预防和治疗移植物抗宿主病(GVHD)的新策略

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1 预防和治疗移植物抗宿主病(GVHD)的新策略
Simrit Parmar, MD Stem Cell Transplant & Cellular Therapy BTG2013, Hong Kong

2 急性 GVHD的危险因素 HLA 配型不合 年龄增加 供受者性别不同 所患疾病类型和状态 放疗数量和移植预处理方案的强度
氨甲蝶林和环孢素或他克莫司的剂量

3 急性 GVHD: 病理生理 1. 受者状况 3. 细胞和 炎症因子效应器 2. 供者 T 性别激活

4 急性 GVHD 急性 GVHD 主要发生于植活前后. 以往错误定义为发生于移植后100天内的GVHD. 3个主要受累器官:
皮肤: 皮疹 胃肠系统: 恶心 / 呕吐和腹泻 肝功异常: 典型的是於胆 (黄疸). 同胞移植发生率为 9-50%. Vigorito et al. Blood 2009

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7 急性 GVHD: 生存与复发 Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0
T H Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0 Grade I — HR 1.5 (95% CI ) Grade II — HR 2.5 (95% CI ) Grade III — HR 5.8 (95% CI ) Grade IV — HR 14.7 (95% CI 11-20) Grade 0 acute GVHD — hazard ratio (HR) for relapse 1.0 Grade I — HR 0.94 (95% CI ) Grade II — HR 0.60 (95% CI ) Grade III — HR 0.48 (95% CI ) Grade IV — HR 0.14 (95% CI ) R E L A P S E

8 “你想接受小剂量 vidaza 或全身皮肤脱落?”
“很好，否则我将送你去做移植” “我告诉你，你做完后，就会带上尿布” “你想接受小剂量 vidaza 或全身皮肤脱落?”

9 GVHD 的预防

10 “没有免费午餐” 原则 GVHD GVHD 复发 排斥 延迟免疫重建

11 HCT 中的免疫功能 临床医学中免疫功能异常很多见 疾病控制的主要机制是依赖 GVT 反应, 然而异基因移植中的 GVHD是主要障碍

12 GVHD 在2个时代的风险 Gooley et al. N. Engl. J Med 363:2091, 2010

13 Absolute light emission
体内供者细胞光发射追踪 CD4+ CD8+ B220+ NK1.1+ Gr-1/Mac-1+ 2x105 cells/well Absolute light emission 0.00 0.05 0.10 0.15 Luciferase 2A eGFP bAct luc+ reporter mouse 异基因 HCT B T M BM Bone Marrow Splenocytes FVB/N WT luc+ Balb/c H-2q/Thy1.1H-2d/Thy1.2

14 急性移植物抗宿主病的发生 Beilhack, A. et al. Blood :1113

15 急性 GVHD 的演变

16 预防 GVHD 的方法 药物 移植物来源 T 细胞去除 免疫调节 CNI/MTX CNI/MTX vs Rapa/MTX
BM vs PBPC MRD vs URD vs UCB T 细胞去除 CD34 选择 ATG, Campath 免疫调节

17 免疫功能的调节 对健康和疾病是最重要的 免疫反应的划分 细胞因子 调节T 细胞 (Treg, NK-T, iTreg, others)
CD4+ T Cell Subsets 调节 反应

18 CD4+CD25+ 调节 T 细胞 调节免疫反应的主要细胞群 表达转录因子FoxP3
混合淋巴反应(MLR)中，细胞接触依赖的异体反应抑制 动物模型中，预防器官特异的自身免疫疾病 (如 IBD, 糖尿病) 体外，IL-10和 TGF- 涉及介导抑制效应

19 调节 T 细胞 Treg 频率随着aGVHD的严重性而降低. aGVHD 发生时 Treg 频率预示疗效.
伴有aGVHD的异基因 HCT 受者 的 Treg 频率低于无aGVHD者的40%. Treg 频率随着aGVHD的严重性而降低. aGVHD 发生时 Treg 频率预示疗效. Magenau et al. BBMT

20 循环中 Tregs 预示OS 63% 38% Magenau et al. BBMT

21 以保留GVL来控制 GVHD d5 Death from d15 GVHD Survival [%] TCD BM only, n = 14
TCD BM + Tcon, n = 15 TCD BM + Tcon + Treg n = 9 Time [d] post BMT Relative Signal Intensity Tcon BM only Tcon + Treg 500 5000 d5 d15 Death from GVHD 100 5000 1000 20000 Edinger et al. Nature Medicine 9:1144, 2003

22 临床移植Treg的挑战 Treg 是细胞群 缺乏临床分级药物分离和可能应用的独立标志 人体临界功能分析 调节需要

23 扩增CB Tregs 显示 FOXP3 去甲基化并抑制 alloMLR

24 3rd Party CB Tregs 防止 GVHD

25 通过GFP和荧光素进行体内跟踪 Treg

26 dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10

27 dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10

28 dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10

29 dorsal Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10

30 ventral Treg Treg+PBPC Day 3 Day 10

31 Treg Doses to be Studied
Dose Cohort Treg Dose Dose Level 1 1 × 105 Tregs/kg Dose Level 2 5 × 105 Tregs/kg Dose Level 3 1 × 106 Tregs/kg Dose Level 4 5 × 106 Tregs/kg Dose Level 5 1 × 107 Tregs/kg

32 下一步: 应用Treg的过继免疫治疗 MMF+Sirolimus Infusion of Ex-vivo Expanded Tregs
Day -6 -5 -4 -3 -2 Day-1 MEL BU Infusion of Ex-vivo Expanded Tregs BMT FLU 40 mg/m2 Day -8 -7 -6 -5 -4 -3 -2 Day-1 +1 +2 +3 +4 +6 BU Test Dose 32mg/m2 Rest BMT Infusion of Ex-vivo Expanded Tregs FLU 40 mg/m2 CY** 50 mg/kg

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34 患者接受Treg 剂量 > 30x105/kg 临床结果

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36 CD34+ cell selected graft
半相合移植计划 (Stanford) Endpoints: Chimerism Immune reconstitution Acute and chronic GVHD EFS, OS CD34+ cell selected graft CD4+CD25+ Treg CD4+/CD8+ Tcon Mel, TT, Flu + Day -10 +14 +16 5-10 x 106/kg 105/kg 3x105/kg 106/kg Cell Dose BB IND13923

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38 选择 CD4+CD25+ Tregs (U. Perugia)
1st step: Depletion of CD8+/CD19+cells Immunomagnetic Selection of CD4+CD25+Cells 2ndstep: Enrichment of CD25+ cells CD25 CD127 CD4 FoxP3 Gate on CD4CD25+high Gate on CD4CD25+ Cells (x109) ( ) ( ) %CD4CD ( ) ( ) N° cells (x 106) 330 ( ) ( ) %CD4CD25high ( ) ( ) N° cells (x 106) ( ) ( ) Starting fraction Final fraction . We employed a fully automated immunomagnetic procedure to select the naturally occurring CD4+cd25+ REGULATORY T CELLS . Starting from a leukophoresis product we first performed depletion of CD8+/cd19+ cells and then positive immunoselection of CD 25 +cells. The final fraction contained between 2 and 400 hundred million cells. Purity in terms of CD4CD25+was over 90% and about one third were 25 high. In the final fraction percentage of FOX P3+ cells was about 70% .WE also observed a profound depletion of CD127 expression.. Fox P3+ cells 71.9 ± 15 % 38

39 免疫重建模型 Recovery of CD4+ and CD8+ T cell subpopulations Spectratyping
Donors In our patients the pattern of immunoreconstitution was very different to what we usually see in the haplo setting. First of all there was a rapid increase in peripheral blood T cell subpopulations. Indeed the CD4 count reached 50 x microliter at median of 34 days and rapidily rose to 200 at median of 70 days. A similar behaviour was observed for the CD8 counts. Spectratyping showed the rapid development of a wide T cell repertoire , which approached donor scores in a few months, Complexity score Months after transplant

40 CMV 再激活情况 Tregs Group Control Group p<0.05 Evaluable Patients
Days after transplant Patients with CMV reactivation CMV reactivation episodes are significantly lower after “Treg-haplo transplant” as compared to our control group p<0.05 Control Group Days after transplant 40 40

41 结果 – U. of Perugia 无事件生存 12/26 (46%) 相关毒性的治疗: 急性 GVHD III-IV级 (2)
静脉阻塞病 (3) 多器官衰竭 (1) 急性 GVHD III-IV级 (2) 严重感染 (7) 复发 (AML 1) 中位追踪 18.5 个月 (范围 ) However the bad new is the high TRM. -The 50% transplant related mortality needs to be viewed in light of the clinical characteristics of this cohort of patients: 4 deaths were due to regimen related toxicity and 3 of these patients have been heavily pretreated with several lines of chemotherapy. The 4 patients who died of aspergillosis had fungal disease in the lung and in the brain before the transplant. On the other hand no fatal infections occurred after the first 2 months post-transplant, confirming the good recovery of immune response against pathogens One patient with AML relapsed 6 months after transplant. At median follow-up of ???? months, 46% of patients are alive and disease free. D’Ianni et al. Blood 2011

42 结论 GVHD 仍是异基因HCT后最重要的并发症 鼠的研究已证实，免疫调节在控制免疫反应功能异常（包括GVHD ）中起重要作用
临床移植正在进行中，并已经有了有前景的早期结果