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-...     

CAR-T Cell Therapy for Breast Cancer: From Basic Research to Clinical Application

Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients'' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions.     

CAR-T cell therapy and its application in clinical cancer treatment

Immunotherapy is regarded as the most significant method for cancer treatment in recent years. Chimeric antigen receptor T cells (CAR-T) technology, one form of target immunotherapy, has made a great breakthrough in hematological malignancies treatment and also a few progress in solid tumor treatment. This article reviews the history and mechanism of CAR-T, as well as the advantages and limitations of CAR-T in clinical cancer treatment. Then the review mainly discussed the clinical trial progress of CAR-T cell therapy in solid tumor treatment. A primary obstacle of CAR-T therapy is the heterogeneity in solid tumors. With an increasing number of solid tumor surface antigens being discovered, different varieties of CARs have been designed to treat solid tumors and have made some progress in clinical trial. In the end, this review puts forward a possible development direction of CAR-T.     

Chimeric antigen receptor T cells in solid tumors: a war against the tumor microenvironment

Chimeric antigen receptor(CAR) T cell is a novel approach, which utilizes anti-tumor immunity for cancer treatment. As compared to the traditional cell-mediated immunity, CAR-T possesses the improved specificity of tumor antigens and independent cytotoxicity from major histocompatibility complex molecules through a monoclonal antibody in addition to the Tcell receptor. CAR-T cell has proven its effectiveness, primarily in hematological malignancies, specifically where the CD19 CAR-T cells were used to treat B-cell acute lymphoblastic leukemia and B-cell lymphomas. Nevertheless, there is little progress in the treatment of solid tumors despite the fact that many CAR agents have been created to target tumor antigens such as CEA,EGFR/EGFRvIII, GD2, HER2, MSLN, MUC1, and other antigens. The main obstruction against the progress of research in solid tumors is the tumor microenvironment, in which several elements, such as poor locating ability, immunosuppressive cells,cytokines, chemokines, immunosuppressive checkpoints, inhibitory metabolic factors, tumor antigen loss, and antigen heterogeneity, could affect the potency of CAR-T cells. To overcome these hurdles, researchers have reconstructed the CAR-T cells in various ways. The purpose of this review is to summarize the current research in this field, analyze the mechanisms of the major barriers mentioned above, outline the main solutions, and discuss the outlook of this novel immunotherapeutic modality.     

细胞治疗的典范：嵌合抗原受体T细胞疗法

嵌合抗原受体T(CAR-T)细胞疗法是一种利用合成受体特异性靶向抗原的过继性细胞疗法(ACT),目前在血液肿瘤的治疗中有极大的临床应用价值。虽然美国食品药品监督管理局(FDA)已经批准两款CAR-T药物上市,但CAR-T疗法在治疗过程中仍然存在一些副作用,如细胞因子释放综合征(CRS)、神经毒性、B细胞功能缺失等。同时,CAR-T疗法在实体瘤治疗中的效果甚微,主要原因是缺乏特异性靶点以及肿瘤微环境对CAR-T细胞功能的抑制等。文中将从CAR的结构设计、临床应用、合成生物学对新型CAR的优化来阐述应用CAR-T细胞疗法治疗肿瘤所面临的挑战及广阔前景。     

Phase 1 clinical trial demonstrated that MUC1 positive metastatic seminal vesicle cancer can be effectively eradicated by modified Anti-MUC1 chimeric antigen receptor transduced T cells

Recent progress in chimeric antigen receptor-modified T-cell(CAR-T cell) technology in cancer therapy is extremely promising, especially in the treatment of patients with B-cell acute lymphoblastic leukemia. In contrast, due to the hostile immunosuppressive microenvironment of a solid tumor, CAR T-cell accessibility and survival continue to pose a considerable challenge, which leads to their limited therapeutic efficacy. In this study, we constructed two anti-MUC1 CAR-T cell lines. One set of CAR-T cells contained SM3 single chain variable fragment(sc Fv) sequence specifically targeting the MUC1 antigen and co-expressing interleukin(IL) 12(named SM3-CAR). The other CAR-T cell line carried the SM3 sc Fv sequence modified to improve its binding to MUC1 antigen(named p SM3-CAR) but did not co-express IL-12. When those two types of CAR-T cells were injected intratumorally into two independent metastatic lesions of the same MUC1+ seminal vesicle cancer patient as part of an interventional treatment strategy, the initial results indicated no side-effects of the MUC1 targeting CAR-T cell approach, and patient serum cytokines responses were positive. Further evaluation showed that p SM3-CAR effectively caused tumor necrosis, providing new options for improved CAR-T therapy in solid tumors.     

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.     

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.     

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.     

CAR-T联合疗法治疗实体瘤的研究进展

CAR-T疗法(chimeric antigen receptor T-cell immunotherapy)即嵌合抗原受体T细胞免疫疗法,是目前肿瘤免疫治疗中较有潜力的策略之一^[1],已成功应用于多种血液肿瘤的治疗。但CAR-T疗法在治疗实体瘤领域进展缓慢,存在肿瘤微环境的限制、细胞因子释放综合征以及严重的脱靶效应等诸多挑战^[2]。与单一靶向治疗相比,CAR-T联合疗法为改进肿瘤治疗方法提供了新的方向。综述目前常用的CAR-T联合治疗策略,针对CAR-T疗法在实体瘤治疗领域面临的主要挑战,探讨提高CAR-T疗效的潜在方案。     

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.     

A review of cancer immunotherapy toxicity

Though chimeric antigen receptor T cell (CAR-T) technology has emerged as an effective breakthrough against hematological malignancies, its application to solid tumors remains challenging and is restricted by the high complicacy of the tumor microenvironment (TME). Macrophages are innate immune cells that are inherently equipped with a wide range of therapeutic effects, including elevated infiltration rate, enhanced phagocytosis and cytotoxicity, mediation of immune suppression, and antigen presentation. In light of these unique biological functions and their ability to penetrate tumors, macrophages have emerged as a promising approach for the treatment of solid tumors. This review initially clarifies the biological characteristics of macrophages and tumor-associated macrophages (TAMs), then reviews macrophage sources and the CAR design structure, outlines the ways to deliver CAR to macrophages and the preparation of CAR-macrophages (CAR-Ms), and finally summarizes the application and prospects for the treatment of solid tumors by CAR-Ms in recent years.     

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。     

CAR T targets and microenvironmental barriers of osteosarcoma

Osteosarcoma (OS) is one of the most common malignancies in children and adolescents. Multimodal chemotherapy and aggressive surgical resection have improved the prognosis of patients with osteosarcoma. However, the prognosis of OS patients with unresectable advanced tumors, distant metastasis or chemotherapy is still poor. Chimeric antigen receptor (CAR) T cells have achieved remarkable success in the treatment of hematologic malignancies, injecting new vitality into the field of adoptive cell therapy. However, the efficacy in solid tumors has been largely limited. The reason for the poor curative effect of solid tumors is mainly the heterogeneity of solid tumor antigen, immune escape, tumor microenvironment barrier, resistance of immunosuppressive cells and inhibitory factors, which lead to the obstruction of CAR T cell infiltration and the aggravation of failure. Potential antigenic targets for osteosarcoma CAR T cell therapy are under continuous exploration. Some of the antigenic targets, such as anti-HER2-CAR T cells, have achieved good results in preclinical studies, and some of them have entered clinical studies and achieved certain clinical effects. In this review, we discuss the research progress of potential antigen targets and osteosarcoma microenvironment of CAR T cells in the treatment of osteosarcoma.     

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.     

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.     

Micro-hydrogel injectables that deliver effective CAR-T immunotherapy against 3D solid tumor spheroids

Chimeric antigen receptor (CAR-) T cells are revolutionizing cancer treatment, as a direct result of their clinical impact on the treatment of hematological malignancies. However for solid tumors, CAR-T cell therapeutic efficacy remains limited, primarily due to the complex immunosuppressive tumor microenvironment, inefficient access to tumor cells and poor persistence of the killer cells. In this in vitro study, an injectable, gelatin-based micro-hydrogel system that can encapsulate and deliver effective CAR-T therapy is investigated. CAR-T cells targeting TAG-72, encapsulated in these microgels possessed high viability (> 87%) after 7 days, equivalent to those grown under normal expansion conditions, with retention of the T cell phenotype and functionality. Microgel recovered CAR-T cells demonstrated potent on-target cytotoxicity against human ovarian cancer in vitro and on three-dimensional tumor spheroids, by completely eliminating tumor cells. The gelatin-based micro-hydrogels have the potential to serve as carrier systems to augment CAR-T immunotherapeutic treatment of solid tumors.     

Novel antigens of CAR T cell therapy: New roads; old destination

Chimeric antigen receptor T cell (CAR-T) therapy has so far proved itself as a reliable therapeutic option for the treatment of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), and mantle cell lymphoma (MCL). However, this picture is not as colorful when it comes to the treatment of solid tumors mainly due to the lack of definitive tumor antigens, as well as the immunosuppressive tumor microenvironments and poor CAR-T infiltration. The recent developments in bioinformatics and cell biology, such as single-cell RNA sequencing, have offered silver linings in the subject of tumor antigen discovery. In the current review, we summarize the development of some CAR-T therapies that target novel tumor antigens, rather than the traditionally CAR-T-targeted ones, and briefly discuss the clinical antitumor achievements of those evaluated in patients, so far. Furthermore, we propose some tumor antigens that might someday be therapeutically beneficial while targeted by CAR-Ts based on the experimental evaluations of their specific monoclonal antibodies.     

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细胞介绍及抗肿瘤临床应用

过继性细胞免疫治疗（adoptive cellular immunotherapy,ACI）是目前较为有效的恶性肿瘤的治疗方法之一。随着技术的日趋成熟,已在多种实体瘤和血液肿瘤的t临床治疗中取得较好疗效。其中,嵌合抗原受体（chimeric antigen receptor,CAR）T细胞技术是近年来发展非常迅速的一种细胞治疗技术。通过基因改造技术,效应T细胞的靶向性、杀伤活性和持久性均较常规应用的免疫细胞高,并可克服肿瘤局部免疫抑制微环境和打破宿主免疫耐受状态。目前,CAR的信号域已从第一代的单一信号分子发展为包含CD28、4—1BB等共刺激分子的多信号结构域（第二、三代）,临床应用广泛。但是,该技术也存在脱靶效应、插入突变等临床应用风险。该文将就CAR—T细胞技术在恶性肿瘤免疫治疗中的应用及可能存在的问题作一综述。