中山大学教授颜光美M1“灭癌”病毒 有望进行临床试验

新型天然溶瘤病毒M1可精确杀灭癌细胞引自“医学论坛网”【期刊名称】《中国肿瘤临床》【年(卷),期】2014(000)020【摘要】“在杀死癌细胞的同时，也杀死了正常细胞。

”这个困惑了全世界医生和癌症病人的怪圈，终于有望打破。

《美国科学院院报》最近发表了一篇重点论文指出，中国科学家发现，一种叫做M1的天然病毒能特异性杀死癌细胞而不伤害正常细胞。

这种新型溶瘤病毒有望成为下一代抗癌利器。

这项研究成果是由中国中山大学中山医学院颜光美团队独立完成并具完全的自主知识产权。

全球癌症发病率呈现快速增长态势，现有的治疗手段远远未能满足临床需求。

该团队历经多年潜心研究，终于从中国海南岛分离得到一种M1的天然病毒。

颜光美团队使用细胞培养方法发现，M1病毒能选择性地感染并杀死包括肝癌、结直肠癌、膀胱癌、黑色素瘤在内的多种癌细胞，而对正常细胞无毒副作用。

整体动物模型证明，M1病毒“像长了眼睛一样准确找到肿瘤组织并将其杀灭”，正常器官则不受影响。

除细胞水平及动物实验之外，课题组还使用大量临床标本进一步证实了上述新型溶瘤病毒的有效性和特异性。

更为重要的是，研究工作还证明了M1病毒作用的分子遗传学机理，找到了特异的负性生物标志物。

一种特别的基因与疗效有关。

这个发现为精准的临床用药和实施个体化疗法提供了可靠的科学依据，也会极大地增加未来临床试验取得成功的机会。

这些结果表明，如同“精确制导”一般的新型天然溶瘤病毒M1，将会安全而有效地治疗癌症，有望成为攻克人类癌症的新一代利器。

【总页数】1页(P1317-1317)【作者】引自“医学论坛网”【作者单位】引自“医学论坛网”【正文语种】中文【相关文献】1.食品天然防腐剂可杀灭癌细胞 [J],2.溶瘤病毒M1诱导宫颈癌细胞C-33A凋亡的作用及机制 [J], 肖晓;周雅思;彭楚茵;邓金清;王来友;朱文博3.携带TRAIL基因的新型溶瘤病毒体外诱导肝癌细胞凋亡 [J], 刘永靖;陈飞虎;苏长青;王星华;钱炎珍;钱其军4.天然病毒M1可杀伤肝癌等癌细胞 [J],5.中山大学分离出一种能杀灭癌细胞的天然病毒 [J],因版权原因，仅展示原文概要，查看原文内容请购买。

“杀灭最后一个癌细胞”只是幻想山东华圣中医肿瘤研究所所长济南华圣医院院长王现军经过长期科学知识的普及和抗癌理念的宣传，许多癌症患者以及他们的亲友，早已走出了“癌症是不治之症”、“癌症等于死亡”的认识误区。

他们认识到，癌症是可防可治的一类疾病，特别是早期发现并早期治疗，会取得非常理想的治疗效果；即使是明确诊断的时候病情已经到了中晚期，只要坚持积极正确的治疗方法，也不是没有临床治愈的可能。

于是，便满怀希望地进行手术，按部就班地接受一次次的放疗和化疗。

随着治疗过程的进展，患者的医学影像检查和生化检验报告不断更新，这些报告显示：患者的瘤体在不断缩小，肿瘤组织在变性、坏死，患者体内的癌细胞在逐步地减少。

在这种情形之下，不少人都会产生一种天真而美好的想法：我们只要坚持不懈地治疗下去，总有一天，会把体内的最后一个癌细胞给杀死，那时候，不就把癌症彻底治愈了吗？而现实却并不这么简单。

癌症是一种全身性的疾病，病灶本身只是局部的集中体现。

手术可以一次性地拿掉大部分的癌细胞，但是无法全部清除。

即使是所谓的“根治术”、“大范围清扫”也只是相对而言，手术范围越大，对人体造成的功能残损就约严重。

放射线和化学药品可以有效地杀灭癌细胞。

但是放射线治疗只能针对病灶局部实施，而且有终生最大剂量的限制，不可以无休止地照射下去；化疗药物可以作用于全身，但是它只是成比例地杀灭癌细胞，同时也对人体正常的组织和器官带来巨大伤害。

化疗药物带来的骨髓抑制、消化道反应、免疫抑制、心肺肝肾损伤、神经系统反应等毒副作用，也使患者无法接受持续的治疗。

由此看来，“杀灭最后一个癌细胞”的想法是难以实现的，坚持这样做的结果，必然是加重痛苦，加速死亡，生命和肿瘤同归于尽。

正确的做法是：对于大部分癌症患者特别是中晚期的患者，在经过治疗取得临床上缓解以后，把治疗的重点转移到调整机体平衡、保护和强化免疫系统功能方面来，扶正祛邪，抑制体内残存癌细胞的分裂增殖，使患者获得更高的生活质量和更长的生存时间。

新型“可复制型活药”溶瘤病毒M1的药动学研究
作者：
来源：《科学中国人》2024年第06期
中山大学中山医学院颜光美/林园团队解释了溶瘤病毒M1作为一种新型“可复制型活药”的药代动力学特征，并基于它的关键调控机制建立了特异性增效策略。

相关成果发表于《药学学报》英文刊（Acta Pharmaceutica Sinica B）。

溶瘤病毒是一类新兴的抗癌“活药”，可选择性感染并杀伤肿瘤细胞，同时不损伤正常组织。

研究人员系统解析了溶瘤病毒M1在免疫健全小鼠体内的药动学特征，并鉴定了对溶瘤病毒M1分布和代谢起关键调控作用的通路。

在此基础上，研究人员通过使用临床药物鲁索替尼成功改善了溶瘤病毒M1的药动学特性，并进一步提升了它的抗腫瘤疗效和机体免疫激活水平。

专利名称：M1病毒变异体及其应用
专利类型：发明专利
发明人：颜光美,林园,郭莉,林子青,吴广恩申请号：CN202010486178.7
申请日：20200601
公开号：CN112011519A
公开日：
20201201
专利内容由知识产权出版社提供
摘要：本发明提供一种M1病毒。

本发明进一步提供所述病毒的一系列应用，所述应用包括但不限于：病毒载体、抗肿瘤剂、药物组合物。

本发明的病毒能够有效抑制多种肿瘤细胞的生长，同时具有肿瘤靶向性，对正常的细胞无毒性；可以经静脉注射的方式给药，具有操作上的便捷性。

申请人：广州威溶特医药科技有限公司
地址：510663 广东省广州市高新技术产业开发区科学城揽月路3号广州国际企业孵化器G区416-428房间
国籍：CN
代理机构：北京市万慧达律师事务所
代理人：谢敏楠
更多信息请下载全文后查看。

Identification and characterization of alphavirus M1as a selective oncolytic virus targeting ZAP-defective human cancersYuan Lin a,1,Haipeng Zhang a,1,Jiankai Liang a,1,Kai Li a,Wenbo Zhu a,Liwu Fu b,Fang Wang b,Xiaoke Zheng c,Huijuan Shi c,Sihan Wu a,Xiao Xiao a,Lijun Chen a,Lipeng Tang a,Min Yan a,Xiaoxiao Yang a,Yaqian Tan a,Pengxin Qiu a,Yijun Huang a,Wei Yin d,Xinwen Su a,Haiyan Hu e,Jun Hu f,2,and Guangmei Yan a,2Departments of a Pharmacology,d Biochemistry,and f Microbiology,Zhongshan School of Medicine and e School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou510080,China;b State Key Laboratory of Oncology in South China,Sun Yat-sen University Cancer Center,Guangzhou510060,China; and c Department of Pathology,First Affiliated Hospital of Sun Yat-sen University,Guangzhou510080,ChinaEdited*by Bernard Roizman,University of Chicago,Chicago,IL,and approved September10,2014(received for review May12,2014)Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents.Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers.Here,we identify a naturally occurring alphavirus(M1)as a novel selective killer tar-geting zinc-finger antiviral protein(ZAP)-deficient cancer cells.In vitro,in vivo,and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1.We showed that the selectivity depends on ZAP deficiency by systematic identification.A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers,suggesting exten-sive application prospects for M1.Additionally,M1killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis.Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses.personalized medicine|unfolded protein response|translational inhibition D espite advances in cancer therapy over the past few decades,cancer is still a major health problem all over the world(1). One innovative class of targeted anticancer strategies is the use of replicating oncolytic viruses with selective tropism for can-cerous cells and tissues(2,3).The tumor selectivity of oncolytic virus is primarily based on the genetic abnormalities of malignant cells,including innate immune defects,aberrant oncogenic sig-naling,and tumor-specific receptors(4–6).The thriving viruses in tumor cells may lead to direct cell lysis,anticancer immune response,or modulation of tumor vasculature(3,7).Moreover, some of the cancer-targeted multimechanistic oncolytic viruses have been proven to be well-tolerated in clinical trials,with patients exhibiting only mild flu-like symptoms,offering great potential for increasing efficacy while eliminating the side effects (8).To date,several oncolytic viruses have been tested in pre-clinical and clinical trials,of which the milestone is a pivotal phase III trial using talimogene laherparepvec for unresected melanoma(2,3,9).Although a few therapeutic viruses are performing well in clinical trials,not all patients showed good response.Novel oncolytic viruses that grow better in some cancer cells in a predictable manner remain to be discovered for po-tentially personalized cancer therapy.M1is a strain of Getah-like alphavirus that was isolated from culicine mosquitoes collected on Hainan Island of China(10,11). Getah virus is transmitted mainly among horses and pigs,and it has not been linked to human illness(12–14).Also,M1does not cause apparent disease symptoms in mice or rats,even on ad-ministration of doses up to3×107pfu per mouse or3×108pfu per rat.Earlier,we reported that M1induces apoptosis in glioma cells(10).Thus,we hypothesized that an apathogenic cancer cell-killing virus could be a candidate for systemic oncolytic therapy.In this study,we sought to investigate the anticancer effectiveness and tumor tropism of M1and uncover the mechanisms,aiming to identify a candidate for personalized oncolytic virotherapy. ResultsSelective Killing of Cancer Cells by Naturally Occurring Alphavirus M1. To explore the oncolytic efficacy of M1,we first examined the effects of M1on the viability of various cultured human cancer cells and normal cells.M1markedly induced cell death in cancer cells in a dose-related fashion(representative data are shown in Fig.1A,all tested cell lines are listed in Table S1,and all tested primary cells are listed in Table S2).Thus,of66cancer cell lines that we screened,29lines showed a more than30%decrease in viability48h after exposure to10pfu virus per cell.In contrast, there was little apparent reduction in primary normal cell via-bility,even after exposure to100pfu virus per cell for96h(Fig. 1A).To test the hypothesis that oncolysis of M1correlated with virus growth,we measured virus titers in a total of38cell lines 36h after exposure to0.1pfu per cell of M1.A positive correlation between M1-induced oncolysis and virus growth wasobserved Author contributions:Y.L.,J.H.,and G.Y.designed research;Y.L.,H.Z.,J.L.,K.L.,W.Z.,X.Z.,H.S.,X.X.,L.C.,L.T.,M.Y.,X.Y.,Y.T.,and X.S.performed research;L.F.,F.W.,S.W.,P.Q., Y.H.,and H.H.contributed new reagents/analytic tools;Y.L.,H.Z.,J.L.,K.L.,X.Z.,H.S.,W.Y.,and J.H.analyzed data;and Y.L.and G.Y.wrote the paper.The authors declare no conflict of interest.*This Direct Submission article had a prearranged editor.Data deposition:The cDNA microarray data reported in this paper have been deposited in the Gene Expression Omnibus(GEO)database,/geo(accession no. GSE54342).1Y.L.,H.Z.,and J.L.contributed equally to this work.2To whom correspondence may be addressed.Email:hujun@ or ygm@ .This article contains supporting information online at /lookup/suppl/doi:10. 1073/pnas.1408759111/-/DCSupplemental./cgi/doi/10.1073/pnas.1408759111PNAS Early Edition|1of9M E D I C A L S C I E N C E S P N A S P L U S(Fig.1B ),indicating that cancer-selective replication leads to the cancer-targeting property of M1.To further evaluate the in vivo antitumor potential of M1,we established three preclinical tumor models,including the Hep3B human hepatocellular carcinoma (HCC)s.c.xenograft model in BALB/c-nu/nu mice (Fig.1C and D ),the 4T1mouse breast cancer orthotopic model in immunocompetent BALB/c mice (Fig.1E ),and the B16mouse skin melanoma s.c.model in C57BL/6mice (Fig.1F ).After palpable tumors formed,mice in each model were randomized to receive either six doses of intratumoral injection (2×106pfu per dose)or two doses of i.v.infusion of M1(3×107pfu per dose).In parallel,mice were treated with vehicle as nega-tive controls.Consistent with the in vitro experiments,evident antitumor effects were observed in M1-treated animals.It is noteworthy that M1-treated mice remained asymptomatic throughout the treatment,and no obvious difference in body weight between control and M1-treated groups was detected (Fig.1C –F ).Moreover,to evaluate the safety and potential toxicity of M1,we i.v.injected two doses of M1(3×107pfu per dose)into immunocompetent BALB/c mice.All eight of the M1-injected mice survived until 27d postinjection,when they were killed.Body weight was measured every 3d.There was no significantdifference in body weight between control and M1-injected groups during the course of the study (Fig.S1A ).After autopsy,histological analyses of vital tissues,including brain,heart,kidney,liver,lung,skeletal muscle,and spleen,were performed by H&E staining.None of the M1-or mock-injected BALB/c mice showed any abnormal pathology (Fig.S1B ).Complete blood count (CBC)analysis showed decreased WBCs after M1injection,whereas other parameters of CBC analysis,including percentage of neu-trophil granulocytes,percentage of lymphocytes,RBC count,and platelet count,remained unchanged (Fig.S1C ).The observations of mortality,body weight,histopathology,and CBC analysis support the conclusion that M1is safe to animals.We proposed that the antitumor activity and safety of M1are based on high tumor tropism.To further assess the in vivo se-lectivity of M1,we established tumor xenografts with two HCC cell lines:Hep3B cells (sensitive to M1)on the left hind flank of nude mice and cell line PLC cells (resistant to M1)on the right hind flank of nude mice.When palpable tumors developed,mice were treated with one dose of i.v.-delivered M1(3×107pfu),and biodistribution of viral RNA genome was quantified within 96h postinfection by quantitative RT-PCR (qRT-PCR).As we expected,M1was more than 1,000times enriched inHep3BFig.1.Selective oncolytic efficacy of M1in vitro and in vivo.(A )Cell viability assays were performed on a panel of cancer cell lines and primary normal cells 48and 96h after exposure to M1,respectively.N,primary normal cell;T,tumor cell.(B )Viral titers (MOI =0.1pfu per cell;36h)and cell viability (MOI =10pfu per cell;48h)in various infected cell lines.Virus was collected from both supernatant and cell lysate;r is the Pearson correlation coefficient.(A and B )Values are means of three independent experiments.(C –F )Tumor growth (solid symbols)and body weight (open symbols)of tumor-bearing mice.(C and D )Nude,(E )BALB/c,and (F )C57BL/6mice were treated with either vehicle or M1intratumorally (i.t.)or i.v.(n =9per group).Data are shown in means ±SDs.(G and H )Biodistribution of systemically delivered M1.Viral RNA was quantified by qRT-PCR and normalized to the expression of β-actin.Means ±SDs are shown (n =6per group).ns,not significant;Tumor_R,PLC;Tumor_S,Hep3B.*represents not detectable results.2of 9|/cgi/doi/10.1073/pnas.1408759111Lin et al.Fig.2.M1triggers prolonged and severe ER stress-mediated apoptosis in susceptible cancer cells.(A)Observation of ER distension in Hep3B cells infected with M1by transmission EM.Middle shows higher magnification from the box in Top.(Scale bars:500nm.)Quantification of ER distension is also presented.(B)Observation of chromatin condensation in infected Hep3B cells by transmission EM.Red arrows,condensed chromatin;white arrows,nuclear envelope. (Scale bars:1μm.)Quantification of condensed nuclei is also presented.(A and B)Means±SDs from three independent experiments are shown.(C–F)The effect of M1on ER stress signal pathways.Western blot analyses of(C)BiP,(D)phosphorylated eIF-2α(S51),(E)phosphorylated JNK(Y183/Y185),and(F) cleaved casepase-12(Clv-casp-12)are shown.GAPDH andα-tubulin served as loading controls.The ratio between phosphorylated eIF-2αandα-tubulin was calculated.Pro-casp-12,pro-caspase-12.(D)Detection of protein synthesis after M1infection(MOI=10,12h)by L-AHA-biotin labeling and Western blot.(G and H)Caspase activity in Hep3B and LoVo cells treated with M1(MOI=1pfu per cell).CTL,control;hpi,hours postinfection.**P<0.05.Lin et al.PNAS Early Edition|3of9M E D I C A L S C I E N C E S P N A S P L U Stumor tissue than any other tissues tested(Fig.1G).Similar results can be observed in immunocompetent C57BL/6mice bearing B16melanoma(Fig.1H).The finding that the high level of viral RNA in sensitive tumor tissue remained stable for at least4d supports the conclusion that M1efficiently targets and selectively replicates in cancer cells.Tumor-Selective Replication of M1Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Cancer Cells.We next explored the mechanism whereby M1killed cancer cells.Transmission EM showed a progressive distension of endoplasmic reticulum(ER) lumen as early as6h post-M1infection in Hep3B cells(Fig.2A). At24h after M1treatment,catastrophic destruction of ER and condensation of chromatin were observed(Fig.2A and B),in-dicating that M1induced a prolonged and severe ER stress that mediated apoptosis(15).One consequence of ER stress is the synthesis of chaperones that help proteins to fold properly(16, 17).Western blot analyses showed that one of these chaperones, BiP(an HSP70molecular chaperone,the induction of which is commonly used as a marker of ER stress),was strongly increased after M1infection(Fig.2C).Another symbolic event of ER stress is the phosphorylation of eIF-2α(eukaryotic translation initiation factor2,subunit alpha)and subsequent translational inhibition,helping to alleviate the load of unfolded proteins(17). To test the effect of M1on host translational machinery,newly synthesized proteins were labeled with L-azidohomoalaine-biotin (L-AHA,which is an analog of methionine)and detected by Western blot using HRP-conjugated antibiotin antibody.We observed significant phosphorylation of eIF-2αand the corre-sponding inhibition of host protein synthesis by M1treatment(Fig. 2D).We also examined the induction kinetics of PERK(protein kinase R-like ER kinase)and PKR(protein kinase R)after M1 infection and observed that PERK expression was significantly induced by M1infection in a time-dependent manner,whereas the induction of PKR expression was not remarkable(Fig.S2),sug-gesting that eIF-2αphosphorylation was mainly stimulated by ER stress-activated PERK.In contrast to the results obtained in sen-sitive cancer cells,M1did not cause increase in BiP,phosphory-lation of eIF-2α,or translation inhibition in L-02normal liver cells (Fig.2C and D).Similarly,pronounced phosphorylation of eIF-2αwas not observed in M1-infected primary normal hepatocytes(Fig. S3).Translation of alphavirus mRNA has been shown to be re-sistant to eIF-2αphosphorylation because of the highly stable RNA hairpin loop located downstream of the AUG initiator co-don(18).Thus,the high production of M1viral protein triggers prolonged and severe ER stress in sensitive cancer cells.We next examined the ER stress-induced apoptotic pathways (19–21).Western blot analyses revealed that the JNK pathway and caspase-12cascades were activated by M1infection(Fig.2E and F)in susceptible cancer cells,whereas C/EBP(CCAAT-enhancer–binding protein)homologous protein was not induced(Fig.S4). Conversely,both JNK signal and caspase-12activity remained unchanged in L-02cells after M1infection,which would be expected by the absence of M1-induced ER stress(Fig.2E and F). We next tested the downstream caspase cascades in M1-suscep-tible cancer cells by detecting the activity of caspase-9and apo-ptotic executioner caspase-3.Both caspases were activated after M1infection(Fig.2G and H),indicating that the M1-induced ER stress leads to apoptotic cell death.Systematic Identification of Host Factors That Contribute to Tumor Tropism of M1.The finding that M1preferentially replicated in and killed cancer cells urged us to probe the molecular mecha-nism of M1selectivity.Given that type I IFNs are well-known factors triggering antiviral effect against a broad range of viruses (22),we investigated whether IFN signal is involved in M1se-lectivity.Indeed,pretreatment with type I IFNs conferred re-sistance to M1in sensitive cancer cells(Fig.S5A).However,type I IFNs were not induced after M1infection in resistant cells(Fig. S5B),because it was reported that type I IFNs were not induced by alphavirus(at least at early time points)(23).Consistently, inhibition of type I IFNs signaling using IFN-αand IFN-βneu-tralizing antibodies or siRNA targeting IFN-αreceptor subunit IFNAR1did not affect the resistance to M1(Fig.S5C and D). These observations suggest that resistant cells do not exploit type I IFNs to establish the antiviral state against M1.We also found that M1viral RNA and protein levels increased dramatically as early as4h after infection in Hep3B cells but not L-02cells(Fig.3A and B),indicating that constitutively expressed intracellular antiviral factors are responsible for the resistanceof Fig.3.Expression profile and RNAi screening identify zinc-finger antiviral protein(ZAP)as a host factor that contributes to tumor tropism of M1.(A)L-02and Hep3B cells were treated with M1(MOI=10pfu per cell)for1–4h.The levels of viral genomic RNA and endogenous controlβ-actin were analyzed by qRT-PCR.The graph shows the means±SDs of the relative level of expression(normalized to endogenous controls)obtained in three independent experiments.(B)Western blot analysis of parallel samples from A.The protein levels of viral structural protein E1and nonstructural protein NS3were determined,and β-actin served as a loading control.(C)Schematic representation of systematic identification of host factors that regulate M1replication.(D)The results of the screen are shown with the siGENOME siRNA pools ranked in order of z score from lowest(decreased cell count)to highest(increased cell count).The position of ZAP is indicated.(E)Phase–contrast images of L-02cells treated first with siRNA(48h)followed by M1infection(MOI=30pfu per cell;48h)and crystal violet(0.1%)staining.CTL,control;DB,database;hpi,hours postinfection;siNC,negative control siRNA.(Scale bars:100μm.)4of9|/cgi/doi/10.1073/pnas.1408759111Lin et al.L-02cells.Therefore,we compared the transcriptional profiles of these two cell lines to identify candidate cell-encoded suppressors of M1replication.Considering that gene products in the IFN pathway are frequently defective in cancer (5,24,25)and IFN-stimulated genes (ISGs)are crucial antiviral effectors against alphavirus (26),we compiled a list of 317IFN-related genes (IRGs)from published data (the inclusion criteria are stated in Fig.3C and Table S3,and the expressions of IRGs are listed in Table S4).The IRGs with twofold-higher basal expression in L-02cells compared with Hep3B cells were submitted to the DAVID bioinformatics online tool (/)(27)for functional anal-ysis;53candidate genes were identified because of their previously reported antiviral effects.To test the antivirus effects against M1,we used an arrayed library of siRNA pools to target 53candidate genes in L-02cells (Fig.3C ).siRNA-transfected cells were either mock-infected or infected with M1(30pfu virus per cell)for 48h.Morphological changes were observed,and cell numbers were counted after crystal violet staining.We analyzed data from three independent screenings.Subtracting those genes with siRNA alone that was cytotoxic,we identified ZAP as a host factor against M1(Fig.3D and E and Table S5).ZAP Deficiency Is Necessary for the High Tumor Specificity of M1.ZAP is an ISG that inhibits the replication of certain viruses by inducing viral RNA degradation and translational inhibition (28–30).To investigate the role of ZAP,we examined the expression of ZAP in eight cell lines,including four M1-resistant cell lines and four M1-sensitive ones.We observed significantly reduced amounts of ZAP mRNA and protein levels in all four susceptible cells (Fig.4A and B ).In light of the evidence that ZAP is defective in M1-susceptible cells,we used gene silencing to address the contribution of ZAP to the antiviral state of resistant cell lines.Specifically,L-02,PLC,or cell line HCT 116cells were transfected with either a ZAP-specific siRNA or a nontargeting siRNA control.After 48h,the cultures were exposed to M1,and virus replication and cell viability were measured 48h after infection.The results showed that de-pletion of ZAP overcomes the resistance to M1in that it leads to increased viral replication,viral RNA,viral protein expression,and M1-induced cell death (Fig.4C –F ).The silencing efficiency was >80%according to Western blot analyses (Fig.4F ).The next question that we posed is whether ectopic expression of ZAP is able to confer resistance to M1.Consistently,susceptible cells transfected with vectors expressing ZAP showed decreased viral yield,decreased viral RNA and protein expressions,and fi-nally,suppressed viral oncolysis compared with negativecontrolFig.4.The sensitivity of cancer cells to M1requires ZAP deficiency.(A )mRNA levels of ZAP in different cell lines normalized to the expressions of β-actin and TBP.Means ±SDs of three independent experiments are shown.(B )Protein levels of ZAP in different cells.β-actin was used as a loading control.(C –F )Resistant cells transfected with siNC or siZAP were infected with M1for 48h.(C )Cell viability evaluated by MTT assay,(D )viral yield determined by TCID 50assay,(E )viral RNA quantified by qRT-PCR,and (F )viral protein analyzed by Western blot are shown.(G –J )Sensitive cells were transfected with plasmids expressing GFP (negative control)or ZAP for 48h and infected with M1for 48h.(G )Cell viability,(H )viral yield,(I )viral RNA,and (J )viral protein were measured by respective methods.Data are means ±SDs from three independent experiments.(C –E and G –I )All are compared with respective control groups.(C and G )Each color represents one cell line.ND,not detectable;ns,not significant;siNC,negative control siRNA;TBP,TATA box binding protein;TCID 50,median tissue culture infective dose.*P <0.05;**P <0.01;#P <0.05;##P <0.01;&P <0.05;&&P <0.01.Lin et al.PNAS Early Edition |5of 9M E D I C A L S C I E N C E SP N A S P L U S(GFP)(Fig.4G–J).Thus,we showed that ZAP is a host inhibitor that restrains M1replication and that M1specifically targets cancer cells carrying ZAP deficiency.Selective Oncolysis of M1Against Human ex Vivo Cancer Tissues Is ZAP Deficiency-Dependent.To validate the ZAP deficiency-dependent antitumor efficacy of M1,we carried out ex vivo experiments on primary human liver and colon tumor surgical samples by tumor histoculture end-point staining computer image analysis(TECIA) (31,32).Consistent with the in vitro and in vivo oncolytic effects, in23of35(66%)liver cancer samples and9of12(75%)colon cancer samples,exposure to M1triggered a decrease in the via-bility of cultured tumor tissue(percentage of inhibition>10%) (Fig.5A and B),supporting the therapeutic potential of M1against human cancers.Additionally,low mRNA levels of ZAP in tumor tissues cor-related to high ex vivo oncolytic efficacy of M1(Fig.5C).This correlation provided additional support to the hypothesis that the selective antineoplastic effect of M1virus is dependent on ZAP deficiency and indicated that ZAP deficiency may serve as a biomarker for response to M1oncolytic virotherapy.ZAP Deficiency Is Common in Human Cancers.To elucidate the po-tential for personalized therapy of M1for human cancers,we conducted a large-scale multicenter molecular pathology study of ZAP expression in various cohorts of human cancer specimens. ZAP immunohistochemistry(IHC)was performed on eight tissue microarrays(TMAs)containing paired tumor and adjacent non-neoplastic clinical specimens from506patients.ZAP expression was represented by mean staining intensity that was calculated using Imagescope software(Aperio).Overall,69%of liver cancer, 52%of colon cancer,and61%of bladder cancer TMAs showed low levels of ZAP in tumor tissue compared with respective noncancer tissue(Fig.6),implying that ZAP may be a biomarker for liver,colon,and bladder cancer and that M1may serve as a potential oncolytic agent for personalized cancer therapy. DiscussionOver the last several decades,increased understanding of mo-lecular virology and oncology has made it possible for us to select and/or tailor novel viruses for anticancer virotherapy(4).Here, we identify a naturally occurring alphavirus M1as a selective oncolytic agent targeting ZAP-deficient cancer cells.The onco-lytic effect of M1is potent and selective in that it kills a diverse range of cancer cell lines without inducing toxicity in primary normal cells.In addition,M1is efficacious in three aggressive, chemotherapy-refractory,preclinical tumor models on systemic infusion or intratumoral injection.To reveal M1as a clinically relevant therapeutic agent,we show that M1inhibits viability of primary human hepatic and colorectal tumor explants.Two factors are critical for oncolytic virotherapy,including effective delivery to tumor tissues and rapid virus growth within tumor sites(33).We have found that i.v.administration of M1is an effective means of delivering virus to tumor,and because of its high tumor tropism,M1thrives only within the tumor tissue. As a consequence,M1is exceptionally safe to the treated animals (M1does not cause mortality in immune-competent mice or rats, and all examined animals remained asymptomatic throughout the treatment)and may target metastatic tumors.It is of great use in the clinic to elucidate the molecular mech-anism of tumor tropism and identify biomarkers that predict an-titumor efficacies for each oncolytic virus(8).However,although plenty of natural and genetically engineered viral oncolytic agents have been developed,only few reports specified the mechanism of selectivity,including that reovirus requires an activated oncogenic Ras signaling(34)and that vesicular stomatitis virus(VSV)re-quires defects in IFN pathway(35).We hereby show that the resis-tance to M1in L-02and HCT116cells is type I IFN-independent (Fig.S5).Instead,we provide conclusive evidence that ZAP de-ficiency,which is common in human cancers,is essential for M1-selective replication and oncolysis in cancer cells,suggesting a great potential for personalized cancer therapy.One of the advantages of M1for human cancer treatment is that tumor biopsies can be prescreened for expression of ZAP,thus decreasing costs and ex-pediting the treatment of cancer patients.Our study also estab-lishes a successful model for identification of predictors of response to oncolytic virus using comparative expression profiling,functional screening,and TMA.We postulate that this model could add the breadth of opportunities for discovering novel markers that predict effectiveness to oncolytic virotherapy.It has been well-studied that ZAP is a host ISG that inhibits the replication of alphaviruses(28),filoviruses(36),and retro-viruses(37)but does not affect growth of other viruses,including VSV,poliovirus,yellow fever virus,and HSV-1(28).ZAP binds to viral RNA and recruits mRNA degradation machinery,lead-ing to decreased levels of viral RNA(38).ZAP also blocks translation of Sindbis viral RNA(28).We have found that both M1viral RNA and protein levels are dampened after ectopic expression of ZAP in susceptible cancer cell lines and vice versa, indicating that the production of M1is caused by the lack of ZAP-mediated antiviral activity(most likely viral RNA degra-dation but not excluding translational inhibition). Nevertheless,antitumor activity of a certain oncolytic virus differs among cancer cell lines(39).Our data also confirm that some cancer cells show poor response to M1(Table S1).Clearly, there are great opportunities to potentiate oncolytic efficiency by practical tactics,including use of chemical molecules tosensitize Fig.5.The ex vivo antineoplastic effect of M1depends on ZAP deficiency.(A and B)Ex vivo antitumor effect of M1on clinical tumor explants.Surgical(A) liver and(B)colon cancer specimens were divided into∼1-mm3particles and treated with M1(2×107pfu),vehicle,or HgCl2.Tissue viability was assessed by TECIA after MTT staining.(C)ZAP mRNA expression(normalized to the expression ofβ-actin)in parallel samples from A and B.Box-and-whisker plots showing median(horizontal line),interquartile range(box),and maximum/minimum range(whiskers)of the data.Resistant,M1-induced inhibition≤10%(n=12); Sensitive,M1-induced inhibition>10%(n=17).6of9|/cgi/doi/10.1073/pnas.1408759111Lin et al.cancer cells to oncolytic virus and exploitation of gene-armed therapeutic viruses.Some studies reported the enhanced oncolytic efficacy through rational design of combinational therapies (40).The use of com-bination therapeutic strategy would largely decrease the dosage of oncolytic virus and chemical drug,thus reducing the side effects and costs.Elucidation of molecular details of oncolytic agents could provide a breakthrough in the development of therapeutic strategies combining oncolytic viruses with small molecules.Some chemovirus combination therapies have been reported,including that histone deacetylase (HDAC)inhibitors enhance oncolysis of HSV or VSV by suppressing innate immunity (40),inositol-requiring enzyme 1(IRE1-α)inhibitors boost oncolytic efficacy of Maraba virus by inhibiting the ER stress response (41),and Smac mimetic compounds promote antitumor efficacy of VSV by exploiting the virus-stimulating cytokine storm (42).Understanding the molecular biology of M1-induced ER stress and subsequent cell death will help us to discover synergic compounds for combination therapy,such as IRE1-αinhibitors inducing ER stress (41).We are currently exploring the use of multiple ER stress inducers,some of which are in clinical trials,to facilitate the effectiveness of M1.Arming oncolytic viruses with therapeutic genes has been proven to be a successful strategy to increase the potency of these viruses (43).Additionally,previous work indicated that expression vectors based on alphaviruses (such as Sindbis virus and Semliki Forest virus)have been used extensively (44),and novel replication-competent vectors are being investigated for potential therapeutic applications (45,46).Thus,M1can be further armed with several complementary therapeutic proteins (e.g.,GM-CSF or IL-12)or noncoding RNAs to enhance the oncolytic efficacy mostly but not exclusively by unleashing anticancer immune response (7,8).Overall,our findings highlight an example of a potentially personalized cancer therapy using a targeted oncolytic virus that can be selectively administered to patients with ZAP-defective tumors.We predict that such agents will form the arsenal for the war on cancer in the future.Materials and MethodsCell Culture.Cell lines were purchased from American Type Culture Collection,Shanghai Institute of Cell Biology,and Guangzhou Institute of Biomedicine and Health.Cells were cultured in DMEM,RPMI-1640,or F-12supplemented with 10%(vol/vol)FBS and 1%penicillin/streptomycin (Life Technologies).Primary normal cells were purchased from ScienCell Research Laboratories and cultured according to instructions.Primary cancer cells were isolated from surgical tumor tissues using 0.1%trypsin.Specimens were obtained from consenting patients who underwent tumor resection.The institutional review board of Sun Yat-sen University Cancer Center has approved all human studies.Virus.M1was grown in Vero cells.Virus titer was determined by TCID 50assay using BHK-21cells and converted to pfu.The variant of M1in this study was described previously (10).Cell Viability Assay.Cells were seeded in 96-well plates at 4,000cells per well in 0.1mL media.After treatment,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was added to cells (1mg/mL final con-centration),and cells were allowed to grow at 37°C for another 3h.MTT-containing media were removed,and MTT precipitate was dissolved in 100μL DMSO.The optical absorbance was determined at 570nm using a microplate reader (iMark;Bio-Rad).Animal Models.This study was approved by the Animal Ethical and Welfare Committee of Sun Yat-sen University.For the intratumoral injection model,5×106Hep3B cells were inoculated s.c.into the hind flank of 4-wk-old female BALB/c-nu/nu mice.After 4d,palpable tumors developed (50mm 3),and mice were randomized to receive six doses of either M1(2×106pfu per dose)or vehicle intratumorally within 10d.Tumor length and width were measured every other day,and the volume was calculated according to the formula (length ×width 2)/2.Mice were weighed every other day.The observers were blinded to the group allocation.For evaluation of systemic antitumor effect,Hep3B s.c.xenografts were developed as described above,2×1064T1mammary carcinoma cells were injected orthotopically into the inguinal mammary fat pads of 6-wk-old fe-male BALB/c mice,and 2×106B16melanoma cells were inoculated s.c.into the hind flank of 6-wk-old female C57BL/6mice.After 3–5d,each animal was injected i.v.two times 3d apart with either M1(3×107pfu per dose)or vehicle.Tumor volume was calculated,and body weight was measured every 3d.The study was randomized and single blind.For the M1biodistribution study,5×106Hep3B and PLC HCC cells were injected s.c.into the left and right hind flanks,respectively,of 4-wk-old female BALB/c-nu/nu mice.After 4d,each animal received i.v.delivery of 3×107pfu M1.Mice were killed 1–4d after M1injection,and presence of virus was quantified by qRT-PCR from tissue samples,including tumors,brain,heart,kidney,liver,lung,muscle,and spleen.Similar experiments were performed with B16melanoma cells in 6-wk-old female C57BL/6mice.For the safety evaluation study,6-wk-old female BALB/c mice were i.v.-injected with two doses of either M1(3×107pfu per dose)or vehicle.Mice were weighed every 3d.After euthanasia,blood samples were submitted to the clinical laboratory of the First Affiliated Hospital of Sun Yat-sen University for CBC analysis,and vital tissues (including brain,heart,kidney,liver,lung,skeletal muscle,and spleen)were histologically analyzed after H&Estaining.Fig.6.Distribution of ZAP deficiency in clinical cancer specimens.(A )Representative cores of ZAP immunostaining in TMA.Higher magnification shown in the box.(Scale bars:50μm.)(B )Statistical analysis of IHC staining intensity.Box-and-whisker plots showing median (horizontal line),interquartile range (box),and 5th –95th percentiles (whiskers)of the data.Dots indicate outliers.***P <0.001.(C )Distribution of ZAP deficiency in cancers.N,nonneoplastic;T,tumor.Lin et al.PNAS Early Edition |7of 9M E D I C A L S C I E N C E SP N A S P L U S。

mAbs：自身免疫性疾病的潜在治疗抗体2014年10月28日讯/生物谷BIOON/ --通过天然免疫选择和靶向蛋白技术的组合，A * STAR研究者已经制造出一种抗体，能够在人类细胞和小鼠实验模型中有效地阻断与疾病相关的炎症通路。

感染或受伤事件发生后，信号蛋白白细胞介素1β（IL-1β）帮助免疫系统进行快速免疫反击。

然而，过量的IL-1β活性可导致有害的炎症，促进疾病状态如各种自身免疫性疾病。

IL-1β抑制剂是药物研发的活跃区域，Cheng-I Wang和同事最近生成IL-1β特异性抗体，这可能是解决炎症性疾病的关键。

哺乳动物免疫系统已经进化能产生抗体，能结合外来分子，并且具有显著的亲和性和特异性。

Wang和同事们利用蛋白质工程，获得一个有前途的抗体，被证明能够结合并抑制人类和小鼠的IL-1β。

研究人员集中在抗体的一小部分氨基酸，即可能有助于抗体结合IL-1β的氨基酸，随机置换这些氨基酸位点并产生抗体变体的一个库。

通过筛选该抗体变体库，Wang和同事获得性能显着提高的抗体，得到了20〜50倍更好亲和力的抗体变体。

这些抗体的最好变体是P2D7，其在抑制IL-1β上比康纳单抗（市售消炎药）更有效。

康纳单抗和P2D7绑定相同的蛋白质，但识别相同分子上不同位点。

此外，P2D7结合至小鼠和猴子版本的IL-1β蛋白，并具有高亲和力的，而康纳单抗却没有此功效。

研究人员利用小鼠模型显示，P2D7能对抗关节炎和腹膜炎症状，甚至延长了已经注射了人骨髓瘤细胞动物的存活。

（生物谷）本文系生物谷原创编译整理，欢迎转载！转载请注明来源并附原文链接。

谢谢！doi:10.4161/mabs.28614PMC:PMID:A novel human anti-interleukin-1βneutralizing monoclonal antibody showing in vivo efficacyGoh, A. X. H., Bertin-Maghit, S., Yeo, S. P., Ho, A., Derks, H. et al.The pro-inflammatory cytokine interleukin (IL)-1βis a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1βhave been approved to treat diseases suchas rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1βwith a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1βcompared with its parent antibody. This anti-human IL-1βIgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1βpathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1βmonoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.Cancer Cell：癌症外染色体微小分子新功能来源：生物谷2014-10-28 18:092014年10月28日讯/生物谷BIOON/ --近日研究发现，通过癌细胞释放的外染色体、微小的病毒型颗粒可以表达导致肿瘤生长的微RNA分子。