靶向肝癌的GPC3-CAR-T细胞的构建与功能鉴定

目的:构建靶向GPC3阳性肝癌细胞的嵌合抗原受体修饰T细胞[chimeric antigen receptor-modified T cell,CAR-T cell],并评估其对GPC3阳性肝癌细胞的杀伤效能.方法:选择偏爱密码子技术改造基因序列,以增强CAR分子表达效率,设计靶向GPC3抗原的CAR分子基因并构建携带GPC3-CAR基因的慢病毒表达载体pCDH-GPC3-CAR,感染T细胞;采用Western blotting、流式细胞术、实时细胞监测和ELISA技术分别检测GPC3-CAR分子在CAR-T细胞的表达、慢病毒感染T细胞效率、GPC3-CAR-T细胞对GPC3阳性肝癌细胞的杀伤活性和特异性.结果:双酶切pCDH-GPC3-CAR重组慢病毒质粒可见分子质量和GPC3-CAR分子一致的基因片段,成功构建pCDH-GPC3-CAR慢病毒质粒.约54.38％的GPC3-T细胞表达GPC3-CAR分子,称为GPC3-CAR-T细胞.GPC3-CAR-T细胞对GPC3阳性的肝癌细胞Huh-7比GPC3阴性的肝癌细胞SK-HEP-1具有更高效的杀伤能力[(78.96±4.76)％ vs (6.87±3.15)％,P＜0.01].与GPC3阳性Huh-7肝癌细胞共培养的肝癌GPC3-CAR-T细胞分泌IFN-γ水平比Mock组高效[(21 371.4±1 808.3) vs (152.8±12.5) pg/ml,P＜0.01],这可能是其高效杀伤肝癌细胞的机制之一.结论:靶向肝癌的GPC3-CAR-T细胞具有高效分泌IFN-γ细胞因子并特异高效杀伤GPC3阳性肝癌细胞的能力,为进一步推进GPC3-CAR-T临床前和临床研究奠定基础.     

MEK抑制剂显著增强EGFR CAR-T细胞对口腔鳞癌的抗肿瘤活性

目的:构建靶向表皮生长因子受体（epidermal growth factor receptor,EGFR）的二代嵌合抗原受体（chimeric antigen receptor,CAR）-T细胞,以探索MEK抑制剂联合EGFR CAR-T细胞是否可以对口腔鳞癌产生显著的抗肿瘤活性。方法:免疫组化法检测口腔鳞癌患者癌和癌旁组织中EGFR的表达;流式细胞术检测口腔鳞癌细胞系中EGFR的表达;慢病毒感染法构建EGFR CAR-T细胞;生物发光法检测EGFR CAR-T细胞联合MEKi对靶细胞的杀伤活性;流式细胞术检测共孵育后效靶细胞的占比情况;活体成像监测小鼠肿瘤的生长;免疫组化法检测小鼠肿瘤组织中CD3的表达;ELISA法检测肿瘤组织中IFN-γ和GrmB的含量。结果:人口腔鳞癌组织较癌旁组织EGFR显著高表达,大部分口腔鳞癌细胞系高表达EGFR。联合MEKi在短时间的共孵育中并不能显著增强EGFR CAR-T细胞对肿瘤细胞系的杀伤活性,但在较长时间的共孵育后,联合MEKi显著增强EGFR CAR-T细胞的增殖（P＜0.01）和对肿瘤细胞的细胞毒性（P＜0.001）。体内实验也表明,联合MEKi较单独使用EGFR CAR-T细胞而言对肿瘤抑制能力更强（P＜0.01）,肿瘤组织中CD3+T细胞的浸润更多（P＜0.001）,肿瘤部位细胞因子IFN-γ（P＜0.001）和GrmB（P＜0.001）的含量也更高。结论:联合MEKi可以在体内外条件下显著增强EGFR CAR-T细胞的抗肿瘤活性,有望成为针对口腔鳞癌的潜在治疗策略。     

Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.     

Anti-tumor efficacy of human anti-c-met CAR-T cells against papillary renal cell carcinoma in an orthotopic model

Chimeric antigen receptor (CAR)-T cell therapy has shown salient efficacy in cancer immunotherapy, particularly in the treatment of B cell malignancies. However, the efficacy of CAR-T for solid tumors remains inadequate. In this study, we displayed that c-met is an appropriate therapeutic target for papillary renal cell carcinoma (PRCC) using clinical samples, developed an anti-human c-met CAR-T cells, and investigated the anti-tumor efficacy of the CAR-T cells using an orthotopic mouse model as pre-clinical research. Administration of the anti-c-met CAR-T cells induced marked infiltration of the CAR-T cells into the tumor tissue and unambiguous suppression of tumor growth. Furthermore, in combination with axitinib, the anti-tumor efficacy of the CAR-T cells was synergistically augmented. Taken together, our current study demonstrated the potential for clinical application of anti-c-met CAR-T cells in the treatment of patients with PRCC.     

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.     

shRNA靶向抑制CD38的抗CD38 CAR-T细胞构建及其功能的初步鉴定

目的：探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法：构建shRNA靶向抑制CD38的抗CD38 CAR-T细胞的CAR分子,利用逆转录病毒载体包装成功后转导人原代T细胞,制备CAR-T细胞。实验分为shRNA1 CD38 CAR-T组、shRNA2 CD38 CAR-T组和对照组（shR-NC-CD38 CAR-T细胞）。采用qPCR法检测CAR-T细胞CD38 mRNA相对表达水平,计算CAR-T细胞培养0～14 d的增殖倍数,CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B淋巴细胞RPMI-8226-luc共培养时的增殖情况,荧光素酶化学发光法检测CAR-T细胞在不同效靶比（1∶1、1∶2、1∶4、1∶8）时对Raji-luc和RPMI-8226-luc细胞的杀伤效率,ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平,FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果：shR-NC-CD38 CAR、shRNA1-...     

Combination therapy of DKK1 inhibition and NKG2D chimeric antigen receptor T cells for the treatment of gastric cancer

Despite the successful application of chimeric antigen receptor (CAR)-T cell therapy in hematological malignancies, the treatment efficacy in solid tumors remains unsatisfactory, largely due to the highly immunosuppressive tumor microenvironment and low density of specific tumor antigens. Natural killer group 2 member D (NKG2D) CAR-T cells have shown promising treatment effects on several cancers such as lymphoma and multiple myeloma. However, the application and efficacy of NKG2D-CAR-T cells in gastric cancer (GC) still needs further exploration. This study identified a novel combination immunotherapy strategy with Dickkopf-1 (DKK1) inhibition and NKG2D-CAR-T cells, exerting synergistic and superior antitumor effect in GC. We show that the baseline expression of NKG2D ligands (NKG2DLs) is at low levels in GC tissues from The Cancer Genome Atlas and multiple GC cell lines including NCI-N87, MGC803, HGC27, MKN45, SGC7901, NUGC4, and AGS. In addition, DKK1 inhibition by WAY-262611 reverses the suppressive tumor immune microenvironment (TIME) and upregulates NKG2DL expression levels in both GC cell lines and GC tissues from a xenograft NCG mouse model. DKK1 inhibition in GC cells markedly improves the immune-activating and tumor-killing ability of NKG2D-CAR-T cells as shown by cytotoxicity assays in vitro. Moreover, the combination therapy of NKG2D-CAR-T and WAY-262611 triggers superior antitumor effects in vivo in a xenograft NCG mouse model. In sum, our study reveals the role of DKK1 in remodeling GC TIME and regulating the expression levels of NKG2DLs in GC. We also provide a promising treatment strategy of combining DKK1 inhibition with NKG2D-CAR-T cell therapy, which could bring new breakthroughs for GC immunotherapy.     

CAR －T 细胞治疗在血液系统恶性肿瘤中的研究进展

嵌合抗原受体T（ CAR－T）细胞是近年来迅速发展的肿瘤过继性免疫治疗新手段。其主要特点是通过基因工程改造获得识别肿瘤抗原特异性受体的T细胞并赋予其靶向性、杀伤性及持久性的治疗方法。1989年首次提出CAR－T,现已经发展到第四代,且多研究中心对CD19－CAR－T细胞治疗技术进行了Ⅰ期临床研究显示出活性。 CAR－T疗法是目前有望治愈复发难治性血液肿瘤的新方法。     

CAR-T细胞治疗实体肿瘤的新策略

[摘要] 嵌合型抗原受体修饰T(chimeric antigen receptor modified T,CAR-T)细胞治疗作为一种新的过继性免疫疗法在血液肿瘤中已取得了较好的疗效。在实体肿瘤中,肿瘤微环境中存在大量免疫抑制细胞、免疫抑制分子以及胞外基质,对迁移的CAR-T细胞的细胞毒性效应产生抑制作用,在实体肿瘤内严重抑制CAR-T细胞的抗肿瘤效力。同时,大多数实体肿瘤存在肿瘤异质性且缺乏相对特异性肿瘤抗原,CAR-T细胞在实体肿瘤部位的归巢能力差并伴随着脱靶效应,使其在实体瘤中的疗效欠佳。与血液肿瘤相比,实体瘤具有复杂的生物学特性,需要针对性的策略才能保证CAR-T细胞抗肿瘤长期有效。本文就CAR-T 细胞的发展、在实体瘤治疗中的困惑及治疗策略的研究进展作一阐述。     

Chimeric antigen receptor T cells derived from CD7 nanobody exhibit robust antitumor potential against CD7-positive malignancies

The great success of chimeric antigen receptor T (CAR-T)-cell therapy in B-cell malignancies has significantly promoted its rapid expansion to other targets and indications, including T-cell malignancies and acute myeloid leukemia. However, owing to the life-threatening T-cell hypoplasia caused by CD7-CAR-T cells specific cytotoxic against normal T cells, as well as CAR-T cell-fratricide caused by the shared CD7 antigen on the T-cell surface, the clinical application of CD7 as a potential target for CD7⁺ malignancies is lagging. Here, we generated ^CD7ΔT cells using an anti-CD7 nanobody fragment coupled with an endoplasmic reticulum/Golgi retention domain and demonstrated that these cells transduced with CD7-CAR could prevent fratricide and achieve expansion. Additionally, ^CD7ΔCD7-CAR-T cells exhibited robust antitumor potiential against CD7⁺ tumors in vitro as well as in cell-line and patient-derived xenograft models of CD7-positive malignancies. Furthermore, we confirmed that the antitumor activity of CD7-CAR-T cells was positively correlated with the antigen density of tumor cells. This strategy adapts well with current clinical-grade CAR-T-cell manufacturing processes and can be rapidly applied for the therapy of patients with CD7⁺ malignancies.     

CAR-T细胞治疗的神经毒性副反应处理新策略

[摘要] 嵌合型抗原受体修饰T (chimeric antigen receptor modified T,CAR-T)细胞疗法是肿瘤免疫治疗的重要手段之一,CAR-T细胞的靶向性、杀伤活性、增殖性和持久性较常规T细胞明显提高,并且经过不断的改进演变,其在血液系统肿瘤治疗中取得了巨大的成效,受到广泛的关注。然而,其治疗过程中出现的神经毒性,也称CAR-T细胞相关脑病综合征(CAR-T cell relevant encephalopathy syndrome,CRES),影响了CAR-T疗法的临床应用。探索CRES的发病机制及其高风险因素、寻找相应的处理策略,对预防和治疗CRES具有重要意义。本文以CD19-CAR-T 细胞治疗为例,就CRES的发病症状、发病机制、高风险因素及应对策略作一综述,为临床治疗提供参考。     

CAR-T细胞治疗多发性骨髓瘤的研究进展

多发性骨髓瘤（MM）是一种源于骨髓浆细胞的恶性肿瘤,截止目前尚无有效的治愈手段。嵌合抗原受体(CAR)-T细胞疗法是一种具有广阔研究前景的MM治疗方法。其设计理念是通过对部分基因序列进行特异性的改造,使CAR特异性地影响靶向肿瘤细胞的抗原,受体与抗原相结合以激活T细胞杀死肿瘤细胞。本文主要阐述CAR-T细胞疗法的设计过程、研究进展及现阶段所面临的缺陷。     

CD19-CAR-T细胞治疗难治复发急性淋巴细胞白血病的研究进展

难治复发急性淋巴细胞白血病（acute lymphocytic leukemia, ALL）预后差,生存期短,其治疗已成为国际难题。嵌合抗原受体基因修饰T（chimeric antigen receptor gene-modified T,CAR-T）细胞是目前最具应用前景的靶向免疫治疗,靶向CD19的CAR-T细胞（CD19-CAR-T）治疗儿童及成人难治复发性ALL的完全缓解率（complete remission,CR）可达90%以上,疗效远高于化疗。然而,细胞因子释放综合征（cytokine release syndrome,CRS）、严重神经毒性（serious neurotoxicity,SNT）、脱靶效应以及疾病复发等严重限制了CAR-T细胞的进一步临床应用。本文主要阐述CAR-T细胞的制备技术、预处理方案、细胞输注剂量及各种并发症防治策略等最新研究进展。     

Study on the Prognostic Value of Aberrant Antigen in Patients With Acute B Lymphocytic Leukemia

BackgroundApproximately 30% to 60% of patients with acute B-lymphocytic leukemia (B-ALL) show as refractory or relapsed, which is one of the major causes of death in patients with B-ALL, but the methods of the treatment for relapsed/refractory B-ALL (R/R B-ALL) are limited. The chimeric antigen receptors redirected T cells (CAR-T cells) have showed a strong anti-leukemia role for B-ALL. About 90% of patients with R/R B-ALL treated with CD19-CAR-T cells achieved complete remission. However, 60% to 70% of patients relapsed after CAR-T cells treatment, which may be related to target antigen reduction or escape. New products are urgently needed to prevent and treat antigenic escapes causing recurrence.Patients and MethodsIn this article, we retrospectively analyzed the immunophenotype of patients with B-ALL initially diagnosed in our center from January 2010 to December 2015 to determine whether aberrant antigen expression was associated with the prognosis of patients in order to find new targets for immunotherapy.ResultsThe results show that disease-free and overall survival in patients without aberrant antigen expression were better than patients with aberrant antigen expression. The most common abnormal antigens were CD123, CD13, and CD56. Correlation analysis showed a negative correlation between aberrant CD123 expression and both disease-free and overall survival.ConclusionTherefore, in the construction of CAR-T cells in patients with R/R B-ALL, conventional CD19 can be combined with aberrant antigens such as CD123 to form CARs with bi-specific antigens or multi-specific antigens may achieve the purpose of improving efficacy. However, more clinical trials are needed.     

Disialoganglioside directed immunotherapy of neuroblastoma

Achieving a cure for metastatic neuroblastoma remains a challenge despite sensitivity to chemotherapy and radiotherapy. Most patients achieve remission, but a failure to eliminate minimal residual disease (MRD) often leads to relapse. Immunotherapy is potentially useful for chemotherapy-resistant disease and may be particularly effective for low levels of MRD that are below the threshold for detection by routine radiological and histological methods. Disialoganglioside (GD2), a surface glycolipid antigen that is ubiquitous and abundant on neuroblastoma cells is an ideal target for immunotherapy. Anti-GD2 monoclonal antibodies currently form the mainstay of neuroblastoma immunotherapy and their safety profile has been well-established. Although responses in patients with gross disease have been observed infrequently, histologic responses of bone marrow disease are consistently achieved in >75 percent of patients with primary refractory neuroblastoma. The advent of highly sensitive and specific molecular assays to measure MRD has confirmed the efficacy anti-GD2 antibody immunotherapy in patients with subclinical disease. Such markers will allow further optimization of other anti-MRD therapies. We review the current status of anti-GD2 clinical trials for neuroblastoma and novel preclinical GD2-targeted strategies for this rare but often lethal childhood cancer.     

Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors

Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.     

多靶点CAR-T细胞治疗肿瘤的研究进展

嵌合抗原受体T细胞（CAR-T）是一种过继细胞免疫疗法,其通过基因手段改造T细胞,从而发挥肿瘤靶向杀伤作用。CAR-T细胞疗法在治疗肿瘤,特别是血液系统恶性肿瘤方面具有显著的作用,但在实体肿瘤中作用不佳。单靶点的CAR-T在应用的过程中容易出现脱靶效应,存在复发或更加难治的风险。双靶点或多靶点CAR-T的开发有望扩大靶细胞抗原覆盖率,有效避免抗原逃逸和防止肿瘤复发,延长患者的生存时间。本文综述了目前多靶点嵌合抗原受体T细胞的研究进展,并探讨了其发展的前景。     

Preclinical development of CD126 CAR-T cells with broad antitumor activity

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive partly due to the restricted use of each CAR construct for specific tumors. Thus, a CAR construct with broad antitumor activity can be advantageous. We identified that CD126 is expressed by many hematologic and solid tumors, including multiple myeloma, lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer, and malignant melanoma among others. CAR-T cells targeting CD126 were generated and shown to kill many tumor cells in an antigen-specific manner and with efficiency directly proportional to CD126 expression. Soluble CD126 did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126 but caused no weight loss or hepatotoxicity in mice. In multiple myeloma and prostate adenocarcinoma xenograft models, intravenously injected CD126 CAR-T cells infiltrated within, expanded, and killed tumor cells without toxicity. Binding of soluble interleukin-6 receptor (sIL-6R) by CAR-T cells could mitigate cytokine release syndrome. Murine SAA-3 levels were lower in mice injected with CD126 CAR-T compared to controls, suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic target for CAR-T cells for many tumors with a low risk of toxicity.Subject terms: Translational research, Immunotherapy     

Immunotherapy for lung cancer: Focusing on chimeric antigen receptor (CAR)-T cell therapy

Besides traditional treatment strategies, including surgery, radiotherapy, and chemotherapy for lung cancer as the leading cause of cancer incidence and death, immunotherapy has also emerged as a new treatment strategy. The goal of immunotherapy is to stimulate the immune system responses against cancer, using various approaches such as therapeutic vaccines, monoclonal antibodies, immune checkpoint inhibitors, and T-cell therapy. Chimeric antigen receptor (CAR)-T cells, one of the most popular cancer immunotherapy approaches in the last decade, are genetically engineered T-cells to redirect patients' immune responses to recognize and eliminate tumor-associated antigens (TAA)-expressing tumor cells. CAR-T cell therapy provides promising benefits in lung tumors. In this review, we summarize different immunotherapy approaches for lung cancer, the structure of CAR-T cells, currently undergoing CARs in clinical trials, and various TAAs are being investigated as potential targets in designing CAR-T cells for lung cancer.     

CAR-NK抗肿瘤研究的现状与发展趋势

作为固有免疫中重要的效应细胞,NK细胞具有强大的抗肿瘤功能,在肿瘤免疫治疗方面具有良好的应用前景.随着嵌合抗原受体(chimeric antigen receptor,CAR)修饰的T细胞(CAR-T)技术的兴起,NK细胞的CAR修饰潜力也受到了关注.CAR-NK的研发既传承了经典CAR-T研发的思路,也实现了诸多基于NK细胞生物特点的创新和发展.外周血NK细胞、NK细胞系和干细胞来源的NK细胞各具生物学特色,在CAR-NK研发中被广泛使用.已有靶向多种血液肿瘤和实体瘤抗原的CAR-NK在体外实验和动物模型中取得显著效果.虽然在临床暂未有数据支持CAR-NK的疗效且仍存在一些瓶颈问题需要攻克,但是CAR-NK有望在治疗实体瘤、简化免疫治疗等方面实现新的突破.