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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,1),Slower tumor growth (,抑制 90%),2) Tumors shrink (,肿瘤体积减小,少见),3) Tumors disappear (,治愈, 极少),治疗,0-30,days,5,x10,5,tumor cells,How to do antitumor experiments,如何做肿瘤治疗效果试验,1) Slower tumor growth (抑制 90,32-,day MCA207 before treatment,32-day MCA207 before treatment,One week after Cy+IL-12,One week after Cy+IL-12,Two weeks after Cy+IL-12,Two weeks after Cy+IL-12,Three weeks after Cy+IL-12,Three weeks after Cy+IL-12,Four weeks after Cy+IL-12,Four weeks after Cy+IL-12,Five weeks after Cy+IL-12,Five weeks after Cy+IL-12,Six weeks after Cy+IL-12,Six weeks after Cy+IL-12,Potentiation of Cy-induced Cancer Regression by IL-12,通过白介素12提高化疗的抗肿瘤效果,no Rx,Cy alone,IL-12 alone,Cy+IL-12,Potentiation of Cy-induced Can,Day-18 peritoneal MCA207 tumors,18,天,腹腔实体,MCA207,肿瘤,Day-18 peritoneal MCA207 tumor,Day-14 experimental lung metastases,14,天,静脉注射建立的肺扩散肿瘤模型,Day-14 experimental lung metas,Antitumor effects of Cy+IL-12 in MCA207 i.p. and i.v. models,Cy+IL-12,在,腹腔及肺扩散肿瘤模型中的治疗效果,route of,inoculation,接种,treatment,(start at day),治疗(,起始时间),cure rate,(survival days),治愈,率(存活天),i.p.,腹腔,i.p.,腹腔,i.v.,肺,扩散,None,无,Cy+IL-12 (18),Cy+IL-12 (14),0/5 (20-27),0/5 (21-31),5/5 (90),8/8 (90),i.v.,肺,扩散,None,无,Antitumor effects of Cy+IL-12,Effects of IL-12 and Cy+IL-12 in the Sa1 ascites tumor modelIL-12,和,Cy+IL-12,在,Sa1,腹水肿瘤模型中的治疗效果,100%,50%,0%,saline,生理盐水,IL-12,Cy,Cy+IL-12,0,10,20,30,40,tumor,接种,treatment,治疗,survival,存活率,days,天数,Effects of IL-12 and Cy+IL-12,The rejection induced by IL-12/Cy+IL-12 is associated with a strong T cell response,与,肿瘤排斥所对应的强免疫反应,CD4,CD8,before,之前,after,之后,The rejection induced by IL-12,Tumor rejection is mediated by a Th1 response,抗,肿瘤作用需要,Th1,型,T,细胞参与,host,宿主,cure rate,治愈率,Normal,正常,TCR,b,KO,T,细胞受体敲除,Nude,裸鼠,IFN-,g,KO,咖玛干扰素敲除,IL-4 KO,白介素-4,敲除,10/10,0/3,0/20,0/20,10/10,Tumor rejection is mediated by,Does Cy+IL-12 work on other tumor models?,环磷酰胺,加白介素是否对所有肿瘤有效?,Responding tumors:,C57B/6: MC203, MCA205, MCA207, FBL-3,BALB/c: CT26, CSA1M, OV-HM,A/J: Sa1,Non-responding tumors:,C57BL/6: MCA101,B16, LLC, Pan02, EL-4,BALB/c: 4T1, S180,Does Cy+IL-12 work on other tu,Question,问题,If immunotherapy is able to eradicate late-stage large tumor burdens, what is the proper condition for it?,如果免疫疗法有可能治愈晚期癌症,条件是什么?,Question 问题If immunotherapy is,Does Cy+IL-12 work on other tumor models?,环磷酰胺,加白介素,12,是否对所有肿瘤有效?,Does Cy+IL-12 work on other tu,Condition#1,:,Pre-existing immunity,条件一:,预存免疫,Condition#1:Pre-existing immu,What is pre-existing immunity?,什么是预存免疫?,Antigen,specific recognition of tumor by the host immune system,宿主对肿瘤抗原有特异性识别,The immune system has responded to the existing tumor prior to therapy start,宿主免疫系统在治疗之前已经对肿瘤有攻击,The host response to the tumor is cell-mediated Th1 type,宿主对肿瘤的应答属于,Th1,型细胞反应,What is pre-existing immunity?,Experimental procedure for adoptive cell transfer,体细胞转导试验步骤,donor,供体,tumor vaccine,瘤苗,tumor challenge,肿瘤接种,tumor-free,排斥接种,recipient,受体,tumor challenge,肿瘤接种,14,day,T cell transfer,输入,T,细胞,T cell,IL-12/Cy+IL-12,治疗,Response,效果?,2,day,Experimental procedure for ado,Tumor-sensitized T cells are necessary for IL-12-induced tumor rejection,肿瘤,特异的,T,细胞在白介素12治疗中的关键作用,donor cells,输入,细胞,treatment,治疗,cure rate,治愈率,none,nave T cells,tumor-immune T cells,IL-12,saline,saline,IL-12,IL-12,Cy+IL-12,0/10,assembly of pre-existing immunity in T cell-deficient host,0/8,0/8,1/10,9/12,10/10,tumor-immune T cells,nave T cells,tumor-immune T cells,nave T cells,Cy+IL-12,0/5,Tumor-sensitized T cells are n,C,condition #2,条件,2,IL-12 should be given during the early phase of recalled pre-existing immunity,白介素,12,最佳给药时间是在预存免疫的回放早期,Ccondition #2 条件2IL-12 shoul,Timing of IL-12 following chemotherapy,白介素,12,的给药最佳时期,time,tumor size,Cy,IL-12,IL-12,IL-12,Timing of IL-12 following chem,Critical timing of IL-12 administration,白介素,12,给药时间的关键性,IL-12 timing following Cy,cure rate,治愈率,day 3-7,(第,3,7,天),100%,day 7-11,第,7,11,天),day 14-18,(第,14,18,天),40%,0%,Large MCA207 model,Cy at 125 mg/kg,IL-12 at 200 ng x 3 (q.o.d.),Critical timing of IL-12 admin,C,condition #3,条件,3chemotherapy must activate antitumor immunity,化疗必须激活一个抗肿瘤免疫反应,Ccondition #3 条件3chemotherap,Chemotherapy to activate antitumor immunity?,化疗引发抗肿瘤免疫反应?,Chemotherapy to activate antit,Immunity is responsible for cure of small tumor by Cy chemotherapy,免疫参与是环磷酰胺化疗治愈小肿瘤的必要条件,host,70-100%,tumor burden,cure rate,normal,normal,no T cell,3-day,7-10-day,8-day,0%,0%,MCA207 small tumor model used,3-day tumor is non-palpable,7-10 tumors are 2-5 mm in size,Cy at 125 mg/kg is used,PEI status,not yet,established,never,Immunity is responsible for cu,Immunity is responsible for significant large tumor regression following chemotherapy,免疫参与加大环磷酰胺化疗疗效,Cy,time,tumor size,normal,no T cell,Immunity is responsible for si,Why can chemotherapy activate antitumor immunity?,为什么化疗可以激活抗肿瘤免疫反应?,Through acute antigen release and recall of pre-existing immunity,通过抗原急性释放达到记忆免疫的回放,If true, then increase antigen presentation at the site of antigen release may increase response to chemotherapy,如果如此,那么在抗原释放位置增加抗原呈递救有可能提高化疗疗效,Why can chemotherapy activate,Increase antigen presentation by DC following chemotherapy enhances tumor responses,提高化疗后的抗原呈递可以提高化疗疗效,cure rate,治愈率,treatment,治疗,Cy alone,单独化疗,DC alone,单独树突细胞,Cy + DC,联合治疗,Responses,应答,regr. relapse,2/10,progression,regression,0/10,10/10,Medium sized (8-11 mm) MCA207 tumor used.,Cy= 120mg/kg given on day 21,DC=cultured immature DC at 1x10,6,given intratumor two days after Cy,Increase antigen presentation,Chemotherapy Responses, Relapse and Resistance to Repeated Therapy,化疗应答,复发及随后的抗药性,Chemotherapy Responses, Relaps,Relapsed tumor is resistant to repeated chemotherapy with Cy in normal mice,肿瘤复发后对二次化疗产生抗药性,1,st,Cy,time,tumor size,2,nd,Cy,normal,no T cell,Relapsed tumor is resistant to,Relapsed tumor following Cy becomes resistant to Cy+IL-12 therapy,复发后的肿瘤对,Cy,IL-12,也产生抗药性,mice bearing,response to,Cy+IL-12,Cy alone,untreated tumor,Cy-treated relapsed tumor,+,100% cure,+,70,),3,1000ng/kg/day,静脉给药,每周,5,天,隔周重复,一共两次,一个,CR,(,melanoma),,一个,PR(RCC),MTD,为,500ng/kg,毒副作用:,感冒症状(发烧,恶心,呕吐,厌食,懒惰),口疮,肝脏转胺酶升高,血液白细胞,淋巴细胞暂时下降(,30,80,),白介素12早期临床试验GI/Wyeth做的一期临床,白介素,12,早期临床试验,Roche,做的准一期临床,10,例黑色素瘤病人,皮下给药,每周一次,,500ng/kg,有混和临床疗效,但达不到,PR,标准,毒副作用:,感冒症状,肝脏转胺酶升高,血液白细胞,淋巴细胞暂时下降,白介素12早期临床试验Roche做的准一期临床,白介素,12,早期临床试验,Roche,做的一期临床,28,例肾癌病人,皮下给药,每周一次,,100-1250ng/kg,MTD,为,1000ng/kg,1PR,(,500ng/kg,),毒副作用:,感冒症状(发烧,恶心,呕吐,厌食,懒惰),口疮,肝脏转胺酶升高,血液白细胞,淋巴细胞暂时下降(,30,80,),白介素12早期临床试验Roche做的一期临床,白介素,12,早期临床试验,Roche,做的二期临床试验,设计,80,例,实际,30,例肾癌病人,皮下给药,每周一次,逐渐上升到,1250ng/kg,2,例,PR,没有明显的毒副作用,试验由于达不到预期的临床疗效而停止,白介素12早期临床试验Roche做的二期临床试验,白介素,12,的二期临床事故,GI,做的二期临床,17,肾癌病人,500ng/kg,静脉给药,每周,5,天,12,个病人在两次注射之后发生,4,级副作用,2,个病人最终死于副作用(胃肠道出血和结肠炎),白介素12的二期临床事故GI做的二期临床,白介素,12,二期临床事故的原因,二期省略了预备注射(,pre-dosing),步骤,预备注射是在连续注射之前一周单独给药,预备注射降低了连续给药时的血液伽马干扰素水平,动物(小鼠和猴子)试验可以证明预备注射降低毒副作用的意义,实际问题是只要控制伽马干扰素水平就可以防止白介素,12,的毒副作用,白介素12二期临床事故的原因二期省略了预备注射(pre-do,白介素,12,后期临床试验,自从事故发生后,两家公司均对高剂量白介素,12,的临床应用产生怀疑,此后的白介素,12,肿瘤临床试验多为学术机构以研究为目的进行的中低剂量试验,新的动物试验表明低剂量的白介素,12,有时也可以起到抗肿瘤效果,白介素12后期临床试验自从事故发生后,两家公司均对高剂量白介,白介素,12,后期临床试验,降低给药频率,但保持,500ng/kg,给药量,28,个肾癌病人,每周两次(第一,四天)静脉注射,一个,PR,三个,SD,停药后仍观察到持续的肿瘤消退,没有严重毒副作用,因为疗效与伽马干扰素的持续诱发相关,认为增加白介素,2,可能会提高疗效(后来证明是错误的),白介素12后期临床试验降低给药频率,但保持500ng/kg给,白介素,12,后期临床试验,皮下注射,降低单次药量,增加频率,15,个肾癌,黑色素癌及大肠癌病人,每周三次,每次,50,,,100,,,300ng/kg,1CR,(,50ng/kg),1PR(300ng/kg),毒副作用:,白细胞,淋巴细胞和中性淋巴细胞抑制,肝脏转胺酶升高,Beta,2-,微球蛋白及,C,反应蛋白升高,(,系统炎症),白介素12后期临床试验皮下注射,降低单次药量,增加频率,白介素,12,后期临床试验,低剂量白介素,12,与疫苗结合,黑色素癌抗原,Melan-A,的片段,IL-12:0,100ng/kg,第一,第,22,天(静脉或皮下),Melan-A,肽段:第,1,,,8,,,15,,,22,,,57,gp100,试验:,IL-12:30ng/kg,与肽段同时,同点皮内注射,gp100:,两周一次(,x2),到四周一次,(x2),到八周一次,没有明显毒副作用,没有明显白介素,12,与临床效果的对应,白介素12后期临床试验低剂量白介素12与疫苗结合,白介素,12,临床失败的主要原因,没有掌握白介素,12,的最佳使用条件,没有预存免疫回放的存在,排除任何接受放化疗的病人,没有给药依据,白介素,12,受体表达不明:没有任何测试,给药剂量错误,高剂量造成,NK,细胞激活,,T,细胞抑止,大量游离伽马干扰素造成系统炎症,白介素12临床失败的主要原因没有掌握白介素12 的最佳使用条,白介素,12,在抗病毒方面的临床试验,抗艾滋病(,HIV,)一期临床试验,65,个艾滋病毒感染者,CD4,细胞数,300x10,6,皮下注射,,30-300ng/kg,一周两次(共,4,周),没有明显毒副作用,没有明显的抗病毒效果,体外细胞特异免疫功能没有明显变化,白介素12在抗病毒方面的临床试验抗艾滋病(HIV)一期临床试,白介素,12,在抗病毒方面的临床试验,抗慢性丙肝病毒(,HCV,)的,I/II,期临床试验(,Roche),60,个慢性丙肝病人,皮下注射,,30-500ng/kg,每周一次,一共,10,周,毒副作用同以前报道过的相同,不严重,与剂量有关,根据剂量有,17-53,的病人病毒,RNA,量下降,50%,肝脏转胺酶没有明显下降,没有完全病毒转阴的情况,疗效比不上,a,干扰素(,10,转阴),白介素12在抗病毒方面的临床试验抗慢性丙肝病毒(HCV)的I,白介素,12,在抗病毒方面的临床试验,抗慢性丙肝病毒(,HCV,)的临床试验(,GI,),225,个抗,a,干扰素的慢性丙肝病人,皮下注射,,500ng/kg,每周两次,一共,12,周,百分之三的病人因副作用退出试验,百分之一的病人中有疗效,肝脏活检没有发现明显变化,肝脏转胺酶水平没有明显下降,白介素12在抗病毒方面的临床试验抗慢性丙肝病毒(HCV)的临,白介素,12,在抗病毒方面的临床试验,抗慢性乙肝病毒(,HBV,)的临床试验(,Rohe,),46,个慢性乙肝病人,皮下注射,,30,250,500ng/kg,每周一次,,12,周,病毒转阴率:,15,左右,病毒转阴伴随,HBeAg,抗原转阴和转胺酶正常化,副作用与剂量相关但没有严重情况出现,白介素12在抗病毒方面的临床试验抗慢性乙肝病毒(HBV)的临,为什么有些乙肝病人有应答而另一些没有?,白介素,12,的主要功能是修饰激活后的,T,细胞,防止耐受,在没有,T,细胞激活的情况下,白介素,12,主要靠,NK,生成的伽马干扰素有一些左右,但不治本,个别病人在治疗期间发生自发的再激活(,reactivation),,造成对白介素,12,的应答,为什么有些乙肝病人有应答而另一些没有?白介素12的主要功能是,白介素,12,的当前处境,基本分子专利于,2010,年到期,两家美国公司已于,2000,年之前放弃自己进行白介素,12,的临床开发,剩余的白介素,12,由,GI,公司交给美国,NIH,使用,从,2005,年,6,月以后,GI,拒绝继续向,NIH,提供临床级白介素,12,目前西方国家没有可供临床试验使用的白介素,12,白介素12的当前处境基本分子专利于2010年到期,肿瘤免疫治疗的新思路课件,重新启动白介素,12,临床应用的机会,美国,Wyeth,公司因为临床事故与缺乏疗效而放弃了白介素,12,的开发,中国药厂指望白介素,12,在美国上市后仿制的道路已经堵死,中国有数家重组白介素,12,的,GMP,生产,中国有庞大的白介素,12,应用适应症人群:肿瘤,乙肝,艾滋病,这些因素与我们最新理念的结合,重新启动白介素12临床应用的机会美国Wyeth公司因为临床事,白介素,12,的临床最佳应用角度,肿瘤治疗方面,常规放化疗对实体瘤的一部分(,10,40,)有抑止但无法根治,无法根治的原因一部分是因为放化疗激活的免疫反应进行不彻底并形成免疫耐受,白介素,12,一方面推进免疫反应的强化和深化,另一方面防止耐受,白介素12的临床最佳应用角度肿瘤治疗方面,实体瘤(肺癌,乳腺,消化道,肝癌等),放化疗,急性免疫回放反应,抗原释放,树突细胞,应答,耐受,复发,死亡,白介素,12,持续应答,治愈或带瘤生存,实体瘤(肺癌,乳腺,消化道,肝癌等)放化疗急性免疫回放反应抗,白介素,12,的临床最佳应用角度,肝炎治疗方面,免疫系统对乙肝病毒有良好识别,病毒和免疫系统之间交叉抑止,慢性乙肝的无法根治的原因是因为抗病毒免疫反应进行不彻底并形成免疫耐受,白介素,12,如果用在抗病毒免疫反应回放发生的早期可以一方面推进免疫反应的强化和深化,另一方面防止耐受,白介素12的临床最佳应用角度肝炎治疗方面,慢性乙肝病人,白介素,12,耐受打破,病毒复制,急性免疫回放,病毒量下降,肝损伤,二次耐受,病毒量持续下降,病毒清除,根治,慢性乙肝病人白介素12耐受打破病毒复制急性免疫回放病毒量下降,白介素,12,临床应用的关键是必须建立在一个新激活的免疫反应之上,白介素12临床应用的关键是必须建立在一个新激活的免疫反应之上,Can we assume that patients experiencing delayed significant response to cytoreduction therapy have activated antitumor immunity behind tumor regression?,我们是否可以假定对放化疗等肿瘤减负手段产生持续应答的病人是由于诱发了抗肿瘤免疫反应?,Can we assume that patients ex,How to detect the presence of the immunity we are looking for?,怎样确定有没有免疫应答的存在?,Direct test: T cell responses to tumor antigen,直接证据:,T,细胞对肿瘤抗原的体外应答,Indirect marker: in vitro T cell responses to IL-12,间接证据:,T,细胞对白介素,12,的体外应答,How to detect the presence of,In vitro T cell response to tumor antigen is the indicator of in vivo immune response,naive,tumor-bearing,Cy,Cy+IL-12,IFN-gamma,spleen from,In vitro T cell response to tu,T cell response to IL-12T,细胞对白介素,12,的应答,Nave or resting T cells do not respond to IL-12 (lack of IL-12 receptor),未活化的,T,细胞不表达白介素,12,受体,因而无应答,Certain activated T cells express high affinity IL-12 receptor,某些活化的,T,细胞表达高敏度白介素,12,受体,Response to IL-12 leads to T cell production of IFN-gamma,对白介素,12,的应答导致,T,细胞分泌伽马干扰素,T cell response to IL-12T细胞对白,T cell response to IL-12 as segregate marker for antitumor immunity,T cell response to IL-12 as se,Chemotherapy enhances T cell response to IL-12,化疗提高,T,细胞对白介素,12,的应答,Chemotherapy enhances T cell r,IL-12 responses by T cells from normal subjects and cancer patients,正常人和癌症病人,T,细胞对白介素,12,的应答,Uno K, Mitsuishi Y, Tanigawa M, et al.,Cancer Immunol Immunother,52: 33-40, 2003,normal,post-surgery,cancer,normal,post-surgery,cancer,IL-12 responses by T cells fro,New clinical approach to cancer immunotherapy,新型免疫治疗的临床应用,Select patients who demonstrate signs of response to cytoreduction therapy,选择对放化疗等减负治疗有应答的肿瘤病人,Screen patients T cell response to IL-12,检测病人,T,细胞对白介素,12,的应答,Give IL-12 to patients who demonstrate significant tumor regression and elevated T cell response to IL-12,对有,T,细胞应答的病人实施白介素,12,的综合治疗,New clinical approach to cance,
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