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.

尽管 CD19 嵌合抗原受体 (CAR) T 细胞疗法对于难治性或复发性 (r/r) B 细胞急性淋巴细胞白血病 (B-ALL) 成年患者具有良好的完全缓解 (CR) 率，范围为 71% 至 93% ）， ¹ 其长期疗效仍然较差，复发率为31%–100%，中位无白血病生存期（LFS）为6.1–11.6个月，中位总生存期（OS） 12.9-18.2 个月。 ¹ CAR T 细胞的耗竭和有限的持久性已被认为是 CD19 CAR T 细胞治疗后复发的主要因素。 ² 树突状细胞 (DC) 疫苗接种具有良好的耐受性，因此是一种与癌症特异性细胞毒性淋巴细胞 (CTL) 相关的有前途的疗法，可以预防复发。此外，DC疫苗接种可以诱导中央记忆T细胞监测和消除微小残留病（MRD）。 ³ 在前期研究中，我们发现表皮生长因子受体途径底物8（EPS8）在白血病患者中高表达，其水平与疾病进展和生存密切相关。 ⁴ Wilms'tumor-1（WT1）已被报道在多种血液系统恶性肿瘤中高表达，且与复发密切相关。 ⁵ 我们之前证明，针对EPS8的DC疫苗接种不仅可以增加中枢记忆T细胞的比例，还可以刺激CAR T细胞的增殖，通过分泌细胞因子，例如增强CAR T细胞的抗白血病功能。 IL-2、TNF-α、IFN-γ 和颗粒酶 B，它们是 T 细胞持续抗肿瘤活性所必需的。 ⁶ 因此，我们设计了一项临床试验，研究 CD19 CAR T 细胞疗法联合 EPS8 或 WT1 靶向 DC 疫苗治疗成人 r/r B-ALL 患者，以探讨该疗法是否改善 LFS。该报告详细介绍了临床研究的安全性和有效性数据。

临床试验方案（图1A）获得南方医科大学珠江医院伦理审查委员会授权（临床试验编号：NCT03291444）。根据《赫尔辛基宣言》涉及人类受试者医学研究的伦理原则，获得了患者的书面知情同意书。表达 HLA-A1101、HLA-A2402 或 HLA-A0201 且 EPS8 或 WT1 高表达的成人 r/r B-ALL 患者符合资格（表 S1）。 T细胞用编码第四代抗CD19 CAR的慢病毒载体转导，该CAR含有嵌合细胞内信号元件（CD28/CD27/CD3z-iCasp9）。 ⁷ EPS8肽衍生DC（EPS8-DC）疫苗用于EPS8阳性患者，WT1肽衍生DC（WT1-DC）疫苗用于EPS8阴性且WT1阳性患者。数据 S1 中提供了有关纳入标准、排除标准、CAR T 细胞生成和 DC 疫苗接种策略的更多详细信息。一旦确认患者适合输注，每天静脉注射包含氟达拉滨（25 mg/m ² ）和环磷酰胺（300 mg/m ² ）的淋巴清除化疗，持续3天。患者在第0天输注第四代抗CD19 CAR T（4sCAR19）细胞。4sCAR19输注1个月后，如果骨髓形态学缓解，则每2周在腹股沟淋巴结附近皮内注射DC疫苗，持续1个月。 4剂。通过流式细胞术、实时 q-PCR 和 IFN-γ ELIspot（数据 S1）对 4sCAR19 细胞和 DC 疫苗进行功能表征和评估。

入组10名成年r/r B-ALL患者并成功接受4sCAR19细胞治疗；两名参与者因 DC 疫苗输注前疾病进展而退出研究。可评估患者的中位年龄为 37 岁（范围 20-69 岁），并且之前接受过三种或更多治疗。补充材料中详细介绍了临床特征、治疗方案和结果（表 S2）。 8 名可评估患者成功接受了一剂 4sCAR19 细胞治疗，中位剂量为 2.26 × 10 ⁶ /kg（范围 6.4 × 10 ⁵ /kg 至 4.46 × 10 ⁶ /kg （范围 2.97 × 10 ⁶ /剂量至 2.68 × 10 ⁷ /剂量）。对于异基因造血干细胞移植（allo-HSCT）后复发的患者，包括P02、P05、P08和P10，使用供体来源的4sCAR19细胞和DC。

第一年每月进行一次随访，第一年后每 6 个月进行一次。输注4sCAR19细胞后，未发生≥3级细胞因子释放综合征（CRS）或免疫效应细胞相关神经毒性综合征（ICANS）（图S1）。 DC疫苗输注期间未发生≥3级事件。只有八名患者中的一名在输注 DC 疫苗后出现局部皮肤反应。所有输注均安全且耐受性良好。输注4sCAR19细胞后，外周血中的血清细胞因子水平增加，尤其是在第7天，但与基线水平没有显着差异（图S2-S9）。这一结果与我们之前的发现一致，主要是由于4SCAR19细胞的安全性提高。 ⁷ DC疫苗接种后细胞因子IL-12p70、IL-10、IFN-γ和TNF-α水平也有所升高，但与基线时没有显着差异，因为患者均已达到CR DC疫苗接种前。在输注 CD19 CAR T 细胞和 DC 疫苗后，接受供体来源细胞治疗的 4 名患者均未出现移植物抗宿主病。

所有八名可评估患者在接受 4sCAR19 细胞治疗后均达到 MRD 阴性 CR。在中位随访 608 天中，中位 LFS 时间为 489 天（图 1B），并且未达到中位 OS（图 1C）。在我们之前仅用4sCAR19细胞治疗难治性B-ALL患者的研究中，中位LFS和OS分别为257天和267天，中位随访时间仅为291天。 ⁷ 因此，接受联合治疗的 r/r B-ALL 患者的中位 LFS 和 OS 可能比接受单独 CD19 CAR T 细胞治疗的患者更长。截至撰写本文时，八名可评估患者中的七名仍然活着（图 1D）。四名 (50%) 患者在截止时间达到连续 MRD 阴性 CR，其中两名 (P02 为 EPS8 ⁺ /P190 ⁺ /HOX11 ⁺ ， P03（有 WT1 和 IKZF1 突变）维持 MRD 阴性 CR 超过 4 年（图 1D）。一名携带 WT1 和 FLT3-ITD 突变的患者 (P05) 维持 MRD 阴性 CR 27 个月，另一名患者 (P10)（EPS8 ⁺ 和 P190 ⁺ ）维持 MRD 阴性CR 在截止时间持续 3 个月（图 1D，表 S2）。 P04 为 EPS8 ⁺ ，具有 IKZF1 突变和复杂的染色体核型异常，在完成 4sCAR19 细胞治疗和 EPS8-DC 疫苗输注后出现 CD19 ⁺ 复发。该患者随后接受VDP方案治疗，但因疾病进展和肺部感染而放弃治疗，LFS和OS分别为7.3和10.4个月。 P07 具有 WT1 突变且染色体正常，在 CD19 ⁺ 复发后接受伊珠单抗奥佐米星 (inotuzumab ozogamicin) 治疗。 P08携带WT1、TP53和SETD2突变，在CD19 ⁺ 复发后接受VP方案联合blinatumomab治疗，2个月后改用inotuzumab ozogamicin。 P09 具有 WT1 突变，p210 ⁺ ，在 4sCAR19 细胞治疗和 WT1-DC 疫苗输注后分子复发，随后接受 allo-HSCT 治疗（表 S2）。在接受供体来源细胞的 4 名患者中，接受 WT1-DC 疫苗的 P08 出现复发，其他 3 名患者实现了 MRD 阴性 CR。根据我们中心之前的研究结果，供体来源的 CAR T 细胞对于异基因造血干细胞移植后成人复发性 B-ALL 患者来说是一种安全且潜在有效的治疗方法，并且优于供体淋巴细胞输注 (DLI)。这项研究的结果表明，使用供体来源的 CAR T 细胞似乎比使用自体来源的 CAR T 细胞更有效。健康的细胞来源保证了 CAR T 细胞的活力。 ⁸

输注 4sCAR19 细胞后第 7 天检测到外周血中 CAR T 细胞扩增的中位峰值（图 1E）。 4sCAR19细胞的中位持续时间为336天（范围84-1549天）。 DC疫苗接种后4sCAR19细胞被重新扩增（图1E，图S10A，B）。对于 LFS 超过 2 年的患者（P02、P03 和 P05），4sCAR19 细胞在 1 年以上仍可检测到，P03 时最长可达 4.2 年（图 S10B）。然而，根据其他报告，成人 B-ALL 患者接受 CD19 CAR T 细胞治疗的中位持续时间为 14-168 天。 ¹ 此外，在我们之前单独使用4sCAR19细胞治疗难治性B-ALL患者的研究中，CAR T细胞在体内的中位持续时间仅为32天（范围19-345天）。 ⁷ 因此，DC疫苗输注后，CAR T细胞在体内的中位持续时间可能会延长。

通过四聚体测定和 IFN-γ ELIspot 测定评估 DC 疫苗接种的治疗效果。生成 HLA/肽四聚体并用于确定 EPS8 或 WT1 特异性 CD8 ⁺ T 细胞的频率。患者的外周血单核细胞被四聚体染色。 LFS 和 OS 超过 2 年的患者的四聚体 ⁺ CD8 ⁺ T 细胞数量显着高于对照组（图 S10C）。通过 IFN-γ ELIspot 测定测量的 CTL 活性表明，DC 疫苗接种后，分泌 IFN-γ 的 CTL 数量显着增加（图 S10D）。接种前 EPS8 的平均表达率为 71.07%，接种 EPS8-DC 后为 2.81%（图 S10E，右图）。 WT1-DC 疫苗接种后 WT1 的表达显着下降（图 S10E，左图）。这些测定表明，疫苗接种后抗原特异性细胞免疫活性增强，从而增强了 MRD 和 CAR T 细胞持久性的免疫监视。由于 CAR T 细胞疗法的疗效有限，抗原阴性复发已成为另一个令人关注的机制。 DC疫苗接种和CAR T细胞的协同作用可以激活内源性免疫系统，从而防止抗原阴性的肿瘤逃逸。 ⁹ 这一发现意味着 DC 疫苗与 CAR T 细胞疗法的组合在体内具有广泛的机制基础，并且不仅限于单独的 CAR T 细胞。

总之，本研究报告了一种针对成人 r/r B-ALL 患者的新型基于 CAR T 细胞的联合免疫治疗策略（CAR T 细胞治疗结合个体化 DC 疫苗接种）。 DC 疫苗接种更安全，并且可以延长 CAR T 细胞在体内的持久性，这一结果与体外研究一致。 ⁶ CAR T细胞疗法联合DC疫苗接种可延长患者的生存时间和生活质量，对于不适合移植或术后复发的r/r ALL成年患者可能是一种潜在的治疗策略移植。