CAR T cells [16, 17], but the clinical data evaluating the third-generation CAR T cells is scarce. Recent studies have reported that third-generation CAR T cells comprising CD28 and 4-1BB costimulatory domains were associated with promising clinical efficacy and remarkably low procedure-specific toxicity in R/R B-ALL [18], as well as R/R chronic lymphocytic leukemia (CLL) [19]. Whether third-generation CARs could induce favorable outcomes and safety in the patients with CNS disease still unknown.
We previously reported a novel third-generation anti-CD19 CAR T cell constuct incorporating intracellular signalling domains derived from CD28, CD3ζ and Toll-like receptor 2 (TLR2) at the cytoplasmic tail, which termed ‘1928zT2’. The addition of the TLR2 intracellulr domain resulted in improved CAR T-cell expansion, greater IFN-γ and GM-CSF production, and enhanced efficacy in vivo [20]. Moreover, early-phase clinical trials have demonstrated that CAR T cells incorporating with TLR2 domain have shown encouraging outcomes and safety in patients with R/R B-cell lymphomas [21, 22], and have the ability to induce complete remission in extramedullary B-ALL tumors [23]. Nevertheless, the safety and feasibility of administration of 1928zT2 for patients with CNS involvement remain unclear. Herein, we report on our institutional experience with 1928ZT2 CAR T-cells administration in 21 consecutive patients with CNS involvement.
Patients and methods
Study design and patients
The phase 2 trial (ClinicalTrials.gov, NCT04605666) was designed to evaluate the efficacy and safety of third-generation 1928zT2 CAR T cells infusions in patients with R/R CD19-positive B-cell malignancies. The study was approved by the Medical Ethics Committee of Nanfang Hospital of Southern Medical University (Approval No. NFEC-2020-098). The trial permitted inclusion of patients with CNS involvement by lymphoma or leukemia at the time of apheresis. We report here on the first 21 enrolled patients with CNS involvement, including 11 patients with B-ALL and 10 patients with B-NHL between July 2020 and May 2023. Briefly, patients underwent lymphodepleting chemotherapy with fludarabine (at a dose of 30 mg per square meter of body-surface area) and cyclophosphamide (at a dose of 300 mg per square meter) daily for 3 consecutive days, followed by a single infusion of the trial 1928zT2 CAR T-cells at 1–2 × 106 cells per kilogram of body weight. Informed consent was obtained from all participants, in compliance with the Declaration of Helsinki.
Third-generation CAR-T cell manufacturing
The third-generation CD19-directed CAR used in this study comprises the murine FMC63 anti-CD19 single-chain variable fragment (scFv), a CD8α hinge, CD28 transmembrane and endodomains, a CD3ζ signaling domain, and the Toll/interleukin-1 receptor (TIR) domain (amino acids 639–784) of TLR2 (1928zT2, Fig. 1B). CAR T-cells were generated as previously described [20, 23]. In brief, T cells were isolated from paitents-derived peripheral blood mononuclear cells (PBMCs) collected by apheresis using a standard density gradient centrifugation method followed by magnetic bead selection to enrich CD3-positive T cells. Then T cells were activated with anti-CD3/anti-CD28 antibodies (Miltenyi Biotec) and transduced with a lentiviral vector incorporating the 1928T2z transgene. CAR-transduced T cells cultured for up to 14 days in the presence of recombinant human IL-2. The range of expansion duration was from 10 to 14 days, with most products being expanded for 12–14 days. Manufacture of 1928zT2 CAR T-cells took place at Guangdong Zhaotai InVivo Biomedicine Co., Ltd according to local Good Manufacturing Practice (GMP) requirements. After meeting release testing criteria, cryopreserved products were transported to the hospital for infusion.
Fig. 1.
Study profile and the 1928zT2 CAR construct. A Study profile. B Schematic diagram showing the composition of the 1928zT2 CAR used in this study. TLR2 signalling domain is inserted at the 3′-end of intracellular regions of the CD3ζ signalling domain (CD3ζ)
Study endpoints and assessments
The primary endpoint was the objective response rate (ORR) including complete remission (CR) or partial response (PR), as determined by assessments of peripheral blood, bone marrow, CSF analysis and diagnostic imaging. The secondary endpoints were overall survival (OS), progression-free survival (PFS), as well as the safety of 1928zT2 CAR T-cells as determined by the incidence of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and other adverse events (AEs). CRS and ICANS were graded according to American Society for Transplantation and Cellular Therapy criteria [24]. Responses were defined according to Lugano criteria and NCCN guidelines for B-NHL and B-ALL patients, respectively [25, 26].
Laboratory assessments
Exploratory analyses include paired peripheral blood (PB) and cerebrospinal fluid (CSF) analyses for 1928zT2 pharmacokinetics, pharmacodynamics, flow cytometry. APC-conjugated anti-mouse FMC63 scFv monoclonal antibody was used to detect 1928zT2 CAR T-cells (defined as CAR+ CD3+) in PB and CSF by flow cytometry. The levels of the cytokines IL-2, IL-4, IL-6, IL-10, IL-171, TNF-α, and IFN-γ in patient serum and CSF were determined by cytometric bead array (CBA) kit (CBA Human Th1/Th2/TH17 Cytokine Kit, BD Biosciences).
Statistical analysis
Patients were followed up for determination of relapse and survival outcomes until the data cutoff. Descriptive statistics included means with standard deviations or median with ranges for continuous variables and frequencies or percentages for categorical variables. The chi-square statistic or Fisher exact test was used for comparisons between categorical variables, and the Mann–Whitney U test was used for continuous variables. PFS and OS were analyzed using the Kaplan–Meier method. P values < 0.05 were considered statistically significant. Analyses were conducted using SPSS statistical software version 20.0.0.
Results
Baseline characteristics
Between 12 July 2020 and 13 May 2023, 21 patients with R/R B-cell malignancies with CNS involvement were enrolled in this study, including 11 with B-ALL and 10 with B-NHL (Fig. 1A). The characteristics of the patients at enrollment are summarized in Table 1 and Additional file 2. The median age of participants with B-ALL was 29 years (14–40), and of those with B-NHL was 56.5 years (26–82). All 11 patients with B-ALL had both bone marrow (BM) and cerebrospinal fluid (CSF) involvement at the time of enrolment; BCR/ABL1 fusion gene was positive in 6 patients (6/11), including 3 patients with T315I mutation. Patients with R/R B-ALL had received a median of 3 prior lines of therapy (2–4), including allogeneic HSCT in 3 patients and prior brain radiotherapy in 3 patients. Of the 10 patients with CNS involvement with R/R B-NHL, 7 had diffuse large B-cell lymphoma (DLBCL) including 1 with Richter’s transformation and 1 with follicular transformation, the other 3 were Burkitt lymphoma, high-grade B-cell lymphoma (HGBL), and B-lymphoblastic lymphoma (B-LBL), respectively. 8 patients had secondary CNS involvement by lymphoma and 2 patients had primary CNS lymphoma. Six of the 10 lymphoma patients had parenchymal brain involvement, six has CSF involvement, two had spinal cord involvement and one had intraocular involvement. 4 of 10 CNS lymphoma patients had concurrent BM involvement by lymphoma. Triple-hit rearrangement was detected in 1 patient (1/10). Patients with R/R B-NHL had received a median of 4 lines of therapy (2–6), including autologous HSCT in 5 patients, and prior radiotherapy in 2 patients.
Table 1.
Baseline characteristics of 21 treated patients and subgroups
| Characteristics | B-ALL (N = 11) | B-NHL (N = 10) |
|---|---|---|
| Median age (range), y | 29 (14–40) | 56.5 (26–82) |
| Male: female | 4: 7 | 5: 3 |
| CNS disease location | ||
|
CSF infiltration Parenchyma Spinal cord Ocular |
11/11 (100.00%) – – – |
6/10 (60.00%) 6/10 (60.00%) 2/10 (20.00%) 1/10 (10.00%) |
| BM involvement | 11/11 (100.00%) | 4/10 (40.00%) |
| Cytogenetic aberration | ||
|
BCR/ABL1 T315I mutation CDKN2A deletion TP53 mutation MYC + BCL2 + BCL6 |
6/11 (54.54%) 3/6 (50.00%) 5/11 (45.45%) 1/11 (9.00%) – |
– – – 1/10 (10.00%) 1/10 (10.00%) |
| Median prior lines of therapy (range) | 3 (2–4) | 4 (2–6) |
| Prior HSCT | ||
|
Allo-HSCT Auto-HSCT |
3/11 (27.27%) – |
– 5/10 (50.00%) |
| Prior radiotherapy | ||
|
Yes No |
3/11 (27.27%) 8/11 (72.72%) |
2/10 (20.00%) 8/10 (80.00%) |
CNS central nervous system, CSF cerebrospinal fluid, BM bone marrow, Allo-HSCT allogenic hematopoietic stem cell transplantation, Auto-HSCT autologous hematopoietic stem cell transplantation
Treatment and response
Of the 11 R/R B-ALL patients, 3 received a single dose of 1 × 106 CAR+ 1928zT2 cells/kg and 8 received 2 × 106 cells/kg. All 10 lymphoma patients received a single dose of 1 × 106 CAR+ 1928zT2 cells/kg. Of the 21 patients treated, the overall response rate (ORR) was 71% (15/21), including 73% (8/11) in B-ALL and 70% (7/10) in B-NHL. 67% (7/11) of patients with B-ALL had a complete remission (CR) in both BM and CNS, while one patient had no response in CNS disease although had a response in the bone marrow. 40% (4/10) of B-NHL patients had a complete response and 30% (3/10) a partial response (PR). There was no difference in the ORR between B-ALL and B-NHL patients. Across all patients, the ORR was 57.1% (12/21), 38.1% (8/21), and 33.3% (7/21) at 3 months, 6 months and 12 months, respectively. The median duration of response (DOR) was 11.1 months (95% CI 2.9–24.4 months). The disease status and survival of the 21 patients are shown in Fig. 2A, B.
Fig. 2.
Clinical responses of patients with CNS involvement following 1928zT2 CAR T-cell infusion. A, B Swimmer plots showing the clinical responses and follow-up of individual patients treated with 1928zT2 CAR T-cells, as indicated with different colors in the swimmer lanes. Each bar represents one patient. Patients A3 and A5 experienced complete remission and underwent allo-HSCT afterwards. Patients A10, N2 and N5 received a second 1928zT2 CAR T-cell infusion following disease progression. CR complete response, PR partial response, SD stable disease, PD progressive disease, Allo-HSCT allogenic hematopoietic stem cell transplantation, 2nd CART received a second 1928zT2 infusion
Three patients with B-ALL disease relapse after the first CAR T-cell (not 1928zT2) infusion received a second administration of CAR T-cells (1928zT2), of which 2 achieved a complete response again. One patient who had Burkitt lymphoma (Patient N10) had a complete remission of CNS disease and extramedullary disease, according to the absence of lesions with fluorodeoxyglucose uptake on positron-emission tomography computed tomography (PET-CT) performed at 1 month after CAR T-cell infusion (Fig. 3A, B). Seven patients died of disease progression after CAR T-cell therapy. At the last assessments, 5 of the 11 B-ALL patients and 5 of 10 the B-NHL patients were alive with sustained CR of both CNS disease and systemic disease.
Fig. 3.
Initial and subsequent imaging of a patient with CNS lymphoma who achieved remission after 1928ZT2 CAR T-cells infusion. A FDG-PET/CT scan demonstrating the lymphoma lesions in ependymal layer of right lateral ventricle (as indicated by the red arrows) and diffuse whole brain hypermetabolism were markedly reduced in a patient (N10) with CNS lymphoma on day 30 after CAR T therapy. B PET/CT scans showed the extramedullary lesions before and after 1928ZT2 CAR T-cells infusion. The red arrows highlight the presence of enhanced glucose metabolism within pericardium, anterior mediastinum, periphery of aortic arch, ascending aorta and pulmonary artery trunk, as well as liver capsule, jejunoileum, ileum, ascending colon, transverse colon and splenic flexure of the colon prior to treatment. These lesions were not dectectable after 1928zT2 CAR T-cell therapy
Long-term survival
The median follow-up duration was 20.4 months (range, 1.0—36.3). Median progression-free survival (PFS) of all patients was 5.8 months (95% CI 1.9–20.4 months), while the median overall survival (OS) was not reached (Fig. 4A, B). The 1-year PFS and OS rate estimates were 41.5% and 61.2%, respectively. At a median follow-up of 25.4 months, median OS of B-ALL was 14.3 months (95% CI 2.5–35.0 months), while median OS of B-NHL was not reached at a median follow-up of 13.2 months. Estimated 12-month OS among patients with R/R B-ALL or B-NHL patients was 54.5% and 68.6%, respectively (P = 0.58). Median PFS in patients with B-ALL and B-NHL was 11.1 months (95% CI, 0.7–35.0 months) and 4.8 months (95% CI 1.0–20.4 months), respectively (P = 0.85), as two of the B-ALL patients underwent allo-HSCT while in CR. The estimated 12-months PFS in B-ALL and B-NHL patients were 41.6% and 42.5%, respectively (P = 0.65). The median duration of response (DOR) was 24.4 months (95% CI, 2.5–36.3 months) and 3.8 months (95% CI 0.9–20.4 months) for B-ALL and B-NHL, respectively (P = 0.09).
Fig. 4.
Overall and progression-free survival of patients with CNS involvement following 1928zT2 CAR T-cell therapy. A, B Kaplan–Meier curves of overall survival and progression-free survival in all patients and the subpopulation with B-ALL and B-NHL
Safety
CRS of any grade was seen in 20 patients (95%), with a median onset at day 3 (2–5) following 1928zT2 CAR T-cell infusion (Table 2). Grade 3 CRS was observed in 3 patients (14%), including 1 patient with B-ALL and 2 patients with B-NHL. No grade 4 CRS was observed. Subgroup analysis found the overall incidence of CRS did not differ between B-ALL and B-NHL patients, but Grade 1 CRS in patients with B-ALL was significantly higher than that of B-NHL patients (81.81% vs 25.00%, P = 0.01). Immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 9 patients (42.8%), including 5 patients with B-ALL and 4 with B-NHL. Median time to ICANS onset was 5 days (2–10) after 1928zT2 CAR T-cell administration. Grade 1–2 ICANS was seen in 3 patients (14.3%), grade 3 ICANS was seen in 5 patients (23.8%), and one patient had grade 4 ICANS (4.8%). ICANS onset was most often associated with the presence and severity of CRS, as Spearman’s analysis showed that there was a significant correlation of neurotoxicity with the incidence and severity of CRS (r = 0.58, P = 0.01, Additional file 1: Figure S1). Of the 9 patients with ICANS, 7 patients (77.8%) received systemic corticosteroids, 2 were treated with tocilizumab, and 3 patients with severe ICANS (grade 3 or 4) resistant to both corticosteroids and tocilizumab received an external ventricular drainage. No patient died of ICANS. All the CRS or ICANS were manageable. There was no significant difference in the incidence of either CRS or ICANS between B-ALL and B-NHL patients.
Table 2.
Rates of CRS and ICANS
| Characteristics | Total (N = 21) | B-ALL (N = 11) | B-NHL (N = 10) | P value |
|---|---|---|---|---|
| CRSa | ||||
|
Any CRS Grade 1 Grade 2 Grade 3 Grade 4 Median time to onset (days) |
20/21 (95.24%) 11/21 (52.38%) 6/21 (28.57%) 3/21 (14.29%) – 3 |
11/11 (100.00%) 9/11 (81.81%) 1/11 (9.09%) 1/11 (9.09%) – 2.5 |
9/10 (87.50%) 2/10 (25.00%) 5/10 (50.00%) 2/10 (12.50%) – 3 |
0.476 0.009 0.064 0.587 – 0.991 |
| ICANSa | ||||
|
Any ICANS Grade 1 Grade 2 Grade 3 Grade 4 Median time to onset (days) |
9/21 (42.86%) 2/21 (9.52%) 1/21 (4.76%) 5/21 (23.81%) 1/21 (4.76%) 5 |
5/11 (45.45%) 2/11 (18.18%) 1/11 (9.09%) 1/11 (9.09%) 1/11 (9.09%) 5 |
4/10 (40.00%) – – 4/10 (40.00%) – 5 |
0.999 0.999 0.999 0.149 0.999 0.999 |
| Treatment of ICANS | ||||
|
Corticosteroids Tocilizumab External ventricular drainage |
7/21 (33.33%) 2/21 (9.52%) 3/21 (14.29%) |
3/11 (27.27%) 2/11 (18.18%) 2/11 (18.18%) |
4/10 (40.00%) – 1/10 (12.50%) |
0.659 0.999 0.999 |
aCRS and ICANS were graded with the use of the American Society of Transplantation and Cellular Therapy 2019 consensus criteria. P value showing the comparison between B-ALL and B-NHL patients
Other AEs within the first 30 days are shown in Additional file 3. Fatal AEs after CAR T-cell infusion occurred in 2 patients: 1 died as a result of acute heart failure on day 76; 1 died due to intracranial infection as Metagenomic next generation sequencing confirmed the presence of Serratia in CSF on day 30 post-infusion.
CSF trafficking and cytokine analysis
The dynamic expansion and persistence of 1928ZT2 CAR T-cells in CSF was monitored in 12 evaluable patients after infusion (Fig. 5A). CAR T cell trafficking into the CSF was noted in patients who demonstrated expansion in peripheral blood (Fig. 5B). All 12 patients exhibited peak expansion of CAR T cells in CSF within two weeks after infusion. Patients who achieved a CR as their best response demonstrated higher levels of CAR T cells in the CSF (Fig. 5C), suggesting that higher CAR T-cell levels in CSF were associated with improved clinical responses. Median time to peak expansion of CAR T cells in the CSF was 9 days post-infusion (6–12). The levels and median onset of peak CAR T cells in the CSF between B-ALL and B-NHL patients were comparable (Additional file 4). The median peak proportion of CAR+ T-cells in CSF among patients with severe ICANS (grade 3–4) was higher than that in those with no or mild ICANS (grade 0–2) (Fig. 5D).
Fig. 5.
CSF expansion of 1928zT2 CAR T-cells and CSF cytokine analysis. A Representative flow cytometric detection of CAR T cells in the CSF of a patient (N10) on day 10 following infusion. Representative flow cytometry plots showing the live CD3+ cells stained with APC-labelled anti-FMC63 scFv monoclonal antibody or IgG Isotype control. B CAR T-cell expansion in CSF was monitored in 12 patients. Data show the percentage of CAR+ T-cells in the CSF of patients with B-ALL (red) and B-NHL (blue) after infusion. C Comparison of peak levels of 1928zT2 cells in CSF between patients who reached a CR or not. D Comparison of peak CSF 1928zT2 CAR T-cells between patients with grade 0–2 ICANS and grade 3–4 ICANS. *P < 0.05. E Cytokine analysis showing peak levels of IL-6, IL-10, IFN-γ and TNF-α in serum and CSF of 6 evaluable patients, as determined at the time of ICANS
In addition, the levels of IL2, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ in both PB and CSF were analyzed after 1928ZT2 CAR T cell infusion (Additional file 1: Figure S2). We observed a notable increase in IL-6 and IFN-γ levels in the cerebrospinal fluid compared to the serum at the time of ICANS (Fig. 5E). This finding further confirmed that the 1928zT2 CAR-T cells had successfully crossed the blood–brain barrier and effectively eradicated tumor cells within the central nervous system.
Discussion
To our knowledge, this is the first and largest prospective study to demonstrate the administration of third-generation CD19-directed CAR T cells for the treatment of CNS B-cell malignancies. The 1928zT2 construct, which incorporates a novel TLR2 co-stimulatory domain, provided promising clinical responses and a manageable safety profile in patients with CNS involvement.
Based on small series of CD19-directed CAR T-cell therapy for patients with CNS disease, overall response rates range from 58 to 87% [11, 13, 27–30]. The overall response rate in this study is comparable at 71% (15/21), and was similar among patients with B-ALL and B-NHL. The estimated 12-months PFS and OS were 41.5% and 61.2%, respectively, consistent with previously reported EFS and OS rates achieved in patients without CNS involvement [6, 31]. Therefore, third-generation CAR T cells 1928zT2 was active at clearing CNS disease and maintaining durable remissions in patients with CNS relapsed or refractory B-ALL or B-NHL.
In our study, the CR rate was 67% (7/11) for B-ALL patients and 40% (4/10) for B-NHL patients. Multiple studies have demonstrated high CR rates (67% to 94%) in B-ALL patients treated with CD19-targeted CAR T cells [32], while CR rates in B-NHL patients are generally lower, ranging from 30 to 69% depending on the subtype and prior treatment status [33]. This disparity can be attributed to several biological and clinical factors that differentiate these two disease entities. The tumor microenvironment in B-NHL can be more immunosuppressive, with higher levels of immune checkpoint molecules, lower levels of CD19 expression and a more complex stromal interaction, which may limit the efficacy of CAR T-cell therapy [34]. Additionally, B-NHL patients are often older and have more extensive prior treatment, which can impair immune system function and reduce CAR T-cell expansion and persistence [35]. Further research and personalized treatment strategies are needed to improve outcomes for B-NHL patients undergoing CAR T-cell therapy.
Due to concerns that CNS involvement may increase the risk of CAR-T-related neurotoxicity, many trials excluded patients with active CNS disease. In prior studies of CAR T-cell therapy in B-cell malignancy without CNS involvement [6, 36, 37], the incidence of ICANS (all grades) ranges from 37 to 67%, and of severe ICANS (grade ≥ 3) is 28–44%. The rates of ICANS in this study is 42.8% and severe ICANS is 28.6% which are consistent with the data reported in non-CNS-involved patients, suggesting the 1928zT2 CAR T cells do not increase the risk of severe neurotoxicity in patients with CNS involvement.
Potential benefits of third-generation CAR constructs over second-generation (2G) CARs have been demonstrated in preclinical studies [17, 20, 38]. Few clinical studies have reported the third-generation anti-CD19 CAR T-cells for the treatment of R/R B-cell malignancies [18, 22, 23]. TLR2 is expressed on activated T cells as a costimulatory receptor, which recognises bacterial cell wall components and mediates enhanced TCR-induced cytokine production and proliferation [39, 40]. Third-generation CAR T-cells incorporating TLR2 domain show greater IFN-γ and GM-CSF production and enhanced efficacy compared with 2G CAR T-cells both in vitro and in vivo [20]. As these cytokines have been associated with CRS and ICANS risk [8], there was a concern that 1928zT2 CAR-T may result in higher rates of CRS and/or ICANS. However, this study finds that CRS and ICANS rates in this study are similar to those reported for the approved CAR T-cell construct 1928z (axi-cel) [28, 41, 42], indicating that the addition of TLR2 domain might improve the clinical efficacy without increasing treatment-relted toxicity.
In this study, the patients with ICANS were treated with corticosteroid, tocilizumab and the other intervention including reducing intracranial pressure and symptomatic support therapy. Of note, 3 patients with severe ICANS who experienced progressive diffuse cerebral edema and resistant to both corticosteroids and tocilizumab, received the treatment of external ventricular drainage (EVD). EVD is a medical procedure used to drain cerebrospinal fluid (CSF) from the brain's ventricles, which can help reduce intracranial pressure and prevent brain damage. The neurological symptoms of these 3 patients were resolved within one week and without causing residual neurological impairment, indicating that EVD treatment may offer a safe and effective therapeutic alternative for those who are refractory to conventional treatments such as corticosteroids and tocilizumab. No deaths resulting from ICANS were reported in this trial and all the ICANS were manageable. Overall, 1928zT2 cell therapy showed a manageable safety profile in the patients with CNS disease.
The onset, severity and duration of CRS and ICANS after CAR T cell therapy were affected by factors related to the host, tumor and/or therapy [8, 43]. Here, we found ICANS was correlated with occurrence and severity of CRS, which is consistent with the findings from prior studies using different CAR constructs [30, 44, 45]. The median time to onset of CRS and ICANS were 3 and 5 days post-infusion, respectively. Our study demonstrated that CAR T cells were able to traffic to the CNS, and the peak CAR T-cell expansion in CSF was associated with the best clinical response in patients, as well as the severity of ICANS. However, no significant correlation between the occurrence of severe ICANS and the clinical efficacy of CAR T cells was found in these patients. Further prospective studies on larger series are needed to confirm these data.
Our study provides valuable insights into the efficacy and safety of third-generation CD19-targeted CAR T-cell therapy in patients with CNS-involved B-cell malignancies. However, the relatively small cohort size and lacks a direct comparator arm of our study, which makes it difficult to draw concrete conclusions about the superiority of the third-generation CAR T-cell construct over previous generations of CAR T-cell therapies. Future studies should consider including larger patient cohorts and comparator arms to better assess the efficacy and safety of third-generation CAR T-cell constructs.
Conclusion
In conclusion, the administration of third-generation 1928zT2 CAR T cells to patients with R/R B-cell malignancies with CNS involvement is associated with high response rates and a manageable safety profile. Our findings support the notion that third-generation CD19-directed CAR T-cell therapy can result in durable remissions for patients with CNS involvement, suggesting that CNS disease should not exclude patients from CAR-T studies.
Fundings
This study was supported by the grants from the Major Program of National Natural Science Foundation of China (82293634), National Natural Science Foundation of China (82300252, 82470171, 81970161, 82170163, 81970147), GuangDong Basic and Applied Basic Research Foundation (2022A1515110828), China Postdoctoral Science Foundation (2022M721498).
Supplementary Information
Acknowledgements
We thank the patients who volunteered to participate in this study, their families and caregivers, the physicians and nurses who gave clinical care, and the staff who coordinated the clinical study. The CAR-T products in this study were provided by Guangdong Zhaotai InVivo Biomedicine Co., Ltd. We thank Dr. Robert Weinkove from the Malaghan Institute of Medical Research in New Zealand for reviewing this manuscript.
Abbreviations
- B-ALL
B-cell acute lymphoblastic leukemia
- B-NHL
B-cell non-Hodgkin lymphoma
- CAR
Chimeric antigen receptor
- CR
Complete remission
- CRS
Cytokine release syndrome
- CNS
Central nervous system
- CSF
Cerebrospinal fluid
- GMP
Good Manufacturing Practice
- HSCT
Hematopoietic stem cell transplantation
- ICANS
Immune effector cell-associated neurotoxicity syndrome
- ORR
Objective response rate
- OS
Overall survival
- PBMCs
Peripheral blood mononuclear cells
- PFS
Progression-free survival
- PR
Partial response
- scFv
Single-chain variable fragment
- TLR2
Toll-like receptor 2
Author contributions
QL, HZ and PL participated in the conception and design of the study. ZW, QW, XL, XW, YW, ZF, FH, XL, JS and LX treated patients and participated in the clinical data collection. BH, RL and NX performed statistical analyses. QL, ZT and PL performed the quality control of the products and performed PK/PD analysis. BH wrote the original draft, and QL reviewed and edited the manuscript. All authors had full access to all the data and verified the underlying data reported in this study and accept responsibility to submit for publication. All authors read and approved the final version of the manuscript.
Data availability
Data are available on reasonable request. The deidentifed participant data of this study are available from the corresponding author (email: liuqifa628@163.com) on reasonable request.
Declarations
Ethics approval and consent to participate
This study involves human participants and was approved by the Medical Ethics Committee of Nanfang Hospital of Southern Medical University (Approval No. NFEC-2020-098). Participants gave informed consent to participate in the study before taking part.
Patient consent for publication
All subjects provided written informed consent before enrollment and samples and data from patients were processed following standard operating procedures approved by the Medical Ethics Committee of Nanfang Hospital of Southern Medical University.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Bailin He, Ren Lin and Na Xu have contributed equally to this work.
Contributor Information
Peng Li, Email: li_peng@gibh.ac.cn.
Hongsheng Zhou, Email: hanson_tcm@126.com.
Qifa Liu, Email: liuqifa628@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data are available on reasonable request. The deidentifed participant data of this study are available from the corresponding author (email: liuqifa628@163.com) on reasonable request.