摘要
目的：1.合成嵌合受体anti-erbB2 scFv-CD28-ξ的基因,并建立其真核表达载体；2.用嵌合受体anti-erbB2 scFv-CD28-ξ基因修饰NK-92细胞；3.建立荷人乳腺癌细胞MDA-MB453(erbB2阳性)、MDA-MB231(erbB2阴性)裸鼠皮下移植瘤模型；4.通过体外及体内实验研究基因修饰的NK-92细胞对erbB2阳性乳腺癌细胞的特异性杀伤。
方法：1.设计嵌合抗原受体的基因序列,由抗erbB2抗体单链可变区(scFv)、c-myc tag、CD8a(ENST00000283635)、CD28(ENST00000374478)及CD3ξ链(ENST00000392122)连接而成,化学合成长度为50-70bp的单链oligo,利用PCR将合成的oligo拼接成完整的序列,将完整的序列经HindⅢ和EcoRI酶切后连接至目的载体PCDNA3.1(+)中。2.用Amaxa Nucleofector技术将嵌合受体anti-erbB2scFv-CD28-ξ基因转入NK-92细胞。3.RT-PCR法检测抗原受体mRNA表达；免疫荧光法检测抗原受体在NK-92细胞表面的表达。4.流式细胞仪检测NK-92-scFv-erbB2-CD28-ξ和NK-92细胞表面CD27、CD158d、NKG2D和CD85j的表达变化。5.用CCK8法检测基因修饰的NK-92细胞对肿瘤细胞的特异性杀伤。6.体内抗瘤试验：第1个模型,MDA-MB453细胞或MDA-MB231细胞接种于BALB／C裸鼠背部皮下,同时尾静脉注射NK-92-scFv-erbB2-CD28-ξ细胞或未转染的NK-92细胞,比较各组成瘤率。第2个模型,MDA-MB453细胞或MDA-MB231细胞接种于BALB／C裸鼠背部皮下建立裸鼠皮下移植瘤模型。荷瘤小鼠随机分为4组,于第1、8天经尾静脉注射NK-92-scFv-erbB2-CD28-ξ细胞或未转染的NK-92细胞。第2、9天分别取小鼠静脉血用ELISA方法检测其中γ-干扰素(IFN-γ)水平。7.实验动物死后取出皮下移植瘤包块和肺脏,福尔马林固定,石蜡包埋,切片常规H&E染色,免疫组化检测CD3、NKG2D、CD56、CD16的表达。
结果：1.成功合成嵌合抗原受体基因并构建其真核表达载体PCDNA3.1(+)-anti-erbB2 scFv-CD28-ξ。2. RT-PCR结果证实了anti-erbB2 scFv-CD28-ξmRNA在NK-92细胞中的表达；流式细胞术检测结果提示NK-92细胞表面嵌合抗原受体anti-erbB2 scFv-CD28-ξ的表达率为18.89%。3.流式细胞术结果显示两种NK-92细胞表面CD27、NKG2D、CD158d、CD85j的表达没有差异。4. anti-erbB2-NK-92细胞对erbB2阳性的MDA-MB453细胞的杀伤率较亲本NK-92细胞至少提高了3倍,这种杀伤作用的增强是抗原特异性的,因为两种NK-92细胞对erbB2阴性的MBA-MD231细胞的杀伤效应没有显著差异。我们还证实Anti-erbB2-NK-92和亲本NK-92细胞对NK细胞敏感的K562细胞的杀伤率同样很高,说明嵌合受体的表达对NK-92细胞的固有细胞毒活性没有影响。5.在体内抗瘤第1种模型里,接种MDA-MB453细胞的裸鼠注射基因修饰的NK-92细胞后成瘤率(第10天)较注射亲本NK-92细胞低,但接种MDA-MB231细胞的裸鼠注射这两种NK-92细胞的成瘤率没有明显差异。在第2种模型里,接受anti-erbB2基因修饰NK-92细胞治疗的荷MDA-MB453肿瘤小鼠的血清IFN-γ水平高于接受亲本NK-92细胞治疗的荷MDA-MB453肿瘤小鼠。而且,与接受亲本NK-92细胞治疗的荷MDA-MB453肿瘤小鼠相比,接受anti-erbB2基因修饰NK-92细胞治疗的荷MDA-MB453肿瘤小鼠的肿瘤体积明显缩小、生存期明显延长、肺转移发生率低、肿瘤组织中有较多淋巴样细胞浸润,经免疫组化证实是NK-92细胞。
结论：1．成功合成嵌合受体anti-erbB2 scFv-CD28-ξ基因并构建其真核表达载体；2.通过Amaxa Nucleofector技术实现嵌合受体anti-erbB2 scFv-CD28-ξ在NK-92细胞的高表达；3.嵌合受体anti-erbB2 scFv-CD28-ξ在NK-92细胞的表达不影响NK-92细胞的表型特征和固有抗瘤活性；4.在体外,基因修饰的NK-92细胞对erbB2阳性乳腺癌细胞有特异性杀伤；5.在体内,基因修饰的NK-92细胞可以特异性抑制erbB2阳性乳腺癌的生长
Objectives:We designed this study to:1. Synthesize the full length gene of recombinants chimeric receptor anti-erbB2 scFv-CD28-ζby means of oligo chemic synthesis and PCR amplification, and construct its eukaryotic expression vector; 2. Genetically engineer NK-92 cells with the scFv anti-erbB2-CD28-ζchimeric recep-tor; 3. Establish models in which subcutaneous xenografts were induced in nude mice using human breast cancer cells MDA-MB453 (erbB2-expressing) and MDA-MB231 (erbB2 negative); 4. Investigate the specific killing of gene modified NK-92 cell to erbB2 expressing breast cancer cell in vitrol and vivo.
Methods:1. The gene sequence of the chimeric antigen receptor comprised anti-erbB2 antibody single-chain variable fragment (scFv), c-myc tag, CD8a (ENST00000283635), CD28 (ENST00000374478) and CD3ζchain, and the restriction sites HindⅢand EcoRⅠwere inducted into this chimeric gene. The single chain oligo with the length of 50-70bp were synthesized chemically according to gene sequencing, and then were ligated into full length gene. The recombinant plasmid PMD-18T-anti-erbB2 scFv-CD28-ζwas digested with HindⅢand EcoRⅠ, then the target gene was inserted to the corresponding restriction site on eukaryotic expression vector PCDNA3.1(+).2. NK-92 cells were gene modified with the scFv anti-erbB2-CD28-ζchimeric receptor by optimized electroporation using the Amaxa Nucleofector system.3. RT-PCR was performed to investigate the expression of scFv-erbB2-CD28-ζmRNA; Immunofluorescence was performed to detect the expression of scFv-erbB2-CD28-ζon NK-92 cell surface.4. Phenotyping of cell surface marker expression on NK-92-scFv-erbB2-CD28-ζand NK-92 cells was determined using FASCan by staining cells with PE-labled Abs specific for CD27, CD158d, NKG2D, and Alexa Fluor647-conjugated Ab specific for CD85j (BioLegend, American).5. The ability of gene-modified NK-92 cells specifically kill tumor targets was assessed in a CCK8 assay.6. In vivo antitumor activity:The ability of gene-modified NK-92 cells expressing the anti erbB2-CD28-ζreceptor to enhance the inhibition of tumor in mice was investigated in the following 2 models. In the first one, MDA-MB453 or MDA-MB231 cells were injected into the flanks of BALB/C mice, NK-92-scFv-erbB2-CD28-ζor parental NK-92 cells (E/T ratio of 5:1) were injected into caudal vein simultaneously. In the second one, MDA-MB453 and MDA-MB231 cells were injected into the flanks of BALB/C mice to establish the mice models bearing transplantation tumor. Mice bearing tumor were then divided into 4 groups randomly and treated on days 1,8 with NK-92-scFv-erbB2-CD28-ζor parental NK-92 cells delivered i.v (caudal vein). Serum levels of IFN-γon day2,9 were determined by ELISA. Forty days after the adoptive transfer, all animals were sacrificed.7. For each animal, upon euthanasia, the hypodermic transplantation block and lung were excised, fixed in formalin, embedded in paraffin, and serially sectioned at 2-μm thickness. Routine H&E staining was done at an interval of every 10 sections. Immunohistochemisty (Dako REAL EnVision Detection System, K8010, American) assay was used to stain tissues with CD3 (F7.2.38, SANTA CRUZ)、NKG2D (14F-2, SANTA CRUZ)、CD56 (IS628, clone 123C3, Dako)、CD16(2Q1240, SANTA CRUZ), for analysis of the infiltration of NK-92 cells in tumor at the unstained paraffin sections.
Results:1. A 1803bp-longed chimeric receptor anti-erbB2 scFv-CD28-ζwas generated by oligo chemic synthesis and PCR amplification, and the eukaryotic expression vector PCDNA3.1(+)-anti-erbB2 scFv-CD28-ζwas constructed. We determined that the output gene was same as target gene in sequence by gene sequencing.2. The expression of anti-erbB2 scFv-CD28-ζmRNA was confirmed by RT-PCR. High level (18.89%) expression of the anti-erbB2 receptor was achieved in NK-92 cells following staining with a c-myc tag mAb specifically recognizing a c-myc tag epitope incorporated into the extracellular domain of the chimeric receptor. 3. We used flow cytometry to compare expression of a number of molecules expressed by anti-erbB2-NK-92 cells and parental NK-92 cells, including activation and inhibition receptors. In three independently performed experiments, we observed no difference in the expression of NK-92 cell markers CD27, NKG2D, CD158d or CD85j between parental NK-92 and anti-erbB2-NK-92 cells. These data indicated that transfection of NK-92 cells with the scFv chimeric receptor had not phenotypically altered expression of a number of important NK-92 cell-associated markers.4. To investigate whether our gene-modified NK-92 cells expressing the anti-erbB2 chimeric receptor could augment cell killing activity to erbB2 expressing targets, we tested two human breast cancer cell lines expressing erbB2 or not in cytotoxiciyt assays with anti-erbB2-NK-92 cells or parental NK-92 cells. We demonstrated at least a three fold increase in the level of killing of erbB2 expressing MDA-MB453 cells by anti-erbB2-NK-92 cells compared with control parental NK-92 cells. This enhanced killing was erbB2 Ag-specific because anti-erbB2-NK-92 and parental NK-92 cells mediated comparable lysis of erbB2 negative MDA-MB231 cells. We also demonstrated that expression of the scFv receptor had no impact on the endogenous cytotoxic ability of NK-92 cells. The cytotoxicity of anti-erbB2-NK-92 and parental NK-92 cells to a NK cell-sensitive target cell line K562 were comparable.5. In the first model, the tumor formation rate of mice in MDA-MB453 & transduced NK-92 cell group was lower than that in MDA-MB453 & parental NK-92 cell group on day 10, whereas similar between MDA-MB231 & transduced NK-92 cell group and MDA-MB231 & parental NK-92 cell group. In the second model, serum IFN-γlevels of mice with MDA-MB453 tumor that received anti-erbB2 gene-modified NK-92 cells were higher than that received parental NK-92 cells. We also demonstrated significantly decreased tumor volume and lung metastasis and increased survival of mice bearing MDA-MB453 tumor that received anti-erbB2 gene-modified NK-92 cells, compared to receiving parental NK-92 cells. In mice bearing MDA-MB453 tumor, there were more lymphocytes infiltrating into tumor tissues when received anti-erbB2 gene-modified NK-92 cells, which were demonstrated as NK-92 cells by immunohistochemisty. These effects were Ag-specific because there were no such significant differences in mice bearing MDA-MB231 tumor.
Conclusions:1. The full length gene of recombinants chimeric receptor anti-erbB2 scFv-CD28-ζwas synthesized, and the construction of its eukaryotic expression vector might provide basis for the study of anti-tumor immunotherapy induced by NK cells.2. High level expression of the anti-erbB2 receptor was achieved in NK-92 cells using Amaxa Nucleofector system.3. Expression of the scFv receptor had no impact on the phenotyping character and endogenous cytotoxic ability of NK-92 cells.4. In vitro, gene-modified NK-92 cells showed Ag-specific cytotoxicity against erbB2-expressing breast cancer cells.5. In vivo, gene-modified NK-92 cells specifically inhibited erbB2-expressing breast cancer growth.
引文
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