【作者】 尚佩沛；

【导师】 侯健；

【作者基本信息】 第二军医大学 ， 内科学， 2012， 博士

【摘要】 多发性骨髓瘤（multiple myeloma, MM）是一种以异常浆细胞增生导致单克隆免疫球蛋白聚集为特征的B细胞恶性肿瘤。近年来，随着我国人口老龄化及诊断水平的进步，该病发病率有不断上升的趋势，而目前常用的化疗及干细胞移植均无法实现治愈，且受制于患者年龄、脏器功能等多方面因素，因此迫切需要寻找更加安全、有效、靶向性强且副作用轻的治疗方法。免疫治疗被认为是理想的治疗方法，但目前常用的独特型诱导的树突状细胞及应用集落刺激因子或白介素等免疫调节疗法临床疗效一般[1,2]，这使得具有多种激活受体系统并在抗病毒和肿瘤细胞免疫中发挥着重要作用的自然杀伤（Natural killer,NK）细胞受到日益广泛的关注[3,4]。NK细胞的杀伤活性不受细胞MHC限制，不需要致敏而且可被多种细胞因子显著增强，上述诸多观点决定了其作为免疫治疗的效应细胞具有的强大优势及潜力[5]。但是，研究发现肿瘤细胞可通过某些机制，如上调经典（HLA-A,B,C）及非经典（HLA-G,E）人类白细胞抗原（HLA）等的表达以逃避NK细胞的免疫监视，这些HLA抗原可通过与NK细胞表面某些免疫球蛋白样受体（killer cellimmunoglobulin-like receptors,KIRs）结合而抑制NK细胞活性，从而产生免疫耐受[6,7]。此外，常用的以细胞因子或体外扩增自体或供体已激活的杀伤细胞[8,9]，或两者联合的方法均无法改善内源性NK细胞活性低的缺陷，且激活后扩增潜力有限，在不同的临床实验中激活的NK细胞亚群的表型也不同，在评价疗效及副反应时就缺乏统一标准，因此进一步开发和改造NK细胞以改进免疫治疗就成为当务之急。NK-92是目前国内外唯一进入I/II期临床用于过继免疫治疗的NK细胞，对恶性肿瘤尤其血液肿瘤具有广谱而强大的细胞毒效应，在有效杀伤肿瘤细胞同时对正常造血细胞无明显毒性。本研究拟通过高亲和力抗CD138杂交瘤细胞系获得CD138特异性单链抗体可变区基因片段（scFv），与编码CD8α铰链区及TCR复合物上CD3ζ信号链的基因重组构成嵌合受体基因，通过慢病毒载体转染NK-92细胞。表达嵌合受体的NK92可靶向性地与骨髓瘤细胞表面高水平表达的CD138分子特异性结合，进而通过CD3ζ链促进内源性信号转导，诱导强烈的抗骨髓瘤细胞免疫应答，加速并增强肿瘤细胞的凋亡和溶解。由于NK-92几乎不含所有抑制性KIRs，避免瘤细胞产生免疫耐受；而且它易于大量扩增，为彻底清除骨髓瘤细胞提供更安全、特异而有效的免疫治疗方法。通过改变嵌合受体基因结构，可将本研究理念扩展应用至其它实体肿瘤的治疗，具有广阔的前景。实验研究包括以下三个方面：第一部分构建慢病毒表达载体并转染NK92细胞的实验研究目的构建慢病毒表达载体并转染NK92细胞方法构建编码包括CD138特异性单链抗体可变区基因（scFv）片段、可弯曲的CD8α铰链区（编码105-165氨基酸，使嵌合抗原受体表达并固定于细胞膜）与CD3ζ信号链的胞内区这三个部分的嵌合抗原受体基因。通过慢病毒载体转染NK-92细胞。结果成功通过RT-PCR由抗CD138杂交瘤细胞系总RNA扩增得到抗CD138抗体的重链和轻链可变区（VH和VL）基因片段,经测序及Ig blast,，符合Ig VH和VL的特征。通过5’RACE反向PCR技术，扩增得到免疫球蛋白重链（Ig）引导肽SP。经SignalP3.0Server预测结果：M G W S S I I L F L V A T A T G V H S为Ig VH重链SP通过酶切连接构建嵌合抗原受体基因，其序列包括Ig重链引导肽、CD138特异性scFv、标记基因Myc-tag、鼠CD8铰链区(编码105-165氨基酸)及CD3ζ链。通过THD-01慢病毒表达载体构建包装纯化嵌合抗原受体基因scFv-(CD138)/hinge/ζ。包装病毒转染NK-92细胞，结果NK-92基因转染效率在15％左右，转染后24h细胞存活率为35-40％。结论构建重组的CD138-scFv-CD3ζ嵌合受体基因并成功转染NK-92细胞，直接激活了NK-92的胞内信号转导系统，加强了免疫治疗的靶向性和有效性。第二部分靶向性NK-92-scFv(CD138)-ζ细胞对骨髓瘤特异性抗肿瘤效应的体外实验研究目的通过体外实验检测NK-92-scFv(CD138)-ζ细胞的特异性抗骨髓瘤效应。方法采用LDH释放法（PROMEGA公司CytoTox96KIT）检测转染后NK-92-scFv(CD138)-ζ细胞在不同效靶比（10:1、5:1、1:1）对骨髓瘤细胞系LP-1、NCI-H929、RPMI-8226及2例经CD138免疫磁珠分选的原代MM细胞的杀伤功能，并与亲代细胞的杀伤功能进行比较。采用流式检测不同效靶比（10:1、5:1、1:1）情况下，NK-92-scFv(CD138)-ζ或亲代NK-92细胞对上述骨髓瘤细胞系及原代MM细胞的杀伤功能。结果LDH结果显示，在不同效靶比（10:1、5:1、1:1）情况下，NK-92-scFv较亲代NK-92的抗骨髓瘤效应均有显著增强，特别在效靶比10:1时，在LP-1、NCI-H929、U266，NK-92-scFv和NK-92杀伤活性分别为57.8±6.2%vs35.9±6.5%、48.8±5.9%vs29.2±4.4%、39.5±6.4%vs23.1±4.6%，NK-92-scFv与NK-92差异显著（P<0.05），同样，2例原代MM细胞在效靶比为10:1时，NK-92-scFv和NK-92杀伤活性也有显著增强，分别为35.5±6.2%vs22.8±5.9%、40.8±7.2%vs15.6±6.6%。流式结果显示，将CFSE标记NK‐92细胞，效靶细胞混合孵育4H,通过Annexin V‐FITC和PI双标法检测CD138阳性细胞群的凋亡率，检测了H929,LP1,U266细胞株和5例原代细胞.结果显示在不同效靶比（1:1、5:1、10:1）情况下，NK‐92‐scFv较亲代NK‐92的抗骨髓瘤效应均有显著增强，效靶比5:1时，在H929、LP‐1、U266,NK‐92‐scFv和NK‐92杀伤活性分别为91.7±2.8%vs21%±5.2%、95.9±5.5%vs15±3.2%、73.2%±4.1vs36.3±6.5%，NK‐92‐scFv与NK‐92差异显著（P<0.05）。通过采用抗CD138封阻抗体预先与MM细胞孵育，可以显著降低NK‐92‐scFv(CD138)‐ζ对骨髓瘤细胞的杀伤效率，从而证实NK‐92‐scFv(CD138)‐ζ较其亲代细胞显著增强的抗MM活性是由细胞表面的嵌合受体scFv(CD138)‐ζ所介导。以LP‐1为例，效靶比5:1和10:1时，预先将靶细胞与封阻抗体共孵育，可使凋亡率分别由47.2±7.7%降至35.6±5.5%、54.4±6.9%降至40.7±3.6%（p<0.05）。结论体外实验证实NK-92-scFv(CD138)-ζ细胞较其亲代NK-92细胞的抗骨髓瘤活性显著增强。第三部分建立多发性骨髓瘤的SCID鼠动物模型进行体内实验研究目的建立多发性骨髓瘤的SCID鼠动物模型，通过体内实验检测NK-92-scFv(CD138)-ζ细胞的特异性抗骨髓瘤效应。方法选取10只6-8周的SCID小鼠，皮下植入兔骨，将分离纯化后的原代细胞用台盼蓝计数，每只小鼠注射5×106浓度的原代细胞，每周取血样一次测试血轻链并保存血样，每周测量瘤体大小两次，拍摄X片观察成瘤情况，记录小鼠生存时间，瘤体直径达到2CM记做死亡。当血清轻链达到成瘤标准（5ug/ml），小鼠分组给药，每次107个NK92细胞，重悬于50-100ulPBS中并加入10000IU IL-2，鼠尾静脉注射，3-4天给药一次，共给药3次，比较NK-92-scFv(CD138)-ζ细胞与亲代NK92细胞抗骨髓瘤效应的=差异。结果成功的建立了多发性骨髓瘤的SCID鼠动物模型。在注射CD138+原代骨髓瘤细胞的10只SCID‐rab小鼠中，3只成瘤，成瘤率30%。其中注射NK‐92的成瘤小鼠生存时间为51天，另两只小鼠中，注射NK‐92的成瘤小鼠目前仍生存（55天），瘤体较前增大，注射NK-92-scFv的成瘤小鼠目前仍生存（55天），瘤体较前未再增大。结论多发性骨髓瘤细胞可以在SCID‐rab模型中形成肿瘤。注射NK-92-scFv细胞可以抑制成瘤小鼠肿瘤的生长。更多还原

【Abstract】 Multiple myeloma(MM) is a neoplasm of malignant plasma cells，characterized byaccumulation of malignant plasma cells in the bone marrow.For the past few years,becauseof the aging of population and progress of diagnose,the mobility is climbing.The treatmentof MM like conventional chemotherapy,hematopoietic stem cell transplant can not cure thedisease,and limited by the patient age、organ function and other factors.So we need to findanother therapeutic method which is more safe,effective, targetable and low sideeffect.Immunotherapy is an ideal therapeutic method,So we pay close attention to thenatural killer cell.The killing activity of natural killer cell can not be limited by MHC,itneed not be sensitized and can be strengthended by a lot of cell factors。So natural killercell has powerful superiority and potential.But research find that tumor cell can escape the immunosurveillance of natural killercell.The antigen of HLA can combine with killer cell immunoglobulin-like receptors whichon the survace of natural killer cell to inhibition its cytoactive,finally it has immunetolerance.Beside that,the endogenous natural killer cell activity is very low which can notbe improved,so the urgent priority is to develop and reform natural killer cell to improvethe immunotherapy.NK-92cell line is the only natural killer cell line currently used for adoptiveimmunotherapy in ongoing I/II phase clinical trial,displaying high cytotoxicity against avariery of malignant cells in vitro,in particular,malignant cells of hematologic origin,andsimultaneously having no observed toxicity against nonmalignant allogeneic cells.Ourstudy is to be intended to transducer NK-92cells with a lentiviral vector encoding achimeric antigen receptor that consists of the CD138-specific scFv antibody fragment,aflexible hinge region of CD8αchain,and the CD3δchain.The genetically modified NK-92cells stably expressing the chimeric antigen receptor specific for CD138,which is highlyexpressed on myeloma cells surface,coule induce intensified anti-myeloma immuneresponse,and reinforce the lysis and apoptosis of myeloma cells.NK-92cells were shownto lack almost all inhibitory KIRs,which diminish the possibility of developingimmunotolerance of tumor cells,and can be easily expanded to large numbers and arereadily available on demand in standardized quality for adoptive therapy.Our study hasextensive applied prospects for a broad spectrum of tumor targets through replacing thescFv portion of the fusion protein for other tumor-associated antigen receptor.Theempirical study include three parts: Part ITo construct lentivirus expression vector and to transfectNK92cellsObjective To construct lentivirus expression vector and to transfect NK92cellsMethods To construct jogged antigen receptor gene which includes specificity CD138single-chain antibody fragment,flexible CD8αhinge region and CD3ζsignal chain.uselentivirus expression vector to transfection NK-92cells.Results Successfully used realtime polymerase chain reaction amplification CD138Hybridoma cell line RNA to obtain variable region of heavy chain and light chain genefragment of anti-CD138antibody,we find it fit the variable region of heavy chain and lightchain gene fragment characteristic by sequencing and immunoglobin blast.We gain the immunoglobulin heavy chain leader peptide by5’RACE oppositepolymerase chain reaction technology. SignalP3.0Server predict results：M G W S S I IL F L V A T A T G V H S is immunoglobulin heavy chain leader peptide.We build jogged antigen receptor gene by enzyme digestion connection,the seriesinclude immunoglobulin heavy chain leader peptide、 specificity CD138single-chainantibody fragment、marker gene Myc-tag、mouse CD8α hinge area and (CD3ζchain.We purification jogged antigen receptor gene scFv-(CD138)/hinge/ζ by lentivirusexpression vector.Packaging virus transfection NK-92cell，the efficiency of transfection isabout15%，after transfection24h cell survival ratio is35-40％.Conclution We successfully constructed custom-crafted CD138-scFv-CD3ζjogged antigenreceptor gene and transfected NK-92cells，directly activated the signal transduction systemof NK-92cells,enhanced the targeting and effectivity of immunotherapy. Part IIIn vitro experimental study of NK-92-scFv(CD138)-ζObjective To use vitro experimental study to detect the specific anti myeloma effect ofNK-92-scFv(CD138)-ζ cells.Methods To use LDH release method to detect the anti myeloma effect of NK-92-scFv(CD138)-ζ cell at different effector cell and target cell ratio（10:1,5:1,1:1），and tocompare with parent cell killing function。We use flow cytometer to detect anti myelomaeffect of NK-92-scFv(CD138)-ζ cell and parent NK92cells at different effector cell andtarget cell ratio（10:1,5:1,1:1）.Results The result of LDH shows that at different effector cells and target cells ratio（10:1、5:1、1:1）,the anti myeloma effect of NK-92-scFv(CD138)-ζ cells are stronger thanparent NK92cells. Especially at effector cell and target cell ratio10:1，the respectivekilling function of NK-92-scFv(CD138)-ζ cell and parent NK92cell to LP-1、NCI-H929and U266is57.8±6.2%vs35.9±6.5%,48.8±5.9%vs29.2±4.4%,39.5±6.4%vs23.1±4.6%，the difference is notable（P<0.05）.Similarly，the respective killing function ofNK-92-scFv(CD138)-ζ cell and parent NK92cell to two case of generation MM cell is35.5±6.2%vs22.8±5.9%,40.8±7.2%vs15.6±6.6%.The result of flow cytometer shows that at different effector cell and target cellratio（10:1、5:1、1:1）he anti myeloma effect of NK-92-scFv(CD138)-ζ cells are strongerthan parent NK92cells. Specially at effector cell and target cell ratio10:1，the respectivekilling function of NK-92-scFv(CD138)-ζ cells and parent NK92cells to NCI-H929、LP-1and U266is91.7±2.8%vs21%±5.2%,95.9±5.5%vs15±3.2%,73.2%±4.1vs36.3±6.5%，the difference is notable（P<0.05）；Similarly，the respective killing function at5:1and10:1of NK-92-scFv(CD138)-ζ cells and parent NK92cells to two case ofgeneration MM cell is30.6±7.2%vs21.8±5.9%,36.2±7.2%vs15.6±6.6%（P<0.05）。Conclusion The vitro experimental study confirm that the anti myeloma effect ofNK-92-scFv(CD138)-ζ cells is stronger than parent NK92cells. Part IIITo construct the SCID animal model of MM to study in vivoObjective To construct the SCID animal model of MM to study in vivoMethods To choose6-8week SCID mice，subcutaneous implantation in rabbit bone，inject5×10~6primary cells to every mice，draw blood every week to detect light chain andto preserve the blood sample, measure the tumor volume two time a week,observe thetumor formation by X ray,record the survival time of mice.When the light chain of serumachieve5ug/ml，inject107NK92cells from the mice caudal vein，compare the differenceof NK-92-scFv(CD138)-ζcells with parent NK92cells.Results We successfully built the SCID animal model of MM. In ten SCID-rab, thetumor formation rate is30%.The tumor burden SCID-rab mouse is survival now, the tumorwhich treatment by NK-92-scFv(CD138)-ζ cells was not further increased.Conclusion The purified primary multiple myeloma cells can form tumor in the SCID-rabmouse。更多还原