CN116036138A - Application of oncolytic virus VG161 in colorectal cancer treatment and sensitivity prediction method - Google Patents
Application of oncolytic virus VG161 in colorectal cancer treatment and sensitivity prediction method Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及医药技术应用领域，具体是指溶瘤病毒VG161在治疗结直肠癌中的应用及敏感性预测方法。The invention relates to the application field of medical technology, in particular to the application and sensitivity prediction method of oncolytic virus VG161 in the treatment of colorectal cancer.
背景技术Background technique
结直肠癌是全球高发和常见的恶性肿瘤，我国结直肠癌的发病率和死亡率呈现逐年上升趋势。远处转移的结直肠癌缺乏较好的治疗手段；结直肠癌的整体疗效提高缓慢，是目前结直肠癌治疗过程中需要迫切解决的两个问题，需要进一步发现新的治疗方式和手段。随着对肠癌免疫学和肿瘤微环境研究的不断深入，免疫治疗有望成为继手术，放疗，化疗和分子靶向治疗后新兴的治疗手段。Colorectal cancer is a high incidence and common malignant tumor in the world, and the incidence and mortality of colorectal cancer in my country are showing an increasing trend year by year. There is a lack of better treatment methods for colorectal cancer with distant metastases; the overall curative effect of colorectal cancer is slowly improving, which are two problems that need to be urgently solved in the current treatment of colorectal cancer, and new treatment methods and methods need to be further discovered. With the deepening of research on colon cancer immunology and tumor microenvironment, immunotherapy is expected to become an emerging treatment after surgery, radiotherapy, chemotherapy and molecular targeted therapy.
溶瘤病毒是通过对自然界存在的一些致病力较弱的病毒进行基因改造制成的特殊的溶瘤病毒，其利用靶细胞中抑癌基因的失活或缺陷从而选择性地感染肿瘤细胞，在其内大量复制并最终摧毁肿瘤细胞。溶瘤病毒还可以通过表达毒性蛋白、诱导炎性细胞因子及T细胞介导的免疫反应提高免疫系统对肿瘤细胞的抗肿瘤效应。这些病毒无法在正常机体内复制而不具有杀伤作用，因此溶瘤病毒的选择性杀伤作用有更高的效率和更低的副作用。通过基因工程技术，我们人为插入免疫细胞刺激因子和免疫检查点激动剂或抑制剂，通过溶瘤病毒的介导在肿瘤细胞内高表达这些基因，提高了治疗的靶向性。溶瘤病毒在肿瘤免疫治疗中的应用：较早溶瘤病毒T-Vec已上市，其用于治疗黑色素瘤，名为Imlygic。T-Vec是经过基因修饰的1型单纯疱疹病毒(HSV-1)，它可以在肿瘤细胞内复制并表达免疫激活蛋白粒细胞-巨噬细胞集落刺激因子(GM-CSF)。将其直接注射到黑色素瘤病灶中可造成肿瘤细胞的溶解，从而使肿瘤细胞破裂，并释放出肿瘤源性抗原和GM-CSF，加速抗肿瘤的免疫应答。Oncolytic virus is a special oncolytic virus made by genetically modifying some weakly pathogenic viruses that exist in nature. It uses the inactivation or defect of tumor suppressor genes in target cells to selectively infect tumor cells. It replicates in large numbers and eventually destroys tumor cells. Oncolytic viruses can also enhance the anti-tumor effect of the immune system on tumor cells by expressing toxic proteins, inducing inflammatory cytokines, and T cell-mediated immune responses. These viruses cannot replicate in the normal body and do not have a killing effect, so the selective killing effect of oncolytic viruses has higher efficiency and lower side effects. Through genetic engineering technology, we artificially insert immune cell stimulators and immune checkpoint agonists or inhibitors, and highly express these genes in tumor cells through the mediation of oncolytic viruses, improving the targeting of treatment. Application of oncolytic virus in tumor immunotherapy: The earlier oncolytic virus T-Vec has been marketed, and it is used to treat melanoma, named Imlygic. T-Vec is a genetically modified herpes simplex virus type 1 (HSV-1) that can replicate in tumor cells and express the immune-activating protein granulocyte-macrophage colony-stimulating factor (GM-CSF). Injecting it directly into melanoma lesions can cause the lysis of tumor cells, thereby rupturing tumor cells, releasing tumor-derived antigens and GM-CSF, and accelerating the anti-tumor immune response.
抗PD1/PDL1在结直肠癌的整体疗效较差，但对于存在微卫星不稳定的结直肠癌患者效果较好，考虑与微卫星不稳定的结直肠癌错配修复基因缺失导致肿瘤抗原性增加相关，因此如何增强PD1/PDL1抑制剂对超过85％的MSS结直肠癌的治疗作用，是目前利PD1/PDL1抑制剂治疗结直肠癌领域的研究重点。The overall effect of anti-PD1/PDL1 in colorectal cancer is poor, but the effect is better for colorectal cancer patients with microsatellite instability, considering that the loss of mismatch repair genes in colorectal cancer with microsatellite instability leads to increased tumor antigenicity Therefore, how to enhance the therapeutic effect of PD1/PDL1 inhibitors on more than 85% of MSS colorectal cancers is the current research focus in the field of using PD1/PDL1 inhibitors to treat colorectal cancers.
此外，研究表明CRC是一种免疫源性疾病，其复杂多变的分子异质性导致了免疫治疗在结直肠癌中的疗效一般。虽然多种药物联用，促进肿瘤裂解，增加肿瘤抗原蛋白的释放，增强CRC免疫原性具有可行性。但是多种药物联合免疫治疗有难以逾越的障碍：多个药物同时给药带来的系统毒性问题；同时使用多个药物产生的高额费用；协调管控多个药物的给药、预防交叉反应和相互作用所带来的复杂性都是我们不可避免要越过的阻碍。In addition, studies have shown that CRC is an immunogenic disease, and its complex and variable molecular heterogeneity leads to the mediocre efficacy of immunotherapy in colorectal cancer. Although it is feasible to promote tumor lysis, increase the release of tumor antigen protein, and enhance the immunogenicity of CRC by combining multiple drugs. However, there are insurmountable obstacles in multi-drug combination immunotherapy: systemic toxicity caused by simultaneous administration of multiple drugs; The complexities that come with interactions are barriers that we inevitably have to jump over.
研究表明，使用牛痘病毒改造的溶瘤病毒感染癌细胞后，激活更多的T细胞进入肿瘤组织，增加了癌细胞PD-L1的表达。同时加入PD-1/PD-L1抑制剂就有可能取得叠加效果，使得那些原本对PD-1/PD-L1抑制剂不敏感的“冷”肿瘤变得敏感，成为“热”肿瘤。溶瘤病毒由于可以同时携带多个免疫调控因子/抗体药物，在瘤内表达能够解决上述难题，因此具有较高的效价比和可行性。Studies have shown that after infecting cancer cells with an oncolytic virus modified by vaccinia virus, more T cells are activated to enter tumor tissue and increase the expression of PD-L1 in cancer cells. Adding PD-1/PD-L1 inhibitors at the same time may achieve additive effects, making those "cold" tumors that were originally insensitive to PD-1/PD-L1 inhibitors sensitive and become "hot" tumors. Oncolytic viruses can carry multiple immune regulatory factors/antibody drugs at the same time, and their expression in tumors can solve the above problems, so they have high potency and feasibility.
发明内容Contents of the invention
本发明针对上述问题，提出溶瘤病毒VG161在治疗结直肠癌中的应用，具体地是，溶瘤病毒VG161对人结直肠癌有增强抑制作用，在人原代结直肠癌3D培养组织和小鼠荷瘤模型，溶瘤病毒VG161可以激活免疫反应，以及增强抗结直肠肿瘤的免疫记忆。In view of the above problems, the present invention proposes the application of oncolytic virus VG161 in the treatment of colorectal cancer, specifically, the oncolytic virus VG161 has an enhanced inhibitory effect on human colorectal cancer, and it can be used in 3D cultured tissues and small In a mouse tumor-bearing model, oncolytic virus VG161 can activate immune responses and enhance immune memory against colorectal tumors.
本发明提供的技术方案为：溶瘤病毒VG161在治疗结直肠癌中的应用。The technical solution provided by the invention is: the application of oncolytic virus VG161 in the treatment of colorectal cancer.
进一步地，所述溶瘤病毒VG161的构建基于1型单纯疱疹病毒。Further, the construction of the oncolytic virus VG161 is based on herpes
进一步地，所述溶瘤病毒VG161经基因改造后携带和表达IL12、IL15/IL15RA和PD-L1阻断肽。Further, the oncolytic virus VG161 is genetically modified to carry and express IL12, IL15/IL15RA and PD-L1 blocking peptides.
进一步地，所述治疗产生免疫记忆。Further, the treatment produces immune memory.
本发明另一方面提供了溶瘤病毒VG161在治疗结直肠癌中的敏感性预测方法，包括如下步骤：Another aspect of the present invention provides a sensitivity prediction method for oncolytic virus VG161 in the treatment of colorectal cancer, comprising the following steps:
步骤一：通过共培养溶瘤病毒VG161与肠癌细胞系，检测肠癌细胞的细胞毒性；Step 1: Detect the cytotoxicity of intestinal cancer cells by co-cultivating oncolytic virus VG161 and intestinal cancer cell lines;
步骤二：VG161感染所述肠癌细胞系之后检测ICP27p(病毒启动子)的复制，进一步筛选对VG161敏感细胞系；Step 2: After VG161 infects the intestinal cancer cell line, detect the replication of ICP27p (virus promoter), and further screen the VG161-sensitive cell line;
步骤三：通过RNA-seq检测敏感细胞系和不敏感细胞系的基因表达谱，寻找病毒易感标志物；Step 3: Detect the gene expression profiles of sensitive cell lines and insensitive cell lines by RNA-seq, and look for virus susceptibility markers;
步骤四：通过qPCR检测病毒易感标志物在肠癌细胞系中的表达谱来预测VG161对于结直肠癌治疗的敏感性。Step 4: Predict the sensitivity of VG161 to the treatment of colorectal cancer by detecting the expression profile of virus susceptibility markers in colorectal cancer cell lines by qPCR.
进一步地，所述病毒易感标志物为NYNRIN。Further, the virus susceptibility marker is NYNRIN.
本发明有益效果在于：溶瘤病毒VG161在人原代结直肠癌3D培养组织和CT26细胞小鼠荷瘤模型中，我们发现携带表达IL12、IL15/IL15RA和抗PD-L1肽的溶瘤病毒VG161对人结直肠癌有增强抑制作用，同时溶瘤病毒VG161可以激活免疫反应，以及增强抗结直肠肿瘤的免疫记忆。通过溶瘤病毒VG161对结直肠癌细胞系的体外杀伤作用，我们筛查出对VG161杀伤敏感细胞系。同时RNA-seq比较敏感细胞系和非敏感细胞系的基因表达谱差异，我们寻找出与敏感系相关的基因标记NYNRIN，并在肠癌细胞系中进行了qPCR实验进一步验证了敏感性。The beneficial effect of the present invention is that: oncolytic virus VG161 was found to carry oncolytic virus VG161 expressing IL12, IL15/IL15RA and anti-PD-L1 peptide in human primary colorectal cancer 3D culture tissue and CT26 cell mouse tumor-bearing model It has an enhanced inhibitory effect on human colorectal cancer, and at the same time, the oncolytic virus VG161 can activate the immune response and enhance the immune memory against colorectal tumors. Through the in vitro killing effect of oncolytic virus VG161 on colorectal cancer cell lines, we screened out cell lines sensitive to VG161 killing. At the same time, RNA-seq compared the gene expression profiles of sensitive and non-sensitive cell lines. We found the gene marker NYNRIN associated with sensitive lines, and performed qPCR experiments in intestinal cancer cell lines to further verify the sensitivity.
附图说明Description of drawings
图1为VG161溶瘤病毒示意图。Figure 1 is a schematic diagram of the VG161 oncolytic virus.
图2、图3、图4、图5为通过RNA-seq检测敏感细胞系和不敏感细胞系的基因表达谱示意图。Figure 2, Figure 3, Figure 4, and Figure 5 are schematic diagrams of the gene expression profiles of sensitive and insensitive cell lines detected by RNA-seq.
图6为溶瘤病毒VG161在小鼠结直肠肿瘤细胞荷瘤动物的杀伤治疗效果对比示意图。Fig. 6 is a schematic diagram showing the comparison of killing and therapeutic effects of oncolytic virus VG161 on mouse colorectal tumor cells in tumor-bearing animals.
图7为在人源类结直肠癌3D培养组织模型中，研究溶瘤病毒对原代结直肠癌细胞的杀伤作用以及治疗效果分析示意图。Figure 7 is a schematic diagram of the study of the killing effect of oncolytic virus on primary colorectal cancer cells and the analysis of therapeutic effect in the 3D cultured tissue model of human colorectal cancer.
具体实施方式Detailed ways
下面结合附图对本发明做进一步的详细说明。The present invention will be described in further detail below in conjunction with the accompanying drawings.
本发明针对现有技术中抗PD1/PDL1在结直肠癌的整体疗效较差的问题，我们构建了基于1型单纯疱疹病毒的溶瘤病毒，通过基因改造，该溶瘤病毒携带和表达IL12、IL15/IL15RA和PD-L1阻断肽，如图1所示，溶瘤病毒VG161在人原代结直肠癌3D培养组织和PDX小鼠模型中，我们发现携带表达IL12、IL15/IL15RA和抗PD-L1肽的溶瘤病毒VG161对人结直肠癌有增强抑制作用，同时溶瘤病毒VG161可以激活免疫反应，以及增强抗结直肠肿瘤的免疫记忆。The present invention aims at the poor overall curative effect of anti-PD1/PDL1 in colorectal cancer in the prior art. We have constructed an oncolytic virus based on herpes
在结直肠癌各细胞系中，研究溶瘤病毒VG161对结直肠癌细胞系的体外杀伤作用，筛查对VG161杀伤敏感细胞系，通过RNA-seq检测敏感细胞系和不敏感细胞系的基因表达谱，寻找病毒易感标志物。通过共培养VG161与肠癌细胞系，检测肠癌细胞的细胞毒性。溶瘤病毒感染之后检测ICP27p(病毒启动子)的复制，筛选对VG161溶瘤病毒敏感细胞系。通过RNA-seq比较敏感细胞系和非敏感细胞系的基因表达谱差异，寻找与敏感系相关的基因标记，通过RNA-seq检测敏感细胞系和不敏感细胞系的基因表达谱示意图参考图2、图3、图4、图5。In various colorectal cancer cell lines, study the killing effect of oncolytic virus VG161 on colorectal cancer cell lines in vitro, screen cell lines sensitive to VG161 killing, and detect the gene expression of sensitive and insensitive cell lines by RNA-seq Spectrum, looking for virus susceptibility markers. The cytotoxicity of intestinal cancer cells was detected by co-culturing VG161 and intestinal cancer cell lines. After oncolytic virus infection, the replication of ICP27p (virus promoter) was detected, and the cell lines sensitive to VG161 oncolytic virus were screened. Use RNA-seq to compare the differences in gene expression profiles between sensitive and non-sensitive cell lines, find gene markers related to sensitive lines, and use RNA-seq to detect the gene expression profiles of sensitive and insensitive cell lines. Refer to Figure 2, Figure 3, Figure 4, Figure 5.
溶瘤病毒VG161在小鼠结直肠肿瘤细胞荷瘤动物的杀伤治疗效果，参考图6。瘤内注射溶瘤病毒VG161，空载体病毒骨架以及PBS，肿瘤组织中和血清中分别检测human IL-12和human IgG4的表达，结果表明IL12和PDL-1B表达有限度上调，但血清中水平与对照相比没有明显变化。The killing effect of oncolytic virus VG161 on mouse colorectal tumor cell tumor-bearing animals, refer to Figure 6. Intratumoral injection of oncolytic virus VG161, empty vector virus backbone and PBS, the expression of human IL-12 and human IgG4 were detected in tumor tissue and serum, respectively. There was no significant change compared to the control.
在人源类结直肠癌3D培养组织模型中，研究溶瘤病毒对原代结直肠癌细胞的杀伤作用以及治疗效果分析，参考图7。In the 3D cultured tissue model of human colorectal cancer, the killing effect of oncolytic virus on primary colorectal cancer cells and the analysis of the therapeutic effect were studied, refer to Figure 7.
本发明及其实施方式进行了描述，这种描述没有限制性，附图中所述的也只是本发明的实施方式之一，实际的结构并不局限于此。总而言之如果本领域的普通技术人员受其启示，在不脱离本发明创造宗旨的情况下，不经创造性的设计出与该技术方案相似的结构方式及实施例，均应属于本发明的保护范围。The present invention and its implementations have been described, but this description is not limiting, and what is described in the drawings is only one of the implementations of the present invention, and the actual structure is not limited thereto. All in all, if a person of ordinary skill in the art is inspired by it, without departing from the inventive concept of the present invention, without creatively designing a structure and an embodiment similar to the technical solution, it shall fall within the scope of protection of the present invention.
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