Dendritic cell vaccines extend <scp>CAR</scp> T‐cell persistence and improve the efficacy of <scp>CD19 CAR</scp> T‐cell therapy in refractory or relapsed adult <scp>B‐ALL</scp> patients
Authors: Sanfang Tu, Lijuan Zhou, Rui Huang, Xuan Zhou, Jilong Yang, Yanjie He, Yuxing Hu, Honghao Zhang, Xiaoling Xie, Yuhua Li
Journal: American Journal of Hematology (2024)

Although CD19 chimeric antigen receptor (CAR) T-cell therapy has a favorable complete response (CR) rate ranging from 71% to 93% for adult patients with refractory or relapsed (r/r) B-cell acute lymphoblastic leukemia (B-ALL),1 its long-term efficacy remains poor, with a 31%–100% relapse rate, a median leukemia-free survival (LFS) of 6.1–11.6 months, and a median overall survival (OS) of 12.9–18.2 months.1 Exhaustion and limited persistence of CAR T cells have been recognized as major factors for relapse after CD19 CAR T-cell therapy.2 Dendritic cell (DC) vaccination is well tolerated and thus is a promising therapy related to cancer-specific cytotoxic lymphocytes (CTLs), which may prevent recurrence. Furthermore, DC vaccination can induce central memory T cells to monitor and eliminate minimal residual disease (MRD).3 In previous studies, we found that epidermal growth factor receptor pathway substrate 8 (EPS8) was highly expressed in leukemia patients, and its levels were closely related to disease progression and survival.4 Wilms' tumor-1 (WT1) has been reported to be highly expressed in a variety of hematologic malignancies and is closely related to recurrence.5 We previously demonstrated that EPS8-targeted DC vaccination not only increased the proportion of central memory T cells but also stimulated the proliferation of CAR T cells and enhanced CAR T-cell antileukemic functions through the secretion of cytokines such as IL-2, TNF-α, IFN-γ, and granzyme B, which are necessary for the sustained antitumor activity of T cells.6 Therefore, we designed a clinical trial to study CD19 CAR T cell therapy combined with EPS8- or WT1-targeted DC vaccines for adult r/r B-ALL patients to explore whether this therapy improves LFS. This report details the safety and efficacy data from the clinical study. The clinical trial protocol (Figure 1A) was authorized by the Ethical Review Board of Zhujiang Hospital of Southern Medical University (clinical trial number: NCT03291444). Written informed consent was obtained from patients in accordance with the ethical principles of the Helsinki Declaration involving medical research in human subjects. Adult r/r B-ALL patients who expressed HLA-A1101, HLA-A2402, or HLA-A0201 and had high expression of EPS8 or WT1 were eligible (Table S1). T cells were transduced with lentiviral vectors encoding a fourth-generation anti-CD19 CAR containing a chimeric intracellular signaling element (CD28/CD27/CD3ζ-iCasp9).7 The EPS8 peptide-derived DC (EPS8-DC) vaccine was used in EPS8-positive patients, while the WT1 peptide-derived DC (WT1-DC) vaccine was used in EPS8-negative patients with WT1 positivity. More details on the inclusion criteria, exclusion criteria, generation of CAR T cells, and DC vaccination strategy are provided in the Data S1. Lymphodepleting chemotherapy comprising fludarabine (25 mg/m2) and cyclophosphamide (300 mg/m2) was administered intravenously daily for 3 days once the patient was confirmed to be eligible for infusion. Patients were infused with fourth-generation anti-CD19 CAR T (4sCAR19) cells on Day 0. After 1 month of 4sCAR19 infusion, if bone marrow morphologic remission had been achieved, DC vaccines were administered intradermally near the inguinal lymph nodes every 2 weeks for 4 doses. The 4sCAR19 cells and DC vaccines were functionally characterized and evaluated by flow cytometry, real-time q-PCR, and IFN-γ ELIspot (Data S1). Ten adult patients with r/r B-ALL were enrolled and successfully received 4sCAR19 cell therapy; two participants were withdrawn from the study due to disease progression before DC vaccine infusion. The evaluable patients had a median age of 37 years (range 20–69 years) and had received three or more previous therapies. The clinical characteristics, therapeutic regimens, and outcomes are detailed in the supplementary material (Table S2). Eight evaluable patients were successfully administered one dose of 4sCAR19 cells at a median dose of 2.26 × 106/kg (range 6.4 × 105/kg to 4.46 × 106/kg) on Day 0. At a median interval of 49 days, all eight patients were infused with DC vaccines biweekly for a total of 4 doses at a median dose of 5.44 × 106/kg (range 2.97 × 106/dose to 2.68 × 107/dose). For patients who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), including P02, P05, P08, and P10, donor-derived 4sCAR19 cells and DCs were used. Follow-up was conducted monthly in the first year and once every 6 months after the first year. No Grade ≥3 cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) occurred after infusion of 4sCAR19 cells (Figure S1). No Grade ≥3 events occurred during DC vaccine infusion. Only one of eight patients experienced local skin reactions after DC vaccine infusion. All infusions were safe and well tolerated. The serum cytokine levels in the peripheral blood increased after infusion of 4sCAR19 cells, especially on Day 7, but were not significantly different from the baseline levels (Figures S2–S9). This result is consistent with our previous findings, mainly due to the increased safety of 4SCAR19 cells.7 The levels of the cytokines IL-12p70, IL-10, IFN-γ, and TNF-α were also increased after DC vaccination but were not significantly different from those at baseline because the patients had all achieved CR before DC vaccination. Graft-versus-host disease did not occur in any of the four patients treated with donor-derived cells after infusion of CD19 CAR T cells and DC vaccine. All eight evaluable patients achieved MRD-negative CR after receiving 4sCAR19 cells. Over a median follow-up of 608 days, the median LFS time was 489 days (Figure 1B), and the median OS was not reached (Figure 1C). In our previous study on the treatment of r/r B-ALL patients with 4sCAR19 cells alone, the median LFS and OS were 257 and 267 days, respectively, with a median follow-up of only 291 days.7 Therefore, the median LFS and OS may be longer in r/r B-ALL patients receiving combination therapy than in those receiving CD19 CAR T-cell therapy alone. Seven of the eight evaluable patients were still alive at the time of writing (Figure 1D). Four (50%) patients achieved continuous MRD-negative CR at the cutoff time, two of whom (P02 was EPS8+/P190+/HOX11+, and P03 had WT1 and IKZF1 mutations) maintained MRD-negative CR for more than 4 years (Figure 1D). One patient (P05) with WT1 and FLT3-ITD mutations maintained MRD-negative CR for 27 months, and another patient (P10) (EPS8+ and P190+) maintained MRD-negative CR for 3 months at the cutoff time (Figure 1D, Table S2). P04, who was EPS8+ and had IKZF1 mutations and complex chromosomal karyotype abnormalities, developed CD19+ relapse after the completion of 4sCAR19 cell therapy and EPS8-DC vaccine infusion. The patient was subsequently treated with the VDP regimen but abandoned treatment due to disease progression and lung infection, and her LFS and OS were 7.3 and 10.4 months, respectively. P07, who had a WT1 mutation and normal chromosomes, was treated with inotuzumab ozogamicin after CD19+ relapse. P08, who had WT1, TP53, and SETD2 mutations, was treated with the VP regimen combined with blinatumomab after CD19+ relapse and was subsequently switched to inotuzumab ozogamicin 2 months later. P09, who had a WT1 mutation and was p210+, molecular relapsed after 4sCAR19 cell therapy and WT1-DC vaccine infusion and was subsequently treated with allo-HSCT (Table S2). Of the four patients who received donor-derived cells, P08, who received the WT1-DC vaccine, experienced relapse, and the other three patients achieved MRD-negative CR. Based on the results of previous studies in our center, donor-derived CAR T cells are a safe and potentially effective treatment for adult recurrent B-ALL patients after allo-HSCT and are superior to donor lymphocyte infusion (DLI). The results of this study show that the use of donor-derived CAR T cells appears to be more effective than the use of autologous-derived CAR T cells. A healthy cell source guarantees CAR T-cell viability.8 The median peak of CAR T-cell expansion in the peripheral blood was detected on Day 7 after infusion of 4sCAR19 cells (Figure 1E). The median persistence time of 4sCAR19 cells was 336 days (range 84–1549 days). 4sCAR19 cells were reamplified after DC vaccination (Figure 1E, Figure S10A,B). For patients with an LFS of more than 2 years (P02, P03, and P05), 4sCAR19 cells were still detectable for more than 1 year, with a maximum of 4.2 years in P03 (Figure S10B). However, the median duration of CD19 CAR T-cell therapy for adult B-ALL patients was 14–168 days according to other reports.1 Furthermore, in our previous study on the treatment of r/r B-ALL patients with 4sCAR19 cells alone, the median persistence of CAR T cells in vivo was only 32 days (range 19–345 days).7 Therefore, the median persistence of CAR T cells in vivo may be prolonged after DC vaccination infusion. The therapeutic effect of DC vaccination was evaluated with a tetramer assay and IFN-γ ELIspot assay. HLA/peptide tetramers were generated and used to determine the frequency of EPS8- or WT1-specific CD8+ T cells. Peripheral blood mononuclear cells from patients were stained with tetramers. Patients with LFS and an OS of more than 2 years had significantly greater numbers of tetramer+ CD8+ T cells than did the control group (Figure S10C). The CTL activity measured by the IFN-γ ELIspot assay showed that the number of IFN-γ-secreting CTLs was significantly increased after DC vaccination (Figure S10D). The mean EPS8 expression was 71.07% before vaccination and 2.81% after EPS8-DC vaccination (Figure S10E, right panel). The expression of WT1 decreased significantly after WT1-DC vaccination (Figure S10E, left panel). These assays showed that antigen-specific cellular immune activity was enhanced after vaccination, which enhanced the immune surveillance of MRD and CAR T-cell persistence. Antigen-negative relapse has become another mechanism of concern because of the limited efficacy of CAR T-cell therapy. The synergistic action of DC vaccination and CAR T cells can activate the endogenous immune system, thus preventing antigen-negative tumor escape.9 This finding means that the combination of DC vaccines with CAR T-cell therapy has a broad mechanistic basis in vivo and is not limited to that CAR T cells alone. In summary, this study reports a novel CAR T-cell-based combination immunotherapy strategy (CAR T-cell therapy combined with individualized DC vaccination) for adult r/r B-ALL patients. DC vaccination is safer and may prolong the persistence of CAR T cells in vivo, a result consistent with in vitro studies.6 CAR T-cell therapy combined with DC vaccination may prolong patient survival time and quality of life and may be a potential therapeutic strategy for adult patients with r/r ALL who are not eligible for transplantation or who relapse after transplantation. Contribution: Yuhua Li, Yanjie He, and Sanfang Tu conceived and designed the study; Sanfang Tu, Jilong Yang, and Rui Huang recruited and managed the subjects; Lijuan Zhou and Yuxing Hu generated the DC vaccine; Lijuan Zhou and Sanfang Tu followed up with the subjects and collected data; Honghao Zhang and Xiaoling Xie assisted Lijuan Zhou in the vaccine-related experiments; Yuxing Hu detected the expression of WT1 and EPS8; Sanfang Tu and Lijuan Zhou drafted the manuscript; Lijuan Zhou analyzed the data and created the figures; and Yuhua Li further revised the manuscript. All authors approved the final version. The authors thank the staff of Shenzhen Geno-Immune Medical Institute for preparing and detecting the 4sCAR19 cells used in this study. The authors thank the staff of the Pharm International Regenerative Medicine Technology Group for providing the GMP manufacturing conditions required for DC vaccine preparation. The authors thank the staff of the Clinical Research Center, Zhujiang Hospital, Southern Medical University, for their guidance in the clinical research. The authors thank Lenghe Zhang for helping with the data analysis. This work was supported by the National Natural Science Foundation of China (No. 82270233 and No. 82200246) and the Guangzhou Municipal Science and Technology Project, China (No. 201704020216). The authors declare no competing interests. Written informed consent was obtained from patients in accordance with the ethical principles of the Helsinki Declaration involving medical research in human subjects in the clinical trial. The study was registered in clinicaltrials.gov (NCT03291444). The main data supporting the results in this study are available within the paper and its Supplementary Information. Eligible researchers can request data sets, including unidentified individual subject data. Data may be available on request from the corresponding author from 12 to 36 months after the trial completion. Data S1. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.