CAR-NK的构建策略及在血液系统肿瘤中的研究进展北大核心

自然杀伤(natural killer cells,NK)细胞是先天免疫系统不可或缺的一部分,为抵御异常细胞攻击人体提供了第一道屏障。嵌合抗原受体T细胞(CAR-T)工程是近年来癌症免疫治疗领域的研讨热点,它改变了血液肿瘤的传统治疗方法,这种新颖的治疗方式已经在血液肿瘤中显示出了令人振奋的结果。然而,CAR-T的临床应用受到了高昂的制造成本和各种副作用的限制。NK细胞由于具有更好的安全性、“现成”生产的可行性和强大的细胞毒性激发了人们开发用于癌症治疗的CAR工程NK细胞(CAR-NK)的兴趣,CAR-NK有望取代CAR-T成为更有前景的抗癌方法。本文中我们描述了当前CAR-NK的构建策略和在血液系统肿瘤中的研究进展。     

CAR-NK在实体瘤治疗中的研究进展

瘤免疫细胞治疗近年来备受瞩目。嵌合抗原受体T(chimeric antigen receptor engineered T-cell,CAR-T)细胞用于治疗血液系统恶性肿瘤已经颇见成效,但在实体瘤治疗方面却存在不足。由于具有不同于T细胞的非特异性杀伤肿瘤细胞的能力,CAR修饰的自然杀伤(nature killer,NK)细胞逐渐成为研究热点。目前,广泛的临床前研究证明了CAR-NK细胞疗法的安全性和有效性,尤其在实体瘤治疗方面,展现出了优于CAR-T细胞的应用前景。然而,CAR-NK细胞疗法仍存在一些局限性,需要进一步研究以期实现其临床转化。     

Chimeric Antigen Receptor-Natural Killer Cells: A New Breakthrough in the Treatment of Solid Tumours

Natural killer (NK) cells can quickly and directly eradicate tumour cells without recognising tumour-specific antigens. NK cells also participate in immune surveillance, which arouses great interest in the development of novel cancer therapies. The chimeric antigen receptor (CAR) family is composed of receptor proteins that give immune cells extra capabilities to target specific antigen proteins or enhance their killing effects. CAR-T cell therapy has achieved initial success in haematological tumours, but is prone to adverse reactions, especially with cytokine release syndrome in clinical applications. Currently, CAR-NK cell therapy has been shown to successfully kill haematological tumour cells with allogeneic NK cells in clinical trials without adverse reactions, proving its potential to become an off-the-shelf product with broad clinical application prospects. Meanwhile, clinical trials of CAR-NK cells for solid tumours are currently underway. Here we will focus on the latest advances in CAR-NK cells, including preclinical and clinical trials in solid tumours, the advantages and challenges of CAR-NK cell therapy and new strategies to improve the safety and efficacy of CAR-NK cell therapy.     

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma

With the advance of genome engineering technology, chimeric antigen receptors (CARs)-based immunotherapy has become an emerging therapeutic strategy for tumors. Although initially designed for T cells in tumor immunotherapy, CARs have been exploited to modify the function of natural killer (NK) cells against a variety of tumors, including hepatocellular carcinoma (HCC). CAR-NK cells have the potential to sufficiently kill tumor antigen-expressing HCC cells, independent of major histocompatibility complex matching or prior priming. In this review, we summarize the recent advances in genetic engineering of CAR-NK cells against HCC and discuss the current challenges and prospects of CAR-NK cells as a revolutionary cellular immunotherapy against HCC.     

Targeted elimination of prostate cancer by genetically directed human T lymphocytes

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.     

人源IL-15转基因NCG小鼠在CAR-NK细胞肿瘤治疗临床前评价中的应用

目的:探讨人源IL-15转基因NCG小鼠(NCG-hIL-15鼠)在CAR-NK细胞肿瘤治疗临床前评价中的作用.方法:通过qPCR结合WB法检测NCG-hIL-15鼠骨髓及主要器官(脾、肝、肺、肾和胰)中的表达丰度.将人PBMC来源的NK(PB-NK)细胞回输到NCG-hIL-15鼠及对照NCG鼠,监测NK细胞的体内扩...     

T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice

Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.     

Bee venom in cancer therapy

Bee venom (BV) (api-toxin) has been widely used in the treatment of some immune-related diseases, as well as in recent times in treatment of tumors. Several cancer cells, including renal, lung, liver, prostate, bladder, and mammary cancer cells as well as leukemia cells, can be targets of bee venom peptides such as melittin and phospholipase A2. The cell cytotoxic effects through the activation of PLA2 by melittin have been suggested to be the critical mechanism for the anti-cancer activity of BV. The induction of apoptotic cell death through several cancer cell death mechanisms, including the activation of caspase and matrix metalloproteinases, is important for the melittin-induced anti-cancer effects. The conjugation of cell lytic peptide (melittin) with hormone receptors and gene therapy carrying melittin can be useful as a novel targeted therapy for some types of cancer, such as prostate and breast cancer. This review summarizes the current knowledge regarding potential of bee venom and its compounds such as melittin to induce cytotoxic, antitumor, immunomodulatory, and apoptotic effects in different tumor cells in vivo or in vitro. The recent applications of melittin in various cancers and a molecular explanation for the antiproliferative properties of bee venom are discussed.     

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.     

Thomsen-Friedenreich (T) antigen expression increases sensitivity of natural killer cell lysis of cancer cells

In this study, we demonstrate a correlation between T antigen expression on a panel of human carcinoma cell lines and their sensitivity to porcine NK cell lysis. Specifically, the more T antigen is expressed, the more sensitive the cancer cells are to porcine NK cell lysis. Furthermore, this correlation also exists for these cells and their ability to induce tumors in vivo. In this porcine animal model, the less T antigen is expressed, the more prolific the tumor growth in vivo and vice versa. Using the human colorectal adenocarcinoma cell line SW-48, we used limiting dilution to clone 2 populations of cells, one expressing high and the other low levels of T antigen, clones 143 and 111, respectively. In these cloned cells, the clone that expressed more T antigen was more NK-sensitive in vitro and weakly induced tumor growth in vivo. Inversely, the clone that expressed less T antigen clone was more NK-resistant in vitro and grew more prolific tumors in vivo. Using soluble T antigen in a competitive inhibition assay, there was a decrease in porcine NK cell killing of the T antigen+ human cell line Colo 320HSR. Taken together, these findings suggest a novel role for T antigen in the NK cell recognition of cancer cells, specifically as markers for NK sensitivity in carcinoma cell lines. The significance of T antigens as targets for NK cell-mediated lysis is novel and identifies NK cell-T antigen interactions as potentially significant in the immunotherapy of cancer and its associated metastases.     

Natural T cell immunity against cancer.

It has long been a matter of debate whether tumors are spontaneously immunogenic in patients. With the availability of sensitive methods, naturally occurring T cells directed against tumor-associated antigens (TAAs) can be frequently detected in cancer patients. In this review, we summarize the current data on T cell responses to TAAs in various malignancies, including melanoma, colorectal cancer, leukemia, and breast cancer. T cell responses against various antigens, including melanoma differentiation antigens, carcinoembryonic antigen, epithelial cell adhesion molecule, her-2/neu, Wilms' tumor protein, proteinase 3, NY-ESO-1, and surviving, have been reported in a substantial number of patients. In contrast, other TAAs, including most antigens of the MAGE family, do not usually elicit spontaneous T cell responses. A distinction between direct ex vivo T cell responses and in vitro-generated T cell responses is provided because in vitro stimulation results in quantitative and functional changes of T cell responses. The possible role of TAA-specific T cells in immunosurveillance and tumor escape and the implications for immunological treatment strategies are discussed. Naturally occurring T cells against TAAs are a common phenomenon in tumor patients. Understanding the mechanisms and behavior of natural TAA-specific T cells could provide crucial information for rational development of more efficient T cell-directed immunotherapy.     

Strategies for antigen loading of dendritic cells to enhance the antitumor immune response

Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze thaw lysis for DC priming. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-gamma release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-gamma production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in two of three cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in two analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge. These data demonstrate that the method of tumor cell preparation clearly influences the ability of DCs to present antigen to T cells. Correlation of in vitro data with the generation of protective immunity in vivo suggests the utility of irradiated tumor-primed DCs as a means to generate protective immunity in patients with solid malignancies.     

Upregulation of DF3, in association with ICAM-1 and MHC class II by IFN-gamma in short-term human mammary carcinoma cell cultures

This study was designed to determine whether in vitro exposure of isolated short-term human primary and metastatic breast tumor cell cultures to interferon-gamma (IFN-gamma) could enhance expression of the breast tumor associated DF3 antigen in association with the intercellular adhesion molecule 1 (ICAM-1) and MHC class II molecules. Cell cultures were established from primary solid tumors and metastatic cells as previously described (Sgagias et al., 1995). Data show that recombinant human IFN-gamma treatment, in vitro, dramatically increased the breast tumor associated DF3 antigen, in association with ICAM-1, and MHC class II antigens in primary breast cancer cell cultures. All primary breast tumor cell cultures constitutively expressed high levels of HLA-class I antigen. Metastatic breast cancer cell cultures expressed high levels of DF3 and recombinant human IFN-gamma treatment, in vitro, upregulated ICAM-1 and MHC class II antigens before and after passage of the metastatic cells through the nude mouse. Metastatic breast cancer cells similar to primary breast cancer cells constitutively expressed high levels of MHC class I antigens. In addition, three LAK cell lines significantly lysed the primary and the metastatic breast tumor cell cultures to the same degree before and after passage of the metastatic cancer cells through the nude mouse. These data indicate the upregulation of the breast tumor associated DF3 antigen in vitro after IFN-gamma treatment and its persistence in vivo, after passage of the metastatic breast cancer cells through the nude mouse. The ability of IFN-gamma to upregulate the breast tumor associated DF3 antigen in association with the ICAM-1 and HLA class II antigens may play an important role in eliciting an immune response which may contribute to the immunodiagnosis, and immunotherapy of breast cancer.     

The role of monoclonal antibodies to the epithelial membrane antigen in the diagnosis of human tumors]

The paper discusses the possibility of the use of the Soviet-made ICO 25 monoclonal antibodies to membrane antigen of lipid globules of the human milk for differential diagnosis of human tumors. ICO 25 monoclonal antibodies reliably detected the above antigens in normal epithelial and breast cancer cells. However, these antigens cannot be considered strictly specific for breast tissue. They were found in various human epithelial tissues, in the majority of epithelial tumors and lymph node metastases. Staining for ICO 25 monoclonal antibodies was negative in non-epithelial tumors. The above antibodies proved a useful marker for the identification of epithelial origin of primary tumors and their metastases showing unclear histology. They can be used to differentiate between epithelial and non-epithelial malignancies as well as to detect micrometastases and areas of microinvasion. Paraffin-embedded samples can be used for immunohistochemical examination.     

Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach?

Intracellular signals, delivered in professional antigen-presenting cells following the engagement of major histocompatibility complex (MHC) class II molecules, activate a variety of cellular functions that also contribute to efficient antigen presentation. As far as human malignancies, the signaling ability of human leukocyte antigens (HLA) class II molecules is a rather well-characterized event in hematologic tumors; in contrast, very limited evidences are available in solid neoplasias of different histotypes that may constitutively express HLA class II antigens. Among solid malignancies, a significant proportion of human cutaneous melanomas have been shown to express HLA class II molecules, and cutaneous melanoma undoubtedly represents a 'model disease' to investigate tumor immunobiology, to unveil the molecular basis underlying the interactions between neoplastic cells and host's immune system, and ultimately to set up new bio-immunotherapeutic approaches. Upcoming preclinical evidences unveil a signaling potential of HLA-DR antigens expressed on melanoma cells, and suggest for the clinical implication of HLA class II molecules as novel therapeutic targets. Therefore, in this review, we will focus on the emerging role of HLA class II antigens as intracellular signal transducing elements in neoplastic cells of the melanocytic lineage, emphasizing their foreseeable role in targeted therapy of human melanoma and potentially of HLA class II antigens-positive tumors of different histology.     

Microbial-based therapy of cancer: a new twist to age old practice

The use of bacteria in the regression of tumors has long been known. Various approaches for using bacteria in cancer therapy include the use of bacteria as sensitizing agents for chemotherapy, as delivery agents for cancer drugs and as agents for gene therapy. The tumor regression stimulated by infecting microorganisms has been attributed to activation of the immune system of the host. However, recent studies indicate that when tumor-harboring mice with defective immune systems are infected with certain microorganisms, the regression of the tumor is still observed, suggesting that there are other host factors contributing to the microbial associated regression of tumors. Since the use of live or attenuated bacteria for tumor regression has associated toxic effects, studies are in progress to identify a pure microbial metabolite or any component of the microbial cell that might have anti-cancer activity. It has now been demonstrated that a redox protein from Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and trigger the apoptotic cell death. In vivo, this cupredoxin can lead to the regression of tumor growth in immunodeficient mice harboring xenografted melanomas and breast cancer tumors without inducing significant toxic effects, suggesting that it has potential anti-cancer activity. This bacterial protein interacts with p53 and modulates mammalian cellular activity. Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancer treatments. Here, we focus on diverse approaches to cancer biotherapy, including bacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.     

NK细胞肿瘤免疫治疗技术的挑战

自然杀伤(NK)细胞具有以MHC I 非依赖识别机制和快速杀伤病变细胞能力、低移植物抗宿主反应(GVHD)风险、可采用异体细胞回输、体内存活周期短和无细胞因子风暴等长期和不可预期风险较低等特点和优势,使其在肿瘤免疫治疗中展现出巨大的应用潜力。虽然外周血单个核细胞（PBMC）来源NK细胞相比干细胞来源NK和NK细胞系在安全性和肿瘤杀伤能力上相对更好,但细胞制备技术的效率、稳定性和安全性仍有待完善;NK细胞被认为是较理想的嵌合抗原受体（chimeric antigen receptor,CAR）载体,但外周血来源NK细胞转染效率较低,影响了CAR-NK的研发进程。由于NK细胞来源和培养技术的多样性,使细胞制品的活性不一,虽然NK细胞在抗血液肿瘤治疗中表现相对突出,但对实体瘤的治疗效果仍有待验证。总之,NK细胞应用开发近年已取得显著进步,但仍面临生产技术和临床疗效等诸多挑战。     

Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival

A wide variety of human carcinomas have low expression of tumor-associated antigen presentation in the context of MHC class I antigens due to defects in the antigen presentation pathway. This immune evasion mechanism renders many tumors unrecognizable by host immune surveillance mechanisms. The present study examines the expression of HLA, tapasin, transporter associated with antigen processing 1 (TAP1), and beta2 microglobulin in human small cell lung carcinoma and non-small cell lung carcinoma. Immunohistochemical staining showed severe impairment of the antigen presentation pathway in all patients. In order to recover tumor immunogenicity, a nonreplicating adenovirus expressing human TAP1 (AdhTAP1) was used to restore the expression of TAP1 in the antigen presentation pathway-deficient mouse lung carcinoma cell line, CMT.64. Infection of CMT.64 cells with AdhTAP1 increased MHC class I antigen surface expression, antigen presentation, and susceptibility to antigen-specific CTLs. Fluorescence-activated cell sorting and ELISPOT analysis showed that AdhTAP1 treatment significantly increased dendritic cell cross-presentation and cross-priming of tumor antigens. Furthermore, ex vivo and in vivo AdhTAP1 treatment significantly retarded tumor growth and increased survival of mice bearing CMT.64 tumors. Fluorescence-activated cell sorting analysis and immunohistochemical staining showed a significant increase in CD8+ and CD4+ T cells and CD11c+ dendritic cells infiltrating the tumors. The results show that TAP should be considered as a part of the immunotherapies for various cancers because it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic composition of the tumor or the MHC haplotypes of the host.     

T-Cell Receptor-Engineered Cells for the Treatment of Hematologic Malignancies

Recent attention in adoptive immunotherapy for hematologic malignancies has focused on lymphocytes expressing chimeric antigen receptors. An alternative technique to redirect the immune system toward cancer cells involves the use of T-cells carrying an engineered tumor-recognizing T-cell receptor (TCR). This approach allows targeting of surface or intracellular/nuclear proteins as long as they are processed and presented on the cell surface by human leukocyte antigen molecules. Several trials in advanced solid tumors, particularly melanoma and synovial sarcoma, support the validity of this strategy, although tumor responses have often been short-lived. Emerging data from patients with multiple myeloma and myeloid neoplasms suggest that the benefit of TCR-modified cells may extend to blood cancers. Methodological refinements may be necessary to increase the in vivo persistence and functionality of these cells. Particularly with affinity-enhanced TCRs, however, more effective therapies may increase the potential for serious toxicity due to the unexpected on- or off-target reactivity.