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.

Human CD4- CD8- Invariant Natural Killer T Cells Promote IgG Secretion from B Cells Stimulated by Cross-Linking of Their Antigen Receptors

Immunoglobulin (Ig) M production can be induced by the interaction of thymus-independent type-2 (TI-2) antigen (Ag) with B cell Ag receptors (BCRs) without the involvement of conventional T cells;for IgG production through the same process, however, a second signal is required. Previous studies have reported that invariant natural killer T (iNKT) cells may be responsible for the second signal involved in IgG production. In the present study, we addressed whether human iNKT cells could participate in the production of Ig against TI-2 Ag in vitro. Two major distinct subsets of human iNKT cells, CD4+ CD8β- (CD4) and CD4- CD8β- [double negative (DN)] cells, were generated from peripheral blood monocytes from a healthy volunteer. BCR engagement, triggered by anti-IgM antibody stimulation, examined here as a model of BCR engagement triggered by TI-2 Ag, induced abundant IgM production by B cells. Both CD4 and DN iNKT cells reduced IgM production and conversely enhanced IgG production in a dose-dependent manner. In addition, IgG production by CD19+CD27- (naïve) and CD19+CD27+ (memory) B cells was predominantly promoted by DNiNKT cells rather than CD4 iNKT cells;nevertheless, IgM production by both B cell subsets was similarly reduced by either subset of iNKT cells. These results suggest that the DN iNKT subsets may preferentially promote Ig class switching by B cells upon stimulation with TI-2 Ag.

HBsAg stimulates NKG2D receptor expression on natural killer cellsand inhibits hepatitis C virus replication

Background： Higher hepatitis B surface antigen （HBsAg） facilitates hepatitis C virus （HCV） clearance inpatients with hepatitis B virus （HBV）/HCV co-infection. We investigated the effect of exogenous HBsAgon the inhibition of HCV replication mediated by natural killer （NK） cells.

Targeted Therapy of CEA-CAR-NK Cells Against Colorectal Cancer Cells

Objective:Investigate the cytotoxic effect of CAR-NK cells targeting CEA on colorectal cancer cells with positive CEA expression.Methods:The mRNA and protein levels of CEA in different CRC cell lines were detected by qRT-PCR and Western blot analysis.Lentiviral transduction was used to construct CAR-NK cells and empty vector CON-NK cells targeting CEA.Fluorescence microscopy and WB were used to determine whether the cells successfully constructed and expressed CAR structures.The effector NK cells were co-cultured with target cells,and the levels of LDH,IFN-γ,and GM-CSF were detected.The killing rate of effector cells was calculated,and the release of cytokines during the killing of target cells by different effector cells was compared.Results:The expression level of CEA in colorectal cancer patients was significantly higher than that in normal samples and other tumor samples,and the prognosis survival time of patients with high CEA expression was lower than that of CRC patients with low or no CEA expression(P<0.05).The CEA expression of the HT29 cell line was significantly higher than that of the SW1116 cell line at both the mRNA and protein levels.CEA-CAR-NK92 cells and CON-NK92 cells expressed green fluorescence under a microscope,and WB results showed that CEA-CAR-NK92 cells successfully expressed the CAR structure.Compared with CON-NK92 cells and NK92 cells,CEA-CAR-NK92 cells effectively killed HT29 cells(P<0.05).CEA-CAR-NK92 cells secreted a large amount of IFN-γand GM-CSF during the killing of HT29 cells,while the cytokine secretion of CON-NK92 cells and NK92 cells was not significant(P<0.05).Conclusion:CAR-NK92 cells targeting CEA can effectively kill CEA-positive colorectal cancer cells.

Investigation of the Cytotoxicity of CAR-NK-92 Cells Targeting CEA Against Gastric Cancer Cells

Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 cells with MKN-45 gastric cancer cells,the killing effect of CAR-NK-92 cells was detected by a lactate dehydrogenase release assay.The secretion levels of gamma interferon and granulocyte-macrophage colony-stimulating factor were measured using an ELISA assay.Results:The lactate dehydrogenase release assay showed that CAR-NK-92 cells had a significant killing effect on MKN-45 cells compared to CON-NK-92 cells,and the difference was statistically significant(P<0.001).ELISA results indicated that the levels of gamma interferon and granulocyte-macrophage colony-stimulating factor secreted by CAR-NK-92 cells and MKN-45 target cells were significantly increased after co-culture(P<0.001).Conclusion:CAR-NK-92 cells targeting CEA exhibit a significant killing effect on CEA-positive gastric cancer cells.

Natural Killer Cell-Based Immunotherapy for Cancer: Advances and Prospects

Natural killer (NK) cells are key innate immune cells that provide the first line of defense against viral infection and cancer. Although NK cells can discriminate between "self" and "non-self," recognize abnormal cells, and eliminate transformed cells and malignancies in real time, tumors develop several strategies to escape from NK cell attack. These strategies include upregulating ligands for the inhibitory receptors of NK cells and producing soluble molecules or immunosuppressive factors. Various types of NK cells are currently being applied in clinical trials, including autologous or allogeneic NK cells, umbilical cord blood (UCB) or induced pluripotent stem cell (iPSC)-derived NK cells, memory-like NK cells, and NK cell line NK-92 cells, for the treatment of different types of tumors. Chimeric antigen receptors (CARs)-NK cells have recently shown great potential due to their redirect specificity and effective antitumor activity. In this review, we summarize the mechanisms of tumor escape from NK cell recognition, the current status and advanced progress of NK cell-based immunotherapy, ways of enhancing the antitumor capacity of NK cells in vivo, and major challenges for clinical practice in this field.

Revolutionizing gastric cancer treatment:The potential of immunotherapy

Gastric cancer,a prevalent malignancy worldwide,ranks sixth in terms of frequency and third in fatality,causing over a million new cases and 769000 annual deaths.Predominant in Eastern Europe and Eastern Asia,risk factors include family medical history,dietary habits,tobacco use,Helicobacter pylori,and Epstein-Barr virus infections.Unfortunately,gastric cancer is often diagnosed at an advanced stage,leading to a grim prognosis,with a 5-year overall survival rate below 5%.Surgical intervention,particularly with D2 Lymphadenectomy,is the mainstay for early-stage cases but offers limited success.For advanced cases,the National Comprehensive Cancer Network recommends chemotherapy,radiation,and targeted therapy.Emerging immunotherapy presents promise,especially for unresectable or metastatic cases,with strategies like immune checkpoint inhibitors,tumor vaccines,adoptive immunotherapy,and nonspecific immunomodulators.In this Editorial,with regards to the article“Advances and key focus areas in gastric cancer immunotherapy:A comprehensive scientometric and clinical trial review”,we address the advances in the field of immunotherapy in gastric cancer and its future prospects.

Emergence of immunotherapy as a novel way to treat hepatocellular carcinoma

Tumor immunity proceeds through multiple processes, which consist of antigen presentation by antigen presenting cells(APCs) to educate effector cells and destruction by the effector cytotoxic cells. However, tumor immunity is frequently repressed at tumor sites. Malignantly transformed cells rarely survive the attack by the immune system, but cells that do survive change their phenotypes to reduce their immunogenicity. The resultant cells evade the attack by the immune system and form clinically discernible tumors. Tumor microenvironments simultaneously contain a wide variety of immune suppressive molecules and cells to dampen tumor immunity. Moreover, the liver microenvironment exhibits immune tolerance to reduce aberrant immune responses to massively-exposed antigens via the portal vein, and immune dysfunction is frequently associated with liver cirrhosis, which is widespread in hepatocellular carcinoma(HCC) patients. Immune therapy aims to reduce tumor burden, but it is also expected to prevent non-cancerous liver lesions from progressing to HCC, because HCC develops or recurs from noncancerous liver lesions with chronic inflammatory states and/or cirrhosis and these lesions cannot be cured and/or eradicated by local and/or systemic therapies. Nevertheless, cancer immune therapy should augment specific tumor immunity by using two distinct measures: enhancing the effector cell functions such as antigen presentation capacity of APCs and tumor cell killing capacity of cytotoxic cells, and reactivating the immune system in immune-suppressive tumor microenvironments. Here, we will summarize the current status and discuss the future perspective on immune therapy for HCC.

Chimeric antigen receptor- and natural killer cell receptor-engineered innate killer cells in cancer immunotherapy

Chimeric antigen receptor(CAR)-engineered T-cell(CAR-T)therapy has demonstrated impressive therapeutic efficacy against hematological malignancies,but multiple challenges have hindered its application,particularly for the eradication of solid tumors.Innate killer cells(IKCs),particularly NK cells,NKT cells,andγδT cells,employ specific antigen-independent innate tumor recognition and cytotoxic mechanisms that simultaneously display high antitumor efficacy and prevent tumor escape caused by antigen loss or modulation.IKCs are associated with a low risk of developing GVHD,thus offering new opportunities for allogeneic“off-the-shelf”cellular therapeutic products.The unique innate features,wide tumor recognition range,and potent antitumor functions of IKCs make them potentially excellent candidates for cancer immunotherapy,particularly serving as platforms for CAR development.In this review,we first provide a brief summary of the challenges hampering CAR-T-cell therapy applications and then discuss the latest CAR-NK-cell research,covering the advantages,applications,and clinical translation of CAR-and NK-cell receptor(NKR)-engineered IKCs.Advances in synthetic biology and the development of novel genetic engineering techniques,such as gene-editing and cellular reprogramming,will enable the further optimization of IKC-based anticancer therapies.

Primary NK/T cell lymphoma of the testis:A case report and review of the literature

Objective： To study the clinical and pathological features of primary NK/T cell lymphoma of testis and to investigate the effective diagnosis and treatment of this disease. Methods： The surgical specimens of a patient with primary NK/T cell lymphoma of the testis were observed by light microscopy, immunohistochemistry and examined by the polymerase chain reaction （PCR） for Epstein-Barr virus （EBV） DNA and T-cell receptor （TCR） gene rearrangement, and the literature were reviewed. Results： The patient presented with left-sided painless testicular enlargement and the lymphoma had a propensity to spread to the contralateral testis, spleen, central nervous system, and so on. The neoplastic cells were positive for CD56, CD45R0 and CD3ε, while the expressions of CD20, CD79α, CD5, Bcl-2 and PLAP were negative. In addition, the EBV DNA was detected in the lymphoma by PCR. And the results of gene rearrangement studies for the y chain of the T-cell receptor were negative. The pathological diagnosis was NK/T cell lymphoma of the left testis. Conclusion： Primary NK/T cell lymphoma of the testis is a rare entity and progressed rapidly. The histopathological, immunohistochemical, EBV examination and TCR gene rearrangement studies should be carried out as soon as possible in order to get the defined diagnosis. Currently, the therapeutic efficacy is poor and the new measures should be investigated to improve the survival rate.

非小细胞肺癌患者血清CA211水平和NK细胞数与预后相关

目的 探究非小细胞肺癌(NSCLC)患者血清糖类抗原211(CA211)表达水平和自然杀伤(NK)细胞数与预后的相关性。方法 选取杭州师范大学附属医院2019年6月至2022年7月收治的132例NSCLC患者作为实验组,另选取同期收治的132例肺部良性病变患者作为对照组。从检验科收集数据分析NSCLC患者血清CA211表达水平和NK细胞数与患者预后关系以及影响NSCLC患者预后的相关因素。结果 实验组中性粒细胞/淋巴细胞值(NLR值)和血清CA211表达水平高于对照组(P<0.05),NK细胞数低于对照组(P<0.05);实验组血清CA211表达水平和NK细胞数呈负相关(r=-0.405,P<0.001);不同血清CA211表达水平和NK细胞数的患者淋巴结转移情况以及TNM分期存在显著性差异(P<0.05);CA211低表达者1年生存率高于CA211高表达者(P<0.05),NK细胞高水平者1年生存率高于NK细胞低水平者(P<0.05);淋巴结转移、TNM分期、CA211是影响NSCLC患者预后的危险因素(P<0.05),NK细胞是NSCLC患者预后的保护因素(P<0.05)。结论 NSCLC患者血清CA211表达水平及NK细胞数与临床病理特征及预后有密切联系。

嵌合抗原受体T细胞免疫疗法在晚期胃癌中的应用进展

胃癌是最常见的恶性肿瘤之一,当前我国胃癌就诊患者仍以进展期为主,晚期患者就诊时多已失去手术治疗的机会,而传统的放疗、化疗和靶向治疗效果并不理想。嵌合抗原受体(CAR)-T细胞(CAR-T)免疫疗法作为一种新的治疗手段,在血液系统恶性肿瘤中疗效显著,也为胃癌的免疫治疗开辟了新途径。然而,由于胃癌的异质性、肿瘤微环境免疫抑制、肿瘤靶抗原逃逸及脱靶毒性等问题,使得CAR-T免疫疗法在胃癌治疗中的应用存在挑战。本文综述了CAR的结构及CAR-T治疗原理、CAR-T治疗晚期胃癌的主要靶点及治疗现状,并探讨了CAR-T治疗胃癌面临的挑战,旨在为晚期胃癌的临床免疫治疗提供新思路。

肝细胞癌过继细胞疗法研究进展

肝细胞癌是严重威胁人类生存的一类疾病,中国肝细胞癌死亡率居世界第三。由于肝细胞癌发病早期无特异性症状,晚期的治疗方案又存在复发率高等的种种弊端,因此寻找行之有效的治疗新方法迫在眉睫。过继细胞疗法是一种细胞免疫疗法,在肝脏多样化和复杂的免疫微环境中,通过结合肿瘤特异性抗原或是非特异性的调节免疫平衡,过继细胞疗法在肝细胞癌的治疗中逐渐发挥出优势。在这篇综述中我们阐述了肝细胞癌靶向治疗常用生物标志物的形态结构及信号通路,探讨总结了近年来过继细胞疗法治疗肝细胞癌的研究手段及取得的临床效果,并对接下来的研究方向进行了展望。

通用型CAR-NK细胞治疗系统性红斑狼疮的应用与展望

系统性红斑狼疮(SLE)的特点是B细胞异常活化产生大量自身抗体,这些自身抗体与自身抗原形成免疫复合物诱发系统性炎症,导致全身多系统多器官受累。现有靶向B细胞治疗策略(如贝利尤单抗、泰它西普及利妥昔单抗等)的临床疗效仍然有限,近年来靶向CD19的嵌合抗原受体(CAR)-T细胞治疗SLE展现出卓越的疗效。但自体CAR-T细胞治疗存在细胞因子释放综合征、T细胞肿瘤、感染等潜在风险,且价格昂贵。CAR-自然杀伤(NK)细胞治疗是现货型细胞疗法,其安全性高,无神经毒性及肿瘤化风险,且治疗费用低。目前,CAR-NK细胞在治疗血液系统肿瘤中取得了显著的研究成果,且CAR-NK细胞治疗SLE的初步临床研究展现出良好的治疗效果、优异的安全性及可预期的持久性。本文将重点阐述新一代通用型CAR-NK细胞的特点及其用于治疗复发难治性SLE的最新临床研究结果,展望其治疗SLE及B细胞相关自身免疫病的广阔前景。

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor(CAR)-engineered natural killer(NK)cells have shown promise in treating various cancers.However,limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications.Here,we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein(FITC)single-chain variable fragment(scFv)to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors.We next genetically engineer human pluripotent stem cells(hPSCs)with optimized CARs and differentiate them into functional dual CAR-NK cells.The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3(pSTAT3)and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptorβ-chain(ΔIL-2Rβ)and STAT3-binding tyrosine-X-X-glutamine(YXXQ)motif.Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells.Collectively,our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Targeting immune checkpoints:how to use natural killer cells for fighting against solid tumors

Natural killer(NK)cells are unique innate immune cells that mediate antiviral and anti-tumor responses.Thus,they might hold great potential for cancer immunotherapy.NK cell adoptive immunotherapy in humans has shown modest efficacy.In particular,it has failed to demonstrate therapeutic efficiency in the treatment of solid tumors,possibly due in part to the immunosuppressive tumor microenvironment(TME),which reduces NK cell immunotherapy’s efficiencies.It is known that immune checkpoints play a prominent role in creating an immunosuppressive TME,leading to NK cell exhaustion and tumor immune escape.Therefore,NK cells must be reversed from their dysfunctional status and increased in their effector roles in order to improve the efficiency of cancer immunotherapy.Blockade of immune checkpoints can not only rescue NK cells from exhaustion but also augment their robust anti-tumor activity.In this review,we discussed immune checkpoint blockade strategies with a focus on chimeric antigen receptor(CAR)-NK cells to redirect NK cells to cancer cells in the treatment of solid tumors.

Down-regulation of human leukocyte antigens class I on peripheral T lymphocytes and NK cells from subjects in region of high-incidence gastrointestinal tumor

Background Many types of human tumors can suppress the immune system to enhance their survival. Loss or down-regulation of human leukocyte antigens （HLA） class I on tumors is considered to be a major mechanism of tumor immune escape. Our previous studies found that HLA class I on peripheral-blood mononuclear cells was significantly lower in gastric cancer patients. The present study made an analysis of HLA class I expression on peripheral-blood T lymphocytes and NK cells from subjects of Lijiadian village, a village with high-incidence gastrointestinal tumor. Methods A total of 181 villagers from Lijiadian village and 153 normal controls from the Department of Health Examination Center were enrolled in this study. Using a multi-tumor markers detection system, these villagers were divided into two groups： high-risk group （tumor markers positive group） and low-risk group （tumor markers negative group）. The percentage of T lymphocytes and NK cells and levels of HLA class I on their surface were determined in these subjects by flow cytometry. Results Percentages of T lymphocytes and NK cells in peripheral-blood mononuclear cells did not vary with age. The expression level of HLA class I on peripheral T lymphocytes and NK cells was not affected by age or gender, but was significantly down-regulated in Lijiadian villagers （P 〈0.05）, with the low-risk group, there was a significant reduction of cells （P 〈0.05） in the high-risk group. especially on the surface of NK cells （P 〈0.01）. Compared HLA class I on peripheral T lymphocytes （P 〈0.05） and NK Conclusions HLA class I on peripheral T lymphocytes and NK cells may be involved in tumorigenesis and development of gastrointestinal tumor, and understanding their changes in expression may provide new insights into the mechanism of tumor immunity.

嵌合抗原受体自然杀伤细胞疗法研究进展及展望

自然杀伤(NK)细胞是先天免疫系统的主要组成部分。基于其对肿瘤细胞的有效杀伤能力和对健康细胞的良好耐受性,NK细胞已成功地用于过继细胞疗法以治疗肿瘤患者。近年来,嵌合抗原受体(CAR)-T细胞的临床成功,证明了基因修饰的免疫细胞在抗肿瘤领域的巨大潜力。NK细胞凭借优越的低免疫原性也迅速成为基于CAR修饰疗法的新型免疫工具细胞。目前已经开展的多项临床试验中,多种来源的CAR-NK细胞均展现出较为优越的治疗效果和安全性,但靶向实体瘤CAR-NK细胞治疗仍处于初始阶段。本文系统梳理了CAR-NK细胞在临床上取得阶段性进展,同时也指出CAR-NK细胞疗法的局限性,旨在为进一步优化和升级CAR-NK细胞疗法提供参考。

Harnessing natural killer cells to develop next-generation cellular immunotherapy

Cellular immunotherapy harnesses the body’’s own immune system to fight cancer by using engineered T cells,macrophages,or natural killer(NK)cells.Compared to chimeric antigen receptor T(CAR-T)cells that are commonly used to treat hematological malignancies,CAR-NK cells have shown remarkable therapeutic effectiveness while exhibiting enhanced safety,reduced risk of graft-versus-host disease,fewer side effects,and amplified antitumor efficacy.Preclinical trials have unveiled the high potential of adoptive CAR-NK cell therapy to curtail or even eliminate both hematological malignancies and solid tumors in animal models.We brought forth herein the design principle of CAR-NK cells,highlighted the latest progress in the preclinical testing and clinical trials of CAR-NK cells,briefly delved into discussed major roadblocks in CAR-NK therapy,and discussed potential solutions to surmount these challenges.Given the accelerated progress in both basic and translational studies on immune cell engineering,CAR-NK cell therapy promises to become a serious contender and important addition to the next-generation cell-based immunotherapy.

Engineered human pluripotent stem cell-derived natural killer cells: the next frontier for cancer immunotherapy

Adoptive immunotherapy using immune effector cells has revolutionized cancer treatments with approval of two autologous chimeric antigen receptor(CAR)T cell therapies by the US FDA.Clinical trials using natural killer(NK)cell-based adoptive immunotherapy have been shown to be safe and effective for treatment of multiple malignancies,especially acute myelogenous leukemia.However,most of these trails use primary NK cells isolated from peripheral or cord blood which can have donor-dependent variability and can be challenging to genetic engineer to improve antitumor functions,limiting the widespread use of this promising new therapy.NK cells can now be routinely produced from human pluripotent stem cells,both human embryonic stem cells(hESCs)and induced pluripotent stem cells(iPSCs).These pluripotent stem cells are homogenous,easy to genetically modify on a clonal level and can be used as unlimited source of NK cells,making them ideal population to develop standardized,off-the-shelf adoptive NK cell therapy products.In this review,we discuss recent advances of obtaining and expanding hESC and iPSC-derived NK cells and novel genetic engineering strategies that are being applied to improve their antitumor functions.