技术领域technical field
本发明属于肿瘤细胞免疫治疗领域，具体涉及一种同时分泌PD-1中和抗体和GM-CSF因子的肿瘤细胞疫苗及其制备方法。The invention belongs to the field of tumor cell immunotherapy, and in particular relates to a tumor cell vaccine that simultaneously secretes PD-1 neutralizing antibodies and GM-CSF factors and a preparation method thereof.
背景技术Background technique
肿瘤疫苗可激活人体自身免疫系统,诱发特异性免疫反应,提高自身免疫力,是一种主动免疫治疗方法。其具有不良反应轻微、耐受性好,兼具预防和治疗作用,成为全球抗肿瘤研究的热点之一。程序性死亡受体1(PD-1)是T细胞调节受体CD28家族中传导抑制性信号的共刺激分子,介导免疫反应的负性调节信号,在肿瘤发生、病毒感染以及自身免疫病中都发挥了特异性的免疫调节作用。PD-1广泛表达于活化的T细胞、记忆性T细胞和调节性T细胞表面,当它与结合配体PD-L1或PD-L2结合后可下调T细胞活性，是产生肿瘤免疫逃逸的重要分子。GM-CSF(Granulocytemacrophagecolony-stimulatingfactor)即粒细胞-巨噬细胞集落刺激因子能够促进单核细胞、巨噬细胞和树突状细胞(DC)增殖、成熟和迁移以及B和T淋巴细胞的扩增、分化来增强抗原引起的免疫反应，常用做佐剂。目前并未见将两者联合用于肿瘤治疗的报道。Tumor vaccines can activate the body's own immune system, induce specific immune responses, and improve autoimmunity. It is an active immunotherapy method. It has mild adverse reactions, good tolerance, and has both preventive and therapeutic effects, and has become one of the hotspots in global anti-tumor research. Programmed death receptor 1 (PD-1) is a costimulatory molecule that conducts inhibitory signals in the CD28 family of T cell regulatory receptors, mediates negative regulatory signals of immune responses, and is involved in tumorigenesis, viral infection and autoimmune diseases. All play a specific immunomodulatory role. PD-1 is widely expressed on the surface of activated T cells, memory T cells and regulatory T cells. When it binds to the binding ligand PD-L1 or PD-L2, it can down-regulate the activity of T cells, which is an important factor for tumor immune escape. molecular. GM-CSF (Granulocyte macrophage colony-stimulating factor) can promote the proliferation, maturation and migration of monocytes, macrophages and dendritic cells (DC), as well as the expansion of B and T lymphocytes, Differentiation to enhance the immune response caused by antigens, often used as adjuvants. At present, there is no report on the combination of the two for tumor treatment.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种能PD-1中和抗体联合GM-CSF基因共同修饰的抗肿瘤细胞疫苗及其制备方法。The technical problem to be solved by the present invention is to provide an anti-tumor cell vaccine capable of co-modifying PD-1 neutralizing antibody and GM-CSF gene and its preparation method.
该抗肿瘤细胞疫苗是由PD-1中和抗体及GM-CSF细胞因子修饰的肿瘤细胞，经X射线辐照制备而成。The anti-tumor cell vaccine is prepared from tumor cells modified by PD-1 neutralizing antibodies and GM-CSF cytokines and irradiated with X-rays.
其中，上述的X射线辐照的剂量为亚致死剂量。Wherein, the above-mentioned dose of X-ray irradiation is a sub-lethal dose.
其中，上述的X射线的照射剂量为总辐射剂量50-150Gy。Wherein, the above-mentioned X-ray irradiation dose is a total radiation dose of 50-150Gy.
其中，上述的PD-1中和抗体的氨基酸序列为SEQIDNO.8所示。Wherein, the amino acid sequence of the above-mentioned PD-1 neutralizing antibody is shown in SEQ ID NO.8.
其中，上述的GM-CSF的氨基酸序列为SEQIDNO.10或SEQIDNO.12所示。Wherein, the above-mentioned amino acid sequence of GM-CSF is shown in SEQ ID NO.10 or SEQ ID NO.12.
其中，上述的PD-1中和抗体的编码基因的核苷酸序列为SEQIDNO.7所示，或者为其简并序列。Wherein, the nucleotide sequence of the coding gene of the above-mentioned PD-1 neutralizing antibody is shown in SEQ ID NO.7, or its degenerate sequence.
其中，上述的GM-CSF的编码基因的核苷酸序列为SEQIDNO.9或SEQIDNO.11所示，或者为两者各自的简并序列。Wherein, the nucleotide sequence of the above-mentioned GM-CSF coding gene is shown in SEQ ID NO.9 or SEQ ID NO.11, or the degenerate sequences of both.
其中，上述的肿瘤细胞是由载有PD-1中和抗体和GM-CSF的编码基因的载体转化后获得能表达PD-1中和抗体和GM-CSF的能力的肿瘤细胞。Wherein, the above-mentioned tumor cells are tumor cells capable of expressing PD-1 neutralizing antibodies and GM-CSF after being transformed with vectors carrying genes encoding PD-1 neutralizing antibodies and GM-CSF.
其中，上述的载体为慢病毒载体。Wherein, the above-mentioned vector is a lentiviral vector.
其中，上述的慢病毒表达载体为Ubi-MCS-3FLAG-IRES-puro。Wherein, the above-mentioned lentiviral expression vector is Ubi-MCS-3FLAG-IRES-puro.
其中，上述的表达LV-PD-1中和抗体、LV-mGM-CSF(鼠GM-CSF)或者LV-hGM-CSF(人GM-CSF)的基因载体是分别由以下步骤制备得到：Wherein, the above-mentioned gene vectors expressing LV-PD-1 neutralizing antibody, LV-mGM-CSF (mouse GM-CSF) or LV-hGM-CSF (human GM-CSF) are prepared by the following steps:
a、过表达慢病毒载体的制备：从含有目的基因的质粒pcDNA3.1-anti-PD-1、pcDNA3.1-mGM-CSF和pcDNA3.1-hGM-CSF中利用PCR法分别扩增anti-PD-1、mGM-CSF或hGM-CSF目的基因,将Ubi-MCS-3FLAG-IRES-puro目的载体进行BamHI/AgeI酶切,酶切产物电泳回收后进行交换,其产物转化细菌感受态细胞。对长出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对正确的即为构建成功的目的质粒。a. Preparation of overexpression lentiviral vectors: PCR method was used to amplify anti- For the PD-1, mGM-CSF or hGM-CSF target gene, the Ubi-MCS-3FLAG-IRES-puro target vector was digested with BamHI/AgeI, and the digested products were recovered by electrophoresis and exchanged, and the products were transformed into bacterial competent cells. Colony PCR identification was performed on the grown clones first, and then the positive clones identified by PCR were sequenced and compared. The correct comparison was the successful construction of the target plasmid.
b、慢病毒包装与滴度检测：将构建好的表达目的基因的重组病毒质粒及其两种辅助包装原件载体质粒,各质粒载体分别进行高纯度无内毒素抽提,按Invitrogen公司Lipofectamine2000使用说明进行转染293T细胞,转染后8h更换为完全培养基,培养48h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。b. Lentivirus packaging and titer detection: The constructed recombinant virus plasmid expressing the target gene and its two auxiliary packaging original vector plasmids, and each plasmid vector were subjected to high-purity and endotoxin-free extraction, according to the instructions of Lipofectamine2000 of Invitrogen Company 293T cells were transfected, and the complete medium was replaced 8 hours after transfection. After 48 hours of culture, the cell supernatant rich in lentivirus particles was collected and concentrated to obtain a high-titer lentivirus concentrate. In 293T cells Measure and standardize the virus titer.
更进一步的，所述扩增PD-1中和抗体、小鼠GM-CSF基因序列和人GM-CSF基因序列的上下游引物序列分别为SEQIDNO.1和2所示、SEQIDNO.3和4所示，以及SEQIDNO.5和6所示。Further, the upstream and downstream primer sequences of the amplified PD-1 neutralizing antibody, mouse GM-CSF gene sequence and human GM-CSF gene sequence are shown in SEQ ID NO.1 and 2, and SEQ ID NO.3 and 4, respectively. Shown, and shown in SEQIDNO.5 and 6.
本发明还提供了一种制备上述的抗肿瘤细胞疫苗的方法，该方法包括以下步骤：The present invention also provides a method for preparing the above-mentioned anti-tumor cell vaccine, the method comprising the following steps:
a、过表达慢病毒载体的制备：从含有目的基因的质粒pcDNA3.1-anti-PD-1、pcDNA3.1-mGM-CSF或pcDNA3.1-hGM-CSF中,利用PCR法分别扩增PD-1中和抗体、mGM-CSF和hGM-CSF的目的基因序列,将Ubi-MCS-3FLAG-IRES-puro载体进行BamHI/AgeI酶切,酶切产物电泳回收后进行交换,其产物转化细菌感受态细胞。对长出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对正确的即为构建成功的目的质粒；a. Preparation of overexpression lentiviral vector: from the plasmid pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF or pcDNA3.1-hGM-CSF containing the target gene, amplify PD by PCR -1 Target gene sequences of neutralizing antibodies, mGM-CSF and hGM-CSF, the Ubi-MCS-3FLAG-IRES-puro vector was digested with BamHI/AgeI, the digested products were recovered by electrophoresis and exchanged, and the products were transformed into bacterial state cells. Colony PCR identification was performed on the grown clones first, and then sequencing and comparative analysis were performed on the positive clones identified by PCR. The correct comparison was the successful construction of the target plasmid;
b、慢病毒包装与滴度检测：将构建好的表达目的基因的重组病毒质粒及其两种辅助包装原件载体质粒,三种质粒载体分别进行高纯度无内毒素抽提,按Invitrogen公司Lipofectamine2000使用说明进行转染293T细胞,转染8h后更换为完全培养基,培养48h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度；b. Lentivirus packaging and titer detection: The constructed recombinant virus plasmid expressing the target gene and its two auxiliary packaging original vector plasmids, and the three plasmid vectors were subjected to high-purity and endotoxin-free extraction respectively, and were used according to Lipofectamine2000 of Invitrogen Company Instructions to transfect 293T cells, replace with complete medium after 8 hours of transfection, after 48 hours of culture, collect the cell supernatant rich in lentivirus particles, concentrate it to obtain a high-titer lentivirus concentrate, in 293T cells Determination and standardization of virus titer;
c、同时稳定表达PD-1中和抗体和GM-CSF的肿瘤细胞株的制备、筛选及表达检测：c. Preparation, screening and expression detection of tumor cell lines stably expressing PD-1 neutralizing antibody and GM-CSF at the same time:
将制备的慢病毒LV-anti-PD-1、LV-GM-CSF感染肿瘤细胞，慢病毒的感染复数范围为1-100,摸索不同肿瘤细胞的最佳感染系数,确定感染系数后先感染LV-anti-PD-1,用嘌呤霉素进行筛选,筛选后收集病毒感染的细胞培养上清液,用检测PD-1中和抗体的表达水平；将稳定感染LV-anti-PD-1的细胞再次感染LV-GM-CSF,用嘌呤霉素继续筛选；收集细胞上清液,检测GM-CSF表达,确认筛选的细胞株能够同时高表达PD-1中和抗体和GM-CSF；Infect tumor cells with the prepared lentiviruses LV-anti-PD-1 and LV-GM-CSF. The multiplicity of infection of the lentiviruses ranges from 1 to 100. Explore the optimal infection coefficients of different tumor cells. After determining the infection coefficients, first infect LV -anti-PD-1, screened with puromycin, collected virus-infected cell culture supernatant after screening, and used to detect the expression level of PD-1 neutralizing antibody; cells stably infected with LV-anti-PD-1 Infect LV-GM-CSF again, and continue to screen with puromycin; collect cell supernatant, detect GM-CSF expression, and confirm that the screened cell line can highly express PD-1 neutralizing antibody and GM-CSF at the same time;
d、制备高表达PD-1中和抗体和GM-CSF因子的肿瘤细胞疫苗：d. Prepare a tumor cell vaccine that highly expresses PD-1 neutralizing antibodies and GM-CSF factors:
将筛选出稳定感染了LV-GM-CSF和LV-anti-PD-1的肿瘤细胞株培养后计数后收集,经总辐射剂量60-160Gy的X射线辐照,并对辐照前后的肿瘤细胞进行计数、形态观察,通过CCK-8细胞增殖实验检测这些辐照前后细胞的增殖情况,利用ELISA检测辐照前后细胞表达PD-1中和抗体和GM-CSF蛋白的水平，筛选得到辐照后失去增殖能力且能高表达PD-1中和抗体和GM-CSF因子的细胞为肿瘤细胞疫苗。The selected tumor cell lines stably infected with LV-GM-CSF and LV-anti-PD-1 were cultured, counted, collected, irradiated with X-rays with a total radiation dose of 60-160Gy, and the tumor cells before and after irradiation were compared. Counting and morphological observation were carried out, and the proliferation of these cells before and after irradiation was detected by CCK-8 cell proliferation assay, and the expression levels of PD-1 neutralizing antibody and GM-CSF protein in cells before and after irradiation were detected by ELISA, and the post-irradiation cells were screened. Cells that lose their ability to proliferate and highly express PD-1 neutralizing antibodies and GM-CSF factors are tumor cell vaccines.
其中，上述的肿瘤为结肠癌、黑色素瘤、乳腺癌或肺癌。Wherein, the aforementioned tumor is colon cancer, melanoma, breast cancer or lung cancer.
本发明为了增强疫苗诱发的抗肿瘤免疫反应，解除肿瘤微环境的免疫抑制状态，将肿瘤疫苗与解除免疫抑制的抗体药物结合起来,这样既可以解除肿瘤微环境的免疫抑制状态,又可以增强抗肿瘤免疫反应,以获得更好的抗肿瘤效果。In order to enhance the anti-tumor immune response induced by the vaccine and relieve the immunosuppressive state of the tumor microenvironment, the present invention combines the tumor vaccine with the antibody drug that relieves the immunosuppression, so that the immunosuppressive state of the tumor microenvironment can be relieved, and the anti-tumor immune suppression state can be enhanced. Tumor immune response for better anti-tumor effect.
本发明的有益效果在于：本发明创造性地将肿瘤细胞疫苗治疗和解除肿瘤免疫抑制的抗体治疗结合起来，所制备的疫苗这样既可以解除肿瘤微环境的免疫抑制状态,又可以增强抗肿瘤免疫反应。在CT26细胞、LL/2细胞、B16细胞、4T1细胞等通用肿瘤模型中，这种同时表达PD-1中和抗体和GM-CSF因子的肿瘤细胞经亚致死剂量X射线(优选为60～120Gy)辐照制备的疫苗对肿瘤细胞的增殖产生了显著的抑制效应。制备的多种疫苗都取得了显著的抗肿瘤效果，其中以PD-1中和抗体和GM-CSF因子修饰的C26和B16-F10疫苗为例，C26疫苗治疗性动物实验结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达70.6％。B16-F10疫苗治疗性动物实验结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达64.9％。C26疫苗过继性动物实验结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达47.2％。B16-F10疫苗过继性动物实验结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达50.8％。可见本发明所制备的同时表达PD-1中和抗体和GM-CSF因子的肿瘤细胞疫苗，抗肿瘤疗效好，具有很好的开发价值，为肿瘤的免疫细胞治疗提供了新的选择。The beneficial effect of the present invention is that: the present invention creatively combines tumor cell vaccine treatment with antibody treatment to relieve tumor immunosuppression, so that the prepared vaccine can not only relieve the immunosuppressive state of the tumor microenvironment, but also enhance anti-tumor immune response . In general tumor models such as CT26 cells, LL/2 cells, B16 cells, and 4T1 cells, the tumor cells expressing both PD-1 neutralizing antibodies and GM-CSF factors were subjected to sublethal doses of X-rays (preferably 60-120Gy). ) irradiated vaccine produced a significant inhibitory effect on the proliferation of tumor cells. A variety of vaccines prepared have achieved significant anti-tumor effects. Taking PD-1 neutralizing antibody and GM-CSF factor-modified C26 and B16-F10 vaccines as examples, the results of therapeutic animal experiments of C26 vaccine showed that compared with the control group Compared with the co-expression of PD-1 neutralizing antibody and GM-CSF group, the tumor inhibition rate reached 70.6%. The results of B16-F10 vaccine therapeutic animal experiments showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 64.9%. The results of adoptive animal experiments of C26 vaccine showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 47.2%. The results of adoptive animal experiments of B16-F10 vaccine showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 50.8%. It can be seen that the tumor cell vaccine prepared by the present invention, which simultaneously expresses PD-1 neutralizing antibody and GM-CSF factor, has good anti-tumor efficacy, has good development value, and provides a new option for immune cell therapy of tumors.
附图说明Description of drawings
图1、Ubi-MCS-3FLAG-IRES-puro载体结构示意图；Figure 1. Schematic diagram of the Ubi-MCS-3FLAG-IRES-puro carrier structure;
图2、C26、4T1、B16-F10肿瘤细胞疫苗辐照前后PD-1中和和GM-CSF表达检测；Figure 2. Detection of PD-1 neutralization and GM-CSF expression before and after irradiation of C26, 4T1, and B16-F10 tumor cell vaccines;
图3、C26体内治疗性动物实验肿瘤生长曲线和生存期。与对照组相比共表达PD-1中和抗体和GM-CSF治疗组的抑瘤效果为70.6％、PD-1中和抗体组的抑瘤效果为38.5％、GM-CSF组的抑瘤效果为30.5％。此外，联合治疗组的小鼠生存期显著延长。Figure 3. The tumor growth curve and survival period of C26 therapeutic animal experiments in vivo. Compared with the control group, the anti-tumor effect of the co-expressed PD-1 neutralizing antibody and GM-CSF treatment group was 70.6%, the anti-tumor effect of the PD-1 neutralizing antibody group was 38.5%, and the anti-tumor effect of the GM-CSF group was 70.6%. was 30.5%. In addition, the survival time of mice in the combined treatment group was significantly prolonged.
图4、B16-F10体内治疗性动物实验肿瘤生长曲线和生存期。与对照组相比共表达PD-1中和抗体和GM-CSF治疗组的抑瘤效果为64.9％、PD-1中和抗体组的抑瘤效果为55.7％、GM-CSF组的抑瘤效果为20.9％。此外，联合治疗组的小鼠生存期显著延长。Figure 4. B16-F10 in vivo therapeutic animal experiment tumor growth curve and survival period. Compared with the control group, the anti-tumor effect of the co-expressed PD-1 neutralizing antibody and GM-CSF treatment group was 64.9%, the anti-tumor effect of the PD-1 neutralizing antibody group was 55.7%, and the anti-tumor effect of the GM-CSF group was 20.9%. In addition, the survival time of mice in the combined treatment group was significantly prolonged.
图5、C26疫苗过继性动物实验肿瘤生长曲线和生存情况。Figure 5. The tumor growth curve and survival status of C26 vaccine adoptive animal experiments.
图6、B16-F10疫苗过继性动物实验肿瘤生长曲线和生存情况。Fig. 6. Tumor growth curve and survival of B16-F10 vaccine adoptive animal experiment.
具体实施方式Detailed ways
以下结合附图通过对具体实施方式的描述以详细说明但不限制本发明。The following describes in detail but does not limit the present invention through the description of specific embodiments in conjunction with the accompanying drawings.
本发明的主要思路是将分别表达PD-1中和抗体和GM-CSF基因的慢病毒载体共同感染肿瘤细胞，筛选得到稳定表达PD-1中和抗体和GM-CSF基因序列的细胞株。将所筛选到同时高表达PD-1中和抗体和GM-CSF蛋白的稳定株细胞经亚致死剂量X射线(50-150Gy，优选60-120Gy)辐照制备而成。其中能表达PD-1中和抗体及GM-CSF基因的载体或宿主细胞是作为主要的活性成分。在本领域，常用X射线制备肿瘤疫苗，而通常通过辐照后使肿瘤细胞丧失增殖活性和成瘤性，但能正常贴壁瘤。这种状态本领域称为亚致死，而能导致肿瘤细胞出现这种状态的辐照剂量称之为亚致死剂量。The main idea of the present invention is to co-infect tumor cells with lentiviral vectors expressing PD-1 neutralizing antibody and GM-CSF gene respectively, and screen to obtain cell lines stably expressing PD-1 neutralizing antibody and GM-CSF gene sequence. It is prepared by irradiating the stable strain cells with high expression of PD-1 neutralizing antibody and GM-CSF protein at the same time and sublethal dose of X-rays (50-150Gy, preferably 60-120Gy). Among them, the vector or host cell capable of expressing PD-1 neutralizing antibody and GM-CSF gene is used as the main active ingredient. In this field, X-rays are commonly used to prepare tumor vaccines, and usually after irradiation, tumor cells lose their proliferative activity and tumorigenicity, but can normally adhere to the tumor. This state is called sublethal in the field, and the radiation dose that can cause tumor cells to appear in this state is called sublethal dose.
将筛选出稳定感染了LV-GM-CSF、LV-anti-PD-1和同时稳定感染了LV-GM-CSF和LV-anti-PD-1的肿瘤细胞株培养后计数后收集,经总辐射剂量60-160Gy的X射线辐照,并对辐照前后的肿瘤细胞进行计数、形态观察,通过CCK-8细胞增殖实验检测这些辐照前后细胞的增殖情况,利用ELISA检测辐照前后细胞表达PD-1中和抗体和GM-CSF蛋白的水平，以优化亚致死辐照剂量。The selected tumor cell lines that were stably infected with LV-GM-CSF, LV-anti-PD-1, and simultaneously stably infected with LV-GM-CSF and LV-anti-PD-1 were collected after culture, counted, and irradiated. X-ray irradiation with a dose of 60-160Gy, and counting and morphological observation of tumor cells before and after irradiation, the proliferation of these cells before and after irradiation were detected by CCK-8 cell proliferation assay, and the expression of PD in cells before and after irradiation was detected by ELISA. -1 levels of neutralizing antibodies and GM-CSF protein to optimize sublethal irradiation dose.
通过优化，各种肿瘤细胞的较优的辐照剂量为：肺癌细胞系辐照剂量80-150Gy，优选120Gy。乳腺癌细胞系辐照剂量50-100Gy，优选60Gy。黑色素瘤细胞系B16-F10辐照剂量60-120Gy，优选100Gy。结肠癌细胞系C26辐照剂量60-120Gy，优选100Gy。Through optimization, the optimal irradiation dose of various tumor cells is: the irradiation dose of lung cancer cell lines is 80-150Gy, preferably 120Gy. The radiation dose of the breast cancer cell line is 50-100Gy, preferably 60Gy. Melanoma cell line B16-F10 is irradiated with a dose of 60-120Gy, preferably 100Gy. The colon cancer cell line C26 is irradiated with a dose of 60-120Gy, preferably 100Gy.
本发明实施例中制备了4种不同的肿瘤细胞疫苗：肺癌细胞系LL/2(辐照剂量80-150Gy，优选120Gy),乳腺癌细胞系4T1(50-100Gy，优选60Gy),黑色素瘤细胞系B16-F10(60-120Gy，优选100Gy),结肠癌C26(60-120Gy，优选100Gy)。慢病毒的感染复数范围为1-100,摸索不同肿瘤细胞的最佳感染系数,确定感染系数后先感染LV-anti-PD-1,用嘌呤霉素进行筛选,筛选后收集病毒感染的细胞培养上清液,用间接ELISA检测PD-1中和抗体的表达水平。将稳定感染LV-anti-PD-1的细胞再次感染LV-GM-CSF,显微镜下观察绿色荧光蛋白表达,用嘌呤霉素继续筛选。收集细胞上清液,用Elisa方法检测GM-CSF表达,确认筛选的细胞株能够同时高表达PD-1中和抗体和GM-CSF。Four different tumor cell vaccines were prepared in the examples of the present invention: lung cancer cell line LL/2 (irradiation dose 80-150Gy, preferably 120Gy), breast cancer cell line 4T1 (50-100Gy, preferably 60Gy), melanoma cell line Line B16-F10 (60-120Gy, preferably 100Gy), colon cancer C26 (60-120Gy, preferably 100Gy). The multiplicity of infection of the lentivirus ranges from 1 to 100. Explore the optimal infection coefficient of different tumor cells. After determining the infection coefficient, first infect LV-anti-PD-1, screen with puromycin, and collect virus-infected cells for culture after screening. In the supernatant, the expression level of PD-1 neutralizing antibody was detected by indirect ELISA. The cells stably infected with LV-anti-PD-1 were re-infected with LV-GM-CSF, the expression of green fluorescent protein was observed under a microscope, and the selection was continued with puromycin. The cell supernatant was collected, and the expression of GM-CSF was detected by Elisa method, and it was confirmed that the screened cell lines could highly express PD-1 neutralizing antibody and GM-CSF at the same time.
需要说明的是，在小鼠动物实验时，故使用的是小鼠GM-CSF。基于人和小鼠在药物实验中的替代性，本领域技术人员根据本发明的实验结果有理由相信使用人GM-CSF仍然具有相近的效果。It should be noted that mouse GM-CSF was used in the mouse experiment. Based on the substitution of human and mouse in drug experiments, those skilled in the art have reason to believe that the use of human GM-CSF still has similar effects based on the experimental results of the present invention.
实施例一PD-1中和抗体和GM-CSF基因的获取以及LV-anti-PD-1、LV-GM-CSF慢病毒载体的构建Example 1 Acquisition of PD-1 neutralizing antibody and GM-CSF gene and construction of LV-anti-PD-1, LV-GM-CSF lentiviral vector
根据美国专利局递交的anti-PD-1基因序列(patentNo:US20030026800A1)，NCBI递交的mGM-CSF基因序列(NM_009969.4)，hGM-CSF基因序列(NM_000758.3)分别合成了anti-PD-1基因(核苷酸序列如SEQIDNO.7所示，编码的蛋白质的氨基酸序列如SEQIDNO.8所示)、mGM-CSF基因(核苷酸序列如SEQIDNO.9所示，编码的蛋白质的氨基酸序列如SEQIDNO.10所示)，hGM-CSF基因(核苷酸序列如SEQIDNO.11所示，编码的蛋白质的氨基酸序列如SEQIDNO.12所示)。并构建成pcDNA3.1-anti-PD-1、pcDNA3.1-mGM-CSF和pcDNA3.1-hGM-CSF质粒(由南京金斯瑞公司合成)。然后利用PCR法分别扩增PD-1中和抗体、mGM-CSF和hGM-CSF目的基因,将Ubi-MCS-3FLAG-IRES-puro目的载体进行BamHI/AgeI酶切,酶切产物电泳回收后进行交换,其产物转化细菌感受态细胞。对长出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对正确的即为构建成功的目的质粒。Anti-PD- 1 gene (the nucleotide sequence is shown in SEQIDNO.7, the amino acid sequence of the encoded protein is shown in SEQIDNO.8), mGM-CSF gene (the nucleotide sequence is shown in SEQIDNO.9, the amino acid sequence of the encoded protein shown in SEQ ID NO.10), hGM-CSF gene (the nucleotide sequence is shown in SEQ ID NO.11, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.12). And construct pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF and pcDNA3.1-hGM-CSF plasmids (synthesized by Nanjing GenScript). Then PCR method was used to amplify the target genes of PD-1 neutralizing antibody, mGM-CSF and hGM-CSF respectively, and the target vector of Ubi-MCS-3FLAG-IRES-puro was digested with BamHI/AgeI, and the digested products were recovered by electrophoresis and carried out Exchange, and its product transforms bacterial competent cells. Colony PCR identification was performed on the grown clones first, and then the positive clones identified by PCR were sequenced and compared. The correct comparison was the successful construction of the target plasmid.
上述构建主要使用的引物序列如下所示：The primer sequences mainly used in the above construction are as follows:
扩增PD-1中和抗体的上游引物(SEQIDNO.1):Amplify the upstream primer of PD-1 neutralizing antibody (SEQ ID NO.1):
5'-AGGTCGACTCTAGAGGATCCCGCCACCATGGCTTGGGTGTGGACCTTGC-3’(下划线表示引入的BamHI酶切位点)5'-AGGTCGACTCTAGAGGATCC CGCCACCATGGCTTGGGTGTGGACCTTGC-3' (the underline indicates the introduced BamHI restriction site)
扩增PD-1中和抗体的下游引物(SEQIDNO.2):Amplify the downstream primer of PD-1 neutralizing antibody (SEQ ID NO.2):
5'-AGTCCATGGTGGCGACCGGACACTCATTCCTGTTGAAGCTC-3(下划线表示引入的AgeI酶切位点)5'-AGTCCATGGTGGCGACCGGA CACTCATTCCTGTTGAAGCTC-3 (the underline indicates the introduced AgeI restriction site)
扩增mGM-CSF的上游引物(SEQIDNO.3):Amplify the upstream primer of mGM-CSF (SEQ ID NO.3):
5'-GAGGATCCCCGGGTACCGGTCGCCACCATGTGGCTGCAGAATTTAC-3’(下划线表示引入的BamHI酶切位点)5'-GAGGATCC CCGGGTACCGGTCGCCACCATGTGGCTGCAGAATTTAC-3' (the underline indicates the introduced BamHI restriction site)
扩增mGM-CSF的下游引物(SEQIDNO.4):Amplify the downstream primer (SEQ ID NO.4) of mGM-CSF:
5'-TCACCATGGTGGCGACCGGTTTTTGGCCTGGTTTTTTGC-3(下划线表示引入的AgeI酶切位点)5'-TCACCATGGTGGCGACCGGT TTTTGGCCTGGTTTTTTGC-3 (the underline indicates the introduced AgeI restriction site)
扩增hGM-CSF的上游引物(SEQIDNO.5):Amplify the upstream primer of hGM-CSF (SEQ ID NO.5):
5'-GAGGATCCCAGAGCCTGCTGCTCTTGG-3’(下划线表示引入的BamHI酶切位点)5'-GAGGATCC CAGAGCCTGCTGCTCTTGG-3' (the underline indicates the introduced BamHI restriction site)
扩增hGM-CSF的下游引物(SEQIDNO.6):Amplify the downstream primer of hGM-CSF (SEQ ID NO.6):
5'-TCACCATGGTGGCGACCGGTCCAGCAGTCAAAGGGGATGA-3(下划线表示引入的AgeI酶切位点)5'-TCACCATGGTGGCGACCGGT CCAGCAGTCAAAGGGGATGA-3 (the underline indicates the introduced AgeI restriction site)
将上述将构建好的表达目的基因的重组病毒质粒及其两种辅助包装原件载体质粒,三种质粒载体分别进行高纯度无内毒素抽提,按Invitrogen公司Lipofectamine2000使用说明进行转染293T细胞,转染后8h更换为完全培养基,培养48h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。得到LV-anti-PD-1、LV-mGM-CSF、LV-hGM-CSF病毒。The above-mentioned recombinant viral plasmid expressing the target gene and its two auxiliary packaging original vector plasmids, and the three plasmid vectors were subjected to high-purity and endotoxin-free extraction respectively, and were transfected into 293T cells according to the instructions of Lipofectamine 2000 of Invitrogen Company. After 8 hours of infection, the complete medium was replaced. After 48 hours of culture, the cell supernatant rich in lentivirus particles was collected and concentrated to obtain a high-titer lentivirus concentrate. The virus titer was measured and calibrated in 293T cells. LV-anti-PD-1, LV-mGM-CSF, and LV-hGM-CSF viruses were obtained.
实施例二本发明产品C26、4T1、B16-F10肿瘤细胞疫苗的制备以及辐照前后PD-1中和抗体和GM-CSF表达水平检测。Example 2 Preparation of C26, 4T1, B16-F10 tumor cell vaccines of the present invention and detection of expression levels of PD-1 neutralizing antibody and GM-CSF before and after irradiation.
使用上述得到的慢病毒感染结肠癌细胞C26(辐照剂量100Gy)、乳腺癌细胞4T1(辐照剂量100Gy)、黑色素瘤细胞B16-F10细胞株(辐照剂量60Gy)，然后使用X射线辐照失去增殖活性。用同样的方法也制备得到了人肿瘤细胞肺癌细胞系A549,乳腺癌细胞系MCF-7,黑色素瘤细胞系A375,结肠癌HCT116的细胞疫苗。Use the lentivirus obtained above to infect colon cancer cell C26 (irradiation dose 100Gy), breast cancer cell 4T1 (irradiation dose 100Gy), melanoma cell line B16-F10 (irradiation dose 60Gy), and then use X-ray irradiation loss of proliferative activity. The cell vaccines of human tumor cell lung cancer cell line A549, breast cancer cell line MCF-7, melanoma cell line A375 and colon cancer HCT116 were also prepared by the same method.
将筛选得到的稳定表达PD-1中和抗体和GM-CSF的结肠癌细胞C26、乳腺癌细胞4T1、黑色素瘤细胞B16-F10细胞株进行培养，48h后收集细胞上清。同时收集细胞辐照后继续培养48h的细胞上清。Elisa检测辐照前后细胞上清中PD-1中和抗体和GM-CSF的表达水平。结果参见图2。The colon cancer cell C26, breast cancer cell 4T1, and melanoma cell B16-F10 cell lines stably expressing PD-1 neutralizing antibody and GM-CSF were cultured, and the cell supernatant was collected after 48 hours. At the same time, the supernatant of cells cultured for 48 h after irradiation was collected. The expression levels of PD-1 neutralizing antibody and GM-CSF in the cell supernatant before and after irradiation were detected by ELISA. See Figure 2 for the results.
实施例三本发明产品C26疫苗治疗性动物实验Embodiment three Therapeutic animal experiment of product C26 vaccine of the present invention
每组20只Balb/c小鼠,在每只小鼠右侧皮下接种5×105C26细胞,每3天测量1次肿瘤体积,于接种C26细胞后第3d、6d、9d用1×106辐照后的肿瘤细胞疫苗连续治疗3次。治疗分组包括:辐照后的C26细胞组、感染LV的C26细胞辐照组、感染LV-GM-CSF的C26细胞辐照组、感染LV-anti-PD-1的C26细胞辐照组、同时感染LV-GM-CSF及LV-anti-PD-1的细胞辐照组,并设立未处理组作为空白对照。观察各组小鼠的肿瘤生长和小鼠的生存期,每个实验重复3次，结果参见图3。结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达70.6％。能显著减小肿瘤体积，增加动物存活率和存活时间。20 Balb/c mice in each group were subcutaneously inoculated with 5×105 C26 cells on the right side of each mouse, and the tumor volume was measured every 3 days.6 Continuous treatment with tumor cell vaccine after irradiation for 3 times. Treatment groups include: C26 cell group after irradiation, C26 cell irradiation group infected with LV, irradiation group of C26 cell infected with LV-GM-CSF, irradiation group of C26 cell infected with LV-anti-PD-1, simultaneously The cells infected with LV-GM-CSF and LV-anti-PD-1 were irradiated group, and the untreated group was set up as blank control. The tumor growth and the survival period of the mice in each group were observed, and each experiment was repeated 3 times. The results are shown in FIG. 3 . The results showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 70.6%. Can significantly reduce tumor volume, increase animal survival rate and survival time.
实施例四本发明产品B16-F10疫苗治疗性动物实验Embodiment 4 Therapeutic animal experiment of product B16-F10 vaccine of the present invention
每组20只C57小鼠,在每只小鼠右侧皮下接种5×105B16-F10细胞,每3天测量1次肿瘤体积,于接种B16-F10细胞后第3d、6d、9d用1×106辐照后的肿瘤细胞疫苗连续治疗3次。治疗分组包括:辐照后的B16-F10细胞组、感染LV的B16-F10细胞辐照组、感染LV-GM-CSF的B16-F10细胞辐照组、感染LV-anti-PD-1的B16-F10细胞辐照组、同时感染LV-GM-CSF及LV-anti-PD-1的细胞辐照组,并设立未处理组作为空白对照。观察各组小鼠的肿瘤生长和小鼠的生存期,每个实验重复3次，结果参见图4。结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达64.9％。能显著减小肿瘤体积，增加动物存活率和存活时间。20 C57 mice in each group were subcutaneously inoculated with 5×105 B16-F10 cells on the right side of each mouse, and the tumor volume was measured every 3 days. ×106 irradiated tumor cell vaccines were treated continuously for 3 times. Treatment groups include: irradiated B16-F10 cell group, LV-infected B16-F10 cell-irradiated group, LV-GM-CSF-infected B16-F10 cell-irradiated group, LV-anti-PD-1 infected B16 -F10 cell irradiation group, cell irradiation group simultaneously infected with LV-GM-CSF and LV-anti-PD-1, and an untreated group was set up as a blank control. The tumor growth and the survival period of the mice in each group were observed, and each experiment was repeated 3 times. The results are shown in FIG. 4 . The results showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 64.9%. Can significantly reduce tumor volume, increase animal survival rate and survival time.
实施例五本发明产品C26疫苗过继性动物实验Embodiment 5 Product C26 vaccine adoptive animal experiment of the present invention
每组10只Balb/c小鼠,按照治疗性免疫方案重复进行动物实验,免疫结束后分离各组小鼠脾脏淋巴细胞,分别在接种后3d、5d、7d、9d将分离后的小鼠脾脏淋巴细胞通过尾静脉注射的方式对荷瘤小鼠进行过继治疗,每组10只小鼠,1×107cells/每只。每只荷瘤小鼠右侧皮下接种5×105C26细胞,每3天测量1次肿瘤体积。观察各组小鼠的肿瘤生长和小鼠的生存期,每个实验重复3次，结果参见图5。结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达47.2％。能显著减小肿瘤体积，增加动物存活率和存活时间。Ten Balb/c mice were in each group, and the animal experiments were repeated according to the therapeutic immunization protocol. After the immunization, the spleen lymphocytes of the mice in each group were isolated, and the isolated mouse spleens were separated 3d, 5d, 7d, and 9d after inoculation, respectively. The tumor-bearing mice were adoptively treated by injecting lymphocytes into the tail vein, with 10 mice in each group, 1×107 cells/mouse. The right side of each tumor-bearing mouse was subcutaneously inoculated with 5×105 C26 cells, and the tumor volume was measured every 3 days. The tumor growth and the survival period of the mice in each group were observed, and each experiment was repeated 3 times. The results are shown in FIG. 5 . The results showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 47.2%. Can significantly reduce tumor volume, increase animal survival rate and survival time.
实施例六本发明产品B16-F10疫苗过继性动物实验Embodiment 6 Adoptive animal experiment of product B16-F10 vaccine of the present invention
每组10只C57小鼠,按照治疗性免疫方案重复进行动物实验,免疫结束后分离各组小鼠脾脏淋巴细胞,分别在接种后3d、5d、7d、9d将分离后的小鼠脾脏淋巴细胞通过尾静脉注射的方式对荷瘤小鼠进行过继治疗,每组10只小鼠,1×107cells/每只。每只荷瘤小鼠右侧皮下接种5×105B16-F10细胞,每3天测量1次肿瘤体积。观察各组小鼠的肿瘤生长和小鼠的生存期,每个实验重复3次，结果参见图6。结果表明，与对照组相比共表达PD-1中和抗体和GM-CSF组的抑瘤率达50.8％。能显著减小肿瘤体积，增加动物存活率和存活时间。Ten C57 mice in each group were subjected to repeated animal experiments according to the therapeutic immunization protocol. After the immunization, the spleen lymphocytes of mice in each group were isolated, and the isolated mouse spleen lymphocytes were collected 3 days, 5 days, 7 days and 9 days after inoculation respectively. Tumor-bearing mice were adoptively treated by tail vein injection, 10 mice in each group, 1×107 cells/mouse. The right side of each tumor-bearing mouse was subcutaneously inoculated with 5×105 B16-F10 cells, and the tumor volume was measured every 3 days. The tumor growth and the survival period of the mice in each group were observed, and each experiment was repeated 3 times. The results are shown in FIG. 6 . The results showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing antibody and GM-CSF group reached 50.8%. Can significantly reduce tumor volume, increase animal survival rate and survival time.
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20151111 | |
| RJ01 | Rejection of invention patent application after publication |