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1、A New Approach to Cancer Immunotherapy肿瘤免疫治疗的新思路斯坦福大学医学院外科宗康拉,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,5x105 tumor cells,How to do antitumor experiments如何做肿瘤治疗效果试验,32-day MCA207 before treatment,One week after Cy+IL-12,Two weeks after Cy+IL
2、-12,Three weeks after Cy+IL-12,Four weeks after Cy+IL-12,Five 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,Day-18 peritoneal MCA207 tumors18天腹腔实体MCA207肿瘤,Day-14 experimental lung metastases14天
3、静脉注射建立的肺扩散肿瘤模型,Antitumor effects of Cy+IL-12 in MCA207 i.p. and i.v. modelsCy+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
4、,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,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
5、前,after之后,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,TCRb KOT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,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-3BALB/c
6、: CT26, CSA1M, OV-HMA/J: Sa1Non-responding tumors:C57BL/6: MCA101,B16, LLC, Pan02, EL-4BALB/c: 4T1, S180,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？,Does Cy+IL-12 work on other tumor models?环磷酰胺加白介素12是否对
7、所有肿瘤有效？,Condition#1：Pre-existing immunity条件一：预存免疫,What is pre-existing immunity?什么是预存免疫？,Antigenspecific 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-me
8、diated Th1 type宿主对肿瘤的应答属于Th1型细胞反应,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,Tumor-sensitized T cells are necessary for I
9、L-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-
10、immune T cells,nave T cells,Cy+IL-12,0/5,Ccondition #2 条件2IL-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,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,Critical timing of IL-12 administration白介素12 给药时间的关
11、键性,IL-12 timing following Cy,cure rate治愈率,day 3-7（第37天）,100%,day 7-11第711天）,day 14-18（第1418天）,40%,0%,Large MCA207 modelCy at 125 mg/kgIL-12 at 200 ng x 3 (q.o.d.),Ccondition #3 条件3chemotherapy must activate antitumor immunity化疗必须激活一个抗肿瘤免疫反应,Chemotherapy to activate antitumor immunity?化疗引发抗肿瘤免疫反应？,Im
12、munity 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 used3-day tumor is non-palpable7-10 tumors are 2-5 mm in sizeCy at 125 mg/kg is used,PEI status,not ye
13、t,established,never,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,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
14、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,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
15、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 21DC=cultured immature DC at 1x106 given intratumor two days after Cy,Chemotherapy Responses, Relapse and Resistance to Repeated Therapy化疗应答，复发及随后的抗药性,Rel
16、apsed 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,1st Cy,time,tumor size,2nd Cy,normal,no T cell,Relapsed tumor following Cy becomes resistant to Cy+IL-12 therapy复发后的肿瘤对CyIL-12也产生抗药性,mice bearing,response to,Cy+IL-12,Cy alone,untreated tumor,Cy-treated relapsed
17、tumor,+,100% cure,+,20% cure,MCA207 large tumor model used+: significant tumor regression following Cy+: transient shallow tumor regression following Cyresponse to Cy+IL-12: cure by standard Cy+IL-12 therapy,What is the reason for relapse-associated resistance to repeated chemotherapy?产生抗药性的原因是什么呢？M
18、DR?,Selection of chemo-resistant tumor cell is NOT the reason for resistance抗药性不是由于筛选了抗药性肿瘤细胞的原因,tumor from,sensitivity to Cy after replanting in nave host,untreated stock,after 1st Cy relapse,after 2nd Cy relapse,100%,100%,100%,Sensitivity to Cy in vivo is measured by complete eradication of small
19、tumors established in nave mice by Cy therapy,Tolerance of antitumor immunity is responsible for relapse-associated resistance to Cy+IL-12抗药性是由于抗肿瘤免疫反应产生耐受,Transfer of chemo-resistance from relapsed host to untreated host化疗抗药性可以通过免疫耐受转导达到,Immune cell donor（脾细胞供体）: Cy-treated tumor relapsed mice（化疗后肿
20、瘤复发的小鼠）Nave mice or （净鼠）Untreated tumor-bearing mice （未治疗过的荷瘤鼠）Immune cell recipient（脾细胞受体）: mice bearing primary day-10 MCA207 tumor （荷瘤鼠）Manipulation: transfer immune cells and then treat with Cy（导入脾细胞然后开始环磷酰胺的治疗）,Transfer of resistance from relapsed host to untreated host抗药性可以从带复发肿瘤的供体通过脾细胞导入未受治疗
21、的荷瘤鼠,time,tumor size,Cy,control spl cells,relapsed spl cells,The new insight into the high efficacy of Cy+IL-12环磷酰胺加白介素12之所以有效的最新解释,Host bearing immunogenic tumor generates pre-existing immunity免疫型肿瘤在其宿主体内诱发预存免疫This immunity is too week and too late to control the primary tumor这个免疫反应太弱太迟，不足以控制原发肿瘤Ch
22、emotherapy kills large number of tumor cells and releases large amount of tumor antigen化疗药物杀死一些肿瘤细胞，释放大量肿瘤抗原,This newly released tumor antigen induces an acute recall immune response 新释放的肿瘤抗原引发急性免疫回放反应The chemotherapy-triggered immunity runs into exhaustion due to autoimmune-protective mechanism and
23、 becomes tolerated这个反应最终由于自身免疫保护机制而停止而产生耐受In the presence of IL-12 during early phase, the response turns into a Th1 type and persists longer白介素12 的存在使这个反应转变成Th1型，持久下去,The new insight into the high efficacy of Cy+IL-12环磷酰胺加白介素12之所以有效的最新解释,Other chemotherapy drugs and other tumor models: The same fin
24、dings apply其他化疗药和肿瘤模型：同样的情况,Doxorubicin vs. Cyclophosphamide阿霉素与环磷酰胺的对比,Cyclophosphamide a pro-drug, must be given systemically （需要代谢，只能全身给药）narrow anti-tumor range （抗肿瘤范围较窄）well-known immune regulator （著名的免疫调节剂）removes suppressive T cells at low dose (去除抑止性功能细胞）enhance type I cytokine for T cell gr
25、owth （提高Th1型因子分泌）create space for T cell expansion （创造T细胞扩增空间）Doxorubicinactive both systemically and locally（可以全身或局部给药）wide anti-tumor range（抗肿瘤范围较广）not known for immune regulatory activity（对免疫调节功能不祥）,Response of established MCA207tumors to Dox+IL-12阿霉素加白介素12对MCA207肿瘤的治疗效果,Dox dose and route阿霉素的剂量和
26、给药方式,cure rate,i.v. at 16mg/kg,i.t. at 10mg/kg,i.t. at 5mg/kg,i.t. at 2.5mg/kg,80%,100%,20%,0%,Large and mid-sized MCA207 modelDox alone failed to cure any tumors,Response of another tumor to Dox+IL-12阿霉素加白介素12对另外一个肿瘤的治疗效果,cure rate（治愈率）,Treatment治疗手段,None无,Dox alone单独阿霉素,IL-12 alone单独白介素12,Dox+IL-1
27、2阿霉素加白介素12,80%,0%,0%,0%,Day 12-14 established MCA203 sc tumorsDox at 5mg/kg i.t.IL-12 started 3 days after Dox,Local gemcitabine+IL-12 in the MCA207 tumor model肿瘤局部洁西它滨加白介素12治疗MCA207肿瘤,cure rate（治愈率）,Treatment治疗手段,None无,Gem alone at 2.5- 10mg/kg,IL-12 alone单独白介素12,Gem-50mg/kg+IL-12,80%,0%,0%,0%,Gem-
28、20mg/kg+IL-12,Gem-10mg/kg+IL-12,20%,40%,Day-14 established MCA207 sc tumorsGemcitabine given intratumorallyIL-12 started 3 days after gemcitabine,A case of a weakly immunogenic tumor model一个弱免疫型肿瘤的模型,Pan02 is a weekly immunogenic tumorPan02是弱免疫型肿瘤Pan02 does not respond to Cy+IL-12 or Dox+IL-12, but
29、respond weakly to Gem+IL-12 (inhibition)白介素12不能提高环磷酰胺和阿霉素的化疗疗效，但可以稍微增加洁西它滨的疗效（肿瘤生长抑止）Small established Pan02 can be cured under the most optimal treatment combination of Gem+DC+IL-12联合应用洁西它滨，树突细胞加白介素12 可以根治生成的小肿瘤,Response of Pan02 tumors to gemcitabine, DC and IL-12 Gem+DC+IL-12在Pan02肿瘤模型的效果,cure ra
30、te治愈率,treatment治疗,Gem alone,IL-12 alone,0/5,0/5,Gem+IL-12,Gem+DC,0/5,0/5,9-day established Pan02 sc tumors (3-7 mm)Gemcitabine given intratumorally (i.t) for 3 days and then weeklyDC and IL-12 started one days after gemcitabine and then weekly,Gem+DC+IL-12,4/5,DC alone,0/5,Not all chemotherapy drugs
31、 can activate antitumor immunity 并非所有的化疗药都能与白介素12配合使用,cure rate,Tumor model,MCA207,0/10,Pan02,vinblastin+IL-12,0/10,0/5,0/20,drug/treatment,MCA207,MCA207,paclitaxal+IL-12,5-FU+IL-12,Cy+IL-12,30-day established MCA207 sc tumorsChemotherapy was given either i.t or ip a single timeIL-12 started 3 days
32、after chemotherapyResults of multiple experiments was combined,Not all cytokines can match the effect of IL-12并非其他白介素因子能够替代白介素12,cure rate,Tumor model,MCA207,10/10,Cy+IL-12,1/10,1/10,0/10,drug/treatment,MCA207,MCA207,Cy+IL-2,Cy+IFN-g,Cy+IL-23,Cy+TNFa,MCA207,MCA207,0/10,Small or large established MCA
33、207 sc tumorsChemotherapy was given ip a single time at 120mg/kgAll cytokines were started 3 days after chemotherapy and lasted for 5-7 daysResults of multiple experiments was combined,The best way to use IL-12 for immunotherapy白介素12的最佳使用环境,对肿瘤和宿主的选择抗原具备较强的免疫原性对化疗药的选择制造大量抗原释放对给药方式的选择诱发强烈的记忆应答对因子的选择使
34、用白介素12调节应答对效果的选择造成 持续的应答及防止耐受生成,Why have the early clinical trials of IL-12 failed？为什么白介素12的早期临床实验失败了？,白介素12早期临床试验,GI/Wyeth做的一期临床40肿瘤病人（肾癌，黑色素癌，直肠癌）身体状况良好（Karnofsky指数70）31000ng/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（3080）,白介素12
35、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做的一期临床28例肾癌病人皮下给药，每周一次，100-1250ng/kgMTD为1000ng/kg1PR（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（3080）,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/kg2例PR没有明显的
36、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
37、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,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,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/kg1CR（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
38、胺酶升高Beta2-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,低剂量白介素12与疫苗结合黑色素癌抗原Melan-A的片段IL-12:0100ng/kg,第一，第22天（静脉或皮下）Melan-A肽段：第1，8，15，22，57gp100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
39、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300 x106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
40、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,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
41、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,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,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
42、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,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（1040）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,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,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
43、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,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,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
44、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？,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,In vitro T c
45、ell response to tumor antigen is the indicator of in vivo immune response,naive,tumor-bearing,Cy,Cy+IL-12,IFN-gamma,spleen from,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
46、 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-12 as segregate marker for antitumor immunity,Chemotherapy enhances T cell response to IL-12化疗提高T细胞对白介素12 的应答,IL-12 responses by T cells from normal subj
47、ects 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,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,
