Manufacturing chimeric antigen receptor T cells: issues and challenges

Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.     

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8⁺ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.     

Blocking CD38-driven fratricide among T cells enables effective antitumor activity by CD38-specific chimeric antigen receptor T cells

Chimeric antigen receptor T-cell(CAR T) therapy is a kind of effective cancer immunotherapy. However,designing CARs remains a challenge because many targetable antigens are shared by T cells and tumor cells. This shared expression of antigens can cause CAR T cell fratricide. CD38-targeting approaches(e.g.,daratumumab) have been used in clinical therapy and have shown promising results. CD38 is a kind of surface glycoprotein present in a variety of cells, such as T lymphocytes and tumor cells. It was previously reported that CD38-based CAR T cells may undergo apoptosis or T cell-mediated killing(fratricide) during cell manufacturing. In this study, a CAR containing a sequence targeting human CD38 was designed to be functional. To avoid fratricide driven by CD38 and ensure the production of CAR T cells, two distinct strategies based on antibodies(clone MM12 T or clone MM27) or proteins(H02 H or H08 H) were used to block CD38 or the CAR single-chain variable fragment(scFv) domain, respectively, on the T cell surface.The results indicated that the antibodies or proteins, especially the antibody MM27, could affect CAR T cells by inhibiting fratricide while promoting expansion and enrichment. Anti-CD38 CAR T cells exhibited robust and specific cytotoxicity to CD38~+ cell lines and tumor cells. Furthermore, the levels of the proinflammatory factors TNF-a, IFN-g and IL-2 were significantly upregulated in the supernatants of A549~(CD38~+) cells. Finally, significant control of disease progression was demonstrated in xenograft mouse models. In conclusion, these findings will help to further enhance the expansion, persistence and function of anti-CD38 CAR T cells in subsequent clinical trials.     

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.     

Short-term culture with IL-2 is beneficial for potent memory chimeric antigen receptor T cell production

Interleukin-2 (IL-2) has been extensively used to boost the body's immune cells, especially T cells. IL-2 is a cytokine that for many years was used to activate and amplify T cells. Due to its potent T cell growth-inducing functions in vitro, for many years, IL-2 was used for the culture and expansion of various T cell products, including tumor-infiltrating lymphocytes (TIL), T cell receptors T cells (TCR T), or genetically engineered cells with chimeric antigen receptors T cells (CAR T). Despite its positive effect on T cell production, the side-effect is not well studied. Here, we reported that long-term culture with IL-2 promotes terminal differentiation and impairs rather than boosts the function of chimeric antigen receptor T cells. However, short-term culture with IL-2 predominantly generates memory CAR T cell favorable for cancer treatment.     

靶向GPC3的第四代嵌合抗原受体T细胞(分泌IL-7和CCL19)的构建以及功能

基于嵌合抗原受体修饰的T细胞(CAR-T)的过继免疫疗法已被证明是治疗恶性肿瘤最有希望的策略之一,但是目前其在实体瘤中的应用依然有限。研究表明磷脂酰肌醇蛋白聚糖3 (GPC3)对肝细胞癌来说是一种有意义的诊断、治疗和预后生物标志物,且已有利用第二代/第三代GPC3靶向的CAR-T细胞治疗肝细胞癌的研究报道。为了进一步提高治疗效果,构建同时表达GPC3 CAR、人源IL-7和CCL19细胞因子的第四代慢病毒载体,转染293T细胞包装慢病毒并感染人T淋巴细胞制备靶向GPC3的第四代CAR-T细胞(GPC3-BBZ-7×19)。利用细胞计数、趋化小室、荧光素酶生物发光法以及流式细胞术等比较其与第二代GPC3 CAR-T细胞(GPC3-BBZ)在增殖、迁移、杀伤以及亚型分布方面的区别,评估GPC3-BBZ-7×19 CAR-T细胞对免疫缺陷小鼠体内GPC3阳性的肝细胞癌腹腔移植瘤生长的作用。结果表明与GPC3-BBZ CAR-T细胞相比,GPC3-BBZ-7×19 CAR-T细胞具备更强的增殖能力、趋化能力以及记忆干细胞(Stem memory T cell,Tscm)组成比(P值均<0...     

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells

Background

The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.

Secretion of human soluble programmed cell death protein 1 by chimeric antigen receptor-modified T cells enhances anti-tumor efficacy

Background aimsChimeric antigen receptor (CAR) T cells have achieved favorable responses in patients with hematologic malignancies, but the outcome has been far from satisfactory in the treatment of tumors with high expression of immunosuppressive molecules. To overcome this limitation, we modified CAR T cells to secrete types of human soluble programmed cell death protein 1 (PD-1) called sPD-1 CAR T cells.MethodsTo compare the effector function between second (conventional second-generation CAR targeting CD19) and sPD-1 CAR T cells, we measured cytotoxicity, cytokine secretion and activation markers incubated with or without tumor cells expressing CD19 and/or programmed cell death ligand 1 (PD-L1). Furthermore, the anti-tumor efficacy of second and sPD-1 CAR T cells was determined using an NSG mouse model bearing NALM-6-PD-L1. Finally, the underlying mechanism was investigated by metabolic parameters and RNA sequencing analysis of different CAR T cells.ResultsCompared with second CAR T cells, sPD-1 CAR T cells enhanced killing efficiency toward CD19⁺PD-L1⁺ tumor cells in vitro. Furthermore, sPD-1 CAR T cells reduced the tumor burden and prolonged overall survival of the NSG (NOD-SCID-IL2rg) mice bearing NALM-6-PD-L1. To explore the effect of soluble PD-1 on CAR T cells, we found that sPD-1 CAR T cells exhibited higher levels of activation and ameliorative profiles of differentiation, exhaustion, glycolysis and apoptosis.ConclusionsWith constitutive soluble PD-1 secretion, sPD-1 CAR T cells have tended to eradicate tumors with a high expression of PD-L1 more effectively than second CAR T cells. This may be due to soluble PD-1 enhancing apoptosis resistance, aerobic metabolism and a more “stem” differentiation of CAR T cells. Overall, our study presents a feasible strategy to increase the efficacy of CAR T cells.     

NKp44-based chimeric antigen receptor effectively redirects primary T cells against synovial sarcoma

BackgroundT-cell receptor-engineered T-cell therapies have achieved promising response rates against synovial sarcoma in clinical trials, but their applicability is limited owing to the HLA matching requirement. Chimeric antigen receptor (CAR) can redirect primary T cells to tumor-associated antigens without requiring HLA matching. However, various obstacles, including the paucity of targetable antigens, must be addressed for synovial sarcoma. Ligands for natural killer (NK) cell-activating receptors are highly expressed by tumor cells.MethodsThe surface expression of ligands for NK cell-activating receptors in synovial sarcoma cell lines was analyzed. We analyzed RNA sequencing data deposited in a public database to evaluate NKp44-ligand expression. Primary T cells retrovirally transduced with CAR targeting NKp44 ligands were evaluated for their functions in synovial sarcoma cells. Alterations induced by various stimuli, including a histone deacetylase inhibitor, a hypomethylating agent, inflammatory cytokines, and ionizing radiation, in the expression levels of NKp44 ligands were investigated.Results: Ligands for NKp44 and NKp30 were expressed in all cell lines. NKG2D ligands were barely expressed in a single cell line. None of the cell lines expressed NKp46 ligand. Primary synovial sarcoma cells expressed the mRNA of the truncated isoform of MLL5, a known cellular ligand for NKp44. NKp44-based CAR T cells specifically recognize synovial sarcoma cells, secrete interferon-γ, and exert suppressive effects on tumor cell growth. No stimulus altered the expression of NKp44 ligands.ConclusionNKp44-based CAR T cells can redirect primary human T cells to synovial sarcoma cells. CAR-based cell therapies may be an option for treating synovial sarcomas.     

Effects of cryopreservation on chimeric antigen receptor T cell functions

Chimeric antigen receptor T (CART) cell therapy has emerged as a potentially curative “drug” for cancer treatment. Cryopreservation of CART cells is necessary for their clinical application. Systematic studies on the effects of cryopreservation on the antitumor function of CART cells are lacking. Therefore, we compared the phenotypes and functions of CART cells that were cryopreserved during ex vivo expansion with those of freshly isolated populations. T cells expressing an anti-B-cell-maturation-antigen (BCMA) chimeric antigen receptor (CAR) were expanded in vitro for 10 days and then cryopreserved. After one month, the cells were resuscitated, and their transduction rates, apoptosis rates and cell subsets were examined via flow cytometry. The results indicated no significant changes in transduction rates or cell subsets, and the survival rate of the resuscitated cells was approximately 90% Furthermore, similar tumoricidal effects and degranulation functions of the resuscitated cells compared with normally cultured cells were verified by calcein release and CD107a assays. A NOD/SCID mouse model was used to estimate the differences in the in vivo antitumor effects of the cryopreserved and normally cultured T cells, but no significant differences were observed. Following co-culture with several target cell types, the cytokines released by the cryopreserved and normally cultured T cells were measured via enzyme-linked immunosorbent assays (ELISAs). The results revealed that the release of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was significantly decreased. These data demonstrated that with the exception of a decrease in cytokine release, the cryopreserved CART cells retained their antitumor functions.     

Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells

Chimeric antigen receptor (CAR)-engineered T cells have a proven efficacy for the treatment of refractory hematological B cell malignancies. While often accompanied by side effects, CAR-T technology is getting more mature and will become an important treatment option for various tumor indications. In this review, we summarize emerging approaches that aim to further evolve CAR-T cell therapy based on combinations of so-called universal or modular CAR-(modCAR-)T cells, and their respective adaptor molecules (CAR-adaptors), which mediate the crosslinking between target and effector cells. The activity of such modCAR-T cells is entirely dependent on binding of the respective CAR-adaptor to both a tumor antigen and to the CAR-expressing T cell. Contrary to conventional CAR-T cells, where the immunological synapse is established by direct interaction of CAR and membrane-bound target, modCAR-T cells provide a highly flexible and customizable development of the CAR-T cell concept and offer an additional possibility to control T cell activity.     

Combined 4-1BB and ICOS co-stimulation improves anti-tumor efficacy and persistence of dual anti-CD19/CD20 chimeric antigen receptor T cells

Chimeric antigen receptor (CAR) T-cell therapy is a promising therapeutic strategy against lymphoma. However, post-treatment relapses due to antigen loss remain a challenge. Here the authors designed a novel bicistronic CAR construct and tested its functions in vitro and in vivo. The CAR construct consisted of individual anti-CD19 and anti-CD20 single-chain fragment variables equipped with ICOS-CD3ζ and 4-1BB-CD3ζ intracellular domains, respectively. The CD19 and CD20 bicistronic CAR T cells exhibited tumor lytic capacities equivalent to corresponding monospecific CAR T cells. Moreover, when stimulated with CD19 and CD20 simultaneously, the bicistronic CAR T cells showed prolonged persistence and enhanced cytokine generation compared with single stimulations. Interestingly, the authors found that the 4-1BB signal was predominant in the signaling profiles of ICOS and 4-1BB doubly activated CAR T cells. In vivo study using a CD19/CD20 double-positive tumor model revealed that the bicistronic CAR T cells were more efficient than monospecific CD19 CAR T cells in eradicating tumors and prolonging mouse survival. The authors’ novel bicistronic CD19/CD20 CAR T cells demonstrate improved anti-tumor efficacy in response to dual antigen stimulations. These data provide optimism that this novel bicistronic CAR construct can improve treatment outcomes in patients with relapsed/refractory B cell malignancy.     

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 CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.     

Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

BackgroundChimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.ResultsWhere the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non–tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.ConclusionsBy combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.     

嵌合抗原受体T细胞免疫疗法治疗B细胞淋巴瘤的研究进展

B细胞淋巴瘤(B cell lymphoma)因其异质性,患者极易复发,因此成为肿瘤治疗的难题之一。嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)免疫疗法有良好的治疗效果且具精准、快速和高效的特点,成为最具前景的治疗癌症的方法之一。目前,CAR-T免疫疗法在治疗B细胞淋巴瘤上得以成功的应用,同时,FDA已批准相关产品应用于治疗B细胞淋巴瘤,一定程度上弥补了常规治疗B细胞淋巴瘤的方法。现就CAR-T免疫疗法的发展历程、主要靶点及其策略研究作一概述,为治疗B细胞淋巴瘤提供可参考的思路。     

嵌合抗原受体T细胞治疗血液肿瘤的研究进展

嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor T-cell immunotherapy,CAR-T)是近年来迅速发展的肿瘤过继免疫治疗方法,其胞外段抗体以非主要组织相容性复合物(major histocompatibility complex,MHC)方式与相应的肿瘤相关抗原结合识别后,使T细胞活化而发挥抗肿瘤效应。CAR-T在血液疾病治疗中取得较好的效果,现就CAR的结构、CAR-T治疗的靶点、出现的不良反应及采取的相应策略等作一概述。