CAR19/22 T cell cocktail therapy for B-ALL relapsed after allogeneic hematopoietic stem cell transplantation

B cell acute lymphocytic leukemia (B-ALL) patients who have relapsed after hematopoietic stem cell transplantation (HSCT) have a poor prognosis, and there is currently no standard approach available. Chimeric antigen receptor (CAR)-T cells induce high rates of initial response and long-term remission among patients with B-cell malignancies, especially B-ALL. Meanwhile, sequential infusion of CAR19/22 T cells has been proven to be effective at preventing tumor immune escape. In the present study, we retrospectively analyzed 23 B-ALL patients who relapsed after allogeneic (allo)-HSCT and underwent sequential infusion of CAR19/22 T cells, including nine donor-derived and 14 recipient-derived, in our center from July 2016 to July 2020, to evaluate the safety and efficacy of the cocktail of two single-specific CAR-T cells in B-ALL patients relapsed after transplantation. Except for one patient refusing evaluation, the remaining 22 patients achieved minimal residual disease (MRD)-negative complete remission within 30 days after CAR-T infusion. Most toxicities were slight and reversible. The estimated 12-month progression-free survival (PFS) rate was 59.2% (95% confidence interval [CI], 35.9% to 76.5%), and the estimated 12-month overall survival (OS) rate was 67.4% (95% CI, 43.2% to 83.1%). Only two patients had CD19-negative recurrence. In addition, early recurrence after transplantation, graft-versus-host disease (GVHD) and severe infection after CAR-T infusion were poor prognostic factors. Our results indicate that sequential infusion of CAR19/22 T cells is safe and effective for relapsed ALL patients after HSCT. This trial was registered at www.chictr.org.cn as #ChiCTR-OPN-16008526.     

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

CD166-specific CAR-T cells potently target colorectal cancer cells

Chimeric antigen receptor (CAR) T-cell therapy is emerging as an effective cancer treatment, such as for hematological malignancies, however its effectiveness as an approach to treat solid tumors, such as in colorectal cancer (CRC), remains to be better developed. One area of intense development has been in the identification and characterization of novel cancer-related ligand receptors for CAR design and evaluation. It is known that the CD6 receptors CD166 and CD318 are highly expressed in CRC, and several CAR-Ts have also been explored in preclinical and clinical studies for the treatment of CRC, with promising safety and efficacy findings. Here, we constructed a CAR based on the extracellular domain of CD6 and demonstrate its cytotoxic effect in target positive human CRC cell lines. Unexpectedly, we found that CD6-CAR-T cells targeted CD166 instead of CD318. Furthermore, CD6-CAR-T cells show robust cytotoxicity to CD166-positive cell lines in a dose-dependent manner with cytokine IFN-γ significantly released. Particularly, CD6-CAR-T cells show potent cytotoxicity targeting CRC cancer stem cells (CSCs), highlighting that CD6-CAR-T is a promising approach for the therapy of CRC.     

Clinical trials of CAR-T cells in China

Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR)-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous and sequential CAR-T cells are being studied for clinical applications. Multi-target CAR-engineered T cells are also entering clinical trials. T cell receptor-engineered CAR-T and universal CAR-T cells represent new frontiers in CAR-T cell development. In this study, we analyzed the characteristics of CAR constructs and registered clinical trials of CAR-T cells in China and provided a quick glimpse of the landscape of CAR-T studies in China.     

通用型CD19 CAR-T的体外构建及初步功能鉴定

CAR-T免疫细胞治疗已经在血液肿瘤领域取得突破性进展.然而,目前上市和国内临床试验的CAR-T细胞均来自肿瘤患者自身,即自体型CAR-T.因受制于患者T细胞的质量和数量、制备周期长且价格昂贵等原因,很难将其进行大规模临床应用.该研究利用CRISPR/Cas9基因编辑技术敲除健康人脐带血来源T细胞的TCR分子和HLA-...     

CD19 CAR-Targeted T Cells Induce Long-Term Remission and B Cell Aplasia in an Immunocompetent Mouse Model of B Cell Acute Lymphoblastic Leukemia

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.     

The efficacy of anti-CD19 chimeric antigen receptor T cells for B-cell malignancies

Immunotherapy with chimeric antigen receptor T (CAR-T) cells has proved remarkably effective in recently published clinical trials. In this meta-analysis, we performed a systematic review in terms of the clinical response treated with CAR-T cells in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and lymphomas patients. Thirty-eight published clinical studies including 665 patients were eligible for response rate (RR) evaluation. The overall pooled RR of CD19-CAR-T cells was 72% (95% confidence interval: 62–77%). The various clinical parameters were analyzed. RR was 81% in ALL, 68% in lymphoma and 70% in CLL. RR in patients who received interleukin (IL)-2 was 70%, whereas in those who did not receive IL-2, it was 74%. RR was 75% with lymphodepletion and 56% without lymphodepletion. RR with autologous cells was 76% and 57% with allogeneic cells. In conclusion, this meta-analysis showed a high clinical RR of CD19-CAR-T cell–based immunotherapy in patients with refractory B-cell malignancies.     

CAR细胞疗法在T细胞-急性淋巴细胞白血病应用的新进展

嵌合抗原受体(chimeric antigen receptors,CAR)细胞疗法已广泛用于白血病、淋巴瘤的治疗, CD19和CD22靶向CAR-T已在复发、难治性急性B淋巴细胞白血病(RR-B-ALL)等血液系统疾病的治疗上取得了显著疗效,而在T细胞肿瘤治疗上进展缓慢。介绍了目前国内外利用CAR细胞技术与CRISPR / Cas9基因编码技术,设计了T-ALL相关的CAR细胞免疫疗法并进行了CAR细胞免疫疗法在T-ALL治疗上的初步探索。     

Targeting CD276 by CAR-T cells induces regression of esophagus squamous cell carcinoma in xenograft mouse models

Esophageal cancer, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), has a poor prognosis and limited therapeutic options. Chimeric antigen receptor (CAR)-T cells represent a potential ESCC treatment. In this study, we examined CD276 expression in healthy and esophageal tumor tissues and explored the tumoricidal potential of CD276-targeting CAR-T cells in ESCC. CD276 was strongly and homogenously expressed in ESCC and EAC tumor lesions but mildly in healthy tissues, representing a good target for CAR-T cell therapy. We generated CD276-directed CAR-T cells with a humanized antigen-recognizing domain and CD28 or 4–1BB co-stimulation. CD276-specific CAR-T cells efficiently killed ESCC tumor cells in an antigen-dependent manner both in vitro and in vivo. In patient-derived xenograft models, CAR-T cells induced tumor regression and extended mouse survival. In addition, CAR-T cells generated from patient T cells demonstrated potent cytotoxicity against autologous tumor cells. Our study indicates that CD276 is an attractive target for ESCC therapy, and CD276-targeting CAR-T cells are worth testing in ESCC clinical trials.     

Genetic engineering of T cells with chimeric antigen receptors for hematological malignancy immunotherapy

The host immune system plays an instrumental role in the surveillance and elimination of tumors by recognizing and destroying cancer cells. In recent decades, studies have mainly focused on adoptive immunotherapy using engineered T cells for the treatment of malignant diseases. Through gene engraftment of the patient’s own T cells with chimeric antigen receptor (CAR), they can recognize tumor specific antigens effectively and eradicate selectively targeted cells in an MHC-independent fashion. To date, CAR-T cell therapy has shown great clinical utility in patients with B-cell leukemias. Owing to different CAR designs and tumor complex microenvironments, genetically redirected T cells may generate diverse biological properties and thereby impact their long-term clinical performance and outcome. Meanwhile some unexpected toxicities that result from CAR-T cell application have been examined and limited the curative effects. Diverse important parameters are closely related with adoptively transferred cell behaviors, including CAR-T cells homing, CAR constitutive signaling, T cell differentiation and exhaustion. Thus, understanding CARs molecular design to improve infused cell efficacy and safety is crucial to clinicians and patients who are considering this novel cancer therapeutics. In this review, the developments in CAR-T cell therapy and the limitations and perspectives in optimizing this technology towards clinical application are discussed.     

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising. Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence. HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy. HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl. HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.     

稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定

实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此,通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系,研究CD19 CAR-T细胞对其的杀伤,能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞,证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实,连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比,CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后,通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明,成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系,且该细胞系能够被CD19 CAR-T细胞特异性杀伤。     

Optimization of manufacturing conditions for chimeric antigen receptor T cells to favor cells with a central memory phenotype

BackgroundChimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion.MethodsWe compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays.ResultsWe have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics.DiscussionWe propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma.     

The progress and perspectives of CAR-T cell therapy

CAR-T cell therapy has already achieved world-renowned clinical effects in the treatment of hematological malignancies. Due to the tumor heterogeneity, immunosuppressive microenvironment, and other factors, CAR-T cell therapy has still not shown obvious clinical efficacy in clinical treatment of solid tumors. However, great progress has been made in the preparation of CAR-T cells in recent years, including T cells redirected for universal cytokine mediated killing, universal CAR -T cells, non-viral vector CAR-T cells, SynNotch technology, SUPRA CAR technology, regulated CAR-T cells, and bi-specific CAR-T cells, etc. Future research and development of CAR-T cell therapy will be focused on these following aspects: the combined application of CAR-T cells with different targets, known as "Cocktail CAR-T cells", is expected to increase efficiency toward solid tumors; based on systemic biology/synthetic biology theories, CAR-T cells are likely to be transformed to robot or intelligent system by introducing sensors, logic gates, and logic circuits. This article mainly comments on research progress and perspectives on CAR-T cell therapy in solid tumor treatment.     

GPC3-targeted CAR-T cells secreting B7H3-targeted BiTE exhibit potent cytotoxicity activity against hepatocellular carcinoma cell in the in vitro assay

Hepatocellular carcinoma (HCC), the most common primary liver cancer has a high mortality in China, and it is usually diagnosed at a late stage, thereby leaving patients with few effective treatment options. Chimeric antigen receptor-T (CAR-T) cell therapy, a novel immunotherapy that has shown promising results in leukemia, lymphoma and multiple myeloma, is also expected to work well in solid tumors, including HCC. However, the ideal therapeutic efficacy has not yet been achieved, in part due to tumor antigen escape caused by antigen heterogeneity. To overcome such challenge, we screened a panel of biomarkers in HCC cell lines and found that GPC3 and B7H3 were highly expressed on HCC with expression heterogeneity. Then we developed a novel bispecific T cell engagers CAR-T (CAR.T-BiTEs) that drives the expression of a CAR specific for GPC3 and BiTEs against CD3 and B7H3, herein referred to as “GPC3-BiTE CAR.” We found that BiTEs promoted the increased activation of untransduced T cells and IFN-γ release. Moreover, BiTEs secreted by GPC3-BiTE CAR-HEK293T cells promoted increased cytotoxicity activity of untransduced T cells against GPC3+/B7H3+ (GPC3 positive/B7H3 positive) and GPC3-/B7H3+(GPC3 negative/B7H3 positive) HCC cell lines. In vitro function assays showed that GPC3-BiTE CAR-T cells exhibited greater cytotoxicity activity against GPC3+/B7H3+ HCC cell lines than GPC3 CAR-T cells (GPC3-targeted CAR-T cells) and B7H3 CAR-T cells (B7H3-targeted CAR-T cells). Furthermore, GPC3-BiTE CAR-T cells exhibited superior cytotoxicity against GPC3 negative HCC cell lines compared with GPC3 CAR T cells. In conclusion, our study showed that GPC3-BiTE CAR T cells exhibited superior antitumor activity than single-target CAR-T cells and can overcome tumor escape induced by antigen heterogeneity, suggesting that this could be a promising therapeutic strategy for HCC.     

Successful application of anti-CD19 CAR-T therapy with IL-6 knocking down to patients with central nervous system B-cell acute lymphocytic leukemia

Few studies have described chimeric antigen receptor–modified T cell (CAR-T) therapy for central nervous system (CNS) B-cell acute lymphocytic leukemia (B-ALL) patients due to life-threatening CAR-T-related encephalopathy (CRES) safety issues. In this study, CAR-Ts targeting CD19 with short hairpin RNA (shRNA)-IL-6 gene silencing technology (ssCART-19s) were prepared. We conducted a phase 1 clinical trial (ClinicalTrials.gov number, NCT03064269). Three patients with relapsed CNS B-ALL were enrolled, conditioned with the fludarabine and cyclophosphamide for lymphocyte depletion and infused with ssCART-19s for three consecutive days. Clinical symptoms and laboratory examinations were monitored. After ssCART-19 treatment, three patients' symptoms resolved almost entirely. Brain leukemic infiltration reduced significantly based on magnetic resonance imaging (MRI), and there were no leukemic blasts in cerebrospinal fluid (CSF), which was confirmed by cytological and molecular examinations. Additionally, increases in the levels of cytokines and immune cells were observed in the CSF of all patients. Only grade 1 cytokine release syndrome (CRS) manifesting as fever was noted in patients. In conclusion, CAR-Ts with shRNA-IL-6 gene knockdown migrated into the CNS, eradicated leukemic cells and elevated cytokines in CSF with mild, acceptable side effects.     

Remote controlling of CAR-T cells and toxicity management: Molecular switches and next generation CARs

Cell-based immunotherapies have been selected for the front-line cancer treatment approaches. Among them, CAR-T cells have shown extraordinary effects in hematologic diseases including chemotherapy-resistant acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). In this approach, autologous T cells isolated from the patient''s body genetically engineered to express a tumor specific synthetic receptor against a tumor antigen, then these cells expanded ex vivo and re-infusion back to the patient body. Recently, significant clinical response and high rates of complete remission of CAR T cell therapy in B-cell malignancies led to the approval of Kymriah and Yescarta (CD19-directed CAR-T cells) were by FDA for treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Despite promising therapeutic outcomes, CAR T cells also can elicit the immune-pathologic effects, such as Cytokine Release Syndrome (CRS), Tumor Lysis Syndrome (TLS), and on-target off-tumor toxicity, that hampered its application. Ineffective control of these highly potent synthetic cells causes discussed potentially life-threatening toxicities, so researchers have developed several mechanisms to remote control CAR T cells. In this paper, we briefly review the introduced toxicities of CAR-T cells, then describe currently existing control approaches and review their procedure, pros, and cons.     

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML.MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells.ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells.ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.     

S. aureus superantigen protein A expands CD4(+)/CD8(+)/CD19(+)/CD34(+) cells in mice: a potential immunorestorer

Protein A (PA) of Staphylococcus aureus is known for its immunostimulatory, anti-cancer, and anti-toxic properties. The present study revealed that PA stimulates specific immunocytes to act as a potential immunorestorer. It has also been shown that the percentage of various cell types bearing different clusters of differentiation markers, e.g., CD4(+), CD8(+), CD19(+), increases considerably after inoculation with PA. It has also been observed that CD34(+) progenitor cells of bone marrow also increased significantly (P < 0.05) upon PA treatment. PA significantly elevated Th-1 cytokines, e.g., IFN-gamma, TNF-alpha, and IL-1alpha. The increased percentages of CD4(+), CD8(+), CD19(+), CD34(+) cells and elevated cytokine levels in PA treated animals may contribute to the reported anti-tumor, anti-fungal, anti-parasitic, and anti-toxic properties of PA. Since in various diseased conditions and during toxic drug therapy lymphocytes bearing such differentiation markers get suppressed, this type of approach could help in immunorestoration of the host. These findings might help in designing therapeutic approaches toward various diseases which cause immunosuppression.     

Evaluation of piggyBac-mediated anti-CD19 CAR-T cells after ex vivo expansion with aAPCs or magnetic beads

Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.