供体来源的靶向T 细胞输注消除异基因造血干细胞移植后对供者淋巴细胞无反应的B 淋巴细胞白血病微小残留病 - 图文 

Author name et al. / Engineering 2(2016) xxx–xxx173表2?CAR T细胞首次输注与患者结果Patient123456

Chemotherapy before infusionCODPCyCOMPNoneCHOPFlu + Cy

CAR T cells

Chimeric

infused (kg)—Full chimeraFull chimeraFull chimera—Full chimera

1.10 × 1082.00 × 1072.24 × 1089.00 × 1071.30 × 1083.66 × 108

Evaluation CRSGVHD

of MRD2311—3a

CRCRCRCR

————

Glu——————

Virus

Bas

MR time Clinical out-(month)come5234—17

Relapse (death)Relapse (death)MR

(CNSL-CR)MRRelapse (death)MR

Survival time after infusion (month)121215 (alive)16 (alive)517 (alive)

Herpes simplex—(12th month)—————

—————

NR 0.07% —(3%)CR

—

Bas: basiliximab; CHOP: cyclophosphamide (750 mg·m–2 on day 1) + vincristine (1.4 mg·m–2 on days 1, 8, 15, 21) + adriamycin (50 mg·m–2 on day 1) + pred-nisone (60 mg·m–2 on days 1–21); CODP: cyclophosphamide (750 mg·m–2 on day 1) + vincristine (1.8 mg·m–2 on days 1, 8, 15, 21) + daunorubicin (40–60 mg·m–2 on days 1–3) + prednisone (60 mg·m–2 on days 1–21); COMP: cyclophosphamide (400 mg·m–2 on days 1, 8) + vincristine (1.4 mg·m–2 on days 1, 8) + mitoxantrone (5 mg·m–2 on days 1–4) + prednisone (60 mg·m–2 on days 1–21); Cy: cyclophosphamide (500 mg·m–2 on days 1–3); Flu: fludarabine (30 mg·m–2 on days 1–3); Glu: glucocorticoid; MR: molecular remission. NR: no responsea

Patient 6 required medication intervention (tocilizumab × 2 on days 8 and 9).表3 CAR T细胞第二次输注与患者结果Patient12345

ChemotherapyCODPAra-CFluNoneFlu + Cy

Evaluation before CAR T cell fusionMRD+MRD+MRD+, CNSLRelapseMRD+

CAR T cells infused (kg)Cy 2.60 × 108—8.00 × 1076.20 × 101.70 × 10

78

CRS00000

EvaluationMRD+MRD+CRCRRelapse

Clinical outcomesRelapse (death), 7 monthsRelapse (death), 12 monthsAliveAlive

Relapse (death), 3 months

种巩固化疗加DLI可以减少累计复发率，增加无白血病存活率以及allo-HSCT后急性白血病复发患者的存活率。三年累计复发率、无白血病率与移植后存活率分别为32.4%、50.3%和51.4%。Ma等[25]报道，采用DLI治疗了allo-HSCT后复发的19名高危疾病患者，并监测是否出现MRD。其中，6名患者（31.58%）在DLI后未出现白血病进展。由此可知，DLI方案对于治疗急性淋巴细胞白血病的疗效有限，且可能导致危及生命的GVHD。此外，约三分之一的MRD患者可能对DLI无反应，且预后可能恶化。这些患者应采用创新方案予以治疗。CAR T细胞代表一种新型的、有发展前景的免疫疗法[26]。我们探讨了人类白细胞抗原相合同胞供者/无关供者移植后供体来源CART19s输注；在患B-ALL的5名患者中，其中，4名（80%）出现了缓解，且未复发aGVHD[14]。我们还开展了供体来源的CD19靶向T细胞输注临床研究，发现此种治疗使在haplo-HSCT后对供体淋巴细胞输注无反应的B-ALL复发患者出现了MRD阴性缓解。6名患者中，有5名（83.33%）出现了MRD阴性缓解；此外，CRS和GVHD也均得到了控制[15]。总之，供体来源的CD19靶向T细胞输注似乎是治疗allo-HSCT后B-ALL复发患者的有效方法。然而，目前尚不清楚CAR T细胞输注对造血高细胞移植后对供体淋巴细胞输注无反应的MRD患者来说是否有效。本研究结果表明，供体来源的CART19s也是HSCT后治疗MRD患者的有效方法。就我们所知，本研究包括allo-HSCT后B-ALL复发患者接受供体来源的CART19s输注的最大研究。本研究结果提供了供体来源的CART19s输注可有效消除MRD的初步证据。一直以来，人们都很关心供体来源的CAR T细胞输注的安全性。在体外实验中，供者CART19s可在不破坏GVHD活性的情况下增加移植物抗白血病活性，同时不影响细胞毒活性[27]。本研究中还从aGVHD与细胞因174—Fever—Fever—Fever, hypotension, dysfunction-al blood coagulationFever—Fever, hypotension, hypoxemiaFever, hypotension, dysfunction-al blood coagulationCRS symptomsAuthor name et al. / Engineering 2(2016) xxx–xxx导致的GVHD的发病率与强度[15]。这种临床现象与对HSCT受体的安全性。无患者出现主要令人担心的Jacoby等[28]报道的结果一致。在体外，鼠类异基因CD19 T细胞表现出有效的抗白血病活性，但也表现出并发症——aGVHD。本研究中的GVHD的发病率与程度低于因先前用于治疗复发患者的CAR T细胞输注而子综合征两个方面评估了供者来源的CAR T细胞输注)em)inIt oAnitocD itehctteetd0e 01drd(( 3 0doolb larehp)iIrepnA oDnit ich dett8ee3tcd(e td8en8d2.0 s91RTA)ICn foAoiDt tc tnesette1(nd 1ot Cs01.0 desufsnlli enc i 4T2. DR1CA/8CDC+65DC11+8DC84)%( +e4gaDtnC04ecrep+ l3leDCC88 s)llgeck8(0 Td1 Re×s Au0fCn1.i1noisfuegnia ttSs1tneitaP1))II)AIA))))A)DIIIII AD)I AAAD AhtDhAtDDDtsD0 0D ts2hh1 3tt((tsst2h 1 0118(t 7282222548(2((((. . 0400 0 01(0. 0001 1–1 1 )––1))–ggg)···gggg·μ)μμg(·I()()μA·I·I()s))ss·IeI)IeAeAsiI)DiieApAAI AhpDpDioA)Dto ho pDD c DI D2ct7chtoh5hAht ct0t th 15(5757(1700(0((7D7 ( th 711A 11 A A A (× th6(3×N×N×N N26178(. . .. 3011D2D6D4D001)) IIAA)))))II DAD)I AAIII dDthDDAAAn 7 DDD2t((s thth tttsss 11441112(2((((3 . .000—( 00000—————————————————————————————————————————————878778880007011100110100× × × ××1 11× 004 × ×× 60200 0...002306222..89..7661..13nnnoisonononionniioiisssoisiosoniusfufuussfufunfufufi nninnnfinfuidii i in n nttdtdtdi tsssss211n21n21n2123456潜在的致命性GVHD。然而，CAR引起的GVHD仅在有白血病细胞的情况下才会发生。另一种主要担忧是CRS。在本研究中，无患者直接死于CRS。此外，本研究尽管有中枢神经系统白血病（CNSL）患者参与，但并未出现与CRS相关的脑水肿病例。因此，供体来源的CAR T细胞输注对于HSCT后B-ALL的MRD是安全的。本研究中，在接受CAR T细胞输注之前，6名MRD患者中，有5名患者（83.33%）在第一次输注后达到了分子完全缓解，有3名患者至今仍在人世。我们先前的研究中，有反应的5名患者中，有4名患者在2~7个月后复发血液病[15]。尽管本研究纳入的病例较少，但确定MRD治疗对复发患者是否有足够的效果值得评估。5. 总结根据临床观察，我们提出供体来源的CAR T细胞输注是HSCT后可消除B细胞恶性肿瘤患者MRD的一种有效与安全的干预手段。而让人印象更深刻的是，本研究观察期间无患者出现GVHD。但是，尚不清楚这种情况出现的机制。需要进一步的实验和更大规模的临床试验来确认CAR T细胞输注是否可以用作消灭对DLI无反应的患者和MRD患者的MRD的一线干预措施。致谢本工作得到了首都医学发展科研基金（ 2018-2-4084）的支持。.noisufnAuthor contributionsir etfa Yifei Cheng and Yuhong Chen analyzed the materi-yad :al and wrote the paper. Shasha Wang collected the data. IADXiangyu Zhao, Chenhua Yan, Yu Wang, Yao Chen, Wei 表4?输入的细胞特性和相关毒性Author name et al. / Engineering 2(2016) xxx–xxx175Han, Lanping Xu, and Xiaohui Zhang performed the re-search. Lungji Chang and Lei Xiao contributed to cell pro-duction. Kaiyan Liu designed the research. Xiaojun Huang designed the research and edited the manuscript.Compliance with ethics guidelinesThe present protocol was evaluated and allowed by the Peking University People’s Hospital review board. All enrolled patients gave informed consent in accordance with the Declaration of Helsinki. Yifei Cheng, Yuhong Chen, Chenhua Yan, Yu Wang, Xiangyu Zhao, Yao Chen, Wei Han, Lanping Xu, Xiaohui Zhang, Kaiyan Liu, Shasha Wang, Lungji Chang, Lei Xiao, and Xiaojun Huang declare that they have no conflict of in-terest or financial conflicts to disclose.NomenclatureaGVHD acute graft-versus-host diseaseallo-HSCT allogeneic hematopoietic stem cell trans-plantationALT elevated alanine transaminaseB-ALL B cell acute lymphoblastic leukemiaBu busulfanCAR chimeric antigen receptorCART19 CD19-directed chimeric antigen receptor-modified T cellCD cluster of differentiationCIR cumulative incidence of relapseCR complete remissionCRS cytokine release syndromeCsA cyclosporine ACy cyclophosphamideDLI donor lymphoblastic infusionEFS event-free survivalFACS fluorescence-activated cell-sortingGVHD graft-versus-host diseaseGVL graft-versus-leukemiahaplo-HSCT haploidentical hematopoietic stem cell transplantationHLA human leukocyte antigenHSCT hematopoietic stem cell transplantationLFS leukemia-free survivalMRD minimal residual diseaseMTX methotrexateOS overall survivalPBMC peripheral blood mononuclear cellqPCR quantitative real-time polymerase chain re-actionAppendix A. 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